Silencing of Integrated Human Papillomavirus Type
http://www.100md.com
病菌学杂志 2005年第7期
Queensland Institute of Medical Research and the University of Queensland, Brisbane, Queensland
Department of Infectious Diseases, University of Sydney, Sydney, New South Wales, Australia
Department of Physiology, University of Maryland School of Medicine, Rockville, Maryland
ABSTRACT
The serine protease inhibitor SerpinB2 (PAI-2), a major product of differentiating squamous epithelial cells, has recently been shown to bind and protect the retinoblastoma protein (Rb) from degradation. In human papillomavirus type 18 (HPV-18)-transformed epithelial cells the expression of the E6 and E7 oncoproteins is controlled by the HPV-18 upstream regulatory region (URR). Here we illustrate that PAI-2 expression in the HPV-18-transformed cervical carcinoma line HeLa resulted in the restoration of Rb expression, which led to the functional silencing of transcription from the HPV-18 URR. This caused loss of E7 protein expression and restoration of multiple E6- and E7-targeted host proteins, including p53, c-Myc, and c-Jun. Rb expression emerged as sufficient for the transcriptional repression of the URR, with repression mediated via the C/EBP?-YY1 binding site (URR 7709 to 7719). In contrast to HeLa cells, where the C/EBP?-YY1 dimer binds this site, in PAI-2- and/or Rb-expressing cells the site was occupied by the dominant-negative C/EBP? isoform liver-enriched transcriptional inhibitory protein (LIP). PAI-2 expression thus has a potent suppressive effect on HPV-18 oncogene transcription mediated by Rb and LIP, a finding with potential implications for prognosis and treatment of HPV-transformed lesions.
INTRODUCTION
SerpinB2, originally described as plasminogen activator inhibitor type 2 (PAI-2), is expressed by a range of cell types including activated macrophages and many tumors and is a major product of differentiating squamous epithelial cells (33, 43). PAI-2 was one of the first identified members of a unique and growing subclass of serine protease inhibitors (serpins) called ovalbumin-like serpins (ov-serpins) (49). Ov-serpin family members often appear to have nucleocytoplasmic distributions (8, 15), and many have intracellular activities: for instance, CrmA and PI9 are involved in apoptosis inhibition, MENT is involved in DNA binding, and Maspin and Headapin are involved in tumor suppression (8, 49). Although extracellular PAI-2 is well documented as an inhibitor of the extracellular protease urokinase-type plasminogen activator (31), PAI-2 was recently shown to have an additional intracellular activity as a retinoblastoma protein (Rb) binding protein (15). PAI-2 was found to bind the C pocket of Rb via a novel binding motif called the PENF homology motif, which is present in the large C-D interhelical loop region of PAI-2. PAI-2 expression resulted in decreased Rb turnover, with the subsequent increase in Rb levels causing an increase in Rb-mediated activities. The PAI-2-mediated increase in Rb protein levels required both Rb binding via the C-D interhelical region of PAI-2 and an intact reactive site loop (RSL), which plays a pivotal role in the known protease inhibitory activity of PAI-2 (15). The new Rb-associated role for intracellular PAI-2 may explain why PAI-2 expression is often able to confer a series of Rb-related phenotypes such as resistance to apoptosis (19, 23, 61), regulation of gene transcription (1, 37, 48), promotion of differentiation (29, 34, 57), and tumor suppression (20, 23, 31, 34, 38, 41, 56).
A dramatic phenotype resulting from stable PAI-2 expression in HeLa cells was recovery of Rb and loss of E7 protein levels in these human papillomavirus type 18 (HPV-18)-transformed cells (15). High-risk HPVs such as HPV-18 are often associated with cervical cancer (16), and cells from such cancers usually constitutively express the HPV oncoproteins E6 and E7 from HPV-derived DNA integrated into the host cell genome (36). E6 targets p53 and c-Myc, and E7 targets Rb and c-Jun for accelerated degradation, with the loss of these host proteins intimately associated with loss of cell cycle control and tumor development (9, 36). The PAI-2-associated loss of E7 expression suggested that PAI-2 expression somehow leads to suppression of oncogene transcription from the integrated HPV-18 DNA.
Transcription of HPV-18 E6-E7 mRNA is regulated by the HPV upstream regulatory region (URR) and is influenced by several cellular transcription factors (7, 39). There are a number of sites within this URR that (i) bind transcription factors known to interact with Rb (37) and (ii) are involved in the regulation of URR-dependent transcription. According to the URR numbering system described by Bednarek et al. (2, 7), such sites include Oct 1 (URR 7721-7735), AP-1 (URR 7791- 7798) (7), SP1 (URR 34-40) (7, 44), YY1 (URR 7846-13) (3), CDP (URR 7866-18) (39), and the C/EBP?-YY1 binding site (URR 7709-7719) originally referred to as the "switch region" (4, 5). This latter region contains a consensus CCAAT enhancer-binding protein ? (C/EBP?) site, which in HeLa cells is bound by a heterodimer comprising C/EBP? and YY1 (4, 5). Both these transcription factors are individually able to bind Rb (11, 40). The C/EBP?-YY1 binding site lies within the enhancer region (2, 7) of the HPV-18 URR, and in HeLa cells C/EBP?-YY1 binding to the C/EBP?-YY1 binding site causes a two- to threefold enhancement in transcription (4, 5). Although mutating the YY1 site (URR 7846-13) in the HPV-18 URR showed no effect on HPV-18 URR transcriptional activity in Rb-negative HeLa cells (44), YY1 binding to Rb is usually associated with changes in transcriptional activity (40). SP1 is also known to bind Rb (37), and naturally occurring mutations in this site (URR 34-40) enhanced transcription three- to fourfold in HeLa cells (44).
Here we provide insight into the mechanism by which PAI-2 expression results in the loss of HPV-18 oncoprotein activity in HeLa cells. Parental HeLa cells, like most HPV-transformed cells, express little or no Rb. However, Rb expression is restored in HeLa cells stably expressing PAI-2 (15). The PAI-2-mediated recovery of Rb expression resulted in transcriptional silencing of the integrated HPV-18 URR and a subsequent recovery of multiple E6- and E7-targeted host proteins. Repression of the integrated URR was due to an Rb-dependent recruitment to the C/EBP?-YY1 binding site of liver-enriched transcriptional inhibitory protein (LIP). C/EBP? mRNA encodes full-length C/EBP? (also known as LAP) and the smaller dominant-negative isoform LIP by virtue of an in-frame internal AUG translation initiation site (17). LIP can also be generated by proteolytic cleavage of C/EBP? (54). LIP lacks a transactivation domain, also binds the C/EBP? consensus site, and is associated with transcriptional repression (30, 32, 58). The C/EBP?/LIP ratio, which often regulates C/EBP?-dependent transcription of cellular genes (26, 52, 55), was not altered in the PAI-2-expressing cells. Regulation of the integrated HPV URR by Rb and LIP as a consequence of PAI-2 expression represents a novel cellular mechanism of HPV oncogene repression and suggests potential PAI-2-based therapeutic and/or diagnostic applications for HPV-transformed lesions.
MATERIALS AND METHODS
Cell culture. HeLa and the transfected HeLa cell lines were generated and maintained as described previously (15); S1a and S1b stably express PAI-2; A2/7 stably expresses antisense PAI-2; C-D PAI-2a and C-D PAI-2b stably express the C-D interhelical mutant of PAI-2; PAI-2 Ala380a and PAI-2 Ala380b stably express the RSL mutant of PAI-2. Weri, Weri Rb, Y79, and Y79 Rb transfectants were a kind gift from M. Madigan, Royal Prince Alfred Hospital, Sydney, Australia, and were induced to express Rb with 50 μM ZnSO4 for 24 h prior to use in experiments (59). Cell cultures were maintained in RPMI 1640 (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (CSL Ltd., Melbourne, Australia). Transfected cells were also maintained in 400 μg of Geneticin (Invitrogen Life Technologies)/ml.
Western blots. Western analysis used antibodies specific for PAI-2 (polyclonal antibody from American Diagnostica, Greenwich, Conn.); Rb (G3-245); p53 (DO-1) (BD PharMingen, Heidelberg, Germany); Waf1/p21 (187); c-Jun (H-79); c-Myc (9E10); HPV-18 E7 (N-19); glyceraldehyde-3-phosphate dehydrogenase (GAPDH; V-18); and actin (C-11), YY1 (H-10), and C/EBP? (H-7, which recognizes the C terminus) (Santa Cruz Biotechnology, Santa Cruz, Calif.). Horseradish peroxidase-conjugated secondary antibodies (Silenus, Melbourne, Australia) were detected using an ECL chemiluminescent detection system (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). Total cell, nuclear, and cytoplasmic extracts were generated as described previously (15). Protein concentrations were determined with the BCA-200 protein assay kit (Pierce, Rockford, Ill.), 20 μg was separated by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE), and proteins were transferred to Hybond-C nitrocellulose membranes (Amersham). For E7 detection 50 μg of cell protein was separated on 4 to 20% gradient gels (Gradipore, North Ryde, Australia) run at 150 V for 1 h.
Quantitative real-time reverse transcription-PCR. cDNA was prepared from 106 cells as described previously (15). PCR analysis used the following nucleotide primers for HPV-18 E7 (Genset, La Jolla, Calif.): 5'-GCTGAACCACAACGTCACAC-3' and 5'-GGTCGTCTGCTGGAGCTTTCT-3'. The amplification reaction mixture of 20 μl contained 0.1 μg of randomly primed cDNA, 0.5 μM (each) primer pair, 2x Platinum Quantitation PCR Supermix-UDG (Gibco BRL), and 10x SYBR Green (Molecular Probes, Eugene, Oreg.). The cycling conditions were one cycle of 96°C for 2 min, followed by 35 cycles of 96°C for 15 s, 56°C for 15 s, and 72°C for 15 s. Real-time PCR was performed using a Rotogene PCR machine (Corbett Research, Mortlake, Australia). PCR products were visualized using SYBR Green dye and analyzed by Rotogene real-time analysis software (Corbett Research). Quantitation was based on a standard curve established using dilutions of parental cell line GAPDH cDNA.
HPV-URR LUC assays. Cells (7 x 105) were transfected with 0.25 μg of the indicated HPV-URR reporter plasmids and 0.25 μg of a pCMV ?-galactosidase reporter plasmid as a transfection control (Promega, Madison, Wis.) with 3 μl of GeneJammer transfection reagent according to the manufacturer's instructions (Stratagene, La Jolla, Calif.). The parent HPV-URR luciferase (LUC) reporter plasmid and the same reporter plasmid with a mutation within the URR 34 to 40 SP1 site (SP1 mutant) or a mutation within the URR 7846 to 13 YY1 site (YY1 mutant) were generated previously (44). The C/EBP?-YY1 binding site mutant was generated by disruption of the C/EBP? site within the C/EBP?-YY1 binding site (URR 7704-7713; TTTTACTTAA to TTCCGCGGAA) as described previously (5), with the GeneTailor site-directed mutagenesis system (Invitrogen Life Technologies). At 72 h posttransfection cell lysates were analyzed using the LUC assay system (Promega), the Turner Designs TD-20/20 luminometer (Sunnyvale, Calif.), and the ?-galactosidase enzyme assay system with reporter lysis buffer (Promega).
EMSA experiments. Protein-DNA complexes were analyzed using the LightShift chemiluminescent electrophoretic mobility shift assay (EMSA) kit (Pierce). Briefly, 20 or 200 fmol of biotinylated double-stranded DNA (dsDNA) probe (Proligo, Australia Pty Ltd.) was added to 20-μl reaction mixtures containing 10 mM Tris, 50 mM KCl, 1 mM dithiothreitol (DTT), 2.5% glycerol, 5 mM MgCl2, 50 ng of poly(dI-dC)/μl, and 5 μg of nuclear extract. For antibody preincubation reactions, 2 μl of 200-μg/ml anti-C/EBP? antibody (H-7; Santa Cruz), anti-YY1 antibody (H-10; Santa Cruz), anti-Rb antibody (G3-245; BD PharMingen), or anti-PAI-2 antibody (American Diagnostica) was added to cell lysate containing the above reaction mixture for 1 h prior to addition of EMSA probes. After 30 min at room temperature complexes were resolved on 6% Tris-borate-EDTA-polyacrylamide gels at 90 V in 0.5% Tris-borate-EDTA buffer: 100 mM Tris HCl, 100 mM borate, and 2 mM EDTA, pH 8.3. Proteins were then transferred to Hybond N+ (Amersham Pharmacia Biotech), and biotinylated DNA was detected by chemiluminescence per the manufacturer's instructions. The C/EBP?-YY1 binding site biotinylated dsDNA oligonucleotides were as follows: forward, 5'-AGTTTGTTTTTACTTAAGCTA-3', and reverse, 5'-TAGCTTAAGTAAAAACAAACT-3' (URR 7701-7721). Mutant C/EBP?-YY1 binding site oligonucleotides containing a mutation in the C/EBP? binding site were as follows: forward, 5'-AGTTTGTTTCCGCGGAAGCTA-3' and reverse 5'-TACTTCCGCGGAAACAAACT-3' (5).
