Selective inhibition of prolactin gene transcription by the ETS-2 repressor factor.

Regulation of prolactin gene transcription requires cooperative interactions between the pituitary-specific POU domain protein Pit-1 and members of the ETS transcription factor family. We demonstrate here that the ETS-2 repressor factor (ERF) is expressed in pituitary tumor cells and that overexpression of recombinant ERF inhibits prolactin promoter activity, but not the closely related growth hormone promoter. In non-pituitary cell lines, coexpression of ERF disrupts the cooperative interactions between Pit-1 and ETS-1 and blocks the induction of Pit-1-dependent prolactin promoter activity by cAMP. The potential role of ERF in the inhibitory response of the prolactin promoter to dopamine was examined using pituitary tumor cells stably expressing dopamine D2 receptors. The inhibitory responses of the prolactin promoter to ERF and dopamine are additive, suggesting that ERF has a complementary role in this hormonal response. A single Pit-1 DNA-binding element from the prolactin promoter is sufficient to reconstitute the inhibitory response to ERF. DNA binding analysis using either a composite Pit-1/ETS protein-binding site or a Pit-1 element with no known affinity for ETS proteins revealed that ERF interferes with Pit-1 binding. Together, these results demonstrate that ERF is a specific inhibitor of basal and hormone-regulated transcription of the prolactin gene and suggest a new level of complexity for the interaction of ETS factors with Pit-1 target genes.

Androgen-dependent cell cycle arrest and apoptotic death in PC-3 prostatic cell cultures expressing a full-length human androgen receptor.

To assess the function of androgen receptor in androgen-independent prostate cancer cells, human PC-3 prostate carcinoma cells, which lack androgen receptor (AR) expression, were transfected with a full length human AR cDNA sequence inserted into an episomal expression vector system. Several clonal lines of transfected cells expressing varying levels of a 110 kDa AR, as determined by immunoblotting and ligand binding assay, were isolated. The expressed ectopic receptors displayed nuclear binding following androgen treatment and mediated androgen inducibility of a mouse mammary tumor virus (MMTV)-luciferase reporter gene construct in a dose-dependent manner. 5 alpha-dihydrotestosterone (DHT) activation of luciferase activity was blocked by the AR antagonist hydroxyflutamide, and was promoter-specific based on the inability of the hormone-insensitive RSV promoter to respond to DHT. Treatment of AR-expressing PC-3 cells with physiological levels of DHT for 3 days resulted in paradoxical inhibition of cell growth. The growth-inhibitory effect was observed in clonal lines expressing low, moderate and high levels of AR, indicating that it was not the result of AR overexpression. To determine whether AR-expressing PC-3 cells had become androgen dependent, albeit with slowed growth, the effect of 1.0 nM DHT on the growth of two clonal lines expressing low and moderate receptor levels (PC-3(AR)13 and PC-3(AR)2, respectively) was examined on over an 18 day period. DHT removed after 3, 6, or 9 days and replaced with steroid-free medium. Surprisingly, after 6 days of DHT treatment, the number of PC-3(AR)2 cells began to decrease such that all cells were dead by 15 days after initiation of DHT treatment. A similar effect was observed in PC-3(AR)13 cells, but required a longer initial period of DHT exposure. PC-3(AR)2 cells were rescued from cell death if DHT was withdrawn 3 days but not 6 or 9 days after initiation of DHT treatment. As determined by DNA cell cycle analysis, the proportion of cells in the G1 phase was enhanced by DHT treatment, accompanied by a decrease in cells in the S and G2M phase of the cell cycle. After 6 days of DHT treatment, the proportion of cells in G1 decreased which was accompanied by an increase in cells in a subG1 population consistent with apoptosis. DNA fragmentation in PC-3(AR)2 cells after 3 or 6 days of DHT treatment was demonstrated by agarose gel electrophoresis, further indicating the cell death was apoptotic. Removal of DHT from PC-3(AR)2 cultures after 3 days, but not after 6 or 9 days, was followed by a large shift in cells from G1 to S and G2M. These data suggest that DHT blocks the progression of AR transfected PC-3 cells through the cell cycle, resulting in growth inhibition and apoptosis.

Selective constraints on the activation domain of transcription factor Pit-1.

