Pituitary-specific Transcription Factor Research Articles

Regulation of the pituitary-specific transcription factor GHF-1/Pit-1 messenger ribonucleic acid levels by growth hormone-secretagogues in rat anterior pituitary cells in monolayer culture

Pituitary-specific expression of the GH gene is dependent on a pituitary-specific transcription factor GH factor-1 (GHF-1), a homeodomain protein also known as pituitary-specific transcription factor-1 (Pit-1). The aim of this study was to investigate the regulation of GHF-1 messenger RNA (mRNA) levels in primary monolayer cultures of rat anterior pituitary cells. Specifically, in addition to direct activators of second messenger signaling systems, we studied the effects of different hormones, all of which are known to be involved in the regulation of somatotroph cell function. We found that GH-releasing hormone (GHRH) increased GHF-1 mRNA levels in a time- and dose-dependent fashion. GHF-1 mRNA levels were increased 2.5-fold (P < 0.01) after incubation for 2 h with 10(-8) M GHRH. Longer incubations (6, 12, or 24 h) with GHRH failed to show a similar stimulatory effect. A significant increase in GHF-1 mRNA concentration (1.7-fold, P < 0.01) was observed after a 2-h treatment with physiological concentrations (10(-11) M) of GHRH. The action of GHRH seems to occur at the transcriptional level without the need of protein synthesis. Thus, treatment of cells with actinomycin D (5 micrograms/ml) completely abolished GHRH-induced increase in GHF-1 mRNA levels. Cycloheximide (23 micrograms/ml) alone increased GHF-1 mRNA levels (6-fold increase after treatment for 12 h, P < 0.01), as well as potentiating GHRH-induced increase in GHF-1 mRNA concentration (9-fold increase after treatment with GHRH plus cycloheximide for 12 h, P < 0.01). The effect of GHRH on GHF-1 mRNA levels could be mimicked by direct activators of second messenger signaling systems such as forskolin (10(-5) M) or the phorbol ester tumor promoter tetradecanoyl phorbol acetate (TPA) (10(-6) M). Other peptides such as pituitary adenylate cyclase activating polypeptide-38 (10(-7) M) but not GHRP-6 (10(-10) to 10(-5) M), were also able to increase GHF-1 mRNA levels. Treatment of the cells with somatostatin (10(-6) M) for either 2 or 48 h failed to modify basal or GHRH-induced GHF-1 mRNA levels. In contrast, pretreatment of the cells with insulin-like growth factor-1 (5 nM) inhibited basal GHF-1 mRNA concentration as well as completely blunting the subsequent response to cells exposed to GHRH for 2 h. These data demonstrate that GHRH, acting at the transcriptional level and through a mechanism not dependent on protein synthesis, plays a stimulatory role on GHF-1 mRNA levels.

The role of estrogen receptor in modulation of chromatin conformation in the 5′ flanking region of the rat prolactin gene

To determine whether the estrogen receptor (ER) has a role in the modification of chromatin structure, we developed cell lines to model discrete stages in the estrogen response. Each cell line carries a population of stably expressed papillomavirus-based minichromosomes containing the 5′ flanking region of the rat prolactin gene. We examined ER effects at the distal enhancer domain of the rat prolactin promoter, using DNaseI to probe for alterations of the nucleoprotein complex. Within 1 h after the start of estrogen treatment, modifications in the chromatin state of the distal enhancer region were detected in a pituitary-derived, permissive cell line (GH 3G1J). In rat-1 fibroblast cell lines that maintain the same stably expressed papillomavirus-based minichromosomes in the absence of ER or pituitary-specific transcription factors (Rat-1.2A2; non-permissive), no estrogen-induced modifications in the chromatin state were detected at 1 or 24 h. In rat-1 fibroblast cell lines that also contained ectopically expressed, functional ER (Rat-1 + ER.8A1), no estrogen-induced modifications in the chromatin state were detected at 1 h, but at 24 h a specific modification in the local structure was induced. These data support a model in which the ER interacts with chromatin to modify local structure in such a way as to induce a permissive state for interactions of transcription factors necessary for hormone-induced activation of gene transcription.

GH and PRL gene expression by nonradioisotopic in situ hybridization in TSH-secreting pituitary adenomas.

