Pulses of Prolactin Promoter Activity Depend on a Noncanonical E-Box that Can Bind the Circadian Proteins CLOCK and BMAL1
http://www.100md.com
内分泌学杂志 2005年第6期
Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina 29425
Address all correspondence and requests for reprints to: Dr. Fredric R. Boockfor, Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, 173 Ashley Avenue, Charleston, South Carolina 29425. E-mail: boockfor@musc.edu.
Abstract
Recent findings from our laboratory and those of others demonstrated that prolactin gene expression (PRL-GE) oscillates in single living mammotropes, but little information is available on the molecular processes that contribute to this phenomenon. To elucidate the source of this activity, we generated a series of constructs containing decreasing lengths of the PRL promoter fused to a luciferase reporter gene. These constructs were injected into single cells and assayed for photonic activity. We found pulse activity with all plasmids tested, even with the smallest promoter fragment of 331 bp. Sequence analysis of this fragment identified two potential E-boxes (elements known to bind CLOCK and BMAL1 circadian proteins). Furthermore, RT-PCR of PRL cells (pituitary, MMQ, and GH3) revealed expression of clock and bmal1 as well as five other clock genes (per1, per2, cry1, cry2, and tim), suggesting that the circadian system may function in PRL cells. Next, we mutated the core sequences of both E-boxes within the 2.5-kb PRL promoter and found that only mutation of the E-box133 completely abolished PRL-GE pulses. EMSAs revealed that CLOCK and BMAL1 were able to bind to the E-box133 site in vitro. Our results demonstrate that PRL-GE pulses are dependent on a specific E-box binding site in the PRL promoter. Moreover, the indication that CLOCK/BMAL1 can bind to this site suggests that these circadian proteins, either alone or in conjunction with other factors, may regulate intermittent PRL promoter activity in mammotropes, perhaps by acting as a temporal switch for the on/off expression of PRL.
Introduction
PROLACTIN (PRL) is involved in a wide range of mammalian physiological processes and is expressed with temporal characteristics that suggest a high degree of regulation (1). This is evidenced by a complex pattern of secretion consisting of repetitive surges that are timed specifically and depend on the physiological state of the animal. For example, in the female rat, PRL release occurs during the afternoon of proestrus and during pregnancy with specifically timed nocturnal and/or diurnal surges. It is generally accepted that the secretion of PRL from mammotropes depends on a variety of factors released by the hypothalamus that impinge on the pituitary and stimulate or inhibit the synthesis and release of PRL (1, 2). Because of this, it is recognized that much of the timed release of PRL depends on the type and level of modulatory factor that reaches the mammotrope.
The possibility that individual mammotrope function may also play a role in the temporal regulation of PRL release was raised with recent evidence from our laboratory demonstrating that PRL gene expression (PRL-GE) is not constant but oscillates in individual living mammotropes obtained from rat pituitaries (3). This oscillatory function of normal pituitary cells was determined in these experiments by use of a PRL promoter-driven luciferase reporter plasmid to monitor transcriptional activity over time. This type of episodic expression was later confirmed by others using a similar reporter system in cells from hamster pituitaries (4). Although the mechanism(s) underlying these pulses remains obscure, a clue as to what may be involved in this process was obtained by McFerran et al. (5) using a PRL::luc reporter construct and a continuous PRL-producing cell line. This group demonstrated that detectable pulses of PRL promoter expression could be induced experimentally in individual GH3 cells by treatment with high concentrations of serum. Because this treatment was effective in inducing circadian gene expression in cultured mammalian cells (6), these investigators proposed that a relationship might exist between circadian clock regulatory function and PRL oscillatory activity. Although intriguing, there was no direct evidence linking these two phenomenon, nor was information available that identified any specific process(es) underlying PRL oscillatory activity. Nevertheless, when these studies are taken together, two issues become clear. First, PRL-GE in individual mammotropes is a dynamic process that is subject to some type of internal timing mechanism. Second, this mechanism is dependent on the PRL promoter. To understand the mechanism(s) that may underlie these oscillations, the present study was conducted to determine the region of the PRL promoter responsible for intermittent function and begin to identify specific regulatory element(s) present that may be involved in this process.
Materials and Methods
Cells and cell culture
Primary cultures of anterior pituitary cells were prepared from primiparous, lactating female rats (6–10 d postpartum; Sprague Dawley, Charles River Laboratories, Inc., Wilmington, MA). Procedures involving these animals were conducted in accordance with protocols approved by the institution and the University Committee on Animal Care. As reported elsewhere (7) dispersed pituitary cells were maintained in phenol red-free medium-199-Earles’ salts/nutrient medium F-12 supplemented with antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin), L-glutamine (2 mM), and 10% fetal bovine serum. All tissue culture supplies were obtained from Invitrogen (Carlsbad, CA). Rat mammotrope MMQ (CRL-10609, American Type Culture Collection, Manassas, VA) and rat mammosomatotrope GH3 (CCL-82.1, American Type Culture Collection) cells were maintained in phenol red-free DMEM/F12 medium (1:1) supplemented with 2 mM L-glutamine, 10 mM HEPES, antibiotics, and 10% fetal bovine serum. The cells were grown in a 5% CO2 environment at 37 C in accordance with routine cell culture procedures.
Real-time measurement of gene expression in living mammotropes and pulse analysis
For bioluminescent imaging, the anterior pituitary cells obtained from lactating female rats were cultured on 25-mm glass photo-etched coverslips (Bellco Glass, Vineland, NJ) coated with poly-L-lysine as described previously (8). After 2 d in culture, individual cells were transfected by microinjection (Eppendorf 5242) with Endofree-purified plasmid DNA (QIAGEN, Valencia, CA) using a concentration of 0.2 μg/μl. Cells were incubated under normal culture conditions for 24–48 h before imaging. Then coverslips containing the cells were assembled in Sykes-Moore chambers and perifused (10 μl/min) with culture medium containing 0.1 mM luciferin (Sigma, St. Louis, MO). A chamber was placed on the heated stage of a photon-capture microscope (Axioskop, Carl Zeiss, Jena, Germany) and photon images were visualized using x10 objective (Neofluar, Carl Zeiss). Photonic signals from single cells were accumulated and quantified continuously in 30-min bins for more than 40 h using a photon-counting camera system (Argus 50, Hamamatsu, Bridgewater, NJ). Photonic images were analyzed off-line as previously described (8, 9). The presence of PRL-GE pulses was identified as described by Shorte et al. (3). A pulse was defined as a significant rise in photonic activity that lasted longer than 2 h with a value greater than 5% of the photonic signal count at the beginning of the pulse plus 2 times the total signal-noise variation (SNV). SNV was calculated by the square root of each value plus the SD of the corresponding background values squared [SNV = ([signal count] + [SD of detected background2])]. For normalization, the photonic activity data were plotted as signal count over SNV as a function of time. Comparisons of pulse activity between cells microinjected with various constructs and expressing luciferase activity were achieved using two-tailed, unpaired t tests. All data are expressed as mean ± SEM.
Preparation of PRL promoter constructs
Construction of the luciferase reporter plasmid pPLDA12 is described elsewhere (3). This plasmid contains 2.5 kb of the rat PRL promoter fused to a modified luciferase coding sequence (LUCODC-DA), which encodes a short-lived luciferase enzyme (10). Generation of a unidirectional nested set of deletions in the PRL promoter was accomplished using the Erase-a-Base system (Promega, Madison, WI) in accordance with the manufacturer’s recommendations. Briefly, the pPLDA12 was digested with NdeI and filled with -phosphorothioate deoxynucleotide triphosphates. The linear plasmid was restricted with AflII (located near the 5' end of the 2.5-kb PRL promoter fragment) and submitted to Exonuclease III digestion for various times. Plasmids were ligated, precipitated, and transformed in Escherichia coli XL1-Blue MRF' cells. The resulting recombinant DNA molecules were extracted using the QIAprep Spin miniprep kit (QIAGEN), and the length of the PRL promoter in each of the clones was determined by restriction digest with two sets of double-enzyme digestions, BamHI/BglI and BamHI/ScaI (New England Biolabs, Beverly, MA). From the collection of PRL promoter deletion constructs obtained, only a subset was subjected to bioluminescent imaging for evaluation of the photonic activity. Automated DNA sequencing of the 331-bp PRL promoter fragment was obtained by the Biotechnology Resource Laboratory of the Medical University of South Carolina (Charleston, SC).
Site-directed mutagenesis of E-box sites
Mutants of the rat PRL promoter sites E-box133 and E-box10 were generated using the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Briefly, 40 μg of Endofree purified pPLDA12 plasmid DNA was used as template for mutant strand synthesis. For the mutagenesis, the primers E-box10F (5'-GGT TTA TAA AGT CAA TGT CTG CCG CGG AGA AAG CAG TGG TTC TCTT), E-box133F (5'-GTC TTC CTG AAT ATG AAT AAG AAA TAA AAT ACC TCG AGA TGT TTA AAA TTA TTG GGG CTA TCT TAA TGAC), and their complementary oligonucleotides were used (Sigma Genosys, Woodlands, TX). These mutagenic primers were engineered in such a way that the mutated nucleotides formed a unique restriction site (SacII for E-box10, and XhoI for E-box133, sequence underlined), facilitating the screening of the mutated clones. The mutations were confirmed by nucleotide sequencing.