The POU transcription factor Pit-1 activates members of the prolactin/growth hormone gene family in specific endocrine cell types of the pituitary gland. Although Pit-1 is structurally conserved among vertebrate species, evolutionary changes in the pattern of Pit-1 RNA splicing have led to a notable "contraction" of the transactivation domain in the mammalian lineage, relative to Pit-1 in salmonid fish. By site-directed mutagenesis we demonstrate that two splice insertions in salmon Pit-1, called beta (29 aa) and gamma (33 aa), are critical for cooperative activation of the salmon prolactin gene. Paradoxically, Pit-1-dependent activation of the prolactin gene in rat is enhanced in the absence of the homologous beta-insert sequence. This apparent divergence in the mechanism of activation of prolactin genes by Pit-1 is target gene specific, as activation of rat and salmon growth hormone genes by Pit-1 splice variants is entirely conserved. Our data suggest that efficient activation of the prolactin gene in the vertebrate pituitary has significantly constrained the pattern of splicing within the Pit-1 transactivation domain. Rapid evolutionary divergence of prolactin gene function may have demanded changes in Pit-1/protein interactions to accommodate new patterns of transcriptional control by developmental or physiological factors.

A dopamine-responsive domain in the N-terminal sequence of Pit-1. Transcriptional inhibition in endocrine cell types.

The POU transcription factor Pit-1 activates the prolactin gene in pituitary lactotrophs and may integrate responses of the gene to external signals. To study the role of Pit-1 in dopaminergic inhibition of the prolactin gene, we transiently transfected Pit-1 and dopamine D2 receptor vectors into a series of heterologous cell lines and examined dopamine regulation of the prolactin gene promoter. Regulation was Pit-1-dependent in all cell lines tested. Moreover, dopamine responsiveness was cell type-specific: stimulatory in fibroblasts (COS-7) and muscle-type cells (P19/Me2SO-induced) and inhibitory in pancreatic endocrine (RIN, InR1-G9) and neural-like (P19/retinoic acid-induced) cells. Because dopaminergic responses in Pit-1-transfected RIN cells paralleled those in pituitary GH4 cells, the islet cell line was used to test for sequences in Pit-1 that mediate negative hormone signals. Dopamine responsiveness of the Pit-1 transactivation domain (residues 8-80) was examined using a chimeric LexA construct. LxPit-1, LxSp1, and Lx-glucocorticoid receptor fusions all activated basal transcription, but only LxPit-1 was regulated by dopamine. Regulatory responses of LxPit-1 and full-length Pit-1 were quantitatively similar. In addition, gain-of-function G alpha mutants that inhibit Pit-1-dependent promoters in GH4 cells also suppressed selectively Pit-1- or LxPit-1-dependent promoters in RIN cells. This demonstrates that Pit-1 can function as a specific target for distinct inhibitory G protein signals. Interestingly, Pit-1 sequences N-terminal to the DNA-binding POU domain appear to be sufficient in mediating regulation by these pathways.

The chinook salmon gonadotropin II beta subunit gene contains a strong minimal promoter with a proximal negative element.

The salmon pituitary expresses two distinct gonadotropins, gonadotropin I (GTHI) and gonadotropin II (GTHII). These two hormones are synthesized in distinct pituitary cells and secreted at different stages during the reproductive cycle. To study the transcriptional regulation of the hormone-specific beta-subunit of GTHII (sGTHII beta) gene, approximately 3.5 kilobases of the 5'-flanking region was characterized and sequenced. The pituitary specificity of sGTHII beta was examined by analyzing sGTHII beta promoter activity in homologous primary pituitary cells derived from spawning rainbow trout and in a collection of heterologous cell lines. Various lengths of the 5'-flanking region of the sGTHII beta gene were ligated into a vector encoding the bacterial chloramphenicol acetyltransferase (CAT) gene, and the resulting sGTHII beta/CAT chimeric constructs were analyzed using transient expression systems. Several constructs (-3500CAT, -1260CAT, -563CAT, and -39CAT) displayed readily detectable CAT activity in the pituitary cells derived from spawning male and female rainbow trout. In contrast, three of the constructs (-3500CAT, -1260CAT, and -563CAT) were expressed only at background levels in a variety of heterologous cell lines, suggesting that the 5'-flanking sequence of sGTHII beta contains information dictating its pituitary specificity. A silencer sequence (-95 to -35, pSil) was identified, which might function to repress sGTHII beta gene expression in the nongonadotropes or in the gonadotropes at developmental stages that precede final maturation and spawning.

