A Pit-1 Binding Site Adjacent to E-box133 in the Rat PRL Promoter is Necessary for Pulsatile Gene Expression Activity.

Recent evidence reveals that prolactin gene expression (PRL-GE) in mammotropes occurs in pulses, but the molecular process(es) underlying this phenomenon remains unclear. Earlier, we have identified an E-box (E-box133) in the rat PRL promoter that binds several circadian elements and is critical for this dynamic process. Preliminary analysis revealed a Pit-1 binding site (P2) located immediately adjacent to this E-box133 raising the possibility that some type of functional relationship may exist between these two promoter regions. In this study, using serum shocked GH3 cell culture system to synchronize PRL-GE activity, we determined that Pit-1 gene expression occurred in pulses with time phases similar to that for PRL. Interestingly, EMSA analysis not only confirmed Pit-1 binding to the P2 site, but also revealed an interaction with factor(s) binding to the adjacent E-box133 promoter element. Additionally, down-regulation of Pit-1 by siRNA reduced PRL levels during pulse periods. Thus, using multiple evidences, our results demonstrate clearly that the Pit-1 P2 site is necessary for PRL-GE elaboration. Furthermore, the proximity of this critical Pit-1 binding site (P2) and the E-box133 element coupled with the evidences of a site-to-site protein interactions suggest that the process of PRL-GE pulse activity might involve more dynamic and intricate cross-talks between promoter elements that may span some, or all, of the proximal region of the PRL promoter in driving its pulsatile expression.

Cryptorchidism induced in normal rats by the relaxin-like factor inhibitor.

Cryptorchidism is a serious problem, which affects 2-5% of the male population. Failure of the testes to descend into the scrotal region impairs germ cell development and is associated with a greater incidence of testicular cancer. The relaxin-like factor (RLF or insulin-like-3) has been shown to be critically important for the timely descent of the testicles in mice. We have discovered that the signal initiation site of the RLF can be eliminated without measurable effects on hormone binding to its receptor and that the resulting RLF derivative is a competitive inhibitor of RLF called RLFi. RLFi administered to pregnant rats causes dose-dependent gonadal retention in the offspring. The ability to control the severity of the syndrome by altering the concentration of RLFi and the timing of administration enables us to study in detail the structural changes that are associated with the action of RLF during critical stages of development. Targeted inhibition of the physiological migration pattern of testicles by RLFi lets one dissect the physiological process such as to find a window for clinical application of RLF and to search for ancillary factors that might play a role during normal development.

Specific GATA-binding elements in the GnRH promoter are required for gene expression pulse activity: role of GATA-4 and GATA-5 in this intermittent process.

Recent evidence reveals that several GATA factors act as versatile transcriptional modulators in neuroendocrine gene expression. The rat GnRH promoter is expressed in an episodic fashion that requires a portion of the promoter termed the neuron-specific enhancer (NSE) for activity. In this study, we examined whether certain GATA regulatory elements in the NSE are necessary for this intermittent activity. When injected into individual living GT1-7 cells, luciferase reporter constructs containing mutations of either GATA-A- or GATA-B-binding sites resulted in a marked reduction in gene expression pulse frequency, while mutations of both sites virtually abolished pulses. In subsequent studies, RT-PCR and western blot analysis revealed for the first time that GATA-5 and GATA-6 were expressed in GT1-7 cells, but electrophoretic mobility shift assays demonstrated further that GATA-5 bound to one of these GATA sites: GATA-A. Chromatin immunoprecipitation analysis revealed that all three factors, GATA-4, GATA-5, and GATA-6, were associated with the GnRH promoter in vivo. Interestingly though, immunoneutralization of GATA-5 or GATA-4 (reported to bind GATA-B) abolished gene expression pulses, but injection of GATA-6 antibody did not, indicating that of these factors just GATA-5 and GATA-4 are critical for intermittent activity. Finally, gel shift competition experiments revealed an interaction between proteins binding at the GATA-A site and those associating with an adjacent OCT1 site, previously shown to be necessary for pulse formation. These findings indicate that episodic GnRH gene expression pulses are mediated by GATA-5 and GATA-4, likely acting through the GATA-binding sites in the GnRH NSE region. Moreover, our observations that factors associated with GATA sites may also interact with OCT1 sites and that both are critical for pulse activity raise the intriguing possibility that GnRH pulse elaboration is a highly complex process that may require the coordinated interaction of several NSE-binding elements of the GnRH promoter.