Total RNA isolation and RT-PCR of the circadian genes
Total RNA was isolated from either clonal pituitary cell cultures (MMQ and GH3) or pituitary glands from female lactating rats using the RNeasy kit (QIAGEN). First-strand cDNA synthesis and reverse transcription reactions were described elsewhere (11). The RNA samples were treated with DNase I (30 Kunitz units) to remove any trace of genomic DNA as specified in the RNeasy kit procedure (QIAGEN). PCR amplifications of the clock cDNA gene complements (clock, bmal1, per1, per2, cry1, cry2, and tim) were performed in a 25-μl reaction volume containing 1x QIAGEN PCR buffer, 1x QIAGEN Q-solution, 0.6 μM primers, 0.2 mM each deoxynucleotide triphosphates (Invitrogen), and 0.6 U HotStar Taq DNA polymerase (QIAGEN). The design of the rat specific primers was based on the sequence of the mouse-specific primers that were used to amplify circadian genes. PCR products obtained with these primers were sequenced and characterized to confirm their identity (12). The rat-specific circadian gene accession numbers, DNA sequences of their corresponding PCR primers, and size of the amplified fragments were as follows: clock (NM_021856, CLK-F1: 5'-GCG AGA ACT TGG CGT TGA GGAG, CLK-R1: 5'-CTG TGT CCA CTC ATT ACA CTC TGT, 770 bp), bmal1 (AB012600, BMAL1-F1: 5'-TCT GGA GCT CGG CGC TCT TTC, BMAL1-R1: 5'-AAC AAC AGT GTT GGT TGA GAC AAT, 602 bp), per1 (XM_340822, PER1-F1: 5'-CCA GGA CCC AGA AAG AAC TC, PER1-R1: 5'-CCT CTG ATT CGG CAG AAGAC, 513 bp), per2 (NM_031678, PER2-F1: 5'-AGA CGT GGA CAT GAG CAGCG, PER2-R1: 5'-CAG GAT CTT CCC AGA GACCA, 519 bp), cry1 (AF545855, CRY1-F1: 5'-TCC CCT CCC CTT TCT CTTTA, CRY1-R1: 5'-ATC CCT TCT TCC CAA CTGAT, 292 bp), cry2 (NM_133405, CRY2-F1: 5'-AAG CTG AAT TCC CGT CTGTT, CRY2-R1: 5'-GTG GTT TCT GCC CAT TCAGT, 237 bp), and tim (NM_031340, TIM-F1: 5'-GGA GAA CTG TTA CAA CCC ACTC, TIM-R1: 5'-GAA CGT GGA TGG TAT CTC TCA, 440 bp). The PCR conditions were as follows: initial incubation at 95 C for 15 min and then 36 cycles of denaturation at 94 C for 30 sec, annealing at 56 C for 1 min, and elongation at 72 C for 40 sec. The PCR products were resolved in 1.2% Tris acetate EDTA (pH 8.0)-agarose gels stained with ethidium bromide.
Nuclear protein extract and EMSAs
The interaction of MMQ nuclear proteins with the DNA fragment containing the putative E-box133 binding site was analyzed by EMSAs using the LightShift Chemiluminescent EMSA Kit (Pierce, Rockford, IL). The 20-bp DNA duplex used in EMSA experiments consisted of the annealed pair of oligonucleotides E-box133F (5'-AAT AAA ATA CCA TTT GAT GT), and E-box133R (5'-ACA TCA AAT GGT ATT TTA TT) (Integrated DNA Technologies, Inc., Coralville, IA). These two oligonucleotides were biotinylated at their 5'-ends to allow chemiluminescent detection. Nuclear protein extracts from MMQ cells were obtained using the NE-PER nuclear and cytoplasmic extraction reagents (Pierce) supplemented with 2x final concentration of the Halt protease inhibitor cocktail kit (Pierce). Then these extracts were purified and concentrated 3-fold with the Microcon centrifugal filter YM-10 (Millipore, Bedford, MA). Protein concentration in the nuclear extracts was determined using the Micro BCA protein assay reagent kit (Pierce). DNA-protein binding reactions consisted of incubation of 15 μg of the concentrated NE-PER nuclear extracts mixed with a reaction buffer composed of 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 50 ng/μl poly (dI · dC), 2.5% glycerol, 10 mM EDTA (pH 8.0), and 0.05% Nonidet P-40 for 10 min at room temperature. This was followed by addition of 100 fmol of 5-biotin-labeled E-box133 DNA to the reaction mixture and incubating for 25 min at room temperature.
For competition experiments, a 60-fold molar excess of unlabeled duplex DNA fragments corresponding to either the E-box133 site (described above) or the stimulating protein-1 (SP1) site (5'-ATT CGA TCG GGG CGG GGC GAGC, Promega) was added to the reaction mixture before addition of the labeled oligonucleotides. For supershift experiments, we added 4 μg of IgG affinity purified goat polyclonal antibody raised against CLOCK (sc-6927), BMAL1 (sc-8550), or a control IgG (DREAM, sc-9309) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to the binding reaction mixture and incubated for 40 min at room temperature. The CLOCK and BMAL1 antibodies were validated for use in EMSAs by Oishi et al. (13). Reaction mixtures were analyzed by nondenaturing 6% PAGE (Invitrogen) and then transferred to Biodyne B nylon membranes (Pierce). The biotin-labeled DNA probes were detected by chemiluminescence using the chemiluminescent nucleic acid detection module kit (Pierce). The membranes were exposed to x-ray films for 1–3 min and developed according to the manufacturer’s instructions.
Results
Identification of the PRL promoter segment responsible for pulsatility
As stated above, a previous investigation from our laboratory clearly identified pulses of PRL promoter-driven gene expression in living pituitary cells (3). In the present study, initial efforts were directed toward identifying the region(s) of the PRL promoter associated with this pulsatile function. This involved unidirectional deletions of the PRL promoter to generate PRL::luc reporter constructs with different promoter lengths (Fig. 1). These constructs were injected into individual cells and then assayed for photonic expression. By evaluating each fragment length progressively from larger to smaller until pulsatile function disappeared, we reasoned that we would be able to identify the minimal promoter region responsible for this pulsatile function. As shown (Fig. 1), we found that constructs with fragment lengths of approximately 1.3, 0.64, 0.44, and 0.33 kb exhibited pulses. Representative photonic profiles of the smaller constructs tested are shown in Fig. 2. As illustrated, the pulse activity observed with these deletion constructs were indistinguishable from those obtained with the unmodified full-length promoter. In fact, we observed pulse activity with these deletion constructs to occur in approximately the same proportion of cells as compared with the full-length promoters (2.5 kb) [64.1 ± 7.5% (n = 28) vs. 70.3 ± 5.2% (n = 86), P > 0.3, various sized deletion vs. full-length, respectively]. From these experiments, it appeared that the smallest fragment of the PRL promoter tested (0.33 kb) contained the minimal elements needed for pulse activity.
FIG. 1. Influence of PRL promoter fragment size on pulse activity. The plasmid pPLDA12 (2.5-kb PRL promoter) was used as a template to generate unidirectional deletions. As shown, each promoter deletion construct consisted of a portion of the promoter fused to the luciferase reporter gene (luc*). Preparations of constructs containing each fragment size were microinjected into single living pituitary cells obtained from lactating rats and photonic activity monitored for a period of 40 h. At least nine expressing cells/construct were analyzed for pulse activity (+). Note that the smallest region tested that was capable of generating pulse activity was confined to the first 0.33 kb of the PRL proximal promoter region.
FIG. 2. Representative photonic profiles from single mammotropes generated by various PRL promoter deletion constructs. Photonic activity was measured from individual mammotropes microinjected with plasmids containing either the full-length 2.5-kb segment (A) or fragment sizes 0.64 (B), 0.44 (C), or 0.33 kb (D) of the rat PRL promoter. Each point represents photonic signal accumulated for 30 min and is expressed as a function of the SNV (see Materials and Methods). Vertical bars indicate SEM. Asterisks represent significant PRL gene expression pulses. These profiles are representative of determinations made on at least nine luciferase-expressing cells per construct.
With attention focused on this 331-bp segment, we attempted to identify any recognizable transcription factor binding sites that may be associated with intermittent or rhythmic activity. In addition to previously identified regulatory sites [such as the pituitary-specific transcription factor (Pit)-1, Pit-2, Pit-3, and Pit-4 binding sites (14)], DNA sequence analysis revealed two putative E-box binding sites (CANNTG), one located close to the transcription start site termed E-box10 (CAGATG, –10 to –5) and the other localized farther upstream between the Pit-2 and Pit-3 binding sites termed E-box133 (CATTTG, –133 to –128) (Fig. 3). E-box binding sites have been shown to affect expression of a large number of genes, many of which are involved in circadian function (15). In the case of several circadian-regulated genes, the canonical E-box site CACGTG has been reported to preferentially bind the regulatory proteins, CLOCK and BMAL1 (16, 17, 18, 19). Also, variations in the E-box recognition sequence have been reported in a number of clock-regulated genes (20, 21, 22) and are believed to function in circadian processes as well. The presence of E-boxes in this promoter region supports the possibility that clock proteins may be involved in the intermittent expression of the PRL promoter.
FIG. 3. Identification of putative E-boxes in the 331-bp proximal promoter region of rat PRL gene. Nucleotide sequence of the –331/+1 region of the rat PRL promoter is presented. The two putative E-box binding sites, E-box10 (CAGATG) and E-box133 (CATTTG), are denoted (boxes and bold characters). For reference, the pituitary-specific transcription factor Pit-1 binding sites are underlined (their relative locations are described in Ref. 45 ).
Expression of circadian genes in pituitary tissues and PRL-producing cell lines
In the next series of experiments, we attempted to identify whether the circadian transcription factors CLOCK and/or BMAL1 or other circadian regulators were expressed in PRL cells. RT-PCR analysis was performed on primary rat pituitary cells, and two PRL-secreting cell lines (MMQ and GH3). As shown in Fig. 4A, bands corresponding to the predicted molecular weight for clock (770 bp) and bmal1 (602 bp) gene amplification products were identified when total RNA from each cell population was used as a template. In addition to clock and bmal1, we observed the expression of five other key clock regulatory genes (per1, per2, cry1, cry2, and tim with the expected molecular weight of 513, 519, 292, 237, and 440 bp, respectively) in each PRL cell type investigated when primers specific for these factors were used in RT-PCRs (Fig. 4B). The expression in PRL cells of such an array of genes associated with temporal regulatory activity raises the possibility that these factors may play some type of role in intermittent PRL-GE activity in mammotropes.
FIG. 4. RT-PCR analysis of core circadian gene expression in PRL-secreting cells. RT-PCRs were conducted for clock, bmal1, per1, per2, cry1, cry2, and tim gene expression in rat pituitary tissue and PRL-secreting clonal cell lines MMQ and GH3. A, Products obtained after RT-PCR amplification using the specific primers for the circadian transcriptional activator clock (770 bp) and bmal1 (602 bp) genes. B, Amplified products obtained when specific primers for the five core clock regulatory genes, per1 (513 bp), per2 (519 bp), cry1 (292 bp), cry2 (237 bp), and tim (440 bp), were used. The negative control (labeled –CTRL) contained water instead of cDNA template. The pBR322 MspI digest was used as a DNA ladder and is shown in the first lanes for rat pituitary and GH3-derived products. For the rat MMQ-PCR products, we used a 100-bp DNA ladder (present in the far left lane) that ranges from 100 to 1517 bp. MW, Molecular weight
Influence of E-box mutations on PRL promoter activity
The two E-box sites identified above in the PRL promoter may play a role in oscillatory activity possibly by interacting with certain circadian core regulatory proteins expressed in mammotropes. To address the impact of these E-boxes on pulses of PRL gene activity, we mutated each site in the 2.5-kb PRL promoter. The E-box133 site was changed from CATTTG to CTCGAG and the E-box10 site was changed from CAGATG to CCGCGG, altering only the core E-box binding sequences. Reporter plasmids containing each of these mutant constructs were microinjected into pituitary cells from lactating rats and assessed for changes in promoter-driven pulse activity. As illustrated, cells injected with plasmids containing mutated E-box10 (E-box10*) site exhibited normal patterns of photonic activity (Fig. 5A). This activity was virtually indistinguishable from that obtained using nonmodified PRL promoter (compare Fig. 5A with Fig. 2A above). Because of this, the E-box10 site was not considered for further analysis. In contrast, use of the plasmid containing the mutated E-box133 (E-box133*) site resulted in a complete abolition of pulses (Fig. 5B). Cells injected with this mutant construct continued to exhibit basal photonic activity. These results demonstrate that the E-box133 binding site is essential for pulses of PRL promoter activity.