Differential recruitment of steroid hormone response elements may dictate the expression of the pituitary gonadotropin II beta subunit gene during salmon maturation.

The role of testosterone (T) and 17 beta-estradiol (E2) in the control of chinook salmon gonadotropin II beta subunit (sGTHII beta) gene was examined. Both E2 and T specifically stimulated GTHII beta gene expression in cultured juvenile rainbow trout pituitary cells. 5'-Flanking regions of the sGTHII beta gene linked to the chloramphenicol acetyltransferase (CAT) expression vector were transfected into these pituitary cells, and cultures were treated with steroid hormones. Estrogen-stimulated CAT activity occurred with constructs containing a 13-base pair estrogen responsive element (ERE) sequence [proximal ERE (pERE)] located at -273 to -260 upstream of its transcriptional start site. Binding specificity of pERE was confirmed by mobility shift and DNA methylation interference assays using the DNA binding domain of the human estrogen receptor. Interestingly, the pERE functioned to derepress the activity of the proximal silencer (pSil) only in the pituitary cells of juvenile trout but not in cells derived from maturing and sexually matured fish. Another potential ERE sequence comprised of three tandemly linked half-ERE palindromes was located from -2736 to -2659 [distal ERE (dERE)] of the sGTHII beta gene. Distal ERE might be responsible for the steroid responsiveness of the longest sGTHII beta/CAT construct (-3500CAT) observed in the pituitary cells of maturing fish. The function of pERE and dERE were further examined in the heterologous HeLa cells by mutagenesis and cotransfection with a rainbow trout estrogen receptor expression vector. Disruption of the palindromic structure of pERE severely impaired its function. When the sequences between pERE and dERE were deleted, a 200-fold increase in CAT activity was observed in response to E2. A model is proposed to describe the regulation of GTHII beta gene expression at different reproductive stages.

G(i) alpha 2- and G(o) alpha-mediated signaling in the Pit-1-dependent inhibition of the prolactin gene promoter. Control of transcription by dopamine D2 receptors.

Dopaminergic signaling in pituitary lactotrophs is dependent on coupling of D2 receptors to several inhibitory G-protein subtypes, resulting in the activation of multiple signaling pathways. In prolactin-secreting GH4 cells that express cloned D2 receptors, dopamine selectively inhibits the activity of the prolactin gene promoter, a response mediated in part by the pituitary transcription factor Pit-1. Transfected gain-of-function mutants of the G alpha subtypes, Gi alpha 2 (Q205L) and G(o) alpha (Q205L), mimic the promoter-specific and Pit-1-dependent inhibition by dopamine. Whereas the activated Gi alpha 2 subtype suppresses cAMP levels, the G(o) alpha mutant does not, demonstrating a cAMP-independent pathway in the inhibition of the prolactin gene. This alternate pathway could involve other regulators, possibly calcium. Interestingly, in Ltk- cells in which cloned D2 receptors modestly suppress cAMP, but elevate [Ca2+]i, the activity of the prolactin promoter is enhanced rather than inhibited by dopamine. The response is promoter-specific, dependent on Pit-1, and completely blocked by low concentrations of EGTA, consistent with a calcium-regulated pathway. Last, in GH4 cells, the absence of additivity between Gi alpha 2 and G(o) alpha mutants suggests a convergent mechanism in the reduction of prolactin promoter activity, in which either signaling pathway may be sufficient for maximum inhibition. This apparent redundancy in inhibitory control mechanisms may be of physiological importance for maintaining efficient tonic suppression of prolactin synthesis.

Cell type-specific expression of the pituitary transcription activator pit-1 in the human pituitary and pituitary adenomas.