TSH-secreting pituitary adenomas are rare. The transcriptional expression (messenger ribonucleic acids: mRNAs) of TSH beta, GH, and PRL in five patients with TSH-secreting pituitary adenoma was studied by the in situ hybridization (ISH) method in order to elucidate their multiple hormone production. These patients showed inappropriately elevated serum TSH and alpha-subunit levels as well as pituitary mass lesions. The tissues from pituitary adenomas were obtained at the time of transsphenoidal surgery and revealed immunohistochemically the expression of alpha-subunit and TSH beta in all patients. Four adenomas were immunohistochemically associated with GH or PRL localization. The presence of pituitary-specific transcriptional factor Pit-1 was demonstrated in all adenomas in the nuclei of many cells. By ISH, signals for TSH beta mRNA were present in all five cases in many adenoma cells. Expression of GH mRNA and PRL mRNA were detected not only in four adenomas in which both hormonal products were immunolocalized but also in one adenoma that was immunohistochemically negative for GH and PRL. Combined staining by ISH and immunohistochemistry revealed the expression of GH mRNA and PRL mRNA in TSH beta-immunoreactive cells. Our findings indicate that TSH-secreting adenomas are multihormone-producing and could arise from precursor or stem cells rather than from differentiated TSH-secreting cells. It is suggested that ISH combined with immunohistochemistry may provide additional detailed information concerning the multidirectional histogenesis of this rare type of adenoma.

Screening forPITIAbnormality by PCR Direct Sequencing Method

PIT1 abnormality is defined as a genetic abnormality in the PIT1 gene, which encodes a pituitary specific transcription factor Pit-1/GHF-1.PIT1 abnormality has been reported in several patients displaying either complete or incomplete deficiency of thyrotropin (TSH), growth hormone (GH), and prolactin (PRL) in either familial or sporadic cases. To see if there are abnormalities in the PIT1 gene in patients with incomplete TSH, GH, and PRL deficiency, we utilized a PCR direct sequencing method to determine the Pit-1/GHF-1 coding sequence. A total of 15 patients, 1 patient from a family with TSH and GH deficiency, 3 patients with TSH, GH, and PRL deficiency, and 11 patients treated with both human GH (hGH) and thyroid hormone were studied. In one patient of combined pituitary hormone deficiency, the Arg-271-Trp mutation was detected. Since both of the parents did not harbor this mutation, it is a de novo germ line mutation. No mutation was detected in the other patients, showing that PIT1 abnormality is not a frequent cause of GH deficiency.

A 33 kDa Pit-1-like protein binds to the distal region of the human thyrotrophin α-subunit gene

Our previous studies demonstrated that at least two DNA regions with upstream limits between positions -223 to -190 and positions -151 to -135 of the human TSH gene are important for transcriptional regulation by TRH in GH3 rat pituitary cells. The proximal region (-151 to -135 bp) including the cAMP-responsive element (CRE) was required for the induction of the TSH gene by TRH, while the distal region (-223 to -190 bp) containing an element similar to the binding site for the pituitary-specific transcription factor, Pit-1, was necessary to amplify the effects of TRH. To determine whether a pituitary-specific nuclear protein, in addition to the CRE-binding protein, is involved in the molecular mechanism of TRH regulation, a gel retardation assay and Southwestern blot analysis were performed on the distal region with GH3 cell nuclear extracts. GH3 extracts generated a distinct DNA-protein complex that was effectively eliminated in the presence of excess unlabelled DNA fragment, and TRH treatment increased the affinity of protein binding remarkably. Excess Pit-1 DNA-binding sequence from the rat prolactin gene inhibited formation of the complex, but mutation of the Pit-1 consensus sequence in the distal region did not eliminate the complex. In addition, Southwestern experiments showed that a 33 kDa nuclear protein present in GH3 cells bound to this region and its binding affinity was increased slightly 2 h after TRH treatment, with the maximal increase (fivefold) at 3 h, which was similar to the results when using gel retardation. Phosphatase treatment of nuclear protein also resulted in a loss of binding affinity. Taken together, these data indicate that the interaction of a pituitary-specific nuclear protein, identical or closely related to Pit-1, with the distal region may be involved in the TRH stimulation of human TSH gene expression.

Functional interaction of c-Ets-1 and GHF-1/Pit-1 mediates Ras activation of pituitary-specific gene expression: mapping of the essential c-Ets-1 domain.

