Epidermal growth factor activates nuclear factor-B in human proximal tubule cells
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《美国生理学杂志》
Nephrology Division, Department of Internal Medicine II, and Department of Internal Medicine I, University of Ulm, Ulm
Department of Internal Medicine II, Technical University of Munich, Munich, Germany
ABSTRACT
The promotion of cell survival and regeneration in acute renal failure (ARF) is important for the restitution of renal function. Epidermal growth factor (EGF) has been implicated in the regulation of cell proliferation. We provide evidence for a direct link between EGF, nuclear factor-B (NF-B), and cell cycle regulation (cyclin D1). EGF was found to stimulate NF-B-dependent gene transcription and DNA binding. In addition, EGF stimulated cyclin D1 promoter activity as well as cyclin D1 expression. Moreover, inhibition of NF-B caused a pronounced reduction of EGF-induced cyclin D1 promoter activity. Furthermore, both EGF-mediated NF-B activation and cyclin D1 expression were inhibited by coexpression of super IB. Taken together, these data identify NF-B and cyclin D1 as downstream targets of EGF and establish a molecular link between stimulation of EGF via activation of NF-B and cyclin D1 expression in human proximal tubular cells.
EGF; cyclin D1
IN CONTRAST TO THE HEART AND brain, where ischemia results in irreversible cell damage, the kidney can completely restore its structure and function after being severely damaged by either ischemia or toxins (32). This reversibility depends on the ability of renal tubule cells to regenerate and rearrange the damaged areas of epithelial cells along the nephron. This process is characterized by an increase in mitotic activity in renal epithelial cells and induction of nucleic acid synthesis (32). A burst of DNA synthesis is typical for all nephrotoxic or ischemic injuries, differing only in its rate and extent (59).
Several growth factors are key mediators of renal repair processes and compensatory renal epithelial growth (18, 21, 33). Epidermal growth factor (EGF) is one of the most effective growth-promoting substances, which has been identified in renal proximal tubule cells so far. Its function has been extensively investigated in experimental animal models (32, 33, 39). When EGF is administered to animals subjected to renal ischemia, it reduces the extent of renal dysfunction and accelerates recovery of the tubule cells (25, 57). To date, little is known about the underlying transcriptional mechanisms leading to tubular regeneration after acute renal failure (ARF).
A candidate pathway, involved in the EGF-induced mitogenic response, leads to NF-B activation. The inducible transcription factor NF-B participates in the regulation of numerous genes, many of which are involved in inflammation and cell proliferation (2, 20). The NF-B/Rel family has five members (p50, p52, p65/RelA, RelB, and c-Rel), which can form various homodimeric or heterodimeric complexes (2, 20). In most unstimulated cells, NF-B is thought to reside in the cytoplasm bound to an inhibitor protein, designated IB (6). Upon stimulation or activation of cells by a diverse array of stimuli, NF-B is able to rapidly dissociate from IB and translocate into the nucleus, an event that involves phosphorylation of IB on serines 32 and 36 (6, 29). Subsequent work has shown that NF-B is crucial for controlling proliferation and differentiation in many cell types and that it is linked to signaling processes that control cell cycle progression (5, 8, 31, 44, 50). In fact, a nuclear NF-B-like DNA binding activity is induced during the G0-to-G1 transition after serum stimulation in mouse fibroblasts (4).
The proliferative response of mammalian cells to extracellular signals is integrated in the mid- to late G1 phase of the cell cycle (26, 63). The cyclin D1 protein is a regulatory subunit of a holoenzyme, which is rapidly induced during G1 phase progression. There, cyclin D1 and its catalytic subunits are key regulators of cell proliferation (63). Furthermore, cyclin D1 links growth factor signaling to the cell cycle machinery. Cyclin D1 expression is regulated transcriptionally as well as via pathways of ubiquitin-proteosome-dependent degradation (14). The cyclin D1 promoter harbors cis regulatory elements for several transcription factors including AP-1, CREB/ATF, SP1, and STATs (61). Furthermore, the serum responsiveness of the cyclin D1 promoter depends on an intact proximal NF-B site in the cyclin D1 promoter (31).
The data presented here indicate that NF-B is a downstream target of the growth factor EGF in proximal tubule cells. We show that NF-B links EGF signaling to a key regulator of cell cycle progression by activating the cyclin D1 promoter. These observations suggest that EGF-induced NF-B activation contributes directly to the proliferative response needed for renal regeneration after ARF.
MATERIALS AND METHODS
Cell culture and treatments. HK-2, an immortalized proximal tubule cell (PTC) line derived from normal adult human kidney (55), was obtained from American Type Culture Collection (ATCC). Cells were maintained in DMEM/F-12 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) in a humidified atmosphere of 5% CO2-95% air at 37°C and passaged every 7 days. For passage, confluent cells were washed with phosphate-buffered saline (PBS), removed with 0.05% trypsin, and plated in DMEM/F-12 medium. EGF was purchased from Saxon and recombinant TNF- from Sigma. EGF and TNF- were freshly dissolved in culture media and added to the cultures at the indicated concentrations and for the indicated time periods. Before stimulation, cells were serum-starved for 24 h.
Cell growth. HK-2 cells were washed twice in PBS, trypsinized, and resuspended in serum-free DMEM/F-12. Cells were plated in six-well culture dishes (Falcon) at a density of 1 x 105 cells in 1 ml of serum-free DMEM/F-12 additionally for 24 h. HK-2 cells were then stimulated with various concentrations of EGF alone for 24 h. The number of cells was determined daily using a cell-counting chamber.
5-Bromo-2-deoxyuridine enzyme-linked immunosorbent assay. To measure DNA synthesis, a 5-bromo-2-deoxyuridine (BrdU) assay (Roche) was used. Twenty-four-hour serum-starved cells were seeded in 96-well microtiter plates, and EGF at various concentrations was added additionally for 24 h. BrdU was then added for 2 h, cells were fixed with ethanol for 30 min, and an anti-BrdU antibody was added for 90 min. After the cells were washed, the anti-BrdU antibody was detected using a microtiter reader.