Pit-1 is a transcription factor that has been shown to be critical for pituitary-specific activation of the GH and PRL genes. In rodents and humans, differentiation and/or maintenance of somatotroph, lactotroph, and thyrotroph phenotypes are dependent on expression of a functional pit-1 gene. In rodents, Pit-1 protein is detectable in only these three cell types; however, pit-1 mRNA transcripts appear to be present at comparable levels in all adenohypophysial cell types, suggesting that translational controls may dictate the pattern of Pit-1 expression. We examined the distribution of pit-1 transcripts in the human pituitary and pituitary adenomas. All tumors were characterized by immunocytochemistry, electron microscopy, and tissue culture for accurate classification. Northern blot analysis demonstrated abundant levels of pit-1 mRNA in somatotroph, mammosomatotroph, and lactotroph adenomas. Two clinically silent adenomas that expressed TSH as well as gonadotropins contained detectable levels of pit-1 mRNA. No pit-1 expression was otherwise detected in corticotroph, gonadotroph, null cell, or oncocytic adenomas. In situ hybridization localized pit-1 mRNA transcripts in adenomas that contained GH, PRL, or TSH, but not in adenomas composed of other cell types. Pit-1 mRNA was also localized to selected subpopulations of the human nontumorous adenohypophysis that contained immunoreactivity for GH, PRL, and/or TSH. Pit-1 protein immunoreactivity was detected in the nuclei of adenomas that expressed pit-1 mRNA, but not in those that were negative for pit-1 mRNA; it was also localized only in cells containing GH, PRL, or TSH beta in the nontumorous adenohypophysis. These data demonstrate selective expression of the human pit-1 gene in adenohypophysial cell types responsible for GH, PRL, and/or TSH synthesis and are consistent with a predominantly pretranslational regulatory mechanism for Pit-1 expression in the human.

Phylogenetic specificity of prolactin gene expression with conservation of Pit-1 function.

In mammals, the pituitary POU homeodomain protein, Pit-1, binds to proximal and distal 5'-flanking sequences of the PRL gene that dictate tissue-specific expression. These DNA sequences are highly conserved among mammals but are dramatically different from PRL 5' sequences in the teleost species, Oncorhynchus tschawytscha (chinook salmon). To analyze the molecular basis for pituitary-specific gene expression in a distantly related vertebrate, we transfected CAT reporter gene constructs containing 2.4 kilobases (kb) 5'-flanking sequence from the salmon PRL (sPRL) gene into various cell types. Expression of the sPRL gene was restricted to pituitary cells, but in rat pituitary GH4 cells levels of expression were at least 90-fold lower than those obtained with a -3 kb rat PRL (rPRL) construct. Conversely, in primary teleost pituitary cells, -2.4 kb sPRL/CAT was expressed at levels about 10-fold higher than -3 kb rPRL/CAT. To determine whether species-specific transactivation by Pit-1 was sufficient to explain these species differences in PRL gene expression, we isolated a cDNA clone encoding the salmon Pit-1 POU domain and constructed a rat Pit-1 expression vector that contained salmon Pit-1 POU domain sequences substituted in frame. The chimeric Pit-1 encoded 14 amino acids unique to salmon. Coexpression of rat Pit-1 with salmon or rat PRL/CAT in transfected HeLa cells resulted in specific and strikingly comparable levels of promoter activation. Moreover, the specificity and efficacy of the chimeric salmon/rat Pit-1 was similar to wild type rat Pit-1 in activating salmon and rat PRL/CAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory control of prolactin and Pit-1 gene promoters by dopamine. Dual signaling pathways required for D2 receptor-regulated expression of the prolactin gene.