The mechanism by which activation of common signal transduction pathways can elicit cell-specific responses remains an important question in biology. To elucidate the molecular mechanism by which the Ras signaling pathway activates a cell-type-specific gene, we have used the pituitary-specific rat prolactin (rPRL) promoter as a target of oncogenic Ras and Raf in GH4 rat pituitary cells. Here we show that expression of either c-Ets-1 or the POU homeo-domain transcription factor GHF-1/Pit-1 enhance the Ras/Raf activation of the rPRL promoter and that coexpression of the two transcription factors results in an even greater synergistic Ras response. By contrast, the related GHF-1-dependent rat growth hormone promoter fails to respond to Ras or Raf, indicating that GHF-1 alone is insufficient to mediate the Ras/Raf effect. Using amino-terminal truncations of c-Ets-1, we have mapped the c-Ets-1 region required to mediate the optimal Ras response to a 40-amino-acid segment which contains a putative mitogen-activated protein kinase site. Finally, dominant-negative Ets and GHF constructs block Ras activation of the rPRL promoter, and each blocks the synergistic activation mediated by the other partner protein, further corroborating that a functional interaction between c-Ets-1 and GHF-1 is required for an optimal Ras response. Thus, the functional interaction of a pituitary-specific transcription factor, GHF-1, with a widely expressed nuclear proto-oncogene product, c-Ets-1, provides one important molecular mechanism by which the general Ras signaling cascade can be interpreted in a cell-type-specific manner.

Reconstitution of protein kinase A regulation of the rat prolactin promoter in HeLa nonpituitary cells: identification of both GHF-1/Pit-1-dependent and -independent mechanisms.

Expression of the rat PRL (rPRL) gene is highly restricted to pituitary lactotroph cells and is induced by the cAMP-dependent protein kinase A (PKA) pathway. Current data indicate that this PKA effect requires at least one of the redundant pituitary-specific elements of the proximal rPRL promoter, suggesting the involvement of the pituitary-specific transcription factor, GHF-1/Pit-1. To directly determine whether GHF-1 is necessary and sufficient to mediate the PKA activation of the rPRL promoter, we established a cotransfection reconstitution assay whereby the activity of an intact and site-specific mutants of the (-425 to +73) rPRL promoter-luciferase reporter gene was reconstituted by cotransfecting expression vectors encoding for either the PKA beta catalytic subunit, GHF-1, or both, into HeLa nonpituitary cells. Cotransfection of PKA beta alone significantly stimulated rPRL promoter activity in HeLa cells in a GHF-1-independent manner, and this PKA beta effect was mapped to the most proximal GHF-1 site [footprint (FP) I; -67/-36]. Site-specific alterations of either FP II (-130/-120), or of the basal transcription element (BTE; -112/-80), did not significantly affect the PKA beta response. As expected, the transactivation effect of cotransfected GHF-1 mapped to the GHF-1/Pit-1 binding sites, FP I and/or FP III, of the rPRL promoter. Finally, cotransfection of PKA beta and GHF-1 resulted in a marked synergistic response of the rPRL promoter, and this response also localized to the FP I site. These data confirm not only that GHF-1/Pit-1 and the FP I site are involved in mediating the PKA response, but also imply that a distinct and possibly ubiquitous factor is involved by binding to FP I and functionally interacting with GHF-1 to modulate PKA beta regulation of the rPRL promoter.

Transcription Factor Pit-1 and Its Clinical Implications

This review presents a transcription factor, Pit-1, and its relevance to clinical practice. Our knowledge of the molecular and genetic basis of disease is growing at an amazing pace. Intranuclear transcription factor proteins, which influence gene expression, are being discovered and characterized. Pit-1, a pituitary-specific transcription factor, is necessary for the development and subsequent function of somatotrophs, lactotrophs, and thyrotrophs of the anterior pituitary gland. In this review we will discuss the molecular mechanisms by which Pit-1 influences gene expression and explore the role of this factor in pituitary diseases such as hypopituitarism and pituitary tumors.

Abnormalities of the Pituitary-specific Transcription Factor-1 Gene and Protein

Abnormalities of the pituitary-specific transcription factor-1 (Pit-1) gene cause combined deficiencies of growth hormone (GH), prolactin and thyrotropin (TSH) by altering Pit-1 protein in several different ways. The simplest are gene deletions that preclude expression of the protein. Nonsense mutations such as R172X that generate truncated proteins that lack POU specific and/or POU homeo domains and cannot bind to regulatory sites. Some missense mutations such as the Snell mouse W261C disable deoxyribnucleic acid (DNA) binding domains. Others have little effect on binding, but prevent transcriptional activation. The A158P mutation is recessive. It corresponds to “positive control” mutants in the lambda repressor that are unable to activate certain target genes, but retain the ability to silence other target genes. The R271W mutation distal to the POU homeo domain exerts dominant negative effects. Mutant proteins apparently participate in heterodimer formation and compete with active, wild type, homodimers. Alternative splicing of Pit-1 messenger ribonucleic acid (mRNA) introduces some interesting variations. Inclusion of parts of intron 1 in the protein may alter specificity of transcriptional activation. Exclusion of exon 4 bypasses the A158P and R172X mutations. If the protein encoded by this alternative splice form has a function in pituitary cell development, it may help explain the phenotypic variability seen with these mutations.