Plasmid constructs. The luciferase reporter 3xBLUC, which contains three tandem B repeats in pGL3 (Promega), has been described previously (68). Point mutations in IB S32A/S36A were introduced by primer-mediated, site-directed mutagenesis using a Quick Change Mutagenesis kit (Stratagene) to generate pcDNA-IB S32A/S36A (43). The cyclin D1 promoter construct pD1LUC and the promoter construct mutant for the NF-B sites D1-B1M, D1-B2M, and D1-B1M+B2M, harboring point mutations in the D1-B1 (CGCGACCCCC), D1-B2 (CGCGAGTTTT), or in both binding sites (introduced point mutations are underlined), were a kind gift from Dr. Claus Scheidereit (31).
Preparation of whole cell lysates and nuclear extracts. Whole cell lysates were prepared by incubating cell pellets for 30 min at 4°C in immunoprecipitation buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 1 mM PMSF, and 5 mM NaF). Insoluble material was removed by centrifugation, and lysates were aliquoted and stored at –80°C. Nuclear extracts from HK-2 cells treated as indicated were prepared according to the method of Schreiber et al. (62) with a few modifications. Briefly, cells were collected, washed in cold PBS, and pelleted by centrifugation. The cell pellet was resuspended in 400 μl of cold buffer A (in mM: 10 HEPES, 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 dithiothreitol, and 0.5 PMSF) and allowed to swell on ice for 20 min. Cells were disrupted by passage through a 26-gauge needle, and nuclei were centrifuged at 500 g in a microcentrifuge. The nuclear pellet was washed twice in buffer A to avoid cytoplasmatic contamination. Finally, the nuclear pellet was resuspended in 50 μl of cold buffer (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM PMSF, and 5 mM NaF) and rocked at 4°C on a shaking platform for 30 min. The lysates were cleared by centrifugation, aliquoted, and stored at –80°C. Protein concentrations were determined by the Bradford method (Bio-Rad Laboratories) using bovine serum albumin as a standard.
Transfection and luciferase assays. Approximately 0.2 x 106 HK-2 cells/well were seeded in six-well dishes and allowed to attach overnight. Cells were washed twice with PBS and transfected with expression plasmids as indicated. Transfections were performed by the calcium phosphate precipitation method as described previously (60). Cells were washed twice with PBS 12 h after transfection and were serum-starved for a further 24 h. Cells were stimulated with EGF in various concentrations for 24 h. The control experiments were performed with 150 U of human TNF-. Cells were harvested in 250 μl of lysis buffer (Promega) and cleared by centrifugation. Cell lysates were normalized for protein content and used to measure luciferase activity using a commercially available kit (Dual Luciferase Assay System, Promega) in a luminometer (Berthold Analytical Instruments). Transfections were performed at least three times in triplicate.
EMSAs. EMSAs were performed as described using the NF-B motif of the mouse Ig- light chain enhancer (5'-CTCAACAGAGGGGACTTTCCGAGAGGCCAT-3') as a probe (42). Briefly, 5 μg of nuclear extracts were equilibrated for 20 min at room temperature in 20 μl binding buffer containing 4% glycerol, 10 mM Tris, 100 mM NaCl, and 100 ng/μl poly (dl-dC). Approximately 50,000 cpm of the -32P-labeled NF-B probe were added, followed by another 30 min of incubation on ice. The DNA binding complexes were separated by electrophoresis on a nondenaturing 5% polyacrylamide gel in 0.5x Tris-glycine-EDTA buffer (TGE) at 150 V. Gels were dried and exposed on X-ray film (Biomax MS, Kodak). Competition experiments were performed by adding NF-B-specific unlabeled oligonucleotide derived from the IL-6 promoter (5'-TATCAAATGTGGATTTTCCCATGAGTCTCA-3') to the reaction mixture in 20-fold or 60-fold molar excess, or by using an AP-1 oligonucleotide (5'-AGCTTACTCAGTACTAGTACG-3'). For supershift assays, antibodies against p65 and p50 subunits (Santa Cruz Biotechnology) were added to the binding buffer.
Western blotting. Whole cell lysates were boiled in 2x SDS sample buffer (100 mM Tris·HCl, 4% SDS, 0.2% bromphenol blue, 20% glycerol, 10% DTT) and separated by SDS-PAGE on 10% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) by semidry blotting. Membranes were blocked in PBS supplemented with 5% skim milk and 0.1% Tween 20 and subsequently incubated with cyclin D1 antibody (BD Pharmingen). Proteins recognized by the antibody were detected by enhanced chemiluminescence (Amersham) using a horseradish peroxidase-coupled secondary antibody.
Immunofluorescence. Serum-starved cultures of HK-2 cells grown on coverslips were incubated with or without EGF (10–9 M). After 1 h of stimulation, cells were fixed for 5 min in 96% ethanol and subsequently incubated for 20 min in 4% formaldehyde at room temperature. Cells were then permeabilized with 0.2% Triton X-100 and stained with a NF-B p65 (BD Transduction Laboratories) antibody for 1 h followed by detection with an Alexa-labeled (568 nm) secondary antibody (Molecular Probes; Leiden, Netherlands). Nuclear costaining was performed with Sytox green (Molecular Probes). Samples were further analyzed by fluorescence microscopy (Olympus IX-71, x100 magnification) connected to a CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan).
Statistical analysis. Data are expressed as means ± SE. Comparisons between groups were analyzed with Student's t-test (for 2 groups) or one-way ANOVA, followed by the Newman-Keuls post hoc test when indicated (for 3 or more conditions). Differences were considered to be significant at P < 0.05.
RESULTS
Effect of EGF on cell growth and proliferation. To examine whether EGF exhibits its well-established growth-stimulating effect in our system of HK-2 cells, a cell culture model system that displays important functional and morphological characteristics of human tubular cells, we treated HK-2 cells with EGF and assessed cell growth and proliferation (61). Growth curves were performed with increasing concentrations of EGF (10–11, 10–10, and 10–9 M) in the serum-starved culture media. FCS (10%) was used as a positive and untreated cells as a negative control. After 72 h of EGF exposure, cell number increased for all concentrations of EGF compared with untreated cells (Fig. 1A).
