Fredric R. Boockfor

The relationship between pulsatile secretion and calcium dynamics in single, living gonadotropin-releasing hormone neurons.

It is well established that pulsatile release of GnRH regulates the reproductive axis, but little is known about the mechanisms underlying this pulsatility. Recent findings that GT1 cells, a line derived from the mouse embryonic hypothalamus, release GnRH in a pulsatile manner indicates that this rhythmic activity is an intrinsic property of GnRH neurons. In several attempts to uncover the intracellular basis for this pulsatile phenomenon, it was revealed that intracellular calcium concentrations change in a rhythmic fashion in GnRH neurons and that cellular depolarization, which triggers a secretory event, is associated with profound calcium changes in the cells. These findings raised the intriguing possibility that periodic alterations in intracellular calcium concentrations may underlie the phenomenon of pulsatile secretion in GnRH neurons. To address this, we first adapted the use of FM1‐ 43 fluorescence to monitor changes of secretion in individual GT1‐7 cells and then combined this approach with simultaneous measurement of intracellular free calcium ([Ca 21 ]i, fura 2 method). In initial validation experiments, we found that stimulation of exocytosis with K 1 (75 mM )o rN-methyl-D-aspartate (NMDA, 100 mM) predictably evoked dynamic increases of both FM1‐ 43 and fura 2 fluorescence. Later measurement of calcium dynamics and exocytotic activity in unstimulated cells revealed that [Ca 21 ]i underwent transitions from quiescence to high oscillatory behavior, and that these shifts were frequently associated with exocytotic events. Moreover, these calcium oscillatory transitions and associated changes in secretory activity occurred synchronously among most adjacent cells and at a frequency similar to that reported for pulsatile release of GnRH by entire cultures of GnRH neurons. Taken together, these results indicate that the intrinsic secretory pulsatility of GnRH neurons appears to be a consequence of coordinated, periodic changes in the pattern of calcium oscillations within individual cells. (Endocrinology 141: 2012‐2017, 2000)

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.

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.

Fibroblast growth factor modulates the release of transferrin from cultured Sertoli cells

The acute and chronic effects of basic fibroblast growth factor (bFGF) on transferrin (TF) secretion from Sertoli cells were investigated by using reverse hemolytic plaque assays which enabled the visualization of release from individual cells in culture. We found that acute treatment with bFGF stimulates the release of TF from some but not al Sertoli cells in cultures obtained from 20-day-old rats. Chronic treatment with this growth factor resulted in increases in overall cell number in cultures from animals of each age tested (8-20 days of age). In contrast, this long-term treatment decreased markedly the proportions of Sertoli cells that secreted TF but only in cultures from 10-day-old animals. When taken together, these findings of acute and chronic influences of bFGF on TF secreting cells support the possibility that bFGF not only contributes to the modulation of the day-to-day release of certain substances from Sertoli cells, but may also influence development of the portions of the cell population that secrete these substances.

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.

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.

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.

Single and dual hormone secretors in GH3 cultures respond differently to hypothalamic factors.

Recent studies using both normal and tumoral pituitary cell cultures have demonstrated that growth hormone (GH) and prolactin (PRL) secreting populations contain cells which release either one or both of these hormones. In order to determine whether these two cell types can be differentially regulated by hypothalamic factors we performed the following study employing plaque assays for GH and PRL. Using cultures of GH3 cells, a rat tumor cell line which contains both of these cell types, we found that the hypothalamic factors vasoactive intestinal peptide (VIP) and thyrotropin releasing hormone (TRH) when used together had a greater influence on plaque formation than when each was used individually. This suggested that cells were present in culture that responded to one peptide but not the other. Estradiol-treated cultures (which contain only dual-secreting cells) were then evaluated for VIP and TRH responsiveness and found to respond to TRH but not VIP. Finally, we assessed the peptide sensitivity of cultures that were exposed to a conjugate of VIP and the A-chain of ricin (a potent cytotoxin). In addition to eliminating VIP-responsive cells, this treatment markedly reduced the proportions of cells secreting GH-only while having no appreciable influence on dual-hormone secretors. When taken together, our findings indicate that single and dual secretors respond differently to at least two hypothalamic secretagogues and suggest that regulatory differences between these cell types may be important in the control of GH and PRL secretion.