Rat Pit-1 Stimulates Transcription in Vitro by Influencing Preinitiation Complex Assembly

The anterior pituitary-specific transcription factor, Pit-1, activates prolactin, growth hormone, TSHβ, growth hormone receptor genes and autoregulates the pit-1 gene. Its mechanism of transcription activation is unknown. Using immobilized DNA templates and order-of-addition transcription assays, it is shown that Pit-1 is required during pre-initiation complex assembly to activate the prolactin gene in vitro. Using prolactin promoters containing point mutations in the distal TATA box, it is also demonstrated that Pit-1 activation in vitro is not mediated simply by repressing the upstream, alternative promoter. Experiments show that a preformed class II pre-initiation complex is refractory to Pit-1 influence. The data indicate that Pit-1, and perhaps other members of the POU-protein family, activate transcription by influencing the type pre-initiation complex assembled on target promoters.

Congenital Deficiency of Growth Hormone and Prolactin

The case is presented of a 25-year-old woman with congenital deficiency of growth hormone (GH) and prolactin (PRL). Her height was 108cm (-9.98 standard deviation (SD)) on admission. She had a bulging forehead and a small nose with a retracted bridge. Basal levels of GH and PRL were low. In stimulation tests of GH secretion, no responses were observed. In a thyrotropin-releasing hormone (TRH) loading test, the response of PRL was poor but that of thyrotropin (TSH) was normal. The levels of other pituitary hormones were normal. magnetic resonance imaging (MRI) of the brain revealed no abnormalities. In examination of the pituitary-specific transcription factor (Pit-1/GHF-1) (Pit-1) gene in this patient by the polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and deoxy ribonucleic acid (DNA) sequencing analysis, neither mutations nor deletions were detected.

A Pit-1 binding site in the human renin gene promoter stimulates activity in pituitary, placental and juxtaglomerular cells

One of the principal aims of our research is to determine the mechanisms which direct renin gene expression to different sites. We recently demonstrated that human renin (hRen) 5'-flanking DNA sequences -148 +/- 11 can drive the transient expression of a linked luciferase reporter gene transfected into pituitary GC cells. This activity was found to be dependent on the binding of Pit-1 to a site approximately 70 bp upstream from the transcription start site. Pit-1 is a pituitary-specific transcription factor which is involved in directing the cell-specific expression of growth hormone (GH) and prolactin (PRL) gene expression to somatotrope and lactotrope cells of the anterior pituitary. Thus, Pit-1 may be play a role in directing the expression of renin to primate lactotrope cells. Renin promoter-driven luciferase or CAT hybrid genes were found to be expressed following transfection into primary, or early passage cell cultures of placental chorionic membranes, and the renin-secreting renal tumor cell line As4.1. As with GC cells, deletion or mutagenesis of the Pit-1 site reduced activity several-fold in both placental and renal cells. These results suggest that members of the POU family of transcription factors, or some other closely related group such as the Hox proteins, participate in directing renin gene expression to placental and juxtaglomerular cells.

Pituitary-specific transcription factor Pit-1 in the rdw rat with growth hormone- and prolactin-deficient dwarfism

The pituitary gland of the rdw rat (gene symbol: rdw) with hereditary dwarfism expresses 30-100 times less GH and prolactin (PRL) mRNA than normal controls. To clarify the features of rdw rats, TSH and the pituitary-specific transcription factor Pit-1, which is involved not only in the gene expression of GH and PRL but in somatotroph, lactotroph and thyrotroph development as well, were examined. The rdw pituitary contained about seven times more TSH beta mRNA than the normal control, whereas Pit-1 mRNA expression in rdw and control was the same. Nucleotide sequencing of PCR-amplified Pit-1 cDNA indicated that the deduced amino acid sequence of rdw Pit-1 was identical with that of the normal rat. Using an antibody against rat Pit-1 protein produced in E. coli, Western blotting analysis demonstrated the presence of the same amount of Pit-1 protein in rdw and normal rat pituitaries. The distribution of Pit-1-positive cells in the anterior pituitary was essentially the same in rdw and normal rats. It follows from these findings that the defective gene in the rdw rat is unrelated to the Pit-1 gene and the normal quantity of Pit-1 protein is insufficient to produce normal amounts of GH and PRL in the rdw pituitary. These and previous results suggest that the reduction in GH and PRL production in the rdw pituitary might be due to that in thyroid hormone production.