Streptavidin pull-down of C/EBP?-YY1-binding-site-bound proteins with biotinylated dsDNA probes. Nuclear cell lysates from 6 x 106 HeLa, S1a, Weri, and Weri Rb cells were isolated as described previously (15). Protein-DNA complexes were generated in 100-μl reaction mixtures consisting of 150 μg of nuclear cell extracts in 10 mM Tris-50 mM KCl-1 mM DTT-2.5% glycerol-5 mM MgCl2-50 ng of poly(dI-dC)/μl, to which 100 μM biotinylated or unlabeled dsDNA C/EBP?-YY1 binding site probe or mutated C/EBP?-YY1 binding site probe (described above) (Proligo, La Jolla, Calif.) was added. The reaction mixture was incubated at room temperature for 1 h, after which 30 μl of streptavidin immobilized on agarose beads (Sigma-Aldrich, Sydney, Australia) was added to the reaction mixture and incubated for a further 2 h at 4°C with rotation. The streptavidin-agarose beads were pelleted, and the supernatant was discarded. The pelleted streptavidin-agarose beads were washed once with 10 mM Tris-50 mM KCl-1 mM DTT-2.5% glycerol-5 mM MgCl2-0.1% NP-40, and DNA-protein complexes were run on an SDS-12% polyacrylamide gel. Proteins were transferred to a Hybond C membrane (Amersham), and bound proteins were detected by Western blotting with the antibodies described above.
siRNA inhibition of Rb transcription. S1a cells were grown to 50% confluence, and 200 mM double-stranded small inhibitory RNA (siRNA) (Proligo) was transfected by using the Oligofectamine transfection reagent according to the manufacturer's instructions (Invitrogen). The sequences of the Rb siRNA were previously described in reference 15. Nuclear cell lysates were analyzed 72 h posttransfection by streptavidin pull-down assays and Western blot assays with antibodies specific for Rb, C/EBP?, YY1, and PAI-2 as described above.
RESULTS
PAI-2 expression in HeLa cells results in the recovery of multiple E6- and E7-targeted proteins and loss of E7 expression. HeLa cells express little or no Rb, p53, c-Jun, or c-Myc, as E6 and E7 target these proteins for degradation. However, HeLa cells stably expressing PAI-2 (S1a and S1b) have readily detectable levels of all these proteins (Fig. 1A), illustrating that PAI-2 expression results in the restoration of multiple E6- and E7-targeted proteins. PAI-2 expression in HeLa cells also resulted in the restoration of the p53-regulated Waf1/p21 (9) (Fig. 1A). Control HeLa cells transfected with antisense PAI-2 (A2/7) behaved essentially like parental HeLa cells (Fig. 1A). HeLa cells stably expressing the RSL mutant of PAI-2 (PAI-2 Ala380a/b) or the C-D interhelical mutant of PAI-2 (C-D PAI-2a/b) failed to show recovery of Rb or p53 expression (Fig. 1B) (15). Importantly, PAI-2 expression was also associated with a loss of E7 protein (Fig. 1B, S1a), which was not observed in cells expressing the RSL or C-D mutants of PAI-2 (Fig. 1B). Thus, wild-type PAI-2 was required for restoration of Rb levels, loss of E7 protein, and recovery of the E6-targeted p53.
To determine whether the PAI-2-associated loss of E7 was due to loss of mRNA encoding E7, HeLa, A2/7, S1a, and S1b cells were analyzed for E7 mRNA by quantitative real-time PCR. In cells expressing PAI-2 (S1a and S1b) E7 mRNA was undetectable, whereas HeLa and A2/7 cells contained comparable levels of E7 mRNA (Fig. 1C).
These data confirmed and extended work from a previous study (15) and suggested that PAI-2 binding to and proteolytic protection of Rb (via the C-D interhelical and RSL regions, respectively) somehow lead to suppression of HPV oncoprotein transcription. The subsequent loss of oncoprotein expression then leads to the recovery of multiple E6- and E7-targeted proteins.
PAI-2 expression is associated with repression of HPV-18 URR transcriptional activity via the C/EBP?-YY1 binding site. To determine whether PAI-2 expression was associated with transcriptional repression of the E6-E7 promoter region (HPV-18 URR), a LUC reporter assay was employed in which LUC expression was controlled by the HPV-18 URR (44) (Fig. 2A). Parental HeLa cells, HeLa cells stably expressing antisense PAI-2 (A2/7), wild-type PAI-2 (S1a and S1b), the PAI-2 C-D interhelical mutant (C-D PAI-2), or the PAI-2 RSL mutant (PAI-2 Ala380) was transiently transfected with the wild-type HPV-18 URR LUC reporter construct. While the control HeLa cell lines (parental HeLa and A2/7) and the PAI-2 mutant cell lines (C-D PAI-2a and PAI-2 Ala380a) all showed similar levels of URR LUC reporter gene activity, PAI-2-expressing S1a and S1b cells showed only minimal transcriptional activity from this wild-type URR (Fig. 2B, wild-type URR). These data demonstrated that PAI-2 expression leads to silencing of transcription from the HPV URR, an observation consistent with the reported loss of the cotranscribed E6-E7 mRNA (15), loss of E7 protein expression (Fig. 1B), and recovery of E6- and E7-targeted proteins (Fig. 1A) in PAI-2-expressing HeLa cells.
The known interaction of PAI-2 and Rb (15) and the repression of the HPV URR in PAI-2-expressing cells (Fig. 2B, wild-type URR) prompted an analysis of transcription factors known both to regulate the HPV-18 URR and to interact with Rb (7). Despite the recovery of c-Jun (Fig. 1A), we were unable to detect a significant difference in AP-1 activity as determined by EMSA and reporter assays (unpublished data). Western analysis also showed that expression of other AP-1 protein family members was not significantly changed (unpublished data). To further investigate DNA response elements that might be involved in the postulated Rb-associated repression of the HPV-18 URR, HPV-18 URR-LUC reporter plasmids were used, in which the C/EBP?-YY1 binding site (URR 7709-7719), the YY1 site (URR 7846-13), or the SP1 site (URR 34-40) was disrupted (Fig. 2A). When these mutated HPV URR constructs were analyzed for LUC expression, PAI-2-mediated repression of the HPV-18 URR was maintained for the SP1 mutant and the YY1 mutant (Fig. 2B), while PAI-2-mediated repression was lost for the HPV-18 URR construct containing a mutated C/EBP?-YY1 binding site (Fig. 2B, C/EBP?-YY1 mutant). Thus, the C/EBP?-YY1 binding site within the enhancer region is implicated as the site of PAI-2-associated repression of the HPV-18 URR in HeLa cells.
The dominant-repressive C/EBP? isoform LIP binds the C/EBP?-YY1 binding site in PAI-2-expressing HeLa cells. To analyze the transcription factors bound to the C/EBP?-YY1 binding site, an assay was developed whereby nuclear lysates were incubated with biotinylated C/EBP?-YY1 binding site dsDNA probes. After incubation with nuclear lysates, the probes and any transcription factors bound to the probe were pulled down with streptavidin beads and analyzed by SDS-PAGE and Western blotting. As expected in HeLa cells (5), both C/EBP? and YY1 were found bound to the C/EBP?-YY1 binding site probe (Fig. 3A, lane 2). Minimal binding to unlabeled probe or to labeled mutated probe was observed (Fig. 3A, lanes 1 and 3), illustrating the specificity of this assay. Importantly, neither C/EBP? nor YY1 was found bound to the C/EBP?-YY1 binding site probe in PAI-2-expressing S1a cells (Fig. 3A, lane 5). Instead, a low-molecular-mass anti-C/EBP?-reactive band was found bound to this probe in the S1a cells (Fig. 3A, lane 5). Although present in S1a lysates (Fig. 3B, lane 2), neither Rb nor PAI-2 was found bound to the C/EBP?-YY1 binding site probe (Fig. 3A, lane 5). Minimal binding to unlabeled probe or to labeled mutated probe was again observed (Fig. 3A, lanes 4 and 6). The low-molecular-mass band on SDS gels (17 to 20 kDa) (51), the antibody reactivity (H-7 is specific for the C terminus of C/EBP? and therefore also reacts with LIP), and the binding to a known C/EBP? site (5) identified the C/EBP?-YY1-binding-site-binding factor in S1a cells as the dominant-negative C/EBP? isoform LIP. The replacement of C/EBP?-YY1 with LIP on this site in S1a cells was not associated with a change in the expression levels of these transcription factors in S1a cells compared with parental HeLa cells (Fig. 3B, lanes 1 and 2).
Rb expression is required for binding of LIP to the C/EBP?-YY1 binding site. The interaction of PAI-2 with Rb (15) and the established binding of Rb to LIP (11) suggested that Rb may be required for LIP recruitment to the C/EBP?-YY1 binding site in S1a cells. To determine if the binding of LIP to the C/EBP?-YY1 binding site (Fig. 3A, lane 2) required the presence of Rb, S1a cells were treated with siRNA specific for Rb. The potent reduction of Rb expression in S1a cells treated with siRNA was demonstrated by Western analysis of nuclear lysates and was not associated with any changes in YY1, C/EBP? LIP, or PAI-2 expression (Fig. 3B, lane 3). Importantly, siRNA-treated S1a cells showed a substantial reduction in LIP binding to the C/EBP?-YY1 binding site probe and restoration of C/EBP? and YY1 binding to this probe (Fig. 3A, lane 8) to levels similar to those for parental HeLa cells. Analysis of S1a cells treated with a control scrambled siRNA did not reproduce these observations, with LIP remaining bound to the C/EBP?-YY1 binding site probe (Fig. 3A, lane 11). These data demonstrated that Rb expression is required for binding of the transcriptionally repressive LIP to the C/EBP?-YY1 binding site, with loss of Rb (mediated by E7 in HeLa cells or siRNA in S1a cells) resulting in loss of LIP binding to this region.
Loss of C/EBP?-YY1 (complex I) binding to the HPV-18 C/EBP?-YY1 binding site in S1a cells. Formation of the C/EBP?-YY1 dimer and subsequent recruitment of this complex to the C/EBP?-YY1 binding site in HeLa cells are well described elsewhere (5, 6). To confirm that PAI-2 expression alters the transcription complexes bound to the HPV-18 C/EBP?-YY1 binding site, nuclear extracts from HeLa cells and PAI-2-expressing HeLa cells (S1a) were analyzed by EMSA for protein-DNA complexes (Fig. 4A). As expected, incubation of the C/EBP?-YY1 binding site probe with nuclear extracts of HeLa cells showed a slow-migrating doublet. The top band was previously identified as the C/EBP?-YY1 dimer, also known as complex I (4-6) (Fig. 4A, lane 5, arrow). Importantly, EMSA analysis of nuclear extracts from S1a cells showed that C/EBP?-YY1 was not bound to the C/EBP?-YY1 binding site in these cells (Fig. 4A, lane 7), consistent with the results shown in Fig. 3A (lane 5). As expected, a mutated C/EBP?-YY1 binding site probe (containing the same mutations as the probe used in Fig. 3) failed to bind this complex in both cell lines (Fig. 4A, lanes 6 and 8).
No novel complex that might contain LIP could be identified in S1a cells with the use of conditions that were optimized to detect C/EBP?-YY1 complex I bound to the C/EBP?-YY1 binding site probe (Fig. 4A). However, when 10-fold-more labeled C/EBP?-YY1 binding site probe was used, a novel, faster-migrating complex could be detected in these cells (Fig. 4B, lane 5, arrow). This complex was absent in HeLa cell lysates (Fig. 4B, lane 3) and S1a cells incubated with the mutant C/EBP?-YY1 binding site probe (Fig. 4B, lane 4). Preincubation of S1a cell lysates prior to the EMSA reaction with an antibody specific for C/EBP?/LIP resulted in loss of the new fast-migrating complex (Fig. 4B, lane 6). Binding of this new complex to the C/EBP?-YY1 binding site was not eliminated by preincubation with anti-YY1 antibody, anti-Rb antibody, or anti-PAI-2 antibody (Fig. 4B, lanes 7, 8, and 9, respectively). These EMSA experiments are in complete agreement with the data shown in Fig. 3, which show LIP but not C/EBP?, YY1, Rb, or PAI-2 bound to the C/EBP?-YY1 binding site probe in S1a cells. They confirm that PAI-2 expression in HeLa cells results in the loss of C/EBP?-YY1 binding to the C/EBP?-YY1 binding site and show that it is replaced by a fast-migrating, C/EBP?/LIP-antibody-reactive transcription factor.
Rb expression is sufficient for repression of transcription from the HPV-18 URR. To further explore the role of Rb in repression of the HPV-18 URR, the HPV-18 URR LUC reporter constructs used in Fig. 2 were tested in the Rb-negative Weri and Y79 retinoblastoma cell lines and the same cell lines stably expressing Rb (Weri Rb and Y79 Rb) (59). As Weri and Y79 cells do not express any HPV oncoproteins or PAI-2 (data not shown), the role of Rb in HPV-18 URR repression could be analyzed in the absence of any influences from E6, E7, or PAI-2. Wild-type HPV-18 URR transcriptional activity from the reporter construct (Fig. 5A) could be readily detected in the Rb-negative Weri and Y79 cell lines (Fig. 5B, wild-type URR). In contrast, transcriptional activity was repressed in Weri Rb and Y79 Rb lines (Fig. 5B, wild-type URR). Thus, Rb expression alone appeared sufficient for repression of HPV-18 transcription.
In a series of experiments that parallel those described in Fig. 2B, mutation of either the SP1 or YY1 site again had no effect on Rb-mediated repression (Fig. 5B). However, mutation of the C/EBP?-YY1 binding site again restored transcriptional activity from the HPV-18 URR in Rb-positive cell lines (Fig. 5B, C/EBP?-YY1 mutant). These data show that, in the absence of PAI-2 and HPV oncoproteins, Rb expression is associated with repression of the HPV-18 URR via the C/EBP?-YY1 binding site.