FIG. 5. Effect of mutation in E-box sites on PRL pulsatile activity. Representative examples of photonic profiles generated by single primary mammotropes microinjected with luciferase reporter constructs containing either mutations in E-box10 (A) or E-box133 (B) binding sites of the rat PRL promoter (2.5 kb) are presented. As described above, each point represents photonic emissions collected for 30 min and expressed as a function of the SNV. Vertical bars indicate SEM. Asterisks represent significant PRL gene expression pulses. Each profile is representative of results obtained from at least 10 cells in six and seven different experiments for E-box10* and E-box133*, respectively.
EMSA analysis of CLOCK/BMAL1 binding to E-box133
Because the sequence of this putative E-box133 diverges from the canonical or perfect clock-related E-box structure (CACGTG), it was important to demonstrate whether CLOCK and/or BMAL1 could bind to the site. This was accomplished by performing EMSAs. The incubation of 5'-biotin-labeled DNA probe (containing an unmodified E-box133 site) with nuclear proteins from MMQ cells resulted in the formation of a specific band representing a complex formed between the E-box133 DNA probe and bound nuclear proteins (Fig. 6). Moreover, addition of an antibody specific for either CLOCK or BMAL1 caused a supershift to occur as evidenced by the formation of a higher molecular weight complex. The absence of this higher molecular band when control IgG was used indicates that the antibody supershift reaction was specific for CLOCK or BMAL1 protein interaction. In additional control experiments, we found specific band formation to disappear when the reaction mixture was incubated with unlabeled E-box133 DNA but to be unaffected when unrelated SP1 DNA oligonucleotides were used, demonstrating that the complexes that form are specific for the E-box133 site. From these results, it is clear that a CLOCK and BMAL1 protein complex can bind to this E-box133 site in vitro, suggesting that these circadian factors are able to interact with the PRL promoter and may play an important role in the regulation of PRL pulse activity.
FIG. 6. EMSA analysis of CLOCK and BMAL1 binding to the E-box133 site of the PRL promoter. EMSA was performed with nuclear protein extracts from the pituitary MMQ cells and biotinylated E-box133 binding site DNA as probe (see Materials and Methods). The first lane shows the results obtained when biotinylated probe was used in the absence of nuclear extracts. The other lanes represent incubations of the labeled probe with MMQ nuclear extracts (MMQ-NE) alone or combined with various antisera or DNA competitors. Protein DNA interactions are denoted by the appearance of high-molecular-weight complexes (Specific Bands). CLOCK and BMAL1 proteins within the complex were identified (MMQ-NE + CLOCK and MMQ-NE + BMAL1, respectively) by the presence of higher-molecular-weight bands (Supershift) when specific antiserum for these proteins were used. Reactions conducted using control IgG (MMQ-NE + IgG) are presented for comparison. In competition experiments, results of 60-fold molar excess of unlabeled specific E-box133 (MMQ-NE + E-Box133) or nonspecific SP1 (MMQ-NE + SP1) DNA probes are also shown. These results are representative of those obtained in four separate experiments.
Discussion
Previously we reported that PRL promoter activity occurs in rhythmic pulses in individual living mammotropes (3). Because the timing of PRL-GE pulses was not consistent with the strict definition of circadian activity (23), we classified these oscillations as noncircadian. In the present work, we have demonstrated that these pulses are dependent on a specific promoter site (E-box133, CATTTG) that can bind two essential clock regulatory proteins. This is evidenced by the observation that mutation of this site (E-box133*) results in complete abolition of pulse activity. Moreover, supershift EMSA experiments using specific antibodies against CLOCK or BMAL1 demonstrates that these two circadian proteins bind to this site in vitro. When taken together, our findings raise the intriguing possibility that even though PRL-GE pulse activity does not follow a strict circadian pattern, the mechanism underlying PRL-GE pulses may involve specific core regulatory elements that are usually associated with the circadian system.
In mammals, the oscillatory mechanism of the suprachiasmatic nucleus (SCN) coordinates behavior and physiological rhythms throughout the whole animal and consists mainly of two activating factors, CLOCK and BMAL1 and two repressing factors, cryptochrome (CRY)1 and CRY2 in addition to other regulatory proteins (24). In this system, a CLOCK/BMAL1 complex binds preferentially to the canonical E-box sequence CACGTG to activate transcription of these cry genes. The CRY proteins feedback and inhibit CLOCK/BMAL1 binding resulting in the inhibition of their own production. The interaction of these clock factors in stimulating and inhibiting gene expression forms the basis for an oscillatory mechanism that regulates the timing of a variety of factors produced in the hypothalamus (for detailed reviews, see Refs. 24 and 25).
The potential of the E-box133 in the prolactin promoter to bind CLOCK and BMAL1, even though its structure is different from the classic type of E-box sequence normally associated with clock gene regulation, may represent a novel mechanism by which these circadian proteins can modulate nonclock gene expression. It has been generally accepted that a specific hexameric DNA sequence (CACGTG; termed the perfect or canonical E-box) serves as a response element for the circadian regulatory proteins CLOCK and BMAL1 (for review, see Refs. 15 and 26). Indeed, these proteins heterodimerize forming a complex that activates gene transcription when bound to a canonical E-box sequence. In our study, no canonical E-box binding site was identified in the PRL promoter sequence. In contrast, we identified two noncanonical E-boxes (CANNTG) that are usually associated with a more ubiquitous E-box family of transcription regulatory sites. These sites have been linked to a variety of transcriptional processes involving such things as cell proliferation, differentiation, transformation, and apoptosis and bind transcription factors mainly of the helix-loop-helix type (15). Of these two noncanonical E-box elements identified in the PRL proximal promoter region, we found only one (E-box133; CATTTG) to be involved in oscillatory activity as evidenced by the complete abolition of pulses when the site was mutated. Although noncanonical E-boxes have been identified in promoters that have circadian activity such as tim in Drosophilia (21) and per4 in zebrafish (20), it was demonstrated that they cooperate with adjacent canonical E-box elements to promote transcription. Moreover, canonical clock E-box elements do not have to be restricted to the promoter region to be active. Recent investigations on the regulation of the circadian transcription factor albumin D-element binding protein revealed E-box elements in the first and second intron of the gene as well as the promoter region (22). These E-boxes consisted of both canonical and noncanonical types that may well interact as described for tim. In our analysis, we were unable to find a canonical E-box anywhere in the rat PRL gene (genomic region covering more than 35 kb including the rat PRL gene on chromosome 17, WGS supercontig, GenBank accession number NW_047491). On the other hand, we identified several noncanonical E-box sites downstream from the PRL transcription start site. However, their role in PRL pulse activity remains in question because elements contained within the 331-bp PRL promoter segment are sufficient to generate pulses. Our results indicate clearly that a noncanonical E-box element (E-box133), which can bind CLOCK and BMAL1, may not have to work in conjunction with a perfect (CACGTG) E-box to induce oscillatory transcriptional activity of the PRL promoter. If such a nonperfect E-box is active in our system, then it is possible that such an element may be active in others. In recent studies, Panda et al. (27) identified nearly 650 cycling genes by microarray analysis from which they retrieved the sequences of 127 genes from both the mouse and human genomes. Of these sequences, they identified only nine genes that contained canonical E-boxes. However, cursory analysis of the 127 sequences reveals that many of these other genes contain one or more noncanonical E-box binding sites. When taken together, our findings of transcriptional oscillatory activity driven by a single noncanonical E-box identifies a novel mechanism by which the clock system may act on the PRL promoter. Moreover, this observation raises the intriguing possibility that such an E-box type (binding CLOCK and/or BMAL1) may provide a means by which the clock gene system can confer oscillatory activity to a wide variety of nonclock genes.
Our observations of clock components and clock-related rhythmic activity in mammotropes confirms previous observations of clock gene expression in pituitary tissue (28) and extends the view that most, if not all, peripheral cells and tissues have an endogenous timing system. Circadian oscillatory elements have been identified in a wide variety of tissues such as skin, bone marrow, pancreas, retina, liver, heart, kidney, and lung (29, 30, 31, 32, 33, 34, 35). Although not all of the clock genes have been investigated, it has been determined that these tissues have many of the same clock proteins that are present in the SCN of the hypothalamus. Most of the evidence obtained to support rhythmic gene expression in these tissues came from static RNA determinations performed over time in synchronized tissues. This suggested strongly that oscillatory activity occurred in peripheral tissues, but it was not until the adaptation of luciferase technology that oscillatory gene activity could be observed in real time. The use of transgenic reporter mouse lines in which mouse period 1 (per1) promoter drives a luciferase reporter revealed circadian rhythms of per1 expression in liver tissues, with oscillatory activity diminishing within two to seven cycles (36). This suggested that gene oscillations were not self-sustaining but were driven by exogenous input. However, more recent experiments of Yoo et al. (37) using a knockin mouse line in which the luciferase reporter was fused to the per2 gene revealed self-sustained per2 rhythms for at least 20 d. These observations strengthened the idea that peripheral tissues contain the components necessary to maintain rhythmic function. However, the reason for this clock gene controlled oscillatory activity in the production and release of a nonclock or output gene such as PRL remains in question. One possibility is that the oscillatory control imposed at the tissue level provides a means to coordinate cells within a tissue (38). This may enable substantial levels of a particular factor such as PRL to be available for immediate release at critical times.