Transcription of the prolactin gene is suppressed by dopaminergic activation of D2 receptors in pituitary lactotrophs. The mechanism of signal transduction at the nuclear level and the cell surface was examined in the dopamine-responsive GH4ZR7 cell line. Dopamine treatment caused a 40-50% decrease in endogenous prolactin mRNA that was specifically blocked by addition of (-)-sulpiride. To define dopamine-responsive elements, plasmids containing 5'-regulatory regions of the prolactin gene fused to the coding sequences for luciferase were transiently or stably transfected into GH4ZR7 cells. Chimeric transcripts initiated at the authentic transcription start site were regulated in a promoter-selective manner; dopamine or the agonist bromocryptine inhibited prolactin promoter (position -422) activity by 70%, but had no regulatory effects on other cellular or viral promoters. A shorter prolactin promoter (position -78) or a prolactin TATAA box linked to heterologous binding sites for transcription factor Pit-1 was sufficient to confer dopamine inhibition (40%). In addition to the prolactin promoter, we found that dopamine inhibited transcriptional activity of the Pit-1 promoter (positions -258 to +8) by 60%. Surprisingly, deletion of two cAMP response elements in the Pit-1 promoter only partially eliminated dopamine responsiveness. These data suggest that sequences in the Pit-1 promoter between positions -92 and +8, which include an autoregulatory Pit-1-binding site and the TATAA box, are sufficient for negative regulation. In this study, we also examined the signal transduction pathways that link D2 receptor activation and the inhibition of prolactin gene transcription. We found, as suggested in earlier studies, that a dopamine-dependent decrease in cAMP may be important for mediating negative regulation of transcription. However, high extracellular K+ concentrations that prevent dopamine effects on membrane potential and [Ca2+]i, but not cAMP levels, completely blocked dopamine regulation of the prolactin promoter. This suggests that two distinct signaling pathways initiated at D2 receptors may be required for transcriptional regulation of the prolactin gene.

A two-base change in a POU factor-binding site switches pituitary-specific to lymphoid-specific gene expression.

The structurally related POU homeo domain proteins Pit-1 and Oct-2 activate pituitary- and lymphoid-specific transcription, respectively, by binding to similar AT-rich motifs in their target genes. In this study we identify bases critical for recognition and activation by Pit-1 and examine how small differences in Pit-1 and Oct-2-binding sites can impart differential transcriptional responses in pituitary and B-lymphoid cells. Scanning mutagenesis of Pit-1 response elements in both the rat prolactin and growth hormone genes reveals a critical binding motif recognized in an identical manner by the native Pit-1 protein and cloned Pit-1 gene product. This motif, ATTATTCCAT, differs by only two bases from the octamer element, ATTTGCAT, required for Oct-2-dependent activation of immunoglobulin genes. Cross recognition of Pit-1 and Oct-2 sites by both factors can be demonstrated in competitive binding assays, in which an oligometric Pit-1 site from the prolactin gene is converted to an Oct-2 site by a double point mutation. In contrast to the binding data, no cross activation of transcription is detectable in cultured cell lines. When inserted immediately 5' to a prolactin TATA box, the wild-type prolactin element enhances transcription strongly in pituitary cells but is inactive in B cells, whereas the octamer variant of the prolactin site activates expression in B cells but is silent in pituitary lines. Both elements are nonfunctional in heterologous cell lines that lack Pit-1 and Oct-2.(ABSTRACT TRUNCATED AT 250 WORDS)

A family of POU-domain and Pit-1 tissue-specific transcription factors in pituitary and neuroendocrine development.

The anterior pituitary gland provides a model for investigating the molecular basis for the appearance of phenotypically distinct cell types, within an organ, a central question in development. The rat prolactin and growth hormone genes are selectively expressed in distinct cell types (lactotrophs and somatotrophs) of the anterior pituitary gland, which reflect differential mechanisms of gene activation or restriction because of interactions of multiple factors binding to these genes. We find that the pituitary-specific 33,000 dalton transcription factor, Pit-1, normally expressed in somatotrophs, lactotrophs, and thyrotrophs, can bind to and activate both growth hormone and prolactin promoters in vitro at levels even tenfold lower than those normally present in pituitary cells. In the case of the prolactin gene, high levels of expression in transgenic animals required two cis-active regions; a distal enhancer (-1.8 to -1.5 kb) and a proximal region (-422 to +33 bp). Each of these regions alone can direct low levels of fusion gene expression to prolactin-producing cell types in transgenic mice, but a synergistic interaction between these regions is necessary for high levels of expression. The initial appearance of the prolactin transgene expression closely follows the appearance of high levels of Pit-1, but later increases in expression coincident with appearance of mature lactotrophs suggest the operation of additional, critical positive factor(s). Unexpectedly, transgenes containing the distal enhancer removed from its normal context are expressed in both the prolactin-producing lactotrophs and the TSH-producing thyrotrophs, thereby suggesting that sequences flanking this enhancer are necessary to restrict expression to the correct cell type within the pituitary. These data indicate that distinct processes of gene activation and restriction are necessary for the fidelity of cell-type specific expression within an organ. Consistent with this model, we find that lactotroph cell lines that cannot express the growth hormone gene contain high levels of functional Pit-1. We suggest a large, highly related POU-domain gene family, potentially exceeding 100 members, has been conserved and expanded in evolution to meet the increasing requirements for more intricate patterns of cell phenotypes. The POU-domain subgroup of the homeodomain gene family, in concert with other homeodomain proteins and with other classes of transcription factors, is likely to contribute to the establishment of the mammalian neuroendocrine system.