LIP is bound to the C/EBP?-YY1 binding site in Weri Rb cells. In a series of experiments that parallel those shown in Fig. 3, we sought to determine whether the transcriptional repression of the HPV-18 URR seen in Weri Rb cells (Fig. 5B) was also associated with the binding of LIP to the C/EBP?-YY1 binding site, as was observed in S1a cells (Fig. 3A, lane 5). Rb expression in the Weri Rb cells is illustrated in Fig. 3B, lane 2. In contrast to HeLa cells (Fig. 3A, lane 2), no C/EBP? or YY1 was pulled down from nuclear lysates of parental Weri cells by the C/EBP?-YY1 binding site probe (Fig. 6A, lane 2). In fact no C/EBP? could be detected in nuclear lysates of Weri or Weri Rb cells (Fig. 6B), suggesting that these cells may not express sufficient C/EBP? to be able to form detectable levels of complex I. As was observed for HeLa and S1a cells (Fig. 3B), the level of LIP and YY1 expression was not significantly different in the Weri and Weri Rb lines (Fig. 6B). The C/EBP?-YY1 binding site probe failed to pull down LIP in the Rb-negative Weri cells (Fig. 6A, lane 2), as was the case for the Rb-negative parental HeLa cells. However, LIP was found bound to the C/EBP?-YY1 binding site probe in Weri Rb cells (Fig. 6A, lane 5), as was the case for the Rb-expressing S1a cells. Thus, Rb expression in both S1a and Weri Rb cells was associated with LIP binding to the C/EBP?-YY1 binding site.
EMSA analysis of Weri and Weri Rb cell extracts with the C/EBP?-YY1 binding site probe. EMSA analysis of Weri cell extracts with the C/EBP?-YY1 binding site probe failed to show the slow-migrating doublet (Fig. 7A, lane 5, arrow) that is present in HeLa cells (Fig. 4A, lane 5, arrow). In HeLa cells the top band of this doublet was identified as complex I comprising C/EBP?-YY1 (5). The presence in Weri cells of only a single band in this region (Fig. 7A, lane 5, arrow) is consistent with the lack of complex I in these cells. This contention is supported by the lack of detectable C/EBP? by Western blotting (Fig. 6B), and the absence of C/EBP? or YY1 binding to the C/EBP?-YY1 binding site probe in these cells (Fig. 6A, lane 2). The identity of the lower band in the doublet remains unknown, and neither band is present in Weri Rb cells (Fig. 7A, lane 7). Importantly, the fast-migrating complex seen in S1a cells (Fig. 4B, lane 5) is also seen in Weri Rb cells (Fig. 7B, lane 5, arrow), and this complex can also be disrupted by the addition of anti-C/EBP?/LIP antibody (Fig. 7B, lane 6). Anti-YY1, -Rb, and -PAI-2 antibodies again failed to disrupt this complex (data not shown). This fast-migrating complex was not present in Weri cells (Fig. 7B, lane 3), nor was it detected in Weri Rb cells with the use of a mutated C/EBP?-YY1 binding site probe (Fig. 7, lane 4).
Thus, in both S1a cells and Weri Rb cells the presence of Rb was associated in the EMSA experiments with the presence of a fast-migrating, C/EBP?/LIP-antibody-reactive complex bound to the C/EBP?-YY1 binding site, which is absent in the Rb-negative HeLa and Weri cells. The only anti-C/EBP?/LIP-antibody-reactive complex pulled down by the C/EBP?-YY1 binding site probe in S1a cells and Weri Rb was identified as LIP (Fig. 3A, lane 5, and 6A, lane 5), and this was absent in HeLa and Weri cells. The fast-migrating complex identified in the EMSA experiments is likely therefore to contain LIP, with LIP binding to the C/EBP?-YY1 binding site associated with Rb expression.
DISCUSSION
PAI-2, although well documented as an inhibitor of the extracellular urokinase-type plasminogen activator (31), also has an important intranuclear role as a regulator of transcription (1, 15, 48), which is likely mediated by its interaction with Rb (15). Here we confirm that PAI-2 expression in HeLa cells results in the recovery of Rb expression (Fig. 1) and show that this leads to transcriptional silencing of the HPV-18 URR (Fig. 2). This in turn leads to the loss of E6 and E7 protein expression and the recovery of multiple E6- and E7-targeted proteins (Fig. 1). Transcriptional silencing of the HPV-18 URR was due to the Rb-dependent recruitment of dominant-negative repressor LIP to the C/EBP?-YY1 binding site (Fig. 3 to 7). In HeLa cells this was also associated with the loss of C/EBP?-YY1 binding to this region. Thus, PAI-2 expression protects Rb from degradation, Rb then causes recruitment of LIP to the C/EBP?-YY1 binding site, HPV oncogene transcription is silenced, and oncoprotein activity is lost.
We have demonstrated in two systems that recruitment of LIP to the C/EBP?-YY1 binding site of the HPV-18 URR is dependent on Rb expression; suppression of Rb translation by the use of siRNA resulted in loss of LIP binding to the C/EBP?-YY1 binding site in S1a cells, and expression of Rb in Weri cells resulted in LIP binding to this region. Rb expression did not cause upregulation of LIP. How Rb expression causes recruitment of LIP to the C/EBP?-YY1 binding site remains to be elucidated but may simply be a consequence of Rb associating with LIP to enhance LIP's DNA binding affinity to exceed that of C/EBP?-YY1. Rb binding to C/EBP? has been shown to enhance the DNA binding affinity of C/EBP? (11, 13), and Rb interacts with C/EBP? via the C-terminal bZIP domain of C/EBP?, which is also present in LIP (13). Although promoting the DNA binding affinity of C/EBP?, Rb does not remain associated with the DNA-bound C/EBP? (11), consistent with our finding that Rb was not found associated with LIP on the C/EBP?-YY1 binding site (Fig. 3A, lane 5, and 6A, lane 5). Alternatively, Rb expression may disrupt C/EBP?-YY1 dimer formation in HeLa cells by binding C/EBP? and/or YY1 (11, 40), allowing the dominant-negative LIP to bind to the C/EBP?-YY1 binding site (58). The Rb-dependent recruitment of LIP to the C/EBP?-YY1 binding site in Weri Rb cells, when Weri cells do not have detectable C/EBP?-YY1, might argue against this model. However, an unidentified, Rb-disruptable complex may be binding this site in Weri cells (Fig. 7A, lane 2, arrow).
The present study illustrates that at physiological levels functional Rb and LIP act together to suppress transcription in HPV-positive cells but that PAI-2 must be present in HPV-transformed cells to preserve Rb expression. The contention that Rb expression may be important for suppressing the HPV-18 URR is supported by a report from Salcedo et al. (47), who showed that overexpression of Rb in HPV-negative cells repressed transcription from an HPV-18 URR reporter construct. Interestingly, HepG2 cells also contain integrated HPV-18 DNA, but the URR is transcriptionally silenced (4, 5), and these cells express Rb (unpublished observation) and can express PAI-2 (21). Overexpression of LIP has also been shown to reduce transcription from HPV-16 reporter plasmids in HPV-18 and HPV-negative cells (51). Furthermore, we have recently determined that PAI-2 expression suppresses transcription from the HPV-16 URR (data not shown), perhaps suggesting a wider role for PAI-2 and Rb-dependent LIP recruitment in suppressing high-risk HPV oncogene transcription.
PAI-2 expression has been associated with improved prognosis in a number of different cancers (20, 31, 38, 41, 56). However, analysis of PAI-2 expression in malignancies often associated with HPV has been limited (14, 25). Nevertheless, transcriptional profiling suggests that reduced PAI-2 expression is associated with high-risk HPV (10, 46). One should also note that the PAI-2 gene is located at chromosomal locus 18q21, and this is one site that has been associated with loss of heterozygosity leading to tumor formation or progression in several studies of HPV-positive malignancies (24, 50, 60). The present study would predict that PAI-2 expression should represent an important suppressive factor in the development of high-risk HPV lesions, so long as Rb and LIP remain functional in these tissues.
The abilities of PAI-2 to protect Rb from E7-mediated degradation (Fig. 1B) and to repress transcription from the HPV-18 URR (Fig. 2B) were dependent on both the C-D interhelical region, which mediates Rb binding, and the RSL, which mediates protease inhibition. How PAI-2 inhibits Rb degradation remains unclear. However, Rb binding alone appears insufficient, since the RSL mutant, which retains Rb binding activity (15), is unable to inhibit Rb degradation (Fig. 1B, PAI-2 Ala380) and fails to inhibit HPV-18 URR transcription (Fig. 2B). The RSL regions of serpins usually act as a pseudosubstrate for target proteases, suggesting that PAI-2 may inhibit a nuclear protease. A number of proteases are known to be active in the nucleus and are known to target the pocket protein family comprising Rb, p130, and p107; these include calpain 1 (28), caspases (12), and Spase (27). We are currently exploring whether these proteases are the target of PAI-2 inhibition in vivo.
Repression of HPV-18 oncogene transcription can also be achieved in HeLa cells by expression of HPV E2, which binds and represses the URR promoter. Like PAI-2 expression, this also results in recovery of p53 and Rb but results in Rb-dependent senescence (42, 53). S1a and S1b cells do show increased G1 arrest (15) and elevated p21 expression (Fig. 1), both features associated with progression to senescence. The stably transfected S1a and S1b cell lines were selected on the basis of growth (18), and therefore any initial PAI-2-mediated senescence would have been overlooked but might be expected given the loss of E6 and E7.
Whether the PAI-2-associated repression, shown here for the integrated HPV-18 URR (Fig. 1) and for reporter plasmids encoding this URR (Fig. 2), also has a role in repressing E6-E7 transcription from full-length episomal HPV DNA remains to be investigated. How E6 and E7 oncoprotein-mediated inhibition of keratinocyte differentiation is overcome to allow later stages of the HPV viral life cycle is currently unclear (22). PAI-2 is absent in basal keratinocytes (35) but is a major product of differentiating keratinocytes (33, 43). PAI-2 probably contributes to promoting keratinocyte differentiation by elevating Rb levels (45), and this might also lead to downregulation of episomal E6 and E7 transcription. Interestingly, E2 has been shown to synergize with C/EBP? to upregulate C/EBP?-dependent gene transcription (22), and C/EBP? has recently been shown to be an important positive regulator of PAI-2 expression (B. W. Stringer et al., unpublished data).
This paper illustrates that PAI-2 expression has a potent suppressive effect on the transcription of oncogenes from integrated HPV-18 DNA that is mediated by Rb and LIP. These observations may have implications for the use of PAI-2-based reagents in diagnosis and/or treatment of HPV-associated lesions.
ACKNOWLEDGMENTS
We thank M. Madigan, Royal Prince Alfred Hospital, Sydney, for supplying the Weri and Y79 cell lines and R. Tindle (Director, Sir Albert Sakzewski Virus Research Centre, Brisbane, Australia) and I. A. Frazer (Director, The Centre for Immunology and Cancer Research, Brisbane, Australia) for their help with the manuscript.
This work was supported by grants from the NH&MRC of Australia, University of Queensland postgraduate scholarship, and the National Institutes of Health, United States (CA098369).
REFERENCES
Antalis, T. M., M. La Linn, K. Donnan, L. Mateo, J. Gardner, J. L. Dickinson, K. Buttigieg, and A. Suhrbier. 1998. The serine proteinase inhibitor (serpin) plasminogen activation inhibitor type 2 protects against viral cytopathic effects by constitutive interferon alpha/beta priming. J. Exp. Med. 187:1799-1811.
Badal, S., V. Badal, I. E. Calleja-Macias, M. Kalantari, L. S. Chuang, B. F. Li, and H. U. Bernard. 2004. The human papillomavirus-18 genome is efficiently targeted by cellular DNA methylation. Virology 324:483-492.
Bauknecht, T., P. Angel, H. D. Royer, and H. zur Hausen. 1992. Identification of a negative regulatory domain in the human papillomavirus type 18 promoter: interaction with the transcriptional repressor YY1. EMBO J. 11:4607-4617.
Bauknecht, T., F. Jundt, I. Herr, T. Oehler, H. Delius, Y. Shi, P. Angel, and H. Zur Hausen. 1995. A switch region determines the cell type-specific positive or negative action of YY1 on the activity of the human papillomavirus type 18 promoter. J. Virol. 69:1-12.
Bauknecht, T., R. H. See, and Y. Shi. 1996. A novel C/EBP ?-YY1 complex controls the cell-type-specific activity of the human papillomavirus type 18 upstream regulatory region. J. Virol. 70:7695-7705.
Bauknecht, T., and Y. Shi. 1998. Overexpression of C/EBP? represses human papillomavirus type 18 upstream regulatory region activity in HeLa cells by interfering with the binding of TATA-binding protein. J. Virol. 72:2113-2124.
Bednarek, P. H., B. J. Lee, S. Gandhi, E. Lee, and B. Phillips. 1998. Novel binding sites for regulatory factors in the human papillomavirus type 18 enhancer and promoter identified by in vivo footprinting. J. Virol. 72:708-716.
Bird, C. H., E. J. Blink, C. E. Hirst, M. S. Buzza, P. M. Steele, J. Sun, D. A. Jans, and P. I. Bird. 2001. Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1. Mol. Cell. Biol. 21:5396-5407.
Chakrabarti, O., and S. Krishna. 2003. Molecular interactions of ‘high risk’ human papillomaviruses E6 and E7 oncoproteins: implications for tumour progression. J. Biosci. 28:337-348.