The rhythmic release of PRL occurs in distinct patterns, depending on the physiological state of the animal (1). An excellent example of this can be observed in the cycling female in which a surge of serum PRL can be detected during the afternoon of proestrus every 4 or 5 d. Upon mating, stimulation of the uterine cervix induces a secretory pattern consisting of specifically timed nocturnal and diurnal surges. This phenomenon clearly reveals that PRL release is subject to a control process that is centrally coordinated. Because of the circadian nature of this release, it is generally accepted that much of this control originates in the SCN of the hypothalamus. Although it has been demonstrated that oxytocin neurons from the paraventricular nucleus display daily rhythms that correlate with PRL release patterns (39), the identification of neuronal connections between the SCN and this other hypothalamic region emphasizes the likely SCN control of this phenomenon (40, 41). The critical nature of the CLOCK protein in PRL control is evidenced by the report of Miller et al. (42) in which clock mutant mice were characterized. In addition to a number of reproductive hormonal changes related to disruption of circadian activity, these investigators demonstrated that the mutant mice showed a decline in progesterone levels at midpregnancy and a shortened duration of pseudopregnancy. In normal animals, PRL is the factor that maintains the ovarian corpora lutea, the structure that produces progesterone levels necessary to maintain pregnancy and extend pseudopregnancy (43). In light of the importance of the CLOCK protein in both peripheral and central processes, it is quite likely that circadian processes are disrupted at both levels in these mutants. A number of factors such as dopamine, TRH, and oxytocin are secreted from the hypothalamus and influence PRL at the cellular level (1). However, the role of these factors on PRL rhythmic activity remains to be determined. Our ability to assess the influence of clock proteins on rhythmic PRL promoter activity in single mammotropes, in conjunction with analysis of the effects of centrally derived modulators on these rhythms, provides a unique model system that can be used to address the role of peripheral and central clock systems in controlling the activity of an output gene such as PRL.
In summary, our findings that the basis for oscillatory behavior of PRL promoter activity in single mammotropes may involve specific core circadian proteins raises important new issues in the study of the control of hormone function and gene regulation. The possibility that such proteins may underlie most, if not all, oscillatory gene expression must be considered, especially in the pituitary. This tissue produces a number of hormones that are released in a specifically timed manner and are critical to the proper functioning of a variety of systems in the body (44). The idea that clock-related binding elements may be more common than previously recognized and that the pituitary contains a wide array of clock proteins increases the prospect that the clock system may be an important regulator of many pituitary hormones. Our approach using single living mammotropes should enable identification of many of the basic aspects of this process.
References
Freeman ME, Kanyicska B, Lerant A, Nagy G 2000 Prolactin: structure, function, and regulation of secretion. Physiol Rev 80:1523–1631
Leong DA, Frawley LS, Neill JD 1983 Neuroendocrine control of prolactin secretion. Annu Rev Physiol 45:109–127
Shorte SL, Leclerc GM, Vazquez-Martinez R, Leaumont DC, Faught WJ, Frawley LS, Boockfor FR 2002 PRL gene expression in individual living mammotropes displays distinct functional pulses that oscillate in a noncircadian temporal pattern. Endocrinology 143:1126–1133
Stirland JA, Seymour ZC, Windeatt S, Norris AJ, Stanley P, Castro MG, Loudon AS, White MR, Davis JR 2003 Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells. J Endocrinol 178:61–69
McFerran DW, Stirland JA, Norris AJ, Khan RA, Takasuka N, Seymour ZC, Gill MS, Robertson WR, Loudon AS, Davis JR, White MR 2001 Persistent synchronized oscillations in prolactin gene promoter activity in living pituitary cells. Endocrinology 142:3255–3260
Balsalobre A, Damiola F, Schibler U 1998 A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93:929–937
Castano JP, Kineman RD, Frawley LS 1996 Dynamic monitoring and quantification of gene expression in single, living cells: a molecular basis for secretory cell heterogeneity. Mol Endocrinol 10:599–605
Villalobos C, Faught WJ, Frawley LS 1998 Dynamic changes in spontaneous intracellular free calcium oscillations and their relationship to prolactin gene expression in single, primary mammotropes. Mol Endocrinol 12:87–95
Villalobos C, Faught WJ, Frawley LS 1999 Dynamics of stimulus-expression coupling as revealed by monitoring of prolactin promoter-driven reporter activity in individual, living mammotropes. Mol Endocrinol 13:1718–1727
Leclerc GM, Boockfor FR, Faught WJ, Frawley LS 2000 Development of a destabilized firefly luciferase enzyme for measurement of gene expression. Biotechniques 29:590–598
Leclerc GM, Leclerc GJ, Shorte SL, Frawley LS, Boockfor FR 2002 Cloning and mRNA expression of the Ca2+-binding DREAM protein in the pituitary. Gen Comp Endocrinol 129:45–55
Gillespie JM, Chan BP, Roy D, Cai F, Belsham DD 2003 Expression of circadian rhythm genes in gonadotropin-releasing hormone-secreting GT1–7 neurons. Endocrinology 144:5285–5292
Oishi K, Shirai H, Ishida N 2005 CLOCK is involved in the circadian transactivation of peroxisome proliferator-activated receptor (PPAR) in mice. Biochem J 386:575–581
Gourdji D, Laverriere JN 1994 The rat prolactin gene: a target for tissue-specific and hormone-dependent transcription factors. Mol Cell Endocrinol 100:133–142
Munoz E, Baler R 2003 The circadian E-box: when perfect is not good enough. Chronobiol Int 20:371–388
Jin X, Shearman LP, Weaver DR, Zylka MJ, de Vries GJ, Reppert SM 1999 A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. Cell 96:57–68
Hogenesch JB, Gu YZ, Jain S, Bradfield CA 1998 The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc Natl Acad Sci USA 95:5474–5479
Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TD, Weitz CJ, Takahashi JS, Kay SA 1998 Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim. Science 280:1599–1603
Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, Weitz CJ 1998 Role of the CLOCK protein in the mammalian circadian mechanism. Science 280:1564–1569
Vallone D, Gondi SB, Whitmore D, Foulkes NS 2004 E-box function in a period gene repressed by light. Proc Natl Acad Sci USA 101:4106–4111
McDonald MJ, Rosbash M, Emery P 2001 Wild-type circadian rhythmicity is dependent on closely spaced E boxes in the Drosophila timeless promoter. Mol Cell Biol 21:1207–1217
Ripperger JA, Shearman LP, Reppert SM, Schibler U 2000 CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP. Genes Dev 14:679–689
Sensi S, Pace Palitti V, Guagnano MT 1993 Chronobiology in endocrinology. Ann Ist Super Sanita 29:613–631
Ripperger JA, Schibler U 2001 Circadian regulation of gene expression in animals. Curr Opin Cell Biol 13:357–362
Lowrey PL, Takahashi JS 2004 Mammalian circadian biology: elucidating genome-wide levels of temporal organization. Annu Rev Genomics Hum Genet 5:407–441
Reppert SM, Weaver DR 2001 Molecular analysis of mammalian circadian rhythms. Annu Rev Physiol 63:647–676
Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, Schultz PG, Kay SA, Takahashi JS, Hogenesch JB 2002 Coordinated transcription of key pathways in the mouse by the circadian clock. Cell 109:307–320
Chappell PE, White RS, Mellon PL 2003 Circadian gene expression regulates pulsatile gonadotropin-releasing hormone (GnRH) secretory patterns in the hypothalamic GnRH-secreting GT1–7 cell line. J Neurosci 23:11202–11213
Sun ZS, Albrecht U, Zhuchenko O, Bailey J, Eichele G, Lee CC 1997 RIGUI, a putative mammalian ortholog of the Drosophila period gene. Cell 90:1003–1011
Sakamoto K, Nagase T, Fukui H, Horikawa K, Okada T, Tanaka H, Sato K, Miyake Y, Ohara O, Kako K, Ishida N 1998 Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain. J Biol Chem 273:27039–27042
Lopez-Molina L, Conquet F, Dubois-Dauphin M, Schibler U 1997 The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior. EMBO J 16:6762–6771
Bjarnason GA, Jordan RC, Wood PA, Li Q, Lincoln DW, Sothern RB, Hrushesky WJ, Ben-David Y 2001 Circadian expression of clock genes in human oral mucosa and skin: association with specific cell-cycle phases. Am J Pathol 158:1793–1801
Zanello SB, Jackson DM, Holick MF 2000 Expression of the circadian clock genes clock and period1 in human skin. J Invest Dermatol 115:757–760
Chen YG, Mantalaris A, Bourne P, Keng P, Wu JH 2000 Expression of mPer1 and mPer2, two mammalian clock genes, in murine bone marrow. Biochem Biophys Res Commun 276:724–728
Allaman-Pillet N, Roduit R, Oberson A, Abdelli S, Ruiz J, Beckmann JS, Schorderet DF, Bonny C 2004 Circadian regulation of islet genes involved in insulin production and secretion. Mol Cell Endocrinol 226:59–66
Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda M, Block GD, Sakaki Y, Menaker M, Tei H 2000 Resetting central and peripheral circadian oscillators in transgenic rats. Science 288:682–685
Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM, Hong HK, Oh WJ, Yoo OJ, Menaker M, Takahashi JS 2004 PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci USA 101:5339–5346
Gachon F, Nagoshi E, Brown SA, Ripperger J, Schibler U 2004 The mammalian circadian timing system: from gene expression to physiology. Chromosoma 113:103–112
Arey BJ, Freeman ME 1992 Activity of oxytocinergic neurons in the paraventricular nucleus mirrors the periodicity of the endogenous stimulatory rhythm regulating prolactin secretion. Endocrinology 130:126–132
Teclemariam-Mesbah R, Kalsbeek A, Pevet P, Buijs RM 1997 Direct vasoactive intestinal polypeptide-containing projection from the suprachiasmatic nucleus to spinal projecting hypothalamic paraventricular neurons. Brain Res 748:71–76
Watts AG, Swanson LW, Sanchez-Watts G 1987 Efferent projections of the suprachiasmatic nucleus: I. Studies using anterograde transport of Phaseolus vulgaris leucoagglutinin in the rat. J Comp Neurol 258:204–229
Miller BH, Olson SL, Turek FW, Levine JE, Horton TH, Takahashi JS 2004 Circadian clock mutation disrupts estrous cyclicity and maintenance of pregnancy. Curr Biol 14:1367–1373
Smith MS 1980 Role of prolactin in mammalian reproduction. Int Rev Physiol 22:249–276
Dorton AM 2000 The pituitary gland: embryology, physiology, and pathophysiology. Neonatal Netw 19:9–17
Gutierrez-Hartmann A, Siddiqui S, Loukin S 1987 Selective transcription and DNase I protection of the rat prolactin gene by GH3 pituitary cell-free extracts. Proc Natl Acad Sci USA 84:5211–5215(Gilles M. Leclerc and Fre)
Address all correspondence and requests for reprints to: Dr. Fredric R. Boockfor, Laboratory of Molecular Dynamics, Department of Cell Biology and Anatomy, Medical University of South Carolina, 173 Ashley Avenue, Charleston, South Carolina 29425. E-mail: boockfor@musc.edu.