To determine the effect of EGF on HK-2 cell proliferation, BrdU incorporation was performed after treatment with different doses of EGF (0, 10–11, 10–10, and 10–9 M). After 24 h (Fig. 1B), the maximal dose of EGF (10–9 M) treatment stimulated DNA synthesis compared with untreated cells (P < 0.001). These data suggest that the HK-2 cell system employed in this study is suitable to analyze the role of EGF.
EGF induces NF-B DNA binding and activates NF-B-dependent transcription. To identify whether EGF, in addition to the known growth-inducing effects, may activate NF-B, HK-2 cells were treated with various doses of EGF and nuclear extracts were prepared. DNA binding activity of NF-B was detected by EMSA with a 32P-labeled NF-B-specific oligonucleotide as a probe. Whereas no specific NF-B binding activity was displayed in unstimulated cells (Fig. 2A, lane 1), EGF induced NF-B binding in a gradual manner (Fig. 2A, lanes 3–5). Maximum activation of inducible DNA binding activity required 10–9 M EGF. Similar results were obtained with TNF- (150 U/ml) (Fig. 2A, lane 2).
To analyze the time-dependent induction of NF-B binding activity, growth-arrested HK-2 cells were stimulated with 10–9 M EGF at different times. EGF-mediated induction of NF-B binding was clearly detectable after 15 min, reached a maximum after 60 min, and declined thereafter (Fig. 2B, lanes 2–6).
The specificity of NF-B binding induced by EGF was confirmed by competition experiments. Pretreatment with an excess (20- and 60-fold) excess of unlabeled B oligonucleotide or related B oligonucleotide derived from the IL-6 promoter (Fig. 3, lanes 2–5) inhibited NF-B complex formation, whereas competition with an unrelated oligonucleotide spanning an AP-1 binding site (Fig. 3, lane 6) did not.
Because NF-B complexes may constitute a variety of different homodimers or heterodimers, the subunit composition of the complexes was analyzed by supershift assays. Antibodies specific for p50 and p65 NF-B subunits were added to nuclear extracts of EGF-treated HK-2 cells. Addition of both antibodies revealed supershifted complexes (Fig. 3, lanes 7 and 8). Addition of antibodies to the p65 subunit of NF-B showed a strong shift in the mobility of the complexes (Fig. 3, lane 7). Addition of p50 antiserum produced a weaker shift in the mobility of the complexes, indicating some DNA binding of p65-p50 heterodimers (Fig. 3, lane 8).
NF-B binding sites in promoters and enhancers of genes serve as response elements that confer activation after treatment with EGF, TNF-, and diverse stimuli. In the following experiments, a luciferase construct under the control of three B sites (3xIgBLuc) was transiently transfected into serum-starved HK-2 cells. Twenty-four hours after transfection, cells were treated for an additional 24 h with EGF. EGF caused a strong dose-dependent increase in luciferase activity (Fig. 4A). The internal positive control we used was TNF- stimulation, which also induced a 10-fold increase in luciferase activity (Fig. 4A).
Activation of NF-B directly depends on the proper functioning of IBs. We cotransfected HK-2 cells with an expression construct bearing a super repressor form of IB, S32A/S36A. In this construct, the lack of serines 32 and 36 prevents the protein from being phosphorylated, thereby blocking its degradation and thus strongly inhibiting inducible NF-B activation. Overexpression of IB S32A/S36A nearly ablated EGF-dependent B-Luc reporter activity (Fig. 4B).
EGF leads to nuclear translocation of p65. In unstimulated cells, NF-B heterodimers are kept in the cytoplasm as inactive complexes by IB. After stimulation, IBs are usually phosphorylated and degraded, and free NF-B translocates into the nucleus. To investigate whether EGF induces nuclear translocation of p65, HK-2 cells were prepared and incubated with or without EGF. The proximal tubule cells were fixed and stained with an NF-B p65 antibody (red fluorescence) and counterstained with Sytox green to stain nuclei (green fluorescence). Overlaying the red fluorescence with the green counterstain results in a yellow nuclear staining indicative of nuclear localization of active p65. In the unstimulated state, p65 was localized exclusively in the cytoplasm (Fig. 5A). Treatment with EGF for 1 h led to translocation of p65 in the nucleus (Fig. 5B).
EGF induces cyclin D1 promoter activity in human proximal tubule cells. To investigate a possible link between NF-B and cell cycle progression, we focused our interest on potential transcriptional targets involved in the regulation of the cell cycle. Growth factors regulate the progression through the cell cycle from G1 to S phase, and this process requires the expression of D-type cyclins. The human cyclin D1 promoter contains at least two putative NF-B binding sites, termed D1-B1 and D1-B2 (Fig. 6A). Induction of EGF in HK-2 cells led to the fivefold stimulation of a luciferase reporter gene under the control of the cyclin D1 promoter (Fig. 6B). To confirm that growth factor-mediated induction of the cyclin D1 promoter depends on NF-B activity, the cyclin D1 promoter construct was cotransfected with increasing amounts of IB S32A/S36A expression vector (Fig. 7A), and cells were subsequently stimulated with EGF. In fact, inhibition of NF-B by the IB S32A/S36A mutant blocked the induction of EGF-mediated cyclin D1 promoter in a dose-dependent manner, indicating that NF-B is required for cyclin D1 transcriptional activation in early G1. To investigate the mechanism of this regulation in more detail, transfections were performed with promoter constructs containing point mutation of the D1-B1 and D1-B2 sites (Fig. 6A). HK-2 cells were stimulated with EGF and luciferase expression was measured (Fig. 7B). Mutations of the distal NF-B binding site (D1-B1M) slightly interfered with EGF-induced activation of the promoter. In contrast, mutation of the proximal NF-B binding site (D1-B2M), or both binding sites, caused a significant reduction in EGF responsiveness of the cyclin D1 promoter (Fig. 7B). These data demonstrate that EGF treatment induces cyclin D1 promoter activity in HK-2 cells and this activation requires intact B binding sites.