Chang, Y. E., and L. A. Laimins. 2000. Microarray analysis identifies interferon-inducible genes and Stat-1 as major transcriptional targets of human papillomavirus type 31. J. Virol. 74:4174-4182.
Charles, A., X. Tang, E. Crouch, J. S. Brody, and Z. X. Xiao. 2001. Retinoblastoma protein complexes with C/EBP proteins and activates C/EBP-mediated transcription. J. Cell. Biochem. 83:414-425.
Chau, B. N., H. L. Borges, T. T. Chen, A. Masselli, I. C. Hunton, and J. Y. Wang. 2002. Signal-dependent protection from apoptosis in mice expressing caspase-resistant Rb. Nat. Cell Biol. 4:757-765.
Chen, P. L., D. J. Riley, S. Chen-Kiang, and W. H. Lee. 1996. Retinoblastoma protein directly interacts with and activates the transcription factor NF-IL6. Proc. Natl. Acad. Sci. USA 93:465-469.
Daneri-Navarro, A., G. Macias-Lopez, A. Oceguera-Villanueva, S. Del Toro-Arreola, A. Bravo-Cuellar, R. Perez-Montfort, and S. Orbach-Arbouys. 1998. Urokinase-type plasminogen activator and plasminogen activator inhibitors (PAI-1 and PAI-2) in extracts of invasive cervical carcinoma and precursor lesions. Eur. J. Cancer 34:566-569.
Darnell, G. A., T. M. Antalis, R. W. Johnstone, B. W. Stringer, S. M. Ogbourne, D. Harrich, and A. Suhrbier. 2003. Inhibition of retinoblastoma protein degradation by interaction with the serpin plasminogen activator inhibitor 2 via a novel consensus motif. Mol. Cell. Biol. 23:6520-6532.
Dell, G., and K. Gaston. 2001. Human papillomaviruses and their role in cervical cancer. Cell. Mol. Life Sci. 58:1923-1942.
Descombes, P., and U. Schibler. 1991. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell 67:569-579.
Dickinson, J. L., E. J. Bates, A. Ferrante, and T. M. Antalis. 1995. Plasminogen activator inhibitor type 2 inhibits tumor necrosis factor alpha-induced apoptosis. Evidence for an alternate biological function. J. Biol. Chem. 270:27894-27904.
Dickinson, J. L., B. J. Norris, P. H. Jensen, and T. M. Antalis. 1998. The C-D interhelical domain of the serpin plasminogen activator inhibitor-type 2 is required for protection from TNF-alpha induced apoptosis. Cell Death Differ. 5:163-171.
Foekens, J. A., F. Buessecker, H. A. Peters, U. Krainick, W. L. van Putten, M. P. Look, J. G. Klijn, and M. D. Kramer. 1995. Plasminogen activator inhibitor-2: prognostic relevance in 1012 patients with primary breast cancer. Cancer Res. 55:1423-1427.
Gohl, G., T. Lehmkoster, P. A. Munzel, D. Schrenk, R. Viebahn, and K. W. Bock. 1996. TCDD-inducible plasminogen activator inhibitor type 2 (PAI-2) in human hepatocytes, HepG2 and monocytic U937 cells. Carcinogenesis 17:443-449.
Hadaschik, D., K. Hinterkeuser, M. Oldak, H. J. Pfister, and S. Smola-Hess. 2003. The papillomavirus E2 protein binds to and synergizes with C/EBP factors involved in keratinocyte differentiation. J. Virol. 77:5253-5265.
Harbour, J. W., and D. C. Dean. 2000. The Rb/E2F pathway: expanding roles and emerging paradigms. Genes Dev. 14:2393-2409.
Harima, Y., S. Sawada, K. Nagata, M. Sougawa, and T. Ohnishi. 2001. Chromosome 6p21.2, 18q21.2 and human papilloma virus (HPV) DNA can predict prognosis of cervical cancer after radiotherapy. Int. J. Cancer 96:286-296.
Hasina, R., K. Hulett, S. Bicciato, C. Di Bello, G. J. Petruzzelli, and M. W. Lingen. 2003. Plasminogen activator inhibitor-2: a molecular biomarker for head and neck cancer progression. Cancer Res. 63:555-559.
Hattori, T., N. Ohoka, H. Hayashi, and K. Onozaki. 2003. C/EBP homologous protein (CHOP) up-regulates IL-6 transcription by trapping negative regulating NF-IL6 isoform. FEBS Lett. 541:33-39.
Irving, J. A., S. S. Shushanov, R. N. Pike, E. Y. Popova, D. Bromme, T. H. Coetzer, S. P. Bottomley, I. A. Boulynko, S. A. Grigoryev, and J. C. Whisstock. 2002. Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation. J. Biol. Chem. 277:13192-13201.
Jang, J. S., S. J. Lee, Y. H. Choi, P. M. Nguyen, J. Lee, S. G. Hwang, M. L. Wu, E. Takano, M. Maki, P. A. Henkart, and J. B. Trepel. 1999. Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease. Oncogene 18:1789-1796.
Jensen, P. J., Q. Wu, P. Janowitz, Y. Ando, and N. M. Schechter. 1995. Plasminogen activator inhibitor type 2: an intracellular keratinocyte differentiation product that is incorporated into the cornified envelope. Exp. Cell Res. 217:65-71.
Jover, R., R. Bort, M. J. Gomez-Lechon, and J. V. Castell. 2002. Down-regulation of human CYP3A4 by the inflammatory signal interleukin-6: molecular mechanism and transcription factors involved. FASEB J. 16:1799-1801.
Kruithof, E. K., M. S. Baker, and C. L. Bunn. 1995. Biological and clinical aspects of plasminogen activator inhibitor type 2. Blood 86:4007-4024.
Kuang, P. P., and R. H. Goldstein. 2003. Regulation of elastin gene transcription by interleukin-1 beta-induced C/EBP beta isoforms. Am. J. Physiol. Cell Physiol. 285:C1349-C1355.
Lian, X., and T. Yang. 2004. Plasminogen activator inhibitor 2: expression and role in differentiation of epidermal keratinocyte. Biol. Cell. 96:109-116.
Lipinski, M. M., and T. Jacks. 1999. The retinoblastoma gene family in differentiation and development. Oncogene 18:7873-7882.
Lyons-Giordano, B., D. Loskutoff, C. S. Chen, G. Lazarus, M. Keeton, and P. J. Jensen. 1994. Expression of plasminogen activator inhibitor type 2 in normal and psoriatic epidermis. Histochemistry 101:105-112.
McMurray, H. R., D. Nguyen, T. F. Westbrook, and D. J. McAnce. 2001. Biology of human papillomaviruses. Int. J. Exp. Pathol. 82:15-33.
Morris, E. J., and N. J. Dyson. 2001. Retinoblastoma protein partners. Adv. Cancer Res. 82:1-54.
Nakamura, M., H. Konno, T. Tanaka, Y. Maruo, N. Nishino, K. Aoki, S. Baba, S. Sakaguchi, Y. Takada, and A. Takada. 1992. Possible role of plasminogen activator inhibitor 2 in the prevention of the metastasis of gastric cancer tissues. Thromb. Res. 65:709-719.
O'Connor, M. J., W. Stunkel, C. H. Koh, H. Zimmermann, and H. U. Bernard. 2000. The differentiation-specific factor CDP/Cut represses transcription and replication of human papillomaviruses through a conserved silencing element. J. Virol. 74:401-410.
Petkova, V., M. J. Romanowski, I. Sulijoadikusumo, D. Rohne, P. Kang, T. Shenk, and A. Usheva. 2001. Interaction between YY1 and the retinoblastoma protein. Regulation of cell cycle progression in differentiated cells. J. Biol. Chem. 276:7932-7936.
Praus, M., K. Wauterickx, D. Collen, and R. D. Gerard. 1999. Reduction of tumor cell migration and metastasis by adenoviral gene transfer of plasminogen activator inhibitors. Gene Ther. 6:227-236.
Psyrri, A., R. A. DeFilippis, A. P. Edwards, K. E. Yates, L. Manuelidis, and D. DiMaio. 2004. Role of the retinoblastoma pathway in senescence triggered by repression of the human papillomavirus E7 protein in cervical carcinoma cells. Cancer Res. 64:3079-3086.
Risse, B. C., H. Brown, R. M. Lavker, J. M. Pearson, M. S. Baker, D. Ginsburg, and P. J. Jensen. 1998. Differentiating cells of murine stratified squamous epithelia constitutively express plasminogen activator inhibitor type 2 (PAI-2). Histochem. Cell Biol. 110:559-569.
Rose, B., G. Steger, X. P. Dong, C. Thompson, Y. Cossart, M. Tattersall, and H. Pfister. 1998. Point mutations in SP1 motifs in the upstream regulatory region of human papillomavirus type 18 isolates from cervical cancers increase promoter activity. J. Gen. Virol. 79:1659-1663.
Ruiz, S., M. Santos, C. Segrelles, H. Leis, J. L. Jorcano, A. Berns, J. M. Paramio, and M. Vooijs. 2004. Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis. Development 131:2737-2748.
Ruutu, M., P. Peitsaro, B. Johansson, and S. Syrjanen. 2002. Transcriptional profiling of a human papillomavirus 33-positive squamous epithelial cell line which acquired a selective growth advantage after viral integration. Int. J. Cancer 100:318-326.
Salcedo, M., E. Garrido, L. Taja, and P. Gariglio. 1995. The retinoblastoma gene product negatively regulates cellular or viral oncogene promoters in vivo. Arch. Med. Res. 26:S157-S162.
Shafren, D. R., J. Gardner, V. H. Mann, T. M. Antalis, and A. Suhrbier. 1999. Picornavirus receptor down-regulation by plasminogen activator inhibitor type 2. J. Virol. 73:7193-7198.
Silverman, G. A., P. I. Bird, R. W. Carrell, F. C. Church, P. B. Coughlin, P. G. Gettins, J. A. Irving, D. A. Lomas, C. J. Luke, R. W. Moyer, P. A. Pemberton, E. Remold-O'Donnell, G. S. Salvesen, J. Travis, and J. C. Whisstock. 2001. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J. Biol. Chem. 276:33293-33296.
Steenbergen, R. D., M. A. Hermsen, J. M. Walboomers, H. Joenje, F. Arwert, C. J. Meijer, and P. J. Snijders. 1995. Integrated human papillomavirus type 16 and loss of heterozygosity at 11q22 and 18q21 in an oral carcinoma and its derivative cell line. Cancer Res. 55:5465-5471.
Struyk, L., E. van der Meijden, R. Minnaar, V. Fontaine, I. Meijer, and J. ter Schegget. 2000. Transcriptional regulation of human papillomavirus type 16 LCR by different C/EBP? isoforms. Mol. Carcinog. 28:42-50.
Su, W. C., H. Y. Chou, C. J. Chang, Y. M. Lee, W. H. Chen, K. H. Huang, M. Y. Lee, and S. C. Lee. 2003. Differential activation of a C/EBP beta isoform by a novel redox switch may confer the lipopolysaccharide-inducible expression of interleukin-6 gene. J. Biol. Chem. 278:51150-51158.
Wells, S. I., B. J. Aronow, T. M. Wise, S. S. Williams, J. A. Couget, and P. M. Howley. 2003. Transcriptome signature of irreversible senescence in human papillomavirus-positive cervical cancer cells. Proc. Natl. Acad. Sci. USA 100:7093-7098.
Welm, A. L., N. A. Timchenko, and G. J. Darlington. 1999. C/EBP regulates generation of C/EBP? isoforms through activation of specific proteolytic cleavage. Mol. Cell. Biol. 19:1695-1704.
Xiong, W., C. C. Hsieh, A. J. Kurtz, J. P. Rabek, and J. Papaconstantinou. 2001. Regulation of CCAAT/enhancer-binding protein-beta isoform synthesis by alternative translational initiation at multiple AUG start sites. Nucleic Acids Res. 29:3087-3098.
Yoshino, H., Y. Endo, Y. Watanabe, and T. Sasaki. 1998. Significance of plasminogen activator inhibitor 2 as a prognostic marker in primary lung cancer: association of decreased plasminogen activator inhibitor 2 with lymph node metastasis. Br. J. Cancer 78:833-839.
Yu, H., F. Maurer, and R. L. Medcalf. 2002. Plasminogen activator inhibitor type 2: a regulator of monocyte proliferation and differentiation. Blood 99:2810-2818.
Zahnow, C. A., P. Younes, R. Laucirica, and J. M. Rosen. 1997. Overexpression of C/EBPbeta-LIP, a naturally occurring, dominant-negative transcription factor, in human breast cancer. J. Natl. Cancer Inst. 89:1887-1891.
Zhang, M., G. Stevens, and M. C. Madigan. 2001. In vitro effects of radiation on human retinoblastoma cells. Int. J. Cancer 96(Suppl.):7-14.
Zhao, M., and X. X. Wu. 2002. [Analysis of genetic instabilities on chromosome loci 3, 6, 11, and 18 in human cervical carcinoma.] Ai Zheng 21:640-643.
Zhou, H. M., I. Bolon, A. Nichols, A. Wohlwend, and J. D. Vassalli. 2001. Overexpression of plasminogen activator inhibitor type 2 in basal keratinocytes enhances papilloma formation in transgenic mice. Cancer Res. 61:970-976.(Grant A. Darnell, Toni M.)