Abstract
Recent findings from our laboratory and those of others demonstrated that prolactin gene expression (PRL-GE) oscillates in single living mammotropes, but little information is available on the molecular processes that contribute to this phenomenon. To elucidate the source of this activity, we generated a series of constructs containing decreasing lengths of the PRL promoter fused to a luciferase reporter gene. These constructs were injected into single cells and assayed for photonic activity. We found pulse activity with all plasmids tested, even with the smallest promoter fragment of 331 bp. Sequence analysis of this fragment identified two potential E-boxes (elements known to bind CLOCK and BMAL1 circadian proteins). Furthermore, RT-PCR of PRL cells (pituitary, MMQ, and GH3) revealed expression of clock and bmal1 as well as five other clock genes (per1, per2, cry1, cry2, and tim), suggesting that the circadian system may function in PRL cells. Next, we mutated the core sequences of both E-boxes within the 2.5-kb PRL promoter and found that only mutation of the E-box133 completely abolished PRL-GE pulses. EMSAs revealed that CLOCK and BMAL1 were able to bind to the E-box133 site in vitro. Our results demonstrate that PRL-GE pulses are dependent on a specific E-box binding site in the PRL promoter. Moreover, the indication that CLOCK/BMAL1 can bind to this site suggests that these circadian proteins, either alone or in conjunction with other factors, may regulate intermittent PRL promoter activity in mammotropes, perhaps by acting as a temporal switch for the on/off expression of PRL.
Introduction
PROLACTIN (PRL) is involved in a wide range of mammalian physiological processes and is expressed with temporal characteristics that suggest a high degree of regulation (1). This is evidenced by a complex pattern of secretion consisting of repetitive surges that are timed specifically and depend on the physiological state of the animal. For example, in the female rat, PRL release occurs during the afternoon of proestrus and during pregnancy with specifically timed nocturnal and/or diurnal surges. It is generally accepted that the secretion of PRL from mammotropes depends on a variety of factors released by the hypothalamus that impinge on the pituitary and stimulate or inhibit the synthesis and release of PRL (1, 2). Because of this, it is recognized that much of the timed release of PRL depends on the type and level of modulatory factor that reaches the mammotrope.
The possibility that individual mammotrope function may also play a role in the temporal regulation of PRL release was raised with recent evidence from our laboratory demonstrating that PRL gene expression (PRL-GE) is not constant but oscillates in individual living mammotropes obtained from rat pituitaries (3). This oscillatory function of normal pituitary cells was determined in these experiments by use of a PRL promoter-driven luciferase reporter plasmid to monitor transcriptional activity over time. This type of episodic expression was later confirmed by others using a similar reporter system in cells from hamster pituitaries (4). Although the mechanism(s) underlying these pulses remains obscure, a clue as to what may be involved in this process was obtained by McFerran et al. (5) using a PRL::luc reporter construct and a continuous PRL-producing cell line. This group demonstrated that detectable pulses of PRL promoter expression could be induced experimentally in individual GH3 cells by treatment with high concentrations of serum. Because this treatment was effective in inducing circadian gene expression in cultured mammalian cells (6), these investigators proposed that a relationship might exist between circadian clock regulatory function and PRL oscillatory activity. Although intriguing, there was no direct evidence linking these two phenomenon, nor was information available that identified any specific process(es) underlying PRL oscillatory activity. Nevertheless, when these studies are taken together, two issues become clear. First, PRL-GE in individual mammotropes is a dynamic process that is subject to some type of internal timing mechanism. Second, this mechanism is dependent on the PRL promoter. To understand the mechanism(s) that may underlie these oscillations, the present study was conducted to determine the region of the PRL promoter responsible for intermittent function and begin to identify specific regulatory element(s) present that may be involved in this process.
Materials and Methods
Cells and cell culture
Primary cultures of anterior pituitary cells were prepared from primiparous, lactating female rats (6–10 d postpartum; Sprague Dawley, Charles River Laboratories, Inc., Wilmington, MA). Procedures involving these animals were conducted in accordance with protocols approved by the institution and the University Committee on Animal Care. As reported elsewhere (7) dispersed pituitary cells were maintained in phenol red-free medium-199-Earles’ salts/nutrient medium F-12 supplemented with antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin), L-glutamine (2 mM), and 10% fetal bovine serum. All tissue culture supplies were obtained from Invitrogen (Carlsbad, CA). Rat mammotrope MMQ (CRL-10609, American Type Culture Collection, Manassas, VA) and rat mammosomatotrope GH3 (CCL-82.1, American Type Culture Collection) cells were maintained in phenol red-free DMEM/F12 medium (1:1) supplemented with 2 mM L-glutamine, 10 mM HEPES, antibiotics, and 10% fetal bovine serum. The cells were grown in a 5% CO2 environment at 37 C in accordance with routine cell culture procedures.
Real-time measurement of gene expression in living mammotropes and pulse analysis
For bioluminescent imaging, the anterior pituitary cells obtained from lactating female rats were cultured on 25-mm glass photo-etched coverslips (Bellco Glass, Vineland, NJ) coated with poly-L-lysine as described previously (8). After 2 d in culture, individual cells were transfected by microinjection (Eppendorf 5242) with Endofree-purified plasmid DNA (QIAGEN, Valencia, CA) using a concentration of 0.2 μg/μl. Cells were incubated under normal culture conditions for 24–48 h before imaging. Then coverslips containing the cells were assembled in Sykes-Moore chambers and perifused (10 μl/min) with culture medium containing 0.1 mM luciferin (Sigma, St. Louis, MO). A chamber was placed on the heated stage of a photon-capture microscope (Axioskop, Carl Zeiss, Jena, Germany) and photon images were visualized using x10 objective (Neofluar, Carl Zeiss). Photonic signals from single cells were accumulated and quantified continuously in 30-min bins for more than 40 h using a photon-counting camera system (Argus 50, Hamamatsu, Bridgewater, NJ). Photonic images were analyzed off-line as previously described (8, 9). The presence of PRL-GE pulses was identified as described by Shorte et al. (3). A pulse was defined as a significant rise in photonic activity that lasted longer than 2 h with a value greater than 5% of the photonic signal count at the beginning of the pulse plus 2 times the total signal-noise variation (SNV). SNV was calculated by the square root of each value plus the SD of the corresponding background values squared [SNV = ([signal count] + [SD of detected background2])]. For normalization, the photonic activity data were plotted as signal count over SNV as a function of time. Comparisons of pulse activity between cells microinjected with various constructs and expressing luciferase activity were achieved using two-tailed, unpaired t tests. All data are expressed as mean ± SEM.
Preparation of PRL promoter constructs
Construction of the luciferase reporter plasmid pPLDA12 is described elsewhere (3). This plasmid contains 2.5 kb of the rat PRL promoter fused to a modified luciferase coding sequence (LUCODC-DA), which encodes a short-lived luciferase enzyme (10). Generation of a unidirectional nested set of deletions in the PRL promoter was accomplished using the Erase-a-Base system (Promega, Madison, WI) in accordance with the manufacturer’s recommendations. Briefly, the pPLDA12 was digested with NdeI and filled with -phosphorothioate deoxynucleotide triphosphates. The linear plasmid was restricted with AflII (located near the 5' end of the 2.5-kb PRL promoter fragment) and submitted to Exonuclease III digestion for various times. Plasmids were ligated, precipitated, and transformed in Escherichia coli XL1-Blue MRF' cells. The resulting recombinant DNA molecules were extracted using the QIAprep Spin miniprep kit (QIAGEN), and the length of the PRL promoter in each of the clones was determined by restriction digest with two sets of double-enzyme digestions, BamHI/BglI and BamHI/ScaI (New England Biolabs, Beverly, MA). From the collection of PRL promoter deletion constructs obtained, only a subset was subjected to bioluminescent imaging for evaluation of the photonic activity. Automated DNA sequencing of the 331-bp PRL promoter fragment was obtained by the Biotechnology Resource Laboratory of the Medical University of South Carolina (Charleston, SC).
Site-directed mutagenesis of E-box sites
Mutants of the rat PRL promoter sites E-box133 and E-box10 were generated using the QuikChange II XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Briefly, 40 μg of Endofree purified pPLDA12 plasmid DNA was used as template for mutant strand synthesis. For the mutagenesis, the primers E-box10F (5'-GGT TTA TAA AGT CAA TGT CTG CCG CGG AGA AAG CAG TGG TTC TCTT), E-box133F (5'-GTC TTC CTG AAT ATG AAT AAG AAA TAA AAT ACC TCG AGA TGT TTA AAA TTA TTG GGG CTA TCT TAA TGAC), and their complementary oligonucleotides were used (Sigma Genosys, Woodlands, TX). These mutagenic primers were engineered in such a way that the mutated nucleotides formed a unique restriction site (SacII for E-box10, and XhoI for E-box133, sequence underlined), facilitating the screening of the mutated clones. The mutations were confirmed by nucleotide sequencing.
Total RNA isolation and RT-PCR of the circadian genes
Total RNA was isolated from either clonal pituitary cell cultures (MMQ and GH3) or pituitary glands from female lactating rats using the RNeasy kit (QIAGEN). First-strand cDNA synthesis and reverse transcription reactions were described elsewhere (11). The RNA samples were treated with DNase I (30 Kunitz units) to remove any trace of genomic DNA as specified in the RNeasy kit procedure (QIAGEN). PCR amplifications of the clock cDNA gene complements (clock, bmal1, per1, per2, cry1, cry2, and tim) were performed in a 25-μl reaction volume containing 1x QIAGEN PCR buffer, 1x QIAGEN Q-solution, 0.6 μM primers, 0.2 mM each deoxynucleotide triphosphates (Invitrogen), and 0.6 U HotStar Taq DNA polymerase (QIAGEN). The design of the rat specific primers was based on the sequence of the mouse-specific primers that were used to amplify circadian genes. PCR products obtained with these primers were sequenced and characterized to confirm their identity (12). The rat-specific circadian gene accession numbers, DNA sequences of their corresponding PCR primers, and size of the amplified fragments were as follows: clock (NM_021856, CLK-F1: 5'-GCG AGA ACT TGG CGT TGA GGAG, CLK-R1: 5'-CTG TGT CCA CTC ATT ACA CTC TGT, 770 bp), bmal1 (AB012600, BMAL1-F1: 5'-TCT GGA GCT CGG CGC TCT TTC, BMAL1-R1: 5'-AAC AAC AGT GTT GGT TGA GAC AAT, 602 bp), per1 (XM_340822, PER1-F1: 5'-CCA GGA CCC AGA AAG AAC TC, PER1-R1: 5'-CCT CTG ATT CGG CAG AAGAC, 513 bp), per2 (NM_031678, PER2-F1: 5'-AGA CGT GGA CAT GAG CAGCG, PER2-R1: 5'-CAG GAT CTT CCC AGA GACCA, 519 bp), cry1 (AF545855, CRY1-F1: 5'-TCC CCT CCC CTT TCT CTTTA, CRY1-R1: 5'-ATC CCT TCT TCC CAA CTGAT, 292 bp), cry2 (NM_133405, CRY2-F1: 5'-AAG CTG AAT TCC CGT CTGTT, CRY2-R1: 5'-GTG GTT TCT GCC CAT TCAGT, 237 bp), and tim (NM_031340, TIM-F1: 5'-GGA GAA CTG TTA CAA CCC ACTC, TIM-R1: 5'-GAA CGT GGA TGG TAT CTC TCA, 440 bp). The PCR conditions were as follows: initial incubation at 95 C for 15 min and then 36 cycles of denaturation at 94 C for 30 sec, annealing at 56 C for 1 min, and elongation at 72 C for 40 sec. The PCR products were resolved in 1.2% Tris acetate EDTA (pH 8.0)-agarose gels stained with ethidium bromide.