EGF stimulates cyclin D1 expression in HK-2 cells. The effect of EGF stimulation on cyclin D1 expression during the G1 phase was analyzed in synchronization experiments. Cells were serum-starved and then released from G0 by adding EGF. Cells were lysed at the indicated times, and protein extracts were analyzed by Western blotting (Fig. 8). Cyclin D1 expression was low in serum-starved HK-2 cells but increased when EGF was added. The maximum level was reached 4 h after the release of HK-2 cells from the growth-arrested state. This suggests that the observed induction of cyclin D1 promoter is a possible mechanism whereby EGF initiates G0/G1 entry and progression through the cell cycle.
DISCUSSION
Recovery of kidney function after ARF involves the entry of differentiated, quiescent tubule cells into the cell cycle in a rapid, closely regulated fashion. This process depends not only on the replacement or regeneration of injured cells but also on protection from programmed cell death (18, 21, 22, 25, 32, 33, 58). Previous studies have suggested that EGF plays an important role in the repair of renal epithelial cell injury (18, 21, 32, 33, 58, 68). Molecular mechanisms, however, by which EGF can maintain or reestablish the normal epithelial function are still unknown. In the study presented here, we provide evidence that EGF leads to activation of transcription factor NF-B in quiescent proximal tubule cells (HK-2) in vitro. In these cells, EGF exhibits growth-inducing properties similar to what has been described before (11, 22, 64, 69), suggesting that the chosen cell system is suitable in examining further effects of EGF in tubular cells. In HK-2 cells, EGF stimulates cyclin D1 transcription via NF-B. These assumptions are based on the following findings: 1) EGF induces activation of a NF-B reporter construct in a concentration-dependent manner, 2) NF-B activity is a result of nuclear translocation of NF-B protein complexes as assessed by immunofluorescence and EMSAs, and 3) EGF treatment leads to cyclin D1 promoter activity. This activation depends on intact NF-B binding sites harbored in the physiological cyclin D1 promoter sequence. Taken together with earlier studies demonstrating the antiapoptotic role of EGF, these data point to a substantial contribution of EGF in proliferative responses required for tubular regeneration after ARF.
Although the kidney is a major site for synthesis of prepro-EGF, and EGF receptor expression is upregulated at the site of injury, it is remarkable that EGF mRNA levels decrease at the initial time points of renal injury (18, 22, 28, 32, 58, 59). The temporary lack of EGF in this context could result in a significant deficit of survival factors (48, 49, 53, 54). This notion is supported by studies in which ischemic, toxic, or obstructively injured renal tubule cells were found to be rescued from programmed cell death by exogenously administered EGF (9–11, 23, 32, 40, 46). This would support the hypothesis that proliferating tubule cells die from injury unless specific survival signals are provided (3, 17, 49, 53, 54, 66).
NF-B as a potential mediator of EGF signaling, however, plays a central role in cell survival and proliferation (28–38, 54). Some studies demonstrated that NF-B activation after ischemia-reperfusion injury in the kidney led to augmented apoptosis and an increase in TNF- expression (15, 45). These studies, investigating NF-B as a key regulator of genes involved in inflammation and cellular stress showed, that inhibition of NF-B by either pyrrolidine dithicarbamate or N-acetyl-L-cystine resulted in a decreased rate of apoptosis in renal tubule cells. However, there are some concerns regarding these NF-B inhibitors, which may have also NF-B-independent effects (41, 52). Consistent with this, it has been shown that both substances (pyrrolidine dithicarbamate and N-acetyl-L-cystine) are able to induce apoptotic as well as antiapoptotic responses in vitro, depending on the examined tissue and cell type (30). More recent data, however, show that the inhibition of NF-B at inflamed sites could have additional and probably undesirable consequences like promotion of unscheduled cell loss, scarring, and loss of organ function (7, 13, 65). In particular, it has been shown that the blockade of NF-B can sensitize cells in the context of regeneration to TNF--induced apoptosis (47, 69). This might suggest a protective effect for this transcription factor (7). Consistent with the "dual-signal" hypothesis postulating coupling of the proliferation pathways with those of cell death, the apoptotic response in this context could also be interpreted as a consequence of proliferation failure (27, 56). Thus the extracellular signals that proximal tubule cells receive after onset of injury would determine whether NF-B induction leads to apoptosis or survival. EGF could act as a survival factor in proximal tubule cells by delivering antiapoptotic signals through expression of protective genes (13, 22, 47, 68). These observations are consistent with the role of NF-B for cell proliferation described in lymphocytes, fibroblasts, and hepatocytes, where NF-B activation correlates with cellular proliferation (4, 12, 19). There is evidence that NF-B proteins control cell survival and proliferation through their ability to regulate the expression and function of cell regulators such as cyclin D1 (16, 24, 68). The induction of cyclin D1 is a reliable marker for cell cycle (G1 phase) progression (35). In the absence of growth factors, cells are blocked in the cell cycle at the G1 restriction point (1, 51). Our data show that EGF is able to induce activation of cyclin D1 promoter activity and leads to an accumulation of cyclin D1 protein in quiescent HK-2 cells via NF-B. Activation of the cyclin D1 promoter is blocked by inhibition of NF-B activation, and mutation of the NF-B binding site in this element was able to eliminate EGF responsiveness.
In summary, the results described here point to a novel target for EGF-mediated signaling in the context of renal tubule cell regeneration and proliferation. EGF has been shown to exert its effects via NF-B and cyclin D1. Therefore, NF-B could represent the critical convergence point for EGF-mediated signals in ARF. The identification of new proliferation pathways in ARF is an exciting prospect, and their elucidation could provide potential new targets for the treatment of ARF.