Department of Infectious Diseases, University of Sydney, Sydney, New South Wales, Australia
Department of Physiology, University of Maryland School of Medicine, Rockville, Maryland
ABSTRACT
The serine protease inhibitor SerpinB2 (PAI-2), a major product of differentiating squamous epithelial cells, has recently been shown to bind and protect the retinoblastoma protein (Rb) from degradation. In human papillomavirus type 18 (HPV-18)-transformed epithelial cells the expression of the E6 and E7 oncoproteins is controlled by the HPV-18 upstream regulatory region (URR). Here we illustrate that PAI-2 expression in the HPV-18-transformed cervical carcinoma line HeLa resulted in the restoration of Rb expression, which led to the functional silencing of transcription from the HPV-18 URR. This caused loss of E7 protein expression and restoration of multiple E6- and E7-targeted host proteins, including p53, c-Myc, and c-Jun. Rb expression emerged as sufficient for the transcriptional repression of the URR, with repression mediated via the C/EBP?-YY1 binding site (URR 7709 to 7719). In contrast to HeLa cells, where the C/EBP?-YY1 dimer binds this site, in PAI-2- and/or Rb-expressing cells the site was occupied by the dominant-negative C/EBP? isoform liver-enriched transcriptional inhibitory protein (LIP). PAI-2 expression thus has a potent suppressive effect on HPV-18 oncogene transcription mediated by Rb and LIP, a finding with potential implications for prognosis and treatment of HPV-transformed lesions.
INTRODUCTION
SerpinB2, originally described as plasminogen activator inhibitor type 2 (PAI-2), is expressed by a range of cell types including activated macrophages and many tumors and is a major product of differentiating squamous epithelial cells (33, 43). PAI-2 was one of the first identified members of a unique and growing subclass of serine protease inhibitors (serpins) called ovalbumin-like serpins (ov-serpins) (49). Ov-serpin family members often appear to have nucleocytoplasmic distributions (8, 15), and many have intracellular activities: for instance, CrmA and PI9 are involved in apoptosis inhibition, MENT is involved in DNA binding, and Maspin and Headapin are involved in tumor suppression (8, 49). Although extracellular PAI-2 is well documented as an inhibitor of the extracellular protease urokinase-type plasminogen activator (31), PAI-2 was recently shown to have an additional intracellular activity as a retinoblastoma protein (Rb) binding protein (15). PAI-2 was found to bind the C pocket of Rb via a novel binding motif called the PENF homology motif, which is present in the large C-D interhelical loop region of PAI-2. PAI-2 expression resulted in decreased Rb turnover, with the subsequent increase in Rb levels causing an increase in Rb-mediated activities. The PAI-2-mediated increase in Rb protein levels required both Rb binding via the C-D interhelical region of PAI-2 and an intact reactive site loop (RSL), which plays a pivotal role in the known protease inhibitory activity of PAI-2 (15). The new Rb-associated role for intracellular PAI-2 may explain why PAI-2 expression is often able to confer a series of Rb-related phenotypes such as resistance to apoptosis (19, 23, 61), regulation of gene transcription (1, 37, 48), promotion of differentiation (29, 34, 57), and tumor suppression (20, 23, 31, 34, 38, 41, 56).
A dramatic phenotype resulting from stable PAI-2 expression in HeLa cells was recovery of Rb and loss of E7 protein levels in these human papillomavirus type 18 (HPV-18)-transformed cells (15). High-risk HPVs such as HPV-18 are often associated with cervical cancer (16), and cells from such cancers usually constitutively express the HPV oncoproteins E6 and E7 from HPV-derived DNA integrated into the host cell genome (36). E6 targets p53 and c-Myc, and E7 targets Rb and c-Jun for accelerated degradation, with the loss of these host proteins intimately associated with loss of cell cycle control and tumor development (9, 36). The PAI-2-associated loss of E7 expression suggested that PAI-2 expression somehow leads to suppression of oncogene transcription from the integrated HPV-18 DNA.
Transcription of HPV-18 E6-E7 mRNA is regulated by the HPV upstream regulatory region (URR) and is influenced by several cellular transcription factors (7, 39). There are a number of sites within this URR that (i) bind transcription factors known to interact with Rb (37) and (ii) are involved in the regulation of URR-dependent transcription. According to the URR numbering system described by Bednarek et al. (2, 7), such sites include Oct 1 (URR 7721-7735), AP-1 (URR 7791- 7798) (7), SP1 (URR 34-40) (7, 44), YY1 (URR 7846-13) (3), CDP (URR 7866-18) (39), and the C/EBP?-YY1 binding site (URR 7709-7719) originally referred to as the "switch region" (4, 5). This latter region contains a consensus CCAAT enhancer-binding protein ? (C/EBP?) site, which in HeLa cells is bound by a heterodimer comprising C/EBP? and YY1 (4, 5). Both these transcription factors are individually able to bind Rb (11, 40). The C/EBP?-YY1 binding site lies within the enhancer region (2, 7) of the HPV-18 URR, and in HeLa cells C/EBP?-YY1 binding to the C/EBP?-YY1 binding site causes a two- to threefold enhancement in transcription (4, 5). Although mutating the YY1 site (URR 7846-13) in the HPV-18 URR showed no effect on HPV-18 URR transcriptional activity in Rb-negative HeLa cells (44), YY1 binding to Rb is usually associated with changes in transcriptional activity (40). SP1 is also known to bind Rb (37), and naturally occurring mutations in this site (URR 34-40) enhanced transcription three- to fourfold in HeLa cells (44).
Here we provide insight into the mechanism by which PAI-2 expression results in the loss of HPV-18 oncoprotein activity in HeLa cells. Parental HeLa cells, like most HPV-transformed cells, express little or no Rb. However, Rb expression is restored in HeLa cells stably expressing PAI-2 (15). The PAI-2-mediated recovery of Rb expression resulted in transcriptional silencing of the integrated HPV-18 URR and a subsequent recovery of multiple E6- and E7-targeted host proteins. Repression of the integrated URR was due to an Rb-dependent recruitment to the C/EBP?-YY1 binding site of liver-enriched transcriptional inhibitory protein (LIP). C/EBP? mRNA encodes full-length C/EBP? (also known as LAP) and the smaller dominant-negative isoform LIP by virtue of an in-frame internal AUG translation initiation site (17). LIP can also be generated by proteolytic cleavage of C/EBP? (54). LIP lacks a transactivation domain, also binds the C/EBP? consensus site, and is associated with transcriptional repression (30, 32, 58). The C/EBP?/LIP ratio, which often regulates C/EBP?-dependent transcription of cellular genes (26, 52, 55), was not altered in the PAI-2-expressing cells. Regulation of the integrated HPV URR by Rb and LIP as a consequence of PAI-2 expression represents a novel cellular mechanism of HPV oncogene repression and suggests potential PAI-2-based therapeutic and/or diagnostic applications for HPV-transformed lesions.
MATERIALS AND METHODS
Cell culture. HeLa and the transfected HeLa cell lines were generated and maintained as described previously (15); S1a and S1b stably express PAI-2; A2/7 stably expresses antisense PAI-2; C-D PAI-2a and C-D PAI-2b stably express the C-D interhelical mutant of PAI-2; PAI-2 Ala380a and PAI-2 Ala380b stably express the RSL mutant of PAI-2. Weri, Weri Rb, Y79, and Y79 Rb transfectants were a kind gift from M. Madigan, Royal Prince Alfred Hospital, Sydney, Australia, and were induced to express Rb with 50 μM ZnSO4 for 24 h prior to use in experiments (59). Cell cultures were maintained in RPMI 1640 (Invitrogen Life Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovine serum (CSL Ltd., Melbourne, Australia). Transfected cells were also maintained in 400 μg of Geneticin (Invitrogen Life Technologies)/ml.
Western blots. Western analysis used antibodies specific for PAI-2 (polyclonal antibody from American Diagnostica, Greenwich, Conn.); Rb (G3-245); p53 (DO-1) (BD PharMingen, Heidelberg, Germany); Waf1/p21 (187); c-Jun (H-79); c-Myc (9E10); HPV-18 E7 (N-19); glyceraldehyde-3-phosphate dehydrogenase (GAPDH; V-18); and actin (C-11), YY1 (H-10), and C/EBP? (H-7, which recognizes the C terminus) (Santa Cruz Biotechnology, Santa Cruz, Calif.). Horseradish peroxidase-conjugated secondary antibodies (Silenus, Melbourne, Australia) were detected using an ECL chemiluminescent detection system (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom). Total cell, nuclear, and cytoplasmic extracts were generated as described previously (15). Protein concentrations were determined with the BCA-200 protein assay kit (Pierce, Rockford, Ill.), 20 μg was separated by sodium dodecyl sulfate (SDS)-10% polyacrylamide gel electrophoresis (PAGE), and proteins were transferred to Hybond-C nitrocellulose membranes (Amersham). For E7 detection 50 μg of cell protein was separated on 4 to 20% gradient gels (Gradipore, North Ryde, Australia) run at 150 V for 1 h.
Quantitative real-time reverse transcription-PCR. cDNA was prepared from 106 cells as described previously (15). PCR analysis used the following nucleotide primers for HPV-18 E7 (Genset, La Jolla, Calif.): 5'-GCTGAACCACAACGTCACAC-3' and 5'-GGTCGTCTGCTGGAGCTTTCT-3'. The amplification reaction mixture of 20 μl contained 0.1 μg of randomly primed cDNA, 0.5 μM (each) primer pair, 2x Platinum Quantitation PCR Supermix-UDG (Gibco BRL), and 10x SYBR Green (Molecular Probes, Eugene, Oreg.). The cycling conditions were one cycle of 96°C for 2 min, followed by 35 cycles of 96°C for 15 s, 56°C for 15 s, and 72°C for 15 s. Real-time PCR was performed using a Rotogene PCR machine (Corbett Research, Mortlake, Australia). PCR products were visualized using SYBR Green dye and analyzed by Rotogene real-time analysis software (Corbett Research). Quantitation was based on a standard curve established using dilutions of parental cell line GAPDH cDNA.
HPV-URR LUC assays. Cells (7 x 105) were transfected with 0.25 μg of the indicated HPV-URR reporter plasmids and 0.25 μg of a pCMV ?-galactosidase reporter plasmid as a transfection control (Promega, Madison, Wis.) with 3 μl of GeneJammer transfection reagent according to the manufacturer's instructions (Stratagene, La Jolla, Calif.). The parent HPV-URR luciferase (LUC) reporter plasmid and the same reporter plasmid with a mutation within the URR 34 to 40 SP1 site (SP1 mutant) or a mutation within the URR 7846 to 13 YY1 site (YY1 mutant) were generated previously (44). The C/EBP?-YY1 binding site mutant was generated by disruption of the C/EBP? site within the C/EBP?-YY1 binding site (URR 7704-7713; TTTTACTTAA to TTCCGCGGAA) as described previously (5), with the GeneTailor site-directed mutagenesis system (Invitrogen Life Technologies). At 72 h posttransfection cell lysates were analyzed using the LUC assay system (Promega), the Turner Designs TD-20/20 luminometer (Sunnyvale, Calif.), and the ?-galactosidase enzyme assay system with reporter lysis buffer (Promega).
EMSA experiments. Protein-DNA complexes were analyzed using the LightShift chemiluminescent electrophoretic mobility shift assay (EMSA) kit (Pierce). Briefly, 20 or 200 fmol of biotinylated double-stranded DNA (dsDNA) probe (Proligo, Australia Pty Ltd.) was added to 20-μl reaction mixtures containing 10 mM Tris, 50 mM KCl, 1 mM dithiothreitol (DTT), 2.5% glycerol, 5 mM MgCl2, 50 ng of poly(dI-dC)/μl, and 5 μg of nuclear extract. For antibody preincubation reactions, 2 μl of 200-μg/ml anti-C/EBP? antibody (H-7; Santa Cruz), anti-YY1 antibody (H-10; Santa Cruz), anti-Rb antibody (G3-245; BD PharMingen), or anti-PAI-2 antibody (American Diagnostica) was added to cell lysate containing the above reaction mixture for 1 h prior to addition of EMSA probes. After 30 min at room temperature complexes were resolved on 6% Tris-borate-EDTA-polyacrylamide gels at 90 V in 0.5% Tris-borate-EDTA buffer: 100 mM Tris HCl, 100 mM borate, and 2 mM EDTA, pH 8.3. Proteins were then transferred to Hybond N+ (Amersham Pharmacia Biotech), and biotinylated DNA was detected by chemiluminescence per the manufacturer's instructions. The C/EBP?-YY1 binding site biotinylated dsDNA oligonucleotides were as follows: forward, 5'-AGTTTGTTTTTACTTAAGCTA-3', and reverse, 5'-TAGCTTAAGTAAAAACAAACT-3' (URR 7701-7721). Mutant C/EBP?-YY1 binding site oligonucleotides containing a mutation in the C/EBP? binding site were as follows: forward, 5'-AGTTTGTTTCCGCGGAAGCTA-3' and reverse 5'-TACTTCCGCGGAAACAAACT-3' (5).