Nuclear protein extract and EMSAs
The interaction of MMQ nuclear proteins with the DNA fragment containing the putative E-box133 binding site was analyzed by EMSAs using the LightShift Chemiluminescent EMSA Kit (Pierce, Rockford, IL). The 20-bp DNA duplex used in EMSA experiments consisted of the annealed pair of oligonucleotides E-box133F (5'-AAT AAA ATA CCA TTT GAT GT), and E-box133R (5'-ACA TCA AAT GGT ATT TTA TT) (Integrated DNA Technologies, Inc., Coralville, IA). These two oligonucleotides were biotinylated at their 5'-ends to allow chemiluminescent detection. Nuclear protein extracts from MMQ cells were obtained using the NE-PER nuclear and cytoplasmic extraction reagents (Pierce) supplemented with 2x final concentration of the Halt protease inhibitor cocktail kit (Pierce). Then these extracts were purified and concentrated 3-fold with the Microcon centrifugal filter YM-10 (Millipore, Bedford, MA). Protein concentration in the nuclear extracts was determined using the Micro BCA protein assay reagent kit (Pierce). DNA-protein binding reactions consisted of incubation of 15 μg of the concentrated NE-PER nuclear extracts mixed with a reaction buffer composed of 10 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM DTT, 50 ng/μl poly (dI · dC), 2.5% glycerol, 10 mM EDTA (pH 8.0), and 0.05% Nonidet P-40 for 10 min at room temperature. This was followed by addition of 100 fmol of 5-biotin-labeled E-box133 DNA to the reaction mixture and incubating for 25 min at room temperature.
For competition experiments, a 60-fold molar excess of unlabeled duplex DNA fragments corresponding to either the E-box133 site (described above) or the stimulating protein-1 (SP1) site (5'-ATT CGA TCG GGG CGG GGC GAGC, Promega) was added to the reaction mixture before addition of the labeled oligonucleotides. For supershift experiments, we added 4 μg of IgG affinity purified goat polyclonal antibody raised against CLOCK (sc-6927), BMAL1 (sc-8550), or a control IgG (DREAM, sc-9309) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to the binding reaction mixture and incubated for 40 min at room temperature. The CLOCK and BMAL1 antibodies were validated for use in EMSAs by Oishi et al. (13). Reaction mixtures were analyzed by nondenaturing 6% PAGE (Invitrogen) and then transferred to Biodyne B nylon membranes (Pierce). The biotin-labeled DNA probes were detected by chemiluminescence using the chemiluminescent nucleic acid detection module kit (Pierce). The membranes were exposed to x-ray films for 1–3 min and developed according to the manufacturer’s instructions.
Results
Identification of the PRL promoter segment responsible for pulsatility
As stated above, a previous investigation from our laboratory clearly identified pulses of PRL promoter-driven gene expression in living pituitary cells (3). In the present study, initial efforts were directed toward identifying the region(s) of the PRL promoter associated with this pulsatile function. This involved unidirectional deletions of the PRL promoter to generate PRL::luc reporter constructs with different promoter lengths (Fig. 1). These constructs were injected into individual cells and then assayed for photonic expression. By evaluating each fragment length progressively from larger to smaller until pulsatile function disappeared, we reasoned that we would be able to identify the minimal promoter region responsible for this pulsatile function. As shown (Fig. 1), we found that constructs with fragment lengths of approximately 1.3, 0.64, 0.44, and 0.33 kb exhibited pulses. Representative photonic profiles of the smaller constructs tested are shown in Fig. 2. As illustrated, the pulse activity observed with these deletion constructs were indistinguishable from those obtained with the unmodified full-length promoter. In fact, we observed pulse activity with these deletion constructs to occur in approximately the same proportion of cells as compared with the full-length promoters (2.5 kb) [64.1 ± 7.5% (n = 28) vs. 70.3 ± 5.2% (n = 86), P > 0.3, various sized deletion vs. full-length, respectively]. From these experiments, it appeared that the smallest fragment of the PRL promoter tested (0.33 kb) contained the minimal elements needed for pulse activity.
FIG. 1. Influence of PRL promoter fragment size on pulse activity. The plasmid pPLDA12 (2.5-kb PRL promoter) was used as a template to generate unidirectional deletions. As shown, each promoter deletion construct consisted of a portion of the promoter fused to the luciferase reporter gene (luc*). Preparations of constructs containing each fragment size were microinjected into single living pituitary cells obtained from lactating rats and photonic activity monitored for a period of 40 h. At least nine expressing cells/construct were analyzed for pulse activity (+). Note that the smallest region tested that was capable of generating pulse activity was confined to the first 0.33 kb of the PRL proximal promoter region.
FIG. 2. Representative photonic profiles from single mammotropes generated by various PRL promoter deletion constructs. Photonic activity was measured from individual mammotropes microinjected with plasmids containing either the full-length 2.5-kb segment (A) or fragment sizes 0.64 (B), 0.44 (C), or 0.33 kb (D) of the rat PRL promoter. Each point represents photonic signal accumulated for 30 min and is expressed as a function of the SNV (see Materials and Methods). Vertical bars indicate SEM. Asterisks represent significant PRL gene expression pulses. These profiles are representative of determinations made on at least nine luciferase-expressing cells per construct.
With attention focused on this 331-bp segment, we attempted to identify any recognizable transcription factor binding sites that may be associated with intermittent or rhythmic activity. In addition to previously identified regulatory sites [such as the pituitary-specific transcription factor (Pit)-1, Pit-2, Pit-3, and Pit-4 binding sites (14)], DNA sequence analysis revealed two putative E-box binding sites (CANNTG), one located close to the transcription start site termed E-box10 (CAGATG, –10 to –5) and the other localized farther upstream between the Pit-2 and Pit-3 binding sites termed E-box133 (CATTTG, –133 to –128) (Fig. 3). E-box binding sites have been shown to affect expression of a large number of genes, many of which are involved in circadian function (15). In the case of several circadian-regulated genes, the canonical E-box site CACGTG has been reported to preferentially bind the regulatory proteins, CLOCK and BMAL1 (16, 17, 18, 19). Also, variations in the E-box recognition sequence have been reported in a number of clock-regulated genes (20, 21, 22) and are believed to function in circadian processes as well. The presence of E-boxes in this promoter region supports the possibility that clock proteins may be involved in the intermittent expression of the PRL promoter.
FIG. 3. Identification of putative E-boxes in the 331-bp proximal promoter region of rat PRL gene. Nucleotide sequence of the –331/+1 region of the rat PRL promoter is presented. The two putative E-box binding sites, E-box10 (CAGATG) and E-box133 (CATTTG), are denoted (boxes and bold characters). For reference, the pituitary-specific transcription factor Pit-1 binding sites are underlined (their relative locations are described in Ref. 45 ).
Expression of circadian genes in pituitary tissues and PRL-producing cell lines
In the next series of experiments, we attempted to identify whether the circadian transcription factors CLOCK and/or BMAL1 or other circadian regulators were expressed in PRL cells. RT-PCR analysis was performed on primary rat pituitary cells, and two PRL-secreting cell lines (MMQ and GH3). As shown in Fig. 4A, bands corresponding to the predicted molecular weight for clock (770 bp) and bmal1 (602 bp) gene amplification products were identified when total RNA from each cell population was used as a template. In addition to clock and bmal1, we observed the expression of five other key clock regulatory genes (per1, per2, cry1, cry2, and tim with the expected molecular weight of 513, 519, 292, 237, and 440 bp, respectively) in each PRL cell type investigated when primers specific for these factors were used in RT-PCRs (Fig. 4B). The expression in PRL cells of such an array of genes associated with temporal regulatory activity raises the possibility that these factors may play some type of role in intermittent PRL-GE activity in mammotropes.
FIG. 4. RT-PCR analysis of core circadian gene expression in PRL-secreting cells. RT-PCRs were conducted for clock, bmal1, per1, per2, cry1, cry2, and tim gene expression in rat pituitary tissue and PRL-secreting clonal cell lines MMQ and GH3. A, Products obtained after RT-PCR amplification using the specific primers for the circadian transcriptional activator clock (770 bp) and bmal1 (602 bp) genes. B, Amplified products obtained when specific primers for the five core clock regulatory genes, per1 (513 bp), per2 (519 bp), cry1 (292 bp), cry2 (237 bp), and tim (440 bp), were used. The negative control (labeled –CTRL) contained water instead of cDNA template. The pBR322 MspI digest was used as a DNA ladder and is shown in the first lanes for rat pituitary and GH3-derived products. For the rat MMQ-PCR products, we used a 100-bp DNA ladder (present in the far left lane) that ranges from 100 to 1517 bp. MW, Molecular weight
Influence of E-box mutations on PRL promoter activity
The two E-box sites identified above in the PRL promoter may play a role in oscillatory activity possibly by interacting with certain circadian core regulatory proteins expressed in mammotropes. To address the impact of these E-boxes on pulses of PRL gene activity, we mutated each site in the 2.5-kb PRL promoter. The E-box133 site was changed from CATTTG to CTCGAG and the E-box10 site was changed from CAGATG to CCGCGG, altering only the core E-box binding sequences. Reporter plasmids containing each of these mutant constructs were microinjected into pituitary cells from lactating rats and assessed for changes in promoter-driven pulse activity. As illustrated, cells injected with plasmids containing mutated E-box10 (E-box10*) site exhibited normal patterns of photonic activity (Fig. 5A). This activity was virtually indistinguishable from that obtained using nonmodified PRL promoter (compare Fig. 5A with Fig. 2A above). Because of this, the E-box10 site was not considered for further analysis. In contrast, use of the plasmid containing the mutated E-box133 (E-box133*) site resulted in a complete abolition of pulses (Fig. 5B). Cells injected with this mutant construct continued to exhibit basal photonic activity. These results demonstrate that the E-box133 binding site is essential for pulses of PRL promoter activity.