FOOTNOTES
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Department of Internal Medicine II, Technical University of Munich, Munich, Germany
ABSTRACT
The promotion of cell survival and regeneration in acute renal failure (ARF) is important for the restitution of renal function. Epidermal growth factor (EGF) has been implicated in the regulation of cell proliferation. We provide evidence for a direct link between EGF, nuclear factor-B (NF-B), and cell cycle regulation (cyclin D1). EGF was found to stimulate NF-B-dependent gene transcription and DNA binding. In addition, EGF stimulated cyclin D1 promoter activity as well as cyclin D1 expression. Moreover, inhibition of NF-B caused a pronounced reduction of EGF-induced cyclin D1 promoter activity. Furthermore, both EGF-mediated NF-B activation and cyclin D1 expression were inhibited by coexpression of super IB. Taken together, these data identify NF-B and cyclin D1 as downstream targets of EGF and establish a molecular link between stimulation of EGF via activation of NF-B and cyclin D1 expression in human proximal tubular cells.
EGF; cyclin D1
IN CONTRAST TO THE HEART AND brain, where ischemia results in irreversible cell damage, the kidney can completely restore its structure and function after being severely damaged by either ischemia or toxins (32). This reversibility depends on the ability of renal tubule cells to regenerate and rearrange the damaged areas of epithelial cells along the nephron. This process is characterized by an increase in mitotic activity in renal epithelial cells and induction of nucleic acid synthesis (32). A burst of DNA synthesis is typical for all nephrotoxic or ischemic injuries, differing only in its rate and extent (59).
Several growth factors are key mediators of renal repair processes and compensatory renal epithelial growth (18, 21, 33). Epidermal growth factor (EGF) is one of the most effective growth-promoting substances, which has been identified in renal proximal tubule cells so far. Its function has been extensively investigated in experimental animal models (32, 33, 39). When EGF is administered to animals subjected to renal ischemia, it reduces the extent of renal dysfunction and accelerates recovery of the tubule cells (25, 57). To date, little is known about the underlying transcriptional mechanisms leading to tubular regeneration after acute renal failure (ARF).
A candidate pathway, involved in the EGF-induced mitogenic response, leads to NF-B activation. The inducible transcription factor NF-B participates in the regulation of numerous genes, many of which are involved in inflammation and cell proliferation (2, 20). The NF-B/Rel family has five members (p50, p52, p65/RelA, RelB, and c-Rel), which can form various homodimeric or heterodimeric complexes (2, 20). In most unstimulated cells, NF-B is thought to reside in the cytoplasm bound to an inhibitor protein, designated IB (6). Upon stimulation or activation of cells by a diverse array of stimuli, NF-B is able to rapidly dissociate from IB and translocate into the nucleus, an event that involves phosphorylation of IB on serines 32 and 36 (6, 29). Subsequent work has shown that NF-B is crucial for controlling proliferation and differentiation in many cell types and that it is linked to signaling processes that control cell cycle progression (5, 8, 31, 44, 50). In fact, a nuclear NF-B-like DNA binding activity is induced during the G0-to-G1 transition after serum stimulation in mouse fibroblasts (4).
The proliferative response of mammalian cells to extracellular signals is integrated in the mid- to late G1 phase of the cell cycle (26, 63). The cyclin D1 protein is a regulatory subunit of a holoenzyme, which is rapidly induced during G1 phase progression. There, cyclin D1 and its catalytic subunits are key regulators of cell proliferation (63). Furthermore, cyclin D1 links growth factor signaling to the cell cycle machinery. Cyclin D1 expression is regulated transcriptionally as well as via pathways of ubiquitin-proteosome-dependent degradation (14). The cyclin D1 promoter harbors cis regulatory elements for several transcription factors including AP-1, CREB/ATF, SP1, and STATs (61). Furthermore, the serum responsiveness of the cyclin D1 promoter depends on an intact proximal NF-B site in the cyclin D1 promoter (31).
The data presented here indicate that NF-B is a downstream target of the growth factor EGF in proximal tubule cells. We show that NF-B links EGF signaling to a key regulator of cell cycle progression by activating the cyclin D1 promoter. These observations suggest that EGF-induced NF-B activation contributes directly to the proliferative response needed for renal regeneration after ARF.
MATERIALS AND METHODS
Cell culture and treatments. HK-2, an immortalized proximal tubule cell (PTC) line derived from normal adult human kidney (55), was obtained from American Type Culture Collection (ATCC). Cells were maintained in DMEM/F-12 medium supplemented with 10% heat-inactivated fetal calf serum (FCS) in a humidified atmosphere of 5% CO2-95% air at 37°C and passaged every 7 days. For passage, confluent cells were washed with phosphate-buffered saline (PBS), removed with 0.05% trypsin, and plated in DMEM/F-12 medium. EGF was purchased from Saxon and recombinant TNF- from Sigma. EGF and TNF- were freshly dissolved in culture media and added to the cultures at the indicated concentrations and for the indicated time periods. Before stimulation, cells were serum-starved for 24 h.
Cell growth. HK-2 cells were washed twice in PBS, trypsinized, and resuspended in serum-free DMEM/F-12. Cells were plated in six-well culture dishes (Falcon) at a density of 1 x 105 cells in 1 ml of serum-free DMEM/F-12 additionally for 24 h. HK-2 cells were then stimulated with various concentrations of EGF alone for 24 h. The number of cells was determined daily using a cell-counting chamber.
5-Bromo-2-deoxyuridine enzyme-linked immunosorbent assay. To measure DNA synthesis, a 5-bromo-2-deoxyuridine (BrdU) assay (Roche) was used. Twenty-four-hour serum-starved cells were seeded in 96-well microtiter plates, and EGF at various concentrations was added additionally for 24 h. BrdU was then added for 2 h, cells were fixed with ethanol for 30 min, and an anti-BrdU antibody was added for 90 min. After the cells were washed, the anti-BrdU antibody was detected using a microtiter reader.