Streptavidin pull-down of C/EBP?-YY1-binding-site-bound proteins with biotinylated dsDNA probes. Nuclear cell lysates from 6 x 106 HeLa, S1a, Weri, and Weri Rb cells were isolated as described previously (15). Protein-DNA complexes were generated in 100-μl reaction mixtures consisting of 150 μg of nuclear cell extracts in 10 mM Tris-50 mM KCl-1 mM DTT-2.5% glycerol-5 mM MgCl2-50 ng of poly(dI-dC)/μl, to which 100 μM biotinylated or unlabeled dsDNA C/EBP?-YY1 binding site probe or mutated C/EBP?-YY1 binding site probe (described above) (Proligo, La Jolla, Calif.) was added. The reaction mixture was incubated at room temperature for 1 h, after which 30 μl of streptavidin immobilized on agarose beads (Sigma-Aldrich, Sydney, Australia) was added to the reaction mixture and incubated for a further 2 h at 4°C with rotation. The streptavidin-agarose beads were pelleted, and the supernatant was discarded. The pelleted streptavidin-agarose beads were washed once with 10 mM Tris-50 mM KCl-1 mM DTT-2.5% glycerol-5 mM MgCl2-0.1% NP-40, and DNA-protein complexes were run on an SDS-12% polyacrylamide gel. Proteins were transferred to a Hybond C membrane (Amersham), and bound proteins were detected by Western blotting with the antibodies described above.
siRNA inhibition of Rb transcription. S1a cells were grown to 50% confluence, and 200 mM double-stranded small inhibitory RNA (siRNA) (Proligo) was transfected by using the Oligofectamine transfection reagent according to the manufacturer's instructions (Invitrogen). The sequences of the Rb siRNA were previously described in reference 15. Nuclear cell lysates were analyzed 72 h posttransfection by streptavidin pull-down assays and Western blot assays with antibodies specific for Rb, C/EBP?, YY1, and PAI-2 as described above.
RESULTS
PAI-2 expression in HeLa cells results in the recovery of multiple E6- and E7-targeted proteins and loss of E7 expression. HeLa cells express little or no Rb, p53, c-Jun, or c-Myc, as E6 and E7 target these proteins for degradation. However, HeLa cells stably expressing PAI-2 (S1a and S1b) have readily detectable levels of all these proteins (Fig. 1A), illustrating that PAI-2 expression results in the restoration of multiple E6- and E7-targeted proteins. PAI-2 expression in HeLa cells also resulted in the restoration of the p53-regulated Waf1/p21 (9) (Fig. 1A). Control HeLa cells transfected with antisense PAI-2 (A2/7) behaved essentially like parental HeLa cells (Fig. 1A). HeLa cells stably expressing the RSL mutant of PAI-2 (PAI-2 Ala380a/b) or the C-D interhelical mutant of PAI-2 (C-D PAI-2a/b) failed to show recovery of Rb or p53 expression (Fig. 1B) (15). Importantly, PAI-2 expression was also associated with a loss of E7 protein (Fig. 1B, S1a), which was not observed in cells expressing the RSL or C-D mutants of PAI-2 (Fig. 1B). Thus, wild-type PAI-2 was required for restoration of Rb levels, loss of E7 protein, and recovery of the E6-targeted p53.
To determine whether the PAI-2-associated loss of E7 was due to loss of mRNA encoding E7, HeLa, A2/7, S1a, and S1b cells were analyzed for E7 mRNA by quantitative real-time PCR. In cells expressing PAI-2 (S1a and S1b) E7 mRNA was undetectable, whereas HeLa and A2/7 cells contained comparable levels of E7 mRNA (Fig. 1C).
These data confirmed and extended work from a previous study (15) and suggested that PAI-2 binding to and proteolytic protection of Rb (via the C-D interhelical and RSL regions, respectively) somehow lead to suppression of HPV oncoprotein transcription. The subsequent loss of oncoprotein expression then leads to the recovery of multiple E6- and E7-targeted proteins.
PAI-2 expression is associated with repression of HPV-18 URR transcriptional activity via the C/EBP?-YY1 binding site. To determine whether PAI-2 expression was associated with transcriptional repression of the E6-E7 promoter region (HPV-18 URR), a LUC reporter assay was employed in which LUC expression was controlled by the HPV-18 URR (44) (Fig. 2A). Parental HeLa cells, HeLa cells stably expressing antisense PAI-2 (A2/7), wild-type PAI-2 (S1a and S1b), the PAI-2 C-D interhelical mutant (C-D PAI-2), or the PAI-2 RSL mutant (PAI-2 Ala380) was transiently transfected with the wild-type HPV-18 URR LUC reporter construct. While the control HeLa cell lines (parental HeLa and A2/7) and the PAI-2 mutant cell lines (C-D PAI-2a and PAI-2 Ala380a) all showed similar levels of URR LUC reporter gene activity, PAI-2-expressing S1a and S1b cells showed only minimal transcriptional activity from this wild-type URR (Fig. 2B, wild-type URR). These data demonstrated that PAI-2 expression leads to silencing of transcription from the HPV URR, an observation consistent with the reported loss of the cotranscribed E6-E7 mRNA (15), loss of E7 protein expression (Fig. 1B), and recovery of E6- and E7-targeted proteins (Fig. 1A) in PAI-2-expressing HeLa cells.
The known interaction of PAI-2 and Rb (15) and the repression of the HPV URR in PAI-2-expressing cells (Fig. 2B, wild-type URR) prompted an analysis of transcription factors known both to regulate the HPV-18 URR and to interact with Rb (7). Despite the recovery of c-Jun (Fig. 1A), we were unable to detect a significant difference in AP-1 activity as determined by EMSA and reporter assays (unpublished data). Western analysis also showed that expression of other AP-1 protein family members was not significantly changed (unpublished data). To further investigate DNA response elements that might be involved in the postulated Rb-associated repression of the HPV-18 URR, HPV-18 URR-LUC reporter plasmids were used, in which the C/EBP?-YY1 binding site (URR 7709-7719), the YY1 site (URR 7846-13), or the SP1 site (URR 34-40) was disrupted (Fig. 2A). When these mutated HPV URR constructs were analyzed for LUC expression, PAI-2-mediated repression of the HPV-18 URR was maintained for the SP1 mutant and the YY1 mutant (Fig. 2B), while PAI-2-mediated repression was lost for the HPV-18 URR construct containing a mutated C/EBP?-YY1 binding site (Fig. 2B, C/EBP?-YY1 mutant). Thus, the C/EBP?-YY1 binding site within the enhancer region is implicated as the site of PAI-2-associated repression of the HPV-18 URR in HeLa cells.
The dominant-repressive C/EBP? isoform LIP binds the C/EBP?-YY1 binding site in PAI-2-expressing HeLa cells. To analyze the transcription factors bound to the C/EBP?-YY1 binding site, an assay was developed whereby nuclear lysates were incubated with biotinylated C/EBP?-YY1 binding site dsDNA probes. After incubation with nuclear lysates, the probes and any transcription factors bound to the probe were pulled down with streptavidin beads and analyzed by SDS-PAGE and Western blotting. As expected in HeLa cells (5), both C/EBP? and YY1 were found bound to the C/EBP?-YY1 binding site probe (Fig. 3A, lane 2). Minimal binding to unlabeled probe or to labeled mutated probe was observed (Fig. 3A, lanes 1 and 3), illustrating the specificity of this assay. Importantly, neither C/EBP? nor YY1 was found bound to the C/EBP?-YY1 binding site probe in PAI-2-expressing S1a cells (Fig. 3A, lane 5). Instead, a low-molecular-mass anti-C/EBP?-reactive band was found bound to this probe in the S1a cells (Fig. 3A, lane 5). Although present in S1a lysates (Fig. 3B, lane 2), neither Rb nor PAI-2 was found bound to the C/EBP?-YY1 binding site probe (Fig. 3A, lane 5). Minimal binding to unlabeled probe or to labeled mutated probe was again observed (Fig. 3A, lanes 4 and 6). The low-molecular-mass band on SDS gels (17 to 20 kDa) (51), the antibody reactivity (H-7 is specific for the C terminus of C/EBP? and therefore also reacts with LIP), and the binding to a known C/EBP? site (5) identified the C/EBP?-YY1-binding-site-binding factor in S1a cells as the dominant-negative C/EBP? isoform LIP. The replacement of C/EBP?-YY1 with LIP on this site in S1a cells was not associated with a change in the expression levels of these transcription factors in S1a cells compared with parental HeLa cells (Fig. 3B, lanes 1 and 2).
Rb expression is required for binding of LIP to the C/EBP?-YY1 binding site. The interaction of PAI-2 with Rb (15) and the established binding of Rb to LIP (11) suggested that Rb may be required for LIP recruitment to the C/EBP?-YY1 binding site in S1a cells. To determine if the binding of LIP to the C/EBP?-YY1 binding site (Fig. 3A, lane 2) required the presence of Rb, S1a cells were treated with siRNA specific for Rb. The potent reduction of Rb expression in S1a cells treated with siRNA was demonstrated by Western analysis of nuclear lysates and was not associated with any changes in YY1, C/EBP? LIP, or PAI-2 expression (Fig. 3B, lane 3). Importantly, siRNA-treated S1a cells showed a substantial reduction in LIP binding to the C/EBP?-YY1 binding site probe and restoration of C/EBP? and YY1 binding to this probe (Fig. 3A, lane 8) to levels similar to those for parental HeLa cells. Analysis of S1a cells treated with a control scrambled siRNA did not reproduce these observations, with LIP remaining bound to the C/EBP?-YY1 binding site probe (Fig. 3A, lane 11). These data demonstrated that Rb expression is required for binding of the transcriptionally repressive LIP to the C/EBP?-YY1 binding site, with loss of Rb (mediated by E7 in HeLa cells or siRNA in S1a cells) resulting in loss of LIP binding to this region.
Loss of C/EBP?-YY1 (complex I) binding to the HPV-18 C/EBP?-YY1 binding site in S1a cells. Formation of the C/EBP?-YY1 dimer and subsequent recruitment of this complex to the C/EBP?-YY1 binding site in HeLa cells are well described elsewhere (5, 6). To confirm that PAI-2 expression alters the transcription complexes bound to the HPV-18 C/EBP?-YY1 binding site, nuclear extracts from HeLa cells and PAI-2-expressing HeLa cells (S1a) were analyzed by EMSA for protein-DNA complexes (Fig. 4A). As expected, incubation of the C/EBP?-YY1 binding site probe with nuclear extracts of HeLa cells showed a slow-migrating doublet. The top band was previously identified as the C/EBP?-YY1 dimer, also known as complex I (4-6) (Fig. 4A, lane 5, arrow). Importantly, EMSA analysis of nuclear extracts from S1a cells showed that C/EBP?-YY1 was not bound to the C/EBP?-YY1 binding site in these cells (Fig. 4A, lane 7), consistent with the results shown in Fig. 3A (lane 5). As expected, a mutated C/EBP?-YY1 binding site probe (containing the same mutations as the probe used in Fig. 3) failed to bind this complex in both cell lines (Fig. 4A, lanes 6 and 8).
No novel complex that might contain LIP could be identified in S1a cells with the use of conditions that were optimized to detect C/EBP?-YY1 complex I bound to the C/EBP?-YY1 binding site probe (Fig. 4A). However, when 10-fold-more labeled C/EBP?-YY1 binding site probe was used, a novel, faster-migrating complex could be detected in these cells (Fig. 4B, lane 5, arrow). This complex was absent in HeLa cell lysates (Fig. 4B, lane 3) and S1a cells incubated with the mutant C/EBP?-YY1 binding site probe (Fig. 4B, lane 4). Preincubation of S1a cell lysates prior to the EMSA reaction with an antibody specific for C/EBP?/LIP resulted in loss of the new fast-migrating complex (Fig. 4B, lane 6). Binding of this new complex to the C/EBP?-YY1 binding site was not eliminated by preincubation with anti-YY1 antibody, anti-Rb antibody, or anti-PAI-2 antibody (Fig. 4B, lanes 7, 8, and 9, respectively). These EMSA experiments are in complete agreement with the data shown in Fig. 3, which show LIP but not C/EBP?, YY1, Rb, or PAI-2 bound to the C/EBP?-YY1 binding site probe in S1a cells. They confirm that PAI-2 expression in HeLa cells results in the loss of C/EBP?-YY1 binding to the C/EBP?-YY1 binding site and show that it is replaced by a fast-migrating, C/EBP?/LIP-antibody-reactive transcription factor.
Rb expression is sufficient for repression of transcription from the HPV-18 URR. To further explore the role of Rb in repression of the HPV-18 URR, the HPV-18 URR LUC reporter constructs used in Fig. 2 were tested in the Rb-negative Weri and Y79 retinoblastoma cell lines and the same cell lines stably expressing Rb (Weri Rb and Y79 Rb) (59). As Weri and Y79 cells do not express any HPV oncoproteins or PAI-2 (data not shown), the role of Rb in HPV-18 URR repression could be analyzed in the absence of any influences from E6, E7, or PAI-2. Wild-type HPV-18 URR transcriptional activity from the reporter construct (Fig. 5A) could be readily detected in the Rb-negative Weri and Y79 cell lines (Fig. 5B, wild-type URR). In contrast, transcriptional activity was repressed in Weri Rb and Y79 Rb lines (Fig. 5B, wild-type URR). Thus, Rb expression alone appeared sufficient for repression of HPV-18 transcription.
In a series of experiments that parallel those described in Fig. 2B, mutation of either the SP1 or YY1 site again had no effect on Rb-mediated repression (Fig. 5B). However, mutation of the C/EBP?-YY1 binding site again restored transcriptional activity from the HPV-18 URR in Rb-positive cell lines (Fig. 5B, C/EBP?-YY1 mutant). These data show that, in the absence of PAI-2 and HPV oncoproteins, Rb expression is associated with repression of the HPV-18 URR via the C/EBP?-YY1 binding site.