FIG. 5. Effect of mutation in E-box sites on PRL pulsatile activity. Representative examples of photonic profiles generated by single primary mammotropes microinjected with luciferase reporter constructs containing either mutations in E-box10 (A) or E-box133 (B) binding sites of the rat PRL promoter (2.5 kb) are presented. As described above, each point represents photonic emissions collected for 30 min and expressed as a function of the SNV. Vertical bars indicate SEM. Asterisks represent significant PRL gene expression pulses. Each profile is representative of results obtained from at least 10 cells in six and seven different experiments for E-box10* and E-box133*, respectively.
EMSA analysis of CLOCK/BMAL1 binding to E-box133
Because the sequence of this putative E-box133 diverges from the canonical or perfect clock-related E-box structure (CACGTG), it was important to demonstrate whether CLOCK and/or BMAL1 could bind to the site. This was accomplished by performing EMSAs. The incubation of 5'-biotin-labeled DNA probe (containing an unmodified E-box133 site) with nuclear proteins from MMQ cells resulted in the formation of a specific band representing a complex formed between the E-box133 DNA probe and bound nuclear proteins (Fig. 6). Moreover, addition of an antibody specific for either CLOCK or BMAL1 caused a supershift to occur as evidenced by the formation of a higher molecular weight complex. The absence of this higher molecular band when control IgG was used indicates that the antibody supershift reaction was specific for CLOCK or BMAL1 protein interaction. In additional control experiments, we found specific band formation to disappear when the reaction mixture was incubated with unlabeled E-box133 DNA but to be unaffected when unrelated SP1 DNA oligonucleotides were used, demonstrating that the complexes that form are specific for the E-box133 site. From these results, it is clear that a CLOCK and BMAL1 protein complex can bind to this E-box133 site in vitro, suggesting that these circadian factors are able to interact with the PRL promoter and may play an important role in the regulation of PRL pulse activity.
FIG. 6. EMSA analysis of CLOCK and BMAL1 binding to the E-box133 site of the PRL promoter. EMSA was performed with nuclear protein extracts from the pituitary MMQ cells and biotinylated E-box133 binding site DNA as probe (see Materials and Methods). The first lane shows the results obtained when biotinylated probe was used in the absence of nuclear extracts. The other lanes represent incubations of the labeled probe with MMQ nuclear extracts (MMQ-NE) alone or combined with various antisera or DNA competitors. Protein DNA interactions are denoted by the appearance of high-molecular-weight complexes (Specific Bands). CLOCK and BMAL1 proteins within the complex were identified (MMQ-NE + CLOCK and MMQ-NE + BMAL1, respectively) by the presence of higher-molecular-weight bands (Supershift) when specific antiserum for these proteins were used. Reactions conducted using control IgG (MMQ-NE + IgG) are presented for comparison. In competition experiments, results of 60-fold molar excess of unlabeled specific E-box133 (MMQ-NE + E-Box133) or nonspecific SP1 (MMQ-NE + SP1) DNA probes are also shown. These results are representative of those obtained in four separate experiments.
Discussion
Previously we reported that PRL promoter activity occurs in rhythmic pulses in individual living mammotropes (3). Because the timing of PRL-GE pulses was not consistent with the strict definition of circadian activity (23), we classified these oscillations as noncircadian. In the present work, we have demonstrated that these pulses are dependent on a specific promoter site (E-box133, CATTTG) that can bind two essential clock regulatory proteins. This is evidenced by the observation that mutation of this site (E-box133*) results in complete abolition of pulse activity. Moreover, supershift EMSA experiments using specific antibodies against CLOCK or BMAL1 demonstrates that these two circadian proteins bind to this site in vitro. When taken together, our findings raise the intriguing possibility that even though PRL-GE pulse activity does not follow a strict circadian pattern, the mechanism underlying PRL-GE pulses may involve specific core regulatory elements that are usually associated with the circadian system.
In mammals, the oscillatory mechanism of the suprachiasmatic nucleus (SCN) coordinates behavior and physiological rhythms throughout the whole animal and consists mainly of two activating factors, CLOCK and BMAL1 and two repressing factors, cryptochrome (CRY)1 and CRY2 in addition to other regulatory proteins (24). In this system, a CLOCK/BMAL1 complex binds preferentially to the canonical E-box sequence CACGTG to activate transcription of these cry genes. The CRY proteins feedback and inhibit CLOCK/BMAL1 binding resulting in the inhibition of their own production. The interaction of these clock factors in stimulating and inhibiting gene expression forms the basis for an oscillatory mechanism that regulates the timing of a variety of factors produced in the hypothalamus (for detailed reviews, see Refs. 24 and 25).
The potential of the E-box133 in the prolactin promoter to bind CLOCK and BMAL1, even though its structure is different from the classic type of E-box sequence normally associated with clock gene regulation, may represent a novel mechanism by which these circadian proteins can modulate nonclock gene expression. It has been generally accepted that a specific hexameric DNA sequence (CACGTG; termed the perfect or canonical E-box) serves as a response element for the circadian regulatory proteins CLOCK and BMAL1 (for review, see Refs. 15 and 26). Indeed, these proteins heterodimerize forming a complex that activates gene transcription when bound to a canonical E-box sequence. In our study, no canonical E-box binding site was identified in the PRL promoter sequence. In contrast, we identified two noncanonical E-boxes (CANNTG) that are usually associated with a more ubiquitous E-box family of transcription regulatory sites. These sites have been linked to a variety of transcriptional processes involving such things as cell proliferation, differentiation, transformation, and apoptosis and bind transcription factors mainly of the helix-loop-helix type (15). Of these two noncanonical E-box elements identified in the PRL proximal promoter region, we found only one (E-box133; CATTTG) to be involved in oscillatory activity as evidenced by the complete abolition of pulses when the site was mutated. Although noncanonical E-boxes have been identified in promoters that have circadian activity such as tim in Drosophilia (21) and per4 in zebrafish (20), it was demonstrated that they cooperate with adjacent canonical E-box elements to promote transcription. Moreover, canonical clock E-box elements do not have to be restricted to the promoter region to be active. Recent investigations on the regulation of the circadian transcription factor albumin D-element binding protein revealed E-box elements in the first and second intron of the gene as well as the promoter region (22). These E-boxes consisted of both canonical and noncanonical types that may well interact as described for tim. In our analysis, we were unable to find a canonical E-box anywhere in the rat PRL gene (genomic region covering more than 35 kb including the rat PRL gene on chromosome 17, WGS supercontig, GenBank accession number NW_047491). On the other hand, we identified several noncanonical E-box sites downstream from the PRL transcription start site. However, their role in PRL pulse activity remains in question because elements contained within the 331-bp PRL promoter segment are sufficient to generate pulses. Our results indicate clearly that a noncanonical E-box element (E-box133), which can bind CLOCK and BMAL1, may not have to work in conjunction with a perfect (CACGTG) E-box to induce oscillatory transcriptional activity of the PRL promoter. If such a nonperfect E-box is active in our system, then it is possible that such an element may be active in others. In recent studies, Panda et al. (27) identified nearly 650 cycling genes by microarray analysis from which they retrieved the sequences of 127 genes from both the mouse and human genomes. Of these sequences, they identified only nine genes that contained canonical E-boxes. However, cursory analysis of the 127 sequences reveals that many of these other genes contain one or more noncanonical E-box binding sites. When taken together, our findings of transcriptional oscillatory activity driven by a single noncanonical E-box identifies a novel mechanism by which the clock system may act on the PRL promoter. Moreover, this observation raises the intriguing possibility that such an E-box type (binding CLOCK and/or BMAL1) may provide a means by which the clock gene system can confer oscillatory activity to a wide variety of nonclock genes.
Our observations of clock components and clock-related rhythmic activity in mammotropes confirms previous observations of clock gene expression in pituitary tissue (28) and extends the view that most, if not all, peripheral cells and tissues have an endogenous timing system. Circadian oscillatory elements have been identified in a wide variety of tissues such as skin, bone marrow, pancreas, retina, liver, heart, kidney, and lung (29, 30, 31, 32, 33, 34, 35). Although not all of the clock genes have been investigated, it has been determined that these tissues have many of the same clock proteins that are present in the SCN of the hypothalamus. Most of the evidence obtained to support rhythmic gene expression in these tissues came from static RNA determinations performed over time in synchronized tissues. This suggested strongly that oscillatory activity occurred in peripheral tissues, but it was not until the adaptation of luciferase technology that oscillatory gene activity could be observed in real time. The use of transgenic reporter mouse lines in which mouse period 1 (per1) promoter drives a luciferase reporter revealed circadian rhythms of per1 expression in liver tissues, with oscillatory activity diminishing within two to seven cycles (36). This suggested that gene oscillations were not self-sustaining but were driven by exogenous input. However, more recent experiments of Yoo et al. (37) using a knockin mouse line in which the luciferase reporter was fused to the per2 gene revealed self-sustained per2 rhythms for at least 20 d. These observations strengthened the idea that peripheral tissues contain the components necessary to maintain rhythmic function. However, the reason for this clock gene controlled oscillatory activity in the production and release of a nonclock or output gene such as PRL remains in question. One possibility is that the oscillatory control imposed at the tissue level provides a means to coordinate cells within a tissue (38). This may enable substantial levels of a particular factor such as PRL to be available for immediate release at critical times.
The rhythmic release of PRL occurs in distinct patterns, depending on the physiological state of the animal (1). An excellent example of this can be observed in the cycling female in which a surge of serum PRL can be detected during the afternoon of proestrus every 4 or 5 d. Upon mating, stimulation of the uterine cervix induces a secretory pattern consisting of specifically timed nocturnal and diurnal surges. This phenomenon clearly reveals that PRL release is subject to a control process that is centrally coordinated. Because of the circadian nature of this release, it is generally accepted that much of this control originates in the SCN of the hypothalamus. Although it has been demonstrated that oxytocin neurons from the paraventricular nucleus display daily rhythms that correlate with PRL release patterns (39), the identification of neuronal connections between the SCN and this other hypothalamic region emphasizes the likely SCN control of this phenomenon (40, 41). The critical nature of the CLOCK protein in PRL control is evidenced by the report of Miller et al. (42) in which clock mutant mice were characterized. In addition to a number of reproductive hormonal changes related to disruption of circadian activity, these investigators demonstrated that the mutant mice showed a decline in progesterone levels at midpregnancy and a shortened duration of pseudopregnancy. In normal animals, PRL is the factor that maintains the ovarian corpora lutea, the structure that produces progesterone levels necessary to maintain pregnancy and extend pseudopregnancy (43). In light of the importance of the CLOCK protein in both peripheral and central processes, it is quite likely that circadian processes are disrupted at both levels in these mutants. A number of factors such as dopamine, TRH, and oxytocin are secreted from the hypothalamus and influence PRL at the cellular level (1). However, the role of these factors on PRL rhythmic activity remains to be determined. Our ability to assess the influence of clock proteins on rhythmic PRL promoter activity in single mammotropes, in conjunction with analysis of the effects of centrally derived modulators on these rhythms, provides a unique model system that can be used to address the role of peripheral and central clock systems in controlling the activity of an output gene such as PRL.