A tissue-specific transcription factor containing a homeodomain specifies a pituitary phenotype.

Multiple related cis-active elements required for cell-specific activation of the rat prolactin gene appear to bind a pituitary-specific positive transcription factor(s), referred to as Pit-1. DNA complementary to Pit-1 mRNA, cloned on the basis of specific binding to AT-rich cell-specific elements in the rat prolactin and growth hormone genes, encodes a 33 kd protein with significant similarity at its carboxyl terminus to the homeodomains encoded by Drosophila developmental genes. Pit-1 mRNA is expressed exclusively in the anterior pituitary gland in both somatotroph and lactotroph cell types, which produce growth hormone and prolactin, respectively. Pit-1 expression in heterologous cells (HeLa) selectively activates prolactin and growth hormone fusion gene expression, suggesting that Pit-1 is sufficient to confer a characteristic pituitary phenotype. The structure of Pit-1 and its recognition elements suggests that metazoan tissue phenotype is controlled by a family of transcription factors that bind to related cis-active elements and contain several highly conserved domains.

Activation of cell-specific expression of rat growth hormone and prolactin genes by a common transcription factor.

In the anterior pituitary gland, there are five phenotypically distinct cell types, including cells that produce either prolactin (lactotrophs) or growth hormone (somatotrophs). Multiple, related cis-active elements that exhibit synergistic interactions appear to be the critical determinants of the transcriptional activation of the rat prolactin and growth hormone genes. A common positive tissue-specific transcription factor, referred to as Pit-1, appears to bind to all the cell-specific elements in each gene and to be required for the activation of both the prolactin and growth hormone genes. The data suggest that, in the course of development, a single tissue-specific factor activates sets of genes that ultimately exhibit restricted cell-specific expression and define cellular phenotype.

Two different cis-active elements transfer the transcriptional effects of both EGF and phorbol esters.

Short cis-active sequences of the rat prolactin or Moloney murine leukemia virus genes transfer transcriptional regulation by both epidermal growth factor and phorbol esters to fusion genes. These sequences act in a position- and orientation-independent manner. Competitive binding analyses with nuclear extracts from stimulated and unstimulated cells suggest that different trans-acting factors associate with the regulatory sequence of each gene. A model is proposed suggesting that both epidermal growth factor and phorbol esters stimulate the transcription of responsive genes via discrete classes of hormone-dependent, enhancer-like elements that bind different trans-acting factors, even in the absence of hormone stimulation.

Role of ornithine decarboxylase in proliferation of prolactin-dependent lymphoma cells.

Mitogenic stimulation of Nb2 lymphoma cells by lactogenic hormones (prolactin, human growth hormone) caused a dramatic early increase in ornithine decarboxylase (ODC) activity that achieved a maximal level by 6-8 h. A marked increase in ODC activity was also generated when cells which had reached a growth plateau were transferred to fresh medium that did not stimulate growth. Furthermore, low concentrations of human growth hormone (20 pg/mL) elicited a proliferative response, but did not cause a detectable early increase in ODC activity. The early peak of ODC activity thus appeared not to be directly involved in mediating lactogen-stimulated growth nor was it required to support the mitogenic response. However, prolonged suppression of ODC activity by DL-alpha-difluoromethylornithine (DFMO) (200 microM) attenuated the growth of Nb2 cells (50-60% inhibition), indicating that normal cell growth was dependent on ODC and polyamine biosynthesis. Under these conditions, putrescine, the enzyme product, or the polyamines spermidine and spermine restored normal cell growth when added at a concentration of 1 microM or greater. Nb2-SP cells, variants which proliferate in the absence of prolactin, were about two times more resistant to the growth suppressive effects of DFMO than prolactin-responsive Nb2 cells.