Calcium influx and DREAM protein are required for GnRH gene expression pulse activity.

Recent evidence using GT1-7 cells indicates that GnRH pulsatility depends on exocytotic-release and gene transcription events. To determine whether calcium or DREAM may play a role in linking these processes, we used an L-type Ca(2+)-blocker (nimodipine) and found that not only GnRH gene expression (GnRH-GE) pulse activity was abolished but also that binding of proteins to OCT1BS-a (essential site for GnRH-GE pulses) was reduced. We further found that only EF-hand forms of DREAM were expressed in GT1-7 and that DREAM was part of the complex binding to OCT1BS-a. Finally, microinjection of DREAM antibody into cells abolished GnRH-GE pulses demonstrating its importance in pulsatility. These results reveal that calcium and DREAM may bridge cytoplasmic and nuclear events enabling temporal coordination of intermittent activity. Expression of DREAM in various cell types coupled with the universal role of calcium raise the possibility that these factors may play similar role in other secretory cells.

Pulses of prolactin promoter activity depend on a noncanonical E-box that can bind the circadian proteins CLOCK and BMAL1.

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 GH(3)) 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.

Identification of a novel OCT1 binding site that is necessary for the elaboration of pulses of rat GnRH promoter activity.

Recent evidence from our laboratory demonstrated that the OCT1 protein was necessary for GnRH gene promoter pulse activity through its interaction with a specific OCT1 binding site (OCT1BS-a, -1,774/-1,781). In light of the importance of this POU homeoprotein in pulsatile function, we focused on two other highly conserved OCT1 sites within this region, OCT1BS-b (-1,694/-1,701, previously AT-b), and OCT1BS-c (-1,569/-1,562). Mutagenesis of these sites revealed that alteration of OCT1BS-c, but not OCT1BS-b, virtually abolished gene expression pulses in GT1-7 cells. EMSAs confirmed that OCT1 can bind to both sites. Taken together, our findings demonstrate clearly that more than one Oct1 binding site is necessary for GnRH promoter pulses. Moreover, the lack of an influence observed with OCT1BS-b on pulse activity indicates that OCT1 action is not general to all OCT1 sites, but specific to certain octamer sequences in the NSE region of the GnRH promoter.

Episodic activation of the rat GnRH promoter: role of the homeoprotein oct-1.

Recent reports demonstrate that the rat GnRH promoter is activated in an episodic fashion in immortalized GnRH neurons, but little information is available on molecular processes that contribute to this phenomenon. In this study, we dissected the regions of the rat GnRH promoter that mediate these effects by testing a series of 5' deletion luciferase reporter constructs on the pattern of photonic emissions from single, living GT1-7 GnRH neuronal cells. Deletion analysis revealed that the region -2012/-1597 that contains the neuron-specific enhancer (NSE) was required for the elaboration of pulses of GnRH promoter activity. The importance of this region was supported by observations that episodic reporter activity could be transferred to a neutral nonpulsatile promoter (Rous sarcoma virus, RSV(180)). Immunoneutralization of Oct-1 as well as mutation of an octamer binding site located at -1787/-1783 (AT-a site) blocked the pulsatile GnRH promoter activity in GT1-7 neuronal cells. Taken together, our findings indicate that episodic GnRH gene expression is a promoter-dependent phenomenon, which is mediated by Oct-1 interaction with regulatory elements in the NSE region.

Calcium dynamics and resting transcriptional activity regulates prolactin gene expression.

Research on the regulation of hormone gene expression by calcium signaling is hampered by the difficulty of monitoring both parameters within the same individual, living cells. Here we achieved concurrent, dynamic measurements of both intracellular Ca(2+) concentration ([Ca(2+)](i)) and prolactin (PRL) gene promoter activity in single, living pituitary cells. Cells were transfected with the luciferase reporter gene under control of the PRL promoter and subjected to bioluminescence and fluorescence imaging before and after presentation of TSH-releasing hormone (TRH), a prototypic regulator of PRL secretion and gene expression that induces a transient Ca(2+) release, followed by sustained Ca(2+) influx. We found that cells displaying specific photonic emissions (i.e. mammotropes) showed heterogeneous calcium and transcriptional responses to TRH. Transcriptionally responsive cells always exhibited a TRH-induced [Ca(2+)](i) increase. In addition, transcriptional responses were related to the rate of Ca(2+) entry but not Ca(2+) release. Finally, cells lacking transcriptional responses (but showing [Ca(2+)](i) rises) exhibited larger levels of resting PRL promoter activity than transcriptionally responsive cells. Thus, our results suggest that the sustained entry of Ca(2+) induced by TRH (but not the Ca(2+) release) regulates transcriptional responsiveness. Superimposed on this regulation, the previous, resting PRL promoter activity also controls transcriptional responses.