LIP is bound to the C/EBP?-YY1 binding site in Weri Rb cells. In a series of experiments that parallel those shown in Fig. 3, we sought to determine whether the transcriptional repression of the HPV-18 URR seen in Weri Rb cells (Fig. 5B) was also associated with the binding of LIP to the C/EBP?-YY1 binding site, as was observed in S1a cells (Fig. 3A, lane 5). Rb expression in the Weri Rb cells is illustrated in Fig. 3B, lane 2. In contrast to HeLa cells (Fig. 3A, lane 2), no C/EBP? or YY1 was pulled down from nuclear lysates of parental Weri cells by the C/EBP?-YY1 binding site probe (Fig. 6A, lane 2). In fact no C/EBP? could be detected in nuclear lysates of Weri or Weri Rb cells (Fig. 6B), suggesting that these cells may not express sufficient C/EBP? to be able to form detectable levels of complex I. As was observed for HeLa and S1a cells (Fig. 3B), the level of LIP and YY1 expression was not significantly different in the Weri and Weri Rb lines (Fig. 6B). The C/EBP?-YY1 binding site probe failed to pull down LIP in the Rb-negative Weri cells (Fig. 6A, lane 2), as was the case for the Rb-negative parental HeLa cells. However, LIP was found bound to the C/EBP?-YY1 binding site probe in Weri Rb cells (Fig. 6A, lane 5), as was the case for the Rb-expressing S1a cells. Thus, Rb expression in both S1a and Weri Rb cells was associated with LIP binding to the C/EBP?-YY1 binding site.
EMSA analysis of Weri and Weri Rb cell extracts with the C/EBP?-YY1 binding site probe. EMSA analysis of Weri cell extracts with the C/EBP?-YY1 binding site probe failed to show the slow-migrating doublet (Fig. 7A, lane 5, arrow) that is present in HeLa cells (Fig. 4A, lane 5, arrow). In HeLa cells the top band of this doublet was identified as complex I comprising C/EBP?-YY1 (5). The presence in Weri cells of only a single band in this region (Fig. 7A, lane 5, arrow) is consistent with the lack of complex I in these cells. This contention is supported by the lack of detectable C/EBP? by Western blotting (Fig. 6B), and the absence of C/EBP? or YY1 binding to the C/EBP?-YY1 binding site probe in these cells (Fig. 6A, lane 2). The identity of the lower band in the doublet remains unknown, and neither band is present in Weri Rb cells (Fig. 7A, lane 7). Importantly, the fast-migrating complex seen in S1a cells (Fig. 4B, lane 5) is also seen in Weri Rb cells (Fig. 7B, lane 5, arrow), and this complex can also be disrupted by the addition of anti-C/EBP?/LIP antibody (Fig. 7B, lane 6). Anti-YY1, -Rb, and -PAI-2 antibodies again failed to disrupt this complex (data not shown). This fast-migrating complex was not present in Weri cells (Fig. 7B, lane 3), nor was it detected in Weri Rb cells with the use of a mutated C/EBP?-YY1 binding site probe (Fig. 7, lane 4).
Thus, in both S1a cells and Weri Rb cells the presence of Rb was associated in the EMSA experiments with the presence of a fast-migrating, C/EBP?/LIP-antibody-reactive complex bound to the C/EBP?-YY1 binding site, which is absent in the Rb-negative HeLa and Weri cells. The only anti-C/EBP?/LIP-antibody-reactive complex pulled down by the C/EBP?-YY1 binding site probe in S1a cells and Weri Rb was identified as LIP (Fig. 3A, lane 5, and 6A, lane 5), and this was absent in HeLa and Weri cells. The fast-migrating complex identified in the EMSA experiments is likely therefore to contain LIP, with LIP binding to the C/EBP?-YY1 binding site associated with Rb expression.
DISCUSSION
PAI-2, although well documented as an inhibitor of the extracellular urokinase-type plasminogen activator (31), also has an important intranuclear role as a regulator of transcription (1, 15, 48), which is likely mediated by its interaction with Rb (15). Here we confirm that PAI-2 expression in HeLa cells results in the recovery of Rb expression (Fig. 1) and show that this leads to transcriptional silencing of the HPV-18 URR (Fig. 2). This in turn leads to the loss of E6 and E7 protein expression and the recovery of multiple E6- and E7-targeted proteins (Fig. 1). Transcriptional silencing of the HPV-18 URR was due to the Rb-dependent recruitment of dominant-negative repressor LIP to the C/EBP?-YY1 binding site (Fig. 3 to 7). In HeLa cells this was also associated with the loss of C/EBP?-YY1 binding to this region. Thus, PAI-2 expression protects Rb from degradation, Rb then causes recruitment of LIP to the C/EBP?-YY1 binding site, HPV oncogene transcription is silenced, and oncoprotein activity is lost.
We have demonstrated in two systems that recruitment of LIP to the C/EBP?-YY1 binding site of the HPV-18 URR is dependent on Rb expression; suppression of Rb translation by the use of siRNA resulted in loss of LIP binding to the C/EBP?-YY1 binding site in S1a cells, and expression of Rb in Weri cells resulted in LIP binding to this region. Rb expression did not cause upregulation of LIP. How Rb expression causes recruitment of LIP to the C/EBP?-YY1 binding site remains to be elucidated but may simply be a consequence of Rb associating with LIP to enhance LIP's DNA binding affinity to exceed that of C/EBP?-YY1. Rb binding to C/EBP? has been shown to enhance the DNA binding affinity of C/EBP? (11, 13), and Rb interacts with C/EBP? via the C-terminal bZIP domain of C/EBP?, which is also present in LIP (13). Although promoting the DNA binding affinity of C/EBP?, Rb does not remain associated with the DNA-bound C/EBP? (11), consistent with our finding that Rb was not found associated with LIP on the C/EBP?-YY1 binding site (Fig. 3A, lane 5, and 6A, lane 5). Alternatively, Rb expression may disrupt C/EBP?-YY1 dimer formation in HeLa cells by binding C/EBP? and/or YY1 (11, 40), allowing the dominant-negative LIP to bind to the C/EBP?-YY1 binding site (58). The Rb-dependent recruitment of LIP to the C/EBP?-YY1 binding site in Weri Rb cells, when Weri cells do not have detectable C/EBP?-YY1, might argue against this model. However, an unidentified, Rb-disruptable complex may be binding this site in Weri cells (Fig. 7A, lane 2, arrow).
The present study illustrates that at physiological levels functional Rb and LIP act together to suppress transcription in HPV-positive cells but that PAI-2 must be present in HPV-transformed cells to preserve Rb expression. The contention that Rb expression may be important for suppressing the HPV-18 URR is supported by a report from Salcedo et al. (47), who showed that overexpression of Rb in HPV-negative cells repressed transcription from an HPV-18 URR reporter construct. Interestingly, HepG2 cells also contain integrated HPV-18 DNA, but the URR is transcriptionally silenced (4, 5), and these cells express Rb (unpublished observation) and can express PAI-2 (21). Overexpression of LIP has also been shown to reduce transcription from HPV-16 reporter plasmids in HPV-18 and HPV-negative cells (51). Furthermore, we have recently determined that PAI-2 expression suppresses transcription from the HPV-16 URR (data not shown), perhaps suggesting a wider role for PAI-2 and Rb-dependent LIP recruitment in suppressing high-risk HPV oncogene transcription.
PAI-2 expression has been associated with improved prognosis in a number of different cancers (20, 31, 38, 41, 56). However, analysis of PAI-2 expression in malignancies often associated with HPV has been limited (14, 25). Nevertheless, transcriptional profiling suggests that reduced PAI-2 expression is associated with high-risk HPV (10, 46). One should also note that the PAI-2 gene is located at chromosomal locus 18q21, and this is one site that has been associated with loss of heterozygosity leading to tumor formation or progression in several studies of HPV-positive malignancies (24, 50, 60). The present study would predict that PAI-2 expression should represent an important suppressive factor in the development of high-risk HPV lesions, so long as Rb and LIP remain functional in these tissues.
The abilities of PAI-2 to protect Rb from E7-mediated degradation (Fig. 1B) and to repress transcription from the HPV-18 URR (Fig. 2B) were dependent on both the C-D interhelical region, which mediates Rb binding, and the RSL, which mediates protease inhibition. How PAI-2 inhibits Rb degradation remains unclear. However, Rb binding alone appears insufficient, since the RSL mutant, which retains Rb binding activity (15), is unable to inhibit Rb degradation (Fig. 1B, PAI-2 Ala380) and fails to inhibit HPV-18 URR transcription (Fig. 2B). The RSL regions of serpins usually act as a pseudosubstrate for target proteases, suggesting that PAI-2 may inhibit a nuclear protease. A number of proteases are known to be active in the nucleus and are known to target the pocket protein family comprising Rb, p130, and p107; these include calpain 1 (28), caspases (12), and Spase (27). We are currently exploring whether these proteases are the target of PAI-2 inhibition in vivo.
Repression of HPV-18 oncogene transcription can also be achieved in HeLa cells by expression of HPV E2, which binds and represses the URR promoter. Like PAI-2 expression, this also results in recovery of p53 and Rb but results in Rb-dependent senescence (42, 53). S1a and S1b cells do show increased G1 arrest (15) and elevated p21 expression (Fig. 1), both features associated with progression to senescence. The stably transfected S1a and S1b cell lines were selected on the basis of growth (18), and therefore any initial PAI-2-mediated senescence would have been overlooked but might be expected given the loss of E6 and E7.
Whether the PAI-2-associated repression, shown here for the integrated HPV-18 URR (Fig. 1) and for reporter plasmids encoding this URR (Fig. 2), also has a role in repressing E6-E7 transcription from full-length episomal HPV DNA remains to be investigated. How E6 and E7 oncoprotein-mediated inhibition of keratinocyte differentiation is overcome to allow later stages of the HPV viral life cycle is currently unclear (22). PAI-2 is absent in basal keratinocytes (35) but is a major product of differentiating keratinocytes (33, 43). PAI-2 probably contributes to promoting keratinocyte differentiation by elevating Rb levels (45), and this might also lead to downregulation of episomal E6 and E7 transcription. Interestingly, E2 has been shown to synergize with C/EBP? to upregulate C/EBP?-dependent gene transcription (22), and C/EBP? has recently been shown to be an important positive regulator of PAI-2 expression (B. W. Stringer et al., unpublished data).
This paper illustrates that PAI-2 expression has a potent suppressive effect on the transcription of oncogenes from integrated HPV-18 DNA that is mediated by Rb and LIP. These observations may have implications for the use of PAI-2-based reagents in diagnosis and/or treatment of HPV-associated lesions.
ACKNOWLEDGMENTS
We thank M. Madigan, Royal Prince Alfred Hospital, Sydney, for supplying the Weri and Y79 cell lines and R. Tindle (Director, Sir Albert Sakzewski Virus Research Centre, Brisbane, Australia) and I. A. Frazer (Director, The Centre for Immunology and Cancer Research, Brisbane, Australia) for their help with the manuscript.
This work was supported by grants from the NH&MRC of Australia, University of Queensland postgraduate scholarship, and the National Institutes of Health, United States (CA098369).
REFERENCES
Antalis, T. M., M. La Linn, K. Donnan, L. Mateo, J. Gardner, J. L. Dickinson, K. Buttigieg, and A. Suhrbier. 1998. The serine proteinase inhibitor (serpin) plasminogen activation inhibitor type 2 protects against viral cytopathic effects by constitutive interferon alpha/beta priming. J. Exp. Med. 187:1799-1811.
Badal, S., V. Badal, I. E. Calleja-Macias, M. Kalantari, L. S. Chuang, B. F. Li, and H. U. Bernard. 2004. The human papillomavirus-18 genome is efficiently targeted by cellular DNA methylation. Virology 324:483-492.
Bauknecht, T., P. Angel, H. D. Royer, and H. zur Hausen. 1992. Identification of a negative regulatory domain in the human papillomavirus type 18 promoter: interaction with the transcriptional repressor YY1. EMBO J. 11:4607-4617.
Bauknecht, T., F. Jundt, I. Herr, T. Oehler, H. Delius, Y. Shi, P. Angel, and H. Zur Hausen. 1995. A switch region determines the cell type-specific positive or negative action of YY1 on the activity of the human papillomavirus type 18 promoter. J. Virol. 69:1-12.
Bauknecht, T., R. H. See, and Y. Shi. 1996. A novel C/EBP ?-YY1 complex controls the cell-type-specific activity of the human papillomavirus type 18 upstream regulatory region. J. Virol. 70:7695-7705.
Bauknecht, T., and Y. Shi. 1998. Overexpression of C/EBP? represses human papillomavirus type 18 upstream regulatory region activity in HeLa cells by interfering with the binding of TATA-binding protein. J. Virol. 72:2113-2124.
Bednarek, P. H., B. J. Lee, S. Gandhi, E. Lee, and B. Phillips. 1998. Novel binding sites for regulatory factors in the human papillomavirus type 18 enhancer and promoter identified by in vivo footprinting. J. Virol. 72:708-716.
Bird, C. H., E. J. Blink, C. E. Hirst, M. S. Buzza, P. M. Steele, J. Sun, D. A. Jans, and P. I. Bird. 2001. Nucleocytoplasmic distribution of the ovalbumin serpin PI-9 requires a nonconventional nuclear import pathway and the export factor Crm1. Mol. Cell. Biol. 21:5396-5407.
Chakrabarti, O., and S. Krishna. 2003. Molecular interactions of ‘high risk’ human papillomaviruses E6 and E7 oncoproteins: implications for tumour progression. J. Biosci. 28:337-348.
Chang, Y. E., and L. A. Laimins. 2000. Microarray analysis identifies interferon-inducible genes and Stat-1 as major transcriptional targets of human papillomavirus type 31. J. Virol. 74:4174-4182.