Renin gene promoter activity in GC cells is regulated by cAMP and thyroid hormone through Pit-1-dependent mechanisms.

Transcriptional activity of human renin gene (hREN) 5'-flanking DNA sequences in pituitary cells is highly dependent on binding of the pituitary-specific transcription factor Pit-1. Pit-1 has been implicated in cAMP regulation of a number of pituitary genes and has also been shown to interact with thyroid hormone (T3) receptors in mediating T3 responsiveness of the rat growth hormone gene. In the present study we examine the effects of forskolin and T3 on the expression of luciferase hybrid genes containing hREN 5'-flanking DNAs (hREN.luc) transiently transfected into the pituitary cell line GC. Basal activities of all hREN.luc constructs transfected into cells grown in media containing serum stripped of hormones were low. Addition of forskolin stimulated expression up to 48-fold, depending on the hREN sequences present. The hREN sequence -148 to +18 was sufficient for both maximal expression and maximal stimulation by forskolin. Mutagenesis of the Pit-1 site between -82 and -58 reduced forskolin induction 4-5-fold. In addition to the Pit-1 site, the sequence between -148 and -98 was also required for maximal activity and forskolin induction. T3 on its own had no effect on hREN promoter activity in GC cells, but suppressed the effects of forskolin. Gel mobility shift and Western blot analyses indicated that forskolin treatment had no effect on Pit-1 DNA binding or Pit-1 levels. However, T3 reduced Pit-1 levels which was reflected in lower DNA binding under the conditions employed. Taken together, these findings emphasize the importance of cAMP-dependent mechanisms in directing renin gene expression.

INSIGHT: Pit-1/GHF-1: a pituitary-specific transcription factor linking general signaling pathways to cell-specific gene expression

INSIGHT: Pit-1/GHF-1: a pituitary-specific transcription factor linking general signaling pathways to cell-specific gene expression

Thyrotropin releasing hormone stimulates transient phosphorylation of the tissue-specific transcription factor, Pit-1.

The hypothalamic hormone, thyrotropin releasing hormone (TRH), stimulates prolactin (PRL) secretion and gene transcription in the GH3 pituitary cell line. Several studies have provided indirect evidence that phosphorylation of the pituitary-specific transcription factor Pit-1 may mediate TRH effects on PRL transcription. In the present study we have investigated the ability of TRH to alter the phosphorylation of Pit-1. In vivo 32P labeling experiments demonstrated that TRH stimulated a transient phosphorylation of Pit-1, reaching a maximum in 5 min and returning to basal levels within 30 min. Phosphopeptide mapping experiments demonstrated that TRH induced the transient phosphorylation of specific sites in Pit-1. TRH-stimulated phosphorylation of Pit-1 was blocked by treatments that deplete the cellular content of protein kinase C. Metabolic labeling and Western blot analysis demonstrate that TRH does not alter the total cellular content or nuclear concentration of Pit-1. TRH-mediated stimulation of a PRL promoter-luciferase fusion gene occurred under conditions that blocked the transient phosphorylation of Pit-1. These studies suggest that phosphorylation of Pit-1 may not be necessary for TRH mediated enhancement of PRL gene transcription.

INSIGHT: Pit-1/GHF-1: a pituitary-specific transcription factor linking general signaling pathways to cell-specific gene expression.

Identification and ordering of the functional cellular components involved in transmitting a hormone signal from the cell membrane to the nuclear compartment have progressed quite rapidly in recent years. The availability of defined gene fragments ligated to reporter constructs has allowed investigators to map the critical promoter regions that mediate a particular hormone response. Using such minimally delimited hormone-responsive DNA elements in a variety of biochemical and recombinant DNA approaches, such as gel shifts and DNA ligand screening, the putative nuclear protein target(s) of specific hormone signaling pathways have been identified and their cDNAs cloned. Indeed, precisely these approaches were used to identify the CAMP-responsive DNA element (CRE: TGACGTCA) and to clone the nuclear factor [CAMP response element binding protein (CREB)], which together mediate many of the transcriptional responses of the PKA signaling pathway (for review, see Refs. 1 and 2). Although most of the CAMPregulated genes that were initially characterized contain a CRE, it became clear that other DNA motifs and nuclear factors (e.g. AP-2) distinct from the CRE and CREB, respectively, could also mediate the transcriptional effects of the cAMP/PKA pathway (1, 2). As if to validate the complexity by which the cAMP/PKA signal may be transduced, studies of two ancestrally related pituitary-specific genes, PRL (PRL) and GH (GH), have consistently implicated DNA binding sites for the POUhomeodomain protein, Pit-l/GHF-1 , as mediating the CAMP response (3-12). The results of Kapiloff et al. (13) which documented that Pit-l/GHF-1 was phosphorylated in vivo at Ser 115 and Thr 220 in response to CAMP or phorbol ester (TPA) treatment of GC rat