In summary, our findings that the basis for oscillatory behavior of PRL promoter activity in single mammotropes may involve specific core circadian proteins raises important new issues in the study of the control of hormone function and gene regulation. The possibility that such proteins may underlie most, if not all, oscillatory gene expression must be considered, especially in the pituitary. This tissue produces a number of hormones that are released in a specifically timed manner and are critical to the proper functioning of a variety of systems in the body (44). The idea that clock-related binding elements may be more common than previously recognized and that the pituitary contains a wide array of clock proteins increases the prospect that the clock system may be an important regulator of many pituitary hormones. Our approach using single living mammotropes should enable identification of many of the basic aspects of this process.
References
Freeman ME, Kanyicska B, Lerant A, Nagy G 2000 Prolactin: structure, function, and regulation of secretion. Physiol Rev 80:1523–1631
Leong DA, Frawley LS, Neill JD 1983 Neuroendocrine control of prolactin secretion. Annu Rev Physiol 45:109–127
Shorte SL, Leclerc GM, Vazquez-Martinez R, Leaumont DC, Faught WJ, Frawley LS, Boockfor FR 2002 PRL gene expression in individual living mammotropes displays distinct functional pulses that oscillate in a noncircadian temporal pattern. Endocrinology 143:1126–1133
Stirland JA, Seymour ZC, Windeatt S, Norris AJ, Stanley P, Castro MG, Loudon AS, White MR, Davis JR 2003 Real-time imaging of gene promoter activity using an adenoviral reporter construct demonstrates transcriptional dynamics in normal anterior pituitary cells. J Endocrinol 178:61–69
McFerran DW, Stirland JA, Norris AJ, Khan RA, Takasuka N, Seymour ZC, Gill MS, Robertson WR, Loudon AS, Davis JR, White MR 2001 Persistent synchronized oscillations in prolactin gene promoter activity in living pituitary cells. Endocrinology 142:3255–3260
Balsalobre A, Damiola F, Schibler U 1998 A serum shock induces circadian gene expression in mammalian tissue culture cells. Cell 93:929–937
Castano JP, Kineman RD, Frawley LS 1996 Dynamic monitoring and quantification of gene expression in single, living cells: a molecular basis for secretory cell heterogeneity. Mol Endocrinol 10:599–605
Villalobos C, Faught WJ, Frawley LS 1998 Dynamic changes in spontaneous intracellular free calcium oscillations and their relationship to prolactin gene expression in single, primary mammotropes. Mol Endocrinol 12:87–95
Villalobos C, Faught WJ, Frawley LS 1999 Dynamics of stimulus-expression coupling as revealed by monitoring of prolactin promoter-driven reporter activity in individual, living mammotropes. Mol Endocrinol 13:1718–1727
Leclerc GM, Boockfor FR, Faught WJ, Frawley LS 2000 Development of a destabilized firefly luciferase enzyme for measurement of gene expression. Biotechniques 29:590–598
Leclerc GM, Leclerc GJ, Shorte SL, Frawley LS, Boockfor FR 2002 Cloning and mRNA expression of the Ca2+-binding DREAM protein in the pituitary. Gen Comp Endocrinol 129:45–55
Gillespie JM, Chan BP, Roy D, Cai F, Belsham DD 2003 Expression of circadian rhythm genes in gonadotropin-releasing hormone-secreting GT1–7 neurons. Endocrinology 144:5285–5292
Oishi K, Shirai H, Ishida N 2005 CLOCK is involved in the circadian transactivation of peroxisome proliferator-activated receptor (PPAR) in mice. Biochem J 386:575–581
Gourdji D, Laverriere JN 1994 The rat prolactin gene: a target for tissue-specific and hormone-dependent transcription factors. Mol Cell Endocrinol 100:133–142
Munoz E, Baler R 2003 The circadian E-box: when perfect is not good enough. Chronobiol Int 20:371–388
Jin X, Shearman LP, Weaver DR, Zylka MJ, de Vries GJ, Reppert SM 1999 A molecular mechanism regulating rhythmic output from the suprachiasmatic circadian clock. Cell 96:57–68
Hogenesch JB, Gu YZ, Jain S, Bradfield CA 1998 The basic-helix-loop-helix-PAS orphan MOP3 forms transcriptionally active complexes with circadian and hypoxia factors. Proc Natl Acad Sci USA 95:5474–5479
Darlington TK, Wager-Smith K, Ceriani MF, Staknis D, Gekakis N, Steeves TD, Weitz CJ, Takahashi JS, Kay SA 1998 Closing the circadian loop: CLOCK-induced transcription of its own inhibitors per and tim. Science 280:1599–1603
Gekakis N, Staknis D, Nguyen HB, Davis FC, Wilsbacher LD, King DP, Takahashi JS, Weitz CJ 1998 Role of the CLOCK protein in the mammalian circadian mechanism. Science 280:1564–1569
Vallone D, Gondi SB, Whitmore D, Foulkes NS 2004 E-box function in a period gene repressed by light. Proc Natl Acad Sci USA 101:4106–4111
McDonald MJ, Rosbash M, Emery P 2001 Wild-type circadian rhythmicity is dependent on closely spaced E boxes in the Drosophila timeless promoter. Mol Cell Biol 21:1207–1217
Ripperger JA, Shearman LP, Reppert SM, Schibler U 2000 CLOCK, an essential pacemaker component, controls expression of the circadian transcription factor DBP. Genes Dev 14:679–689
Sensi S, Pace Palitti V, Guagnano MT 1993 Chronobiology in endocrinology. Ann Ist Super Sanita 29:613–631
Ripperger JA, Schibler U 2001 Circadian regulation of gene expression in animals. Curr Opin Cell Biol 13:357–362
Lowrey PL, Takahashi JS 2004 Mammalian circadian biology: elucidating genome-wide levels of temporal organization. Annu Rev Genomics Hum Genet 5:407–441
Reppert SM, Weaver DR 2001 Molecular analysis of mammalian circadian rhythms. Annu Rev Physiol 63:647–676
Panda S, Antoch MP, Miller BH, Su AI, Schook AB, Straume M, Schultz PG, Kay SA, Takahashi JS, Hogenesch JB 2002 Coordinated transcription of key pathways in the mouse by the circadian clock. Cell 109:307–320
Chappell PE, White RS, Mellon PL 2003 Circadian gene expression regulates pulsatile gonadotropin-releasing hormone (GnRH) secretory patterns in the hypothalamic GnRH-secreting GT1–7 cell line. J Neurosci 23:11202–11213
Sun ZS, Albrecht U, Zhuchenko O, Bailey J, Eichele G, Lee CC 1997 RIGUI, a putative mammalian ortholog of the Drosophila period gene. Cell 90:1003–1011
Sakamoto K, Nagase T, Fukui H, Horikawa K, Okada T, Tanaka H, Sato K, Miyake Y, Ohara O, Kako K, Ishida N 1998 Multitissue circadian expression of rat period homolog (rPer2) mRNA is governed by the mammalian circadian clock, the suprachiasmatic nucleus in the brain. J Biol Chem 273:27039–27042
Lopez-Molina L, Conquet F, Dubois-Dauphin M, Schibler U 1997 The DBP gene is expressed according to a circadian rhythm in the suprachiasmatic nucleus and influences circadian behavior. EMBO J 16:6762–6771
Bjarnason GA, Jordan RC, Wood PA, Li Q, Lincoln DW, Sothern RB, Hrushesky WJ, Ben-David Y 2001 Circadian expression of clock genes in human oral mucosa and skin: association with specific cell-cycle phases. Am J Pathol 158:1793–1801
Zanello SB, Jackson DM, Holick MF 2000 Expression of the circadian clock genes clock and period1 in human skin. J Invest Dermatol 115:757–760
Chen YG, Mantalaris A, Bourne P, Keng P, Wu JH 2000 Expression of mPer1 and mPer2, two mammalian clock genes, in murine bone marrow. Biochem Biophys Res Commun 276:724–728
Allaman-Pillet N, Roduit R, Oberson A, Abdelli S, Ruiz J, Beckmann JS, Schorderet DF, Bonny C 2004 Circadian regulation of islet genes involved in insulin production and secretion. Mol Cell Endocrinol 226:59–66
Yamazaki S, Numano R, Abe M, Hida A, Takahashi R, Ueda M, Block GD, Sakaki Y, Menaker M, Tei H 2000 Resetting central and peripheral circadian oscillators in transgenic rats. Science 288:682–685
Yoo SH, Yamazaki S, Lowrey PL, Shimomura K, Ko CH, Buhr ED, Siepka SM, Hong HK, Oh WJ, Yoo OJ, Menaker M, Takahashi JS 2004 PERIOD2::LUCIFERASE real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proc Natl Acad Sci USA 101:5339–5346
Gachon F, Nagoshi E, Brown SA, Ripperger J, Schibler U 2004 The mammalian circadian timing system: from gene expression to physiology. Chromosoma 113:103–112
Arey BJ, Freeman ME 1992 Activity of oxytocinergic neurons in the paraventricular nucleus mirrors the periodicity of the endogenous stimulatory rhythm regulating prolactin secretion. Endocrinology 130:126–132
Teclemariam-Mesbah R, Kalsbeek A, Pevet P, Buijs RM 1997 Direct vasoactive intestinal polypeptide-containing projection from the suprachiasmatic nucleus to spinal projecting hypothalamic paraventricular neurons. Brain Res 748:71–76
Watts AG, Swanson LW, Sanchez-Watts G 1987 Efferent projections of the suprachiasmatic nucleus: I. Studies using anterograde transport of Phaseolus vulgaris leucoagglutinin in the rat. J Comp Neurol 258:204–229
Miller BH, Olson SL, Turek FW, Levine JE, Horton TH, Takahashi JS 2004 Circadian clock mutation disrupts estrous cyclicity and maintenance of pregnancy. Curr Biol 14:1367–1373
Smith MS 1980 Role of prolactin in mammalian reproduction. Int Rev Physiol 22:249–276
Dorton AM 2000 The pituitary gland: embryology, physiology, and pathophysiology. Neonatal Netw 19:9–17
Gutierrez-Hartmann A, Siddiqui S, Loukin S 1987 Selective transcription and DNase I protection of the rat prolactin gene by GH3 pituitary cell-free extracts. Proc Natl Acad Sci USA 84:5211–5215(Gilles M. Leclerc and Fre)