Charles, A., X. Tang, E. Crouch, J. S. Brody, and Z. X. Xiao. 2001. Retinoblastoma protein complexes with C/EBP proteins and activates C/EBP-mediated transcription. J. Cell. Biochem. 83:414-425.
Chau, B. N., H. L. Borges, T. T. Chen, A. Masselli, I. C. Hunton, and J. Y. Wang. 2002. Signal-dependent protection from apoptosis in mice expressing caspase-resistant Rb. Nat. Cell Biol. 4:757-765.
Chen, P. L., D. J. Riley, S. Chen-Kiang, and W. H. Lee. 1996. Retinoblastoma protein directly interacts with and activates the transcription factor NF-IL6. Proc. Natl. Acad. Sci. USA 93:465-469.
Daneri-Navarro, A., G. Macias-Lopez, A. Oceguera-Villanueva, S. Del Toro-Arreola, A. Bravo-Cuellar, R. Perez-Montfort, and S. Orbach-Arbouys. 1998. Urokinase-type plasminogen activator and plasminogen activator inhibitors (PAI-1 and PAI-2) in extracts of invasive cervical carcinoma and precursor lesions. Eur. J. Cancer 34:566-569.
Darnell, G. A., T. M. Antalis, R. W. Johnstone, B. W. Stringer, S. M. Ogbourne, D. Harrich, and A. Suhrbier. 2003. Inhibition of retinoblastoma protein degradation by interaction with the serpin plasminogen activator inhibitor 2 via a novel consensus motif. Mol. Cell. Biol. 23:6520-6532.
Dell, G., and K. Gaston. 2001. Human papillomaviruses and their role in cervical cancer. Cell. Mol. Life Sci. 58:1923-1942.
Descombes, P., and U. Schibler. 1991. A liver-enriched transcriptional activator protein, LAP, and a transcriptional inhibitory protein, LIP, are translated from the same mRNA. Cell 67:569-579.
Dickinson, J. L., E. J. Bates, A. Ferrante, and T. M. Antalis. 1995. Plasminogen activator inhibitor type 2 inhibits tumor necrosis factor alpha-induced apoptosis. Evidence for an alternate biological function. J. Biol. Chem. 270:27894-27904.
Dickinson, J. L., B. J. Norris, P. H. Jensen, and T. M. Antalis. 1998. The C-D interhelical domain of the serpin plasminogen activator inhibitor-type 2 is required for protection from TNF-alpha induced apoptosis. Cell Death Differ. 5:163-171.
Foekens, J. A., F. Buessecker, H. A. Peters, U. Krainick, W. L. van Putten, M. P. Look, J. G. Klijn, and M. D. Kramer. 1995. Plasminogen activator inhibitor-2: prognostic relevance in 1012 patients with primary breast cancer. Cancer Res. 55:1423-1427.
Gohl, G., T. Lehmkoster, P. A. Munzel, D. Schrenk, R. Viebahn, and K. W. Bock. 1996. TCDD-inducible plasminogen activator inhibitor type 2 (PAI-2) in human hepatocytes, HepG2 and monocytic U937 cells. Carcinogenesis 17:443-449.
Hadaschik, D., K. Hinterkeuser, M. Oldak, H. J. Pfister, and S. Smola-Hess. 2003. The papillomavirus E2 protein binds to and synergizes with C/EBP factors involved in keratinocyte differentiation. J. Virol. 77:5253-5265.
Harbour, J. W., and D. C. Dean. 2000. The Rb/E2F pathway: expanding roles and emerging paradigms. Genes Dev. 14:2393-2409.
Harima, Y., S. Sawada, K. Nagata, M. Sougawa, and T. Ohnishi. 2001. Chromosome 6p21.2, 18q21.2 and human papilloma virus (HPV) DNA can predict prognosis of cervical cancer after radiotherapy. Int. J. Cancer 96:286-296.
Hasina, R., K. Hulett, S. Bicciato, C. Di Bello, G. J. Petruzzelli, and M. W. Lingen. 2003. Plasminogen activator inhibitor-2: a molecular biomarker for head and neck cancer progression. Cancer Res. 63:555-559.
Hattori, T., N. Ohoka, H. Hayashi, and K. Onozaki. 2003. C/EBP homologous protein (CHOP) up-regulates IL-6 transcription by trapping negative regulating NF-IL6 isoform. FEBS Lett. 541:33-39.
Irving, J. A., S. S. Shushanov, R. N. Pike, E. Y. Popova, D. Bromme, T. H. Coetzer, S. P. Bottomley, I. A. Boulynko, S. A. Grigoryev, and J. C. Whisstock. 2002. Inhibitory activity of a heterochromatin-associated serpin (MENT) against papain-like cysteine proteinases affects chromatin structure and blocks cell proliferation. J. Biol. Chem. 277:13192-13201.
Jang, J. S., S. J. Lee, Y. H. Choi, P. M. Nguyen, J. Lee, S. G. Hwang, M. L. Wu, E. Takano, M. Maki, P. A. Henkart, and J. B. Trepel. 1999. Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease. Oncogene 18:1789-1796.
Jensen, P. J., Q. Wu, P. Janowitz, Y. Ando, and N. M. Schechter. 1995. Plasminogen activator inhibitor type 2: an intracellular keratinocyte differentiation product that is incorporated into the cornified envelope. Exp. Cell Res. 217:65-71.
Jover, R., R. Bort, M. J. Gomez-Lechon, and J. V. Castell. 2002. Down-regulation of human CYP3A4 by the inflammatory signal interleukin-6: molecular mechanism and transcription factors involved. FASEB J. 16:1799-1801.
Kruithof, E. K., M. S. Baker, and C. L. Bunn. 1995. Biological and clinical aspects of plasminogen activator inhibitor type 2. Blood 86:4007-4024.
Kuang, P. P., and R. H. Goldstein. 2003. Regulation of elastin gene transcription by interleukin-1 beta-induced C/EBP beta isoforms. Am. J. Physiol. Cell Physiol. 285:C1349-C1355.
Lian, X., and T. Yang. 2004. Plasminogen activator inhibitor 2: expression and role in differentiation of epidermal keratinocyte. Biol. Cell. 96:109-116.
Lipinski, M. M., and T. Jacks. 1999. The retinoblastoma gene family in differentiation and development. Oncogene 18:7873-7882.
Lyons-Giordano, B., D. Loskutoff, C. S. Chen, G. Lazarus, M. Keeton, and P. J. Jensen. 1994. Expression of plasminogen activator inhibitor type 2 in normal and psoriatic epidermis. Histochemistry 101:105-112.
McMurray, H. R., D. Nguyen, T. F. Westbrook, and D. J. McAnce. 2001. Biology of human papillomaviruses. Int. J. Exp. Pathol. 82:15-33.
Morris, E. J., and N. J. Dyson. 2001. Retinoblastoma protein partners. Adv. Cancer Res. 82:1-54.
Nakamura, M., H. Konno, T. Tanaka, Y. Maruo, N. Nishino, K. Aoki, S. Baba, S. Sakaguchi, Y. Takada, and A. Takada. 1992. Possible role of plasminogen activator inhibitor 2 in the prevention of the metastasis of gastric cancer tissues. Thromb. Res. 65:709-719.
O'Connor, M. J., W. Stunkel, C. H. Koh, H. Zimmermann, and H. U. Bernard. 2000. The differentiation-specific factor CDP/Cut represses transcription and replication of human papillomaviruses through a conserved silencing element. J. Virol. 74:401-410.
Petkova, V., M. J. Romanowski, I. Sulijoadikusumo, D. Rohne, P. Kang, T. Shenk, and A. Usheva. 2001. Interaction between YY1 and the retinoblastoma protein. Regulation of cell cycle progression in differentiated cells. J. Biol. Chem. 276:7932-7936.
Praus, M., K. Wauterickx, D. Collen, and R. D. Gerard. 1999. Reduction of tumor cell migration and metastasis by adenoviral gene transfer of plasminogen activator inhibitors. Gene Ther. 6:227-236.
Psyrri, A., R. A. DeFilippis, A. P. Edwards, K. E. Yates, L. Manuelidis, and D. DiMaio. 2004. Role of the retinoblastoma pathway in senescence triggered by repression of the human papillomavirus E7 protein in cervical carcinoma cells. Cancer Res. 64:3079-3086.
Risse, B. C., H. Brown, R. M. Lavker, J. M. Pearson, M. S. Baker, D. Ginsburg, and P. J. Jensen. 1998. Differentiating cells of murine stratified squamous epithelia constitutively express plasminogen activator inhibitor type 2 (PAI-2). Histochem. Cell Biol. 110:559-569.
Rose, B., G. Steger, X. P. Dong, C. Thompson, Y. Cossart, M. Tattersall, and H. Pfister. 1998. Point mutations in SP1 motifs in the upstream regulatory region of human papillomavirus type 18 isolates from cervical cancers increase promoter activity. J. Gen. Virol. 79:1659-1663.
Ruiz, S., M. Santos, C. Segrelles, H. Leis, J. L. Jorcano, A. Berns, J. M. Paramio, and M. Vooijs. 2004. Unique and overlapping functions of pRb and p107 in the control of proliferation and differentiation in epidermis. Development 131:2737-2748.
Ruutu, M., P. Peitsaro, B. Johansson, and S. Syrjanen. 2002. Transcriptional profiling of a human papillomavirus 33-positive squamous epithelial cell line which acquired a selective growth advantage after viral integration. Int. J. Cancer 100:318-326.
Salcedo, M., E. Garrido, L. Taja, and P. Gariglio. 1995. The retinoblastoma gene product negatively regulates cellular or viral oncogene promoters in vivo. Arch. Med. Res. 26:S157-S162.
Shafren, D. R., J. Gardner, V. H. Mann, T. M. Antalis, and A. Suhrbier. 1999. Picornavirus receptor down-regulation by plasminogen activator inhibitor type 2. J. Virol. 73:7193-7198.
Silverman, G. A., P. I. Bird, R. W. Carrell, F. C. Church, P. B. Coughlin, P. G. Gettins, J. A. Irving, D. A. Lomas, C. J. Luke, R. W. Moyer, P. A. Pemberton, E. Remold-O'Donnell, G. S. Salvesen, J. Travis, and J. C. Whisstock. 2001. The serpins are an expanding superfamily of structurally similar but functionally diverse proteins. Evolution, mechanism of inhibition, novel functions, and a revised nomenclature. J. Biol. Chem. 276:33293-33296.
Steenbergen, R. D., M. A. Hermsen, J. M. Walboomers, H. Joenje, F. Arwert, C. J. Meijer, and P. J. Snijders. 1995. Integrated human papillomavirus type 16 and loss of heterozygosity at 11q22 and 18q21 in an oral carcinoma and its derivative cell line. Cancer Res. 55:5465-5471.
Struyk, L., E. van der Meijden, R. Minnaar, V. Fontaine, I. Meijer, and J. ter Schegget. 2000. Transcriptional regulation of human papillomavirus type 16 LCR by different C/EBP? isoforms. Mol. Carcinog. 28:42-50.
Su, W. C., H. Y. Chou, C. J. Chang, Y. M. Lee, W. H. Chen, K. H. Huang, M. Y. Lee, and S. C. Lee. 2003. Differential activation of a C/EBP beta isoform by a novel redox switch may confer the lipopolysaccharide-inducible expression of interleukin-6 gene. J. Biol. Chem. 278:51150-51158.
Wells, S. I., B. J. Aronow, T. M. Wise, S. S. Williams, J. A. Couget, and P. M. Howley. 2003. Transcriptome signature of irreversible senescence in human papillomavirus-positive cervical cancer cells. Proc. Natl. Acad. Sci. USA 100:7093-7098.
Welm, A. L., N. A. Timchenko, and G. J. Darlington. 1999. C/EBP regulates generation of C/EBP? isoforms through activation of specific proteolytic cleavage. Mol. Cell. Biol. 19:1695-1704.
Xiong, W., C. C. Hsieh, A. J. Kurtz, J. P. Rabek, and J. Papaconstantinou. 2001. Regulation of CCAAT/enhancer-binding protein-beta isoform synthesis by alternative translational initiation at multiple AUG start sites. Nucleic Acids Res. 29:3087-3098.
Yoshino, H., Y. Endo, Y. Watanabe, and T. Sasaki. 1998. Significance of plasminogen activator inhibitor 2 as a prognostic marker in primary lung cancer: association of decreased plasminogen activator inhibitor 2 with lymph node metastasis. Br. J. Cancer 78:833-839.
Yu, H., F. Maurer, and R. L. Medcalf. 2002. Plasminogen activator inhibitor type 2: a regulator of monocyte proliferation and differentiation. Blood 99:2810-2818.
Zahnow, C. A., P. Younes, R. Laucirica, and J. M. Rosen. 1997. Overexpression of C/EBPbeta-LIP, a naturally occurring, dominant-negative transcription factor, in human breast cancer. J. Natl. Cancer Inst. 89:1887-1891.
Zhang, M., G. Stevens, and M. C. Madigan. 2001. In vitro effects of radiation on human retinoblastoma cells. Int. J. Cancer 96(Suppl.):7-14.
Zhao, M., and X. X. Wu. 2002. [Analysis of genetic instabilities on chromosome loci 3, 6, 11, and 18 in human cervical carcinoma.] Ai Zheng 21:640-643.
Zhou, H. M., I. Bolon, A. Nichols, A. Wohlwend, and J. D. Vassalli. 2001. Overexpression of plasminogen activator inhibitor type 2 in basal keratinocytes enhances papilloma formation in transgenic mice. Cancer Res. 61:970-976.(Grant A. Darnell, Toni M.)