Plasmid constructs. The luciferase reporter 3xBLUC, which contains three tandem B repeats in pGL3 (Promega), has been described previously (68). Point mutations in IB S32A/S36A were introduced by primer-mediated, site-directed mutagenesis using a Quick Change Mutagenesis kit (Stratagene) to generate pcDNA-IB S32A/S36A (43). The cyclin D1 promoter construct pD1LUC and the promoter construct mutant for the NF-B sites D1-B1M, D1-B2M, and D1-B1M+B2M, harboring point mutations in the D1-B1 (CGCGACCCCC), D1-B2 (CGCGAGTTTT), or in both binding sites (introduced point mutations are underlined), were a kind gift from Dr. Claus Scheidereit (31).
Preparation of whole cell lysates and nuclear extracts. Whole cell lysates were prepared by incubating cell pellets for 30 min at 4°C in immunoprecipitation buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 10% glycerol, 1 mM dithiothreitol, 1 mM PMSF, and 5 mM NaF). Insoluble material was removed by centrifugation, and lysates were aliquoted and stored at –80°C. Nuclear extracts from HK-2 cells treated as indicated were prepared according to the method of Schreiber et al. (62) with a few modifications. Briefly, cells were collected, washed in cold PBS, and pelleted by centrifugation. The cell pellet was resuspended in 400 μl of cold buffer A (in mM: 10 HEPES, 10 KCl, 0.1 EDTA, 0.1 EGTA, 1 dithiothreitol, and 0.5 PMSF) and allowed to swell on ice for 20 min. Cells were disrupted by passage through a 26-gauge needle, and nuclei were centrifuged at 500 g in a microcentrifuge. The nuclear pellet was washed twice in buffer A to avoid cytoplasmatic contamination. Finally, the nuclear pellet was resuspended in 50 μl of cold buffer (20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 1 mM PMSF, and 5 mM NaF) and rocked at 4°C on a shaking platform for 30 min. The lysates were cleared by centrifugation, aliquoted, and stored at –80°C. Protein concentrations were determined by the Bradford method (Bio-Rad Laboratories) using bovine serum albumin as a standard.
Transfection and luciferase assays. Approximately 0.2 x 106 HK-2 cells/well were seeded in six-well dishes and allowed to attach overnight. Cells were washed twice with PBS and transfected with expression plasmids as indicated. Transfections were performed by the calcium phosphate precipitation method as described previously (60). Cells were washed twice with PBS 12 h after transfection and were serum-starved for a further 24 h. Cells were stimulated with EGF in various concentrations for 24 h. The control experiments were performed with 150 U of human TNF-. Cells were harvested in 250 μl of lysis buffer (Promega) and cleared by centrifugation. Cell lysates were normalized for protein content and used to measure luciferase activity using a commercially available kit (Dual Luciferase Assay System, Promega) in a luminometer (Berthold Analytical Instruments). Transfections were performed at least three times in triplicate.
EMSAs. EMSAs were performed as described using the NF-B motif of the mouse Ig- light chain enhancer (5'-CTCAACAGAGGGGACTTTCCGAGAGGCCAT-3') as a probe (42). Briefly, 5 μg of nuclear extracts were equilibrated for 20 min at room temperature in 20 μl binding buffer containing 4% glycerol, 10 mM Tris, 100 mM NaCl, and 100 ng/μl poly (dl-dC). Approximately 50,000 cpm of the -32P-labeled NF-B probe were added, followed by another 30 min of incubation on ice. The DNA binding complexes were separated by electrophoresis on a nondenaturing 5% polyacrylamide gel in 0.5x Tris-glycine-EDTA buffer (TGE) at 150 V. Gels were dried and exposed on X-ray film (Biomax MS, Kodak). Competition experiments were performed by adding NF-B-specific unlabeled oligonucleotide derived from the IL-6 promoter (5'-TATCAAATGTGGATTTTCCCATGAGTCTCA-3') to the reaction mixture in 20-fold or 60-fold molar excess, or by using an AP-1 oligonucleotide (5'-AGCTTACTCAGTACTAGTACG-3'). For supershift assays, antibodies against p65 and p50 subunits (Santa Cruz Biotechnology) were added to the binding buffer.
Western blotting. Whole cell lysates were boiled in 2x SDS sample buffer (100 mM Tris·HCl, 4% SDS, 0.2% bromphenol blue, 20% glycerol, 10% DTT) and separated by SDS-PAGE on 10% polyacrylamide gels. Proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore) by semidry blotting. Membranes were blocked in PBS supplemented with 5% skim milk and 0.1% Tween 20 and subsequently incubated with cyclin D1 antibody (BD Pharmingen). Proteins recognized by the antibody were detected by enhanced chemiluminescence (Amersham) using a horseradish peroxidase-coupled secondary antibody.
Immunofluorescence. Serum-starved cultures of HK-2 cells grown on coverslips were incubated with or without EGF (10–9 M). After 1 h of stimulation, cells were fixed for 5 min in 96% ethanol and subsequently incubated for 20 min in 4% formaldehyde at room temperature. Cells were then permeabilized with 0.2% Triton X-100 and stained with a NF-B p65 (BD Transduction Laboratories) antibody for 1 h followed by detection with an Alexa-labeled (568 nm) secondary antibody (Molecular Probes; Leiden, Netherlands). Nuclear costaining was performed with Sytox green (Molecular Probes). Samples were further analyzed by fluorescence microscopy (Olympus IX-71, x100 magnification) connected to a CCD camera (Hamamatsu Photonics, Hamamatsu City, Japan).
Statistical analysis. Data are expressed as means ± SE. Comparisons between groups were analyzed with Student's t-test (for 2 groups) or one-way ANOVA, followed by the Newman-Keuls post hoc test when indicated (for 3 or more conditions). Differences were considered to be significant at P < 0.05.