PRL gene expression in individual living mammotropes displays distinct functional pulses that oscillate in a noncircadian temporal pattern.

PRL gene expression in the anterior pituitary has been the focus of intensive investigation for many years, but very little information is available on the actual dynamics by which this process occurs in individual mammotrope cells. Here, we used single cell bioluminescent imaging microscopy and a recently refined reporter gene strategy to measure PRL promoter-driven gene expression (PRL-GE) in individual living primary mammotropes. Using this approach we report a new phenomenon involving repetitive on/off gene expression bursts that occurred in a distinctly noncircadian oscillatory pattern. Furthermore, we demonstrate a functional basis for these gene expression oscillations, inasmuch as PRL-GE pulses were sensitive to calcium-dependent modulation, which we show arose exclusively as changes in the shape of individual pulse episodes. Our results provide the first clear evidence that PRL-GE, in its homologous cell environment, displays oscillatory bursts of activity. Moreover, they strongly support the idea that these discrete on/off bursts of activity serve as an important determinant of the timing and level of PRL-GE under both basal and stimulated conditions.

Effects of 4-tert-octylphenol given in drinking water for 4 months on the male reproductive system of Fischer 344 rats.

The environmental pollutant 4-tert-octylphenol (OP) is both toxic and estrogenic to mammalian cells, and injection of OP into adult male rats has devastating effects on their reproductive system. We now report the effects of OP in drinking water ( 1 x 10(-5), 1 x 10(-7) or 1 x 10 (-9) M) on the male reproductive system. Exposure of adult male rats for 4 months to any tested dose of OP had no significant effect on water or food consumption; body weight gain; hematocrit; reproductive organ weights; mean serum LH, FSH or testosterone concentrations; germ cell yield or relative numbers of different classes of testicular cells; or testicular sperm number. In contrast, all doses of OP caused an increase in epididymal sperm with tail abnormalities that would interfere with sperm motility, and the highest dose decreased epididymal sperm number. Our findings raise the possibility that consumption of OP in drinking water may adversely influence male reproductive fertility.

Episodes of prolactin gene expression in GH3 cells are dependent on selective promoter binding of multiple circadian elements.

Prolactin (PRL) gene expression in mammotropes occurs in pulses, but the mechanism(s) underlying this dynamic process remains obscure. Recent findings from our laboratory of an E-box in the rat PRL promoter (E-box133) that can interact with the circadian factors, circadian locomoter output cycles kaput (CLOCK) and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein (BMAL)-1, and was necessary for pulse activity raised the intriguing possibility that the circadian system may be central to this oscillatory process. In this study, we used serum-shocked GH(3) cells, established previously to synchronize PRL pulses between cells in culture, to reveal that pulses of PRL mRNA are linked temporally to the expression of bmal1, cry1, per1, and per3 mRNA in these cells. Moreover, we found that each of these circadian factors binds to the rat PRL promoter by chromatin immunoprecipitation analysis. Using EMSA analysis, we observed that two sites present in the proximal promoter region, E-box133 and E-box10, bind circadian factors differentially (E-box133 interacted with BMAL1, cryptochrome-1, period (PER)-1, and PER3 but not PER2 and E-box10 bound BMAL1, cryptochrome-1, PER2, PER3 but not PER1). More importantly, down-regulation of any factor binding E-box133 significantly reduced PRL mRNA levels during pulse periods. Our results demonstrate clearly that certain circadian elements binding to the E-box133 site are required for episodes of PRL mRNA expression in serum-shocked GH(3) cultures. Moreover, our findings of binding-related differences between functionally distinct E-boxes demonstrate not only that E-boxes can bind different components but suggest that the number and type of circadian elements that bind to an E-box is central in dictating its function.

Administration of connexin43 siRNA abolishes secretory pulse synchronization in GnRH clonal cell populations.