A sequence in the rat Pit-1 gene promoter confers synergistic activation by glucocorticoids and protein kinase-C.

The 5'-flanking region of the gene for Pit-1, a pituitary-specific transcription factor, was isolated from a rat liver genomic library and sequenced. Expression of a reporter construct containing Pit-1 promoter sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene was assessed by transient transfection in rat pituitary GH4C1 cells. Treatment of transfected cells with either dexamethasone (DEX) for 48 h or the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) for the final 20 h of the 48-h posttransfection period had minimal effects on CAT expression. However, CAT activity was elevated about 20-fold when transfected cells were treated with both DEX and TPA. This apparent synergistic activation was lost when DEX treatment was also limited to the final 20 h of the 48-h posttransfection period, suggesting that a time-dependent accumulation of a DEX-induced gene product might be involved. This putative DEX-induced product appeared to be relatively stable, because synergistic activation was observed in cells treated with DEX alone for 36 h, followed by a 10-h incubation without DEX before the addition of TPA. The Pit-1 gene promoter region between -210 and -142 from the transcription start site conferred synergistic regulation by DEX and TPA when placed upstream of position -105 in the herpes viral thymidine kinase promoter.(ABSTRACT TRUNCATED AT 250 WORDS)

Pit-1 gene expression in the human pituitary and pituitary adenomas.

The cellular basis for pituitary neoplasia is poorly understood. The POU domain protein Pit-1 is a pituitary-specific transcription factor involved in the generation, differentiation, and proliferation of three pituitary cell types: lactotrophs, somatotrophs, and thyrotrophs. In this study, we analyzed the expression of Pit-1 gene in a series of 15 different human pituitary tumors and compared it with that observed in normal tissue. Pit-1 transcripts, identical in size (2.4 and 4.5 kilobases) and sequence to those observed in normal tissue were evidenced in PRL-, GH-, and TSH-secreting tumors. Pit-1 is overexpressed (2.5- to 5-fold) in the PRL- and GH-secreting tumors, but to an extent consistent with the predominant cellular type of these adenomas. An isoform of Pit-1, with an insertion of 26 amino acids in the trans-activation domain as a result of alternative splicing, is also present in both normal and tumoral tissues. It is concluded that human pituitary tumorigenesis does not seem to be associated with a gross alteration of Pit-1 gene expression.

An alternatively spliced form of Pit-1 represses prolactin gene expression.

The pituitary-specific transcription factor Pit-1 is required for expression of the PRL gene. Transcription of the PRL gene in the anterior pituitary is both activated and repressed in response to neuroendocrine signals. The molecular events that mediate repression are unknown. Transplantation of GH3 pituitary tumor cells from culture to female Wistar-Furth rats resulted in repression of PRL gene expression. When the transplanted cells were returned to culture, PRL gene expression was rapidly activated. We used this model to study potential mechanisms by which PRL gene expression was silenced. In addition to the appropriate size Pit-1 proteins of 33 and 31 kilodaltons, smaller forms of the transcription factor, migrating at approximately 27 and 24 kilodaltons, were found in transplanted cells in which PRL gene expression was repressed. These smaller forms of Pit-1 protein were observed to disappear when transplanted cells were returned to culture, coincident with the activation of PRL gene expression. A transcript approximately 170 base pairs shorter than expected for that encoding full-length Pit-1 was detected in transplanted GH3 cell RNA by polymerase chain reaction. The shorter Pit-1 transcript was abundant only in GH3 cells after in vivo passage and was not readily detected in transplanted cells after as little as 12 h in culture. This shorter transcript was found to result from excision of sequence corresponding to exon IV and encodes a Pit-1 protein lacking 54 amino acids of the POU-specific domain. Gene transfer studies demonstrated this alternative form of Pit-1 inhibited PRL promoter activity.(ABSTRACT TRUNCATED AT 250 WORDS)