RESULTS
Effect of EGF on cell growth and proliferation. To examine whether EGF exhibits its well-established growth-stimulating effect in our system of HK-2 cells, a cell culture model system that displays important functional and morphological characteristics of human tubular cells, we treated HK-2 cells with EGF and assessed cell growth and proliferation (61). Growth curves were performed with increasing concentrations of EGF (10–11, 10–10, and 10–9 M) in the serum-starved culture media. FCS (10%) was used as a positive and untreated cells as a negative control. After 72 h of EGF exposure, cell number increased for all concentrations of EGF compared with untreated cells (Fig. 1A).
To determine the effect of EGF on HK-2 cell proliferation, BrdU incorporation was performed after treatment with different doses of EGF (0, 10–11, 10–10, and 10–9 M). After 24 h (Fig. 1B), the maximal dose of EGF (10–9 M) treatment stimulated DNA synthesis compared with untreated cells (P < 0.001). These data suggest that the HK-2 cell system employed in this study is suitable to analyze the role of EGF.
EGF induces NF-B DNA binding and activates NF-B-dependent transcription. To identify whether EGF, in addition to the known growth-inducing effects, may activate NF-B, HK-2 cells were treated with various doses of EGF and nuclear extracts were prepared. DNA binding activity of NF-B was detected by EMSA with a 32P-labeled NF-B-specific oligonucleotide as a probe. Whereas no specific NF-B binding activity was displayed in unstimulated cells (Fig. 2A, lane 1), EGF induced NF-B binding in a gradual manner (Fig. 2A, lanes 3–5). Maximum activation of inducible DNA binding activity required 10–9 M EGF. Similar results were obtained with TNF- (150 U/ml) (Fig. 2A, lane 2).
To analyze the time-dependent induction of NF-B binding activity, growth-arrested HK-2 cells were stimulated with 10–9 M EGF at different times. EGF-mediated induction of NF-B binding was clearly detectable after 15 min, reached a maximum after 60 min, and declined thereafter (Fig. 2B, lanes 2–6).
The specificity of NF-B binding induced by EGF was confirmed by competition experiments. Pretreatment with an excess (20- and 60-fold) excess of unlabeled B oligonucleotide or related B oligonucleotide derived from the IL-6 promoter (Fig. 3, lanes 2–5) inhibited NF-B complex formation, whereas competition with an unrelated oligonucleotide spanning an AP-1 binding site (Fig. 3, lane 6) did not.
Because NF-B complexes may constitute a variety of different homodimers or heterodimers, the subunit composition of the complexes was analyzed by supershift assays. Antibodies specific for p50 and p65 NF-B subunits were added to nuclear extracts of EGF-treated HK-2 cells. Addition of both antibodies revealed supershifted complexes (Fig. 3, lanes 7 and 8). Addition of antibodies to the p65 subunit of NF-B showed a strong shift in the mobility of the complexes (Fig. 3, lane 7). Addition of p50 antiserum produced a weaker shift in the mobility of the complexes, indicating some DNA binding of p65-p50 heterodimers (Fig. 3, lane 8).
NF-B binding sites in promoters and enhancers of genes serve as response elements that confer activation after treatment with EGF, TNF-, and diverse stimuli. In the following experiments, a luciferase construct under the control of three B sites (3xIgBLuc) was transiently transfected into serum-starved HK-2 cells. Twenty-four hours after transfection, cells were treated for an additional 24 h with EGF. EGF caused a strong dose-dependent increase in luciferase activity (Fig. 4A). The internal positive control we used was TNF- stimulation, which also induced a 10-fold increase in luciferase activity (Fig. 4A).
Activation of NF-B directly depends on the proper functioning of IBs. We cotransfected HK-2 cells with an expression construct bearing a super repressor form of IB, S32A/S36A. In this construct, the lack of serines 32 and 36 prevents the protein from being phosphorylated, thereby blocking its degradation and thus strongly inhibiting inducible NF-B activation. Overexpression of IB S32A/S36A nearly ablated EGF-dependent B-Luc reporter activity (Fig. 4B).
EGF leads to nuclear translocation of p65. In unstimulated cells, NF-B heterodimers are kept in the cytoplasm as inactive complexes by IB. After stimulation, IBs are usually phosphorylated and degraded, and free NF-B translocates into the nucleus. To investigate whether EGF induces nuclear translocation of p65, HK-2 cells were prepared and incubated with or without EGF. The proximal tubule cells were fixed and stained with an NF-B p65 antibody (red fluorescence) and counterstained with Sytox green to stain nuclei (green fluorescence). Overlaying the red fluorescence with the green counterstain results in a yellow nuclear staining indicative of nuclear localization of active p65. In the unstimulated state, p65 was localized exclusively in the cytoplasm (Fig. 5A). Treatment with EGF for 1 h led to translocation of p65 in the nucleus (Fig. 5B).
EGF induces cyclin D1 promoter activity in human proximal tubule cells. To investigate a possible link between NF-B and cell cycle progression, we focused our interest on potential transcriptional targets involved in the regulation of the cell cycle. Growth factors regulate the progression through the cell cycle from G1 to S phase, and this process requires the expression of D-type cyclins. The human cyclin D1 promoter contains at least two putative NF-B binding sites, termed D1-B1 and D1-B2 (Fig. 6A). Induction of EGF in HK-2 cells led to the fivefold stimulation of a luciferase reporter gene under the control of the cyclin D1 promoter (Fig. 6B). To confirm that growth factor-mediated induction of the cyclin D1 promoter depends on NF-B activity, the cyclin D1 promoter construct was cotransfected with increasing amounts of IB S32A/S36A expression vector (Fig. 7A), and cells were subsequently stimulated with EGF. In fact, inhibition of NF-B by the IB S32A/S36A mutant blocked the induction of EGF-mediated cyclin D1 promoter in a dose-dependent manner, indicating that NF-B is required for cyclin D1 transcriptional activation in early G1. To investigate the mechanism of this regulation in more detail, transfections were performed with promoter constructs containing point mutation of the D1-B1 and D1-B2 sites (Fig. 6A). HK-2 cells were stimulated with EGF and luciferase expression was measured (Fig. 7B). Mutations of the distal NF-B binding site (D1-B1M) slightly interfered with EGF-induced activation of the promoter. In contrast, mutation of the proximal NF-B binding site (D1-B2M), or both binding sites, caused a significant reduction in EGF responsiveness of the cyclin D1 promoter (Fig. 7B). These data demonstrate that EGF treatment induces cyclin D1 promoter activity in HK-2 cells and this activation requires intact B binding sites.