GnRH is released from hypothalamic neurons in coordinated pulses, but the cellular basis for this process is poorly understood. Previously, we found that secretory pulses from GT1-7 cells became synchronized with time in culture. Using this culture model, we investigated whether the gap junction proteins connexin43 (Cx43) and/or connexin26 (Cx26) are involved in this synchronization. Our results reveal that cytoplasmic densities immunoreactive for Cx43, and mRNA or protein for Cx43 increase with time in culture. Also, microinjection of day-3 cultures with siRNA for Cx43 abolished synchronized activity at day 7. Interestingly, cytoplasmic plaques, mRNA, or protein for Cx26 remained stable with culture time and Cx26 siRNA administration did not alter secretory activity. Our findings demonstrate that Cx43, but not Cx26 is necessary for synchronized secretory activity in these GT1-7 cultures and raise the possibility that Cx43-related gap junctions may be important in GnRH neuronal coordination in the hypothalamus.

Reverse hemolytic plaque assays: versatility in the study of secretion.

The reverse hemolytic plaque assay has been used for several years to study hormone release from various endocrine cell types. The basic method utilizes a monolayer (consisting of indicator erythrocytes and the cells under study) that is fixed to the floor of an incubation chamber. Antibody directed against a peptide or protein is added to the chamber. Peptides released from the cells under study complex with the antibody and bind to protein-A on the surface of the indicator erythrocytes. The addition of complement causes the indicator cells to lyse, forming a "plaque" or zone of hemolysis surrounding the secreting cells. The size or rate of formation of these plaques can be used as indices to monitor peptide or protein release. In addition to this standard procedure, the plaque assay can be modified by using loose or unattached indicator cells and is termed the loose plaque assay (LPA). The LPA for a particular peptide can be used alone, sequentially with an assay directed toward another peptide, or repeatedly on the same cells to monitor release over time. In light of the fact that plaque assays do not compromise the function of living cells, it is possible to combine these plaque assays with other procedures such as immunocytochemistry, in situ hybridization, fluorescent microscopy, electrophysiology, and electron microscopy to explore other facets of the secretory process in conjunction with release. When taken together, the plaque assay has been quite useful in the study of endocrine cell secretion. Moreover, with the many adaptations possible, it should be particularly valuable in the future for the study of peptide release in other cell types such as neurons.

Cloning and mRNA expression of the Ca2+-binding DREAM protein in the pituitary.

It is well recognized that the level of intracellular calcium governs several cellular processes such as gene expression and secretion in the pituitary. Recently, a novel gene has been identified in neuroendocrine cells that encodes DREAM, a calcium-binding protein that acts as a transcriptional repressor by binding specific downstream regulatory elements (DRE) on DNA. To explore the possibility that DREAM may be expressed in the rat pituitary and may function in endocrine activity, we analyzed its mRNA expression by RT-PCR. Using oligonucleotide primers derived from the mouse DREAM cDNA, we amplified, cloned, and characterized a 852-bp RT-PCR product from rat pituitary tissue. Two splice variants of the rat DREAM gene differing by four nucleotides (tetramer ACAG) were identified. The ACAG(+) variant (ORF1) consisted of 768bp encoding a protein of 256 residues with an estimated molecular weight of 29.5kDa. Amino acid sequence analysis of ORF1 indicated 92.6% and 98.1% identity to the DREAM gene product from human and mouse, respectively. The second variant, ACAG(-) (ORF2), was 567-bp long and was predicted to encode a peptide of 189 residues with a molecular mass of about 20.8kDa. To determine which endocrine pituitary cells were expressing DREAM, we evaluated several different clonal populations containing cells that expressed specific pituitary hormones. We found that both DREAM splice variants were expressed in each pituitary cell types examined, which included the mammotropes (MMQ cells), somatotropes (GC cells), mammosomatotropes (GH(3) cells), gonadotropes (LbetaT2 cells), thyrotropes (TalphaT1 cells), and corticotropes (AtT-20 cells). Interestingly, the levels of the two variants differed between the cell types tested with the ACAG(+) variant comprising about two-thirds of the DREAM expression for the mammotropes, somatotropes, mammosomatotropes, and corticotropes as compared to less than one-half for the thyrotropes and the gonadotropes. Our initial attempts to identify pituitary-specific genes regulated by DREAM revealed that prolactin gene expression was not influenced by DREAM suggesting that an action of DREAM may involve other pituitary hormones or be mediated by other cell processes. When taken together, our findings of DREAM expression in the pituitary in a manner specific to pituitary endocrine cell type raises the possibility that this protein may play a role in determining specific pituitary cell function.