EGF stimulates cyclin D1 expression in HK-2 cells. The effect of EGF stimulation on cyclin D1 expression during the G1 phase was analyzed in synchronization experiments. Cells were serum-starved and then released from G0 by adding EGF. Cells were lysed at the indicated times, and protein extracts were analyzed by Western blotting (Fig. 8). Cyclin D1 expression was low in serum-starved HK-2 cells but increased when EGF was added. The maximum level was reached 4 h after the release of HK-2 cells from the growth-arrested state. This suggests that the observed induction of cyclin D1 promoter is a possible mechanism whereby EGF initiates G0/G1 entry and progression through the cell cycle.
DISCUSSION
Recovery of kidney function after ARF involves the entry of differentiated, quiescent tubule cells into the cell cycle in a rapid, closely regulated fashion. This process depends not only on the replacement or regeneration of injured cells but also on protection from programmed cell death (18, 21, 22, 25, 32, 33, 58). Previous studies have suggested that EGF plays an important role in the repair of renal epithelial cell injury (18, 21, 32, 33, 58, 68). Molecular mechanisms, however, by which EGF can maintain or reestablish the normal epithelial function are still unknown. In the study presented here, we provide evidence that EGF leads to activation of transcription factor NF-B in quiescent proximal tubule cells (HK-2) in vitro. In these cells, EGF exhibits growth-inducing properties similar to what has been described before (11, 22, 64, 69), suggesting that the chosen cell system is suitable in examining further effects of EGF in tubular cells. In HK-2 cells, EGF stimulates cyclin D1 transcription via NF-B. These assumptions are based on the following findings: 1) EGF induces activation of a NF-B reporter construct in a concentration-dependent manner, 2) NF-B activity is a result of nuclear translocation of NF-B protein complexes as assessed by immunofluorescence and EMSAs, and 3) EGF treatment leads to cyclin D1 promoter activity. This activation depends on intact NF-B binding sites harbored in the physiological cyclin D1 promoter sequence. Taken together with earlier studies demonstrating the antiapoptotic role of EGF, these data point to a substantial contribution of EGF in proliferative responses required for tubular regeneration after ARF.
Although the kidney is a major site for synthesis of prepro-EGF, and EGF receptor expression is upregulated at the site of injury, it is remarkable that EGF mRNA levels decrease at the initial time points of renal injury (18, 22, 28, 32, 58, 59). The temporary lack of EGF in this context could result in a significant deficit of survival factors (48, 49, 53, 54). This notion is supported by studies in which ischemic, toxic, or obstructively injured renal tubule cells were found to be rescued from programmed cell death by exogenously administered EGF (9–11, 23, 32, 40, 46). This would support the hypothesis that proliferating tubule cells die from injury unless specific survival signals are provided (3, 17, 49, 53, 54, 66).
NF-B as a potential mediator of EGF signaling, however, plays a central role in cell survival and proliferation (28–38, 54). Some studies demonstrated that NF-B activation after ischemia-reperfusion injury in the kidney led to augmented apoptosis and an increase in TNF- expression (15, 45). These studies, investigating NF-B as a key regulator of genes involved in inflammation and cellular stress showed, that inhibition of NF-B by either pyrrolidine dithicarbamate or N-acetyl-L-cystine resulted in a decreased rate of apoptosis in renal tubule cells. However, there are some concerns regarding these NF-B inhibitors, which may have also NF-B-independent effects (41, 52). Consistent with this, it has been shown that both substances (pyrrolidine dithicarbamate and N-acetyl-L-cystine) are able to induce apoptotic as well as antiapoptotic responses in vitro, depending on the examined tissue and cell type (30). More recent data, however, show that the inhibition of NF-B at inflamed sites could have additional and probably undesirable consequences like promotion of unscheduled cell loss, scarring, and loss of organ function (7, 13, 65). In particular, it has been shown that the blockade of NF-B can sensitize cells in the context of regeneration to TNF--induced apoptosis (47, 69). This might suggest a protective effect for this transcription factor (7). Consistent with the "dual-signal" hypothesis postulating coupling of the proliferation pathways with those of cell death, the apoptotic response in this context could also be interpreted as a consequence of proliferation failure (27, 56). Thus the extracellular signals that proximal tubule cells receive after onset of injury would determine whether NF-B induction leads to apoptosis or survival. EGF could act as a survival factor in proximal tubule cells by delivering antiapoptotic signals through expression of protective genes (13, 22, 47, 68). These observations are consistent with the role of NF-B for cell proliferation described in lymphocytes, fibroblasts, and hepatocytes, where NF-B activation correlates with cellular proliferation (4, 12, 19). There is evidence that NF-B proteins control cell survival and proliferation through their ability to regulate the expression and function of cell regulators such as cyclin D1 (16, 24, 68). The induction of cyclin D1 is a reliable marker for cell cycle (G1 phase) progression (35). In the absence of growth factors, cells are blocked in the cell cycle at the G1 restriction point (1, 51). Our data show that EGF is able to induce activation of cyclin D1 promoter activity and leads to an accumulation of cyclin D1 protein in quiescent HK-2 cells via NF-B. Activation of the cyclin D1 promoter is blocked by inhibition of NF-B activation, and mutation of the NF-B binding site in this element was able to eliminate EGF responsiveness.
In summary, the results described here point to a novel target for EGF-mediated signaling in the context of renal tubule cell regeneration and proliferation. EGF has been shown to exert its effects via NF-B and cyclin D1. Therefore, NF-B could represent the critical convergence point for EGF-mediated signals in ARF. The identification of new proliferation pathways in ARF is an exciting prospect, and their elucidation could provide potential new targets for the treatment of ARF.
FOOTNOTES
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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