Pit-1 Protein Research Articles

Pit-1 is expressed in normal and tumorous human breast and regulates GH secretion and cell proliferation

The transcription factor pituitary-1 (Pit-1) is mainly expressed in the pituitary gland, where it has critical roles in cell differentiation and as a transcriptional factor for GH and prolactin (PRL). It is also expressed in human extrapituitary tissues (placenta, lymphoid and haematopoietic tissues) and cell lines (human breast adenocarcinoma cells, MCF-7). Despite the widely suggested roles of GH and PRL in the progression of proliferative mammary disorders, Pit-1 expression in human mammary gland has not yet been reported. To evaluate the expression of Pit-1 in human breast and, using the MCF-7 cell line, to investigate whether Pit-1 overexpression regulates GH expression and increases cell proliferation. Using real-time RT-PCR, western blotting and immunohistochemistry, we evaluated the expression of Pit-1 mRNA and protein in seven normal human breasts and 14 invasive ductal mammary carcinomas. GH regulation by Pit-1 in MCF-7 cells was evaluated using RT-PCR, western blotting, ELISA and transfection assays. Cell proliferation was evaluated using bromodeoxyuridine. We found expression of Pit-1 mRNA and protein in both normal and tumorous human breast. We also found that Pit-1 mRNA levels were significantly increased in breast carcinoma compared with normal breast. In MCF-7 cells, Pit-1 overexpression increased GH mRNA and protein concentrations and significantly increased cell proliferation. These findings indicate that Pit-1 is expressed in human breast, that it regulates endogenous human mammary GH secretion, and that it increases cell proliferation. This suggests that, depending on its level of expression, Pit-1 may be involved in normal mammary development, breast disorders, or both.

Association of a PIT1 gene polymorphism with growth hormone mRNA levels in pig pituitary glands

Fourty-six non-castrated, halothane-free, male Landrace pigs were genotyped by PCR-RFLP for the Rsa I polymorphism in the PIT1 gene and classified into AA and AB genotypes. Total RNA was extracted from the pituitaries and the relative quantities of growth hormone (GH) mRNA were determined by semi-quantitative RT-PCR. Pigs with the AB genotype had higher levels of GH mRNA than those with the AA genotype (p = 0.034; Kruskal-Wallis test). This result suggests that the Rsa I polymorphism may be involved in Pit-1 protein expression or function, which in turn may influence GH transcription and expression. Thus, the Rsa I PIT1 gene polymorphism in this pig line may be used as a molecular marker to identify higher GH expression and possibly select for carcass and performance traits affected by GH.

Multistep differentiation of GH-producing cells from their immature cells

In order to study GH cell differentiation, we used the clonal cell lines called MtT/E and MtT/S cells, which were derived from a rat mammotrophic pituitary tumor. Although MtT/E cells are non-hormone-producing ones, Pit-1 protein is present in their nuclei, which suggests that MtT/E cells are progenitor cells of the Pit-1 cell lineage and have the potential to differentiate into hormone-producing cells. On the other hand, MtT/S cells produce GH; however, the responsiveness to GH-releasing hormone (GHRH) is weak and only a small number of secretory granules are present in their cytoplasm, which suggests that MtT/S cells are premature GH cells. In order to differentiate into GH cells from MtT/E cells as a progenitor cell, we examined several differentiation factors and found that retinoic acid (RA) induced the differentiation of MtT/E cells into GH-producing cells. RA-induced GH cells partially matured with the glucocorticoid treatment; however, the responsiveness to GHRH on GH secretion was incomplete. In order to elucidate the mechanism underlying full differentiation of GH cells, we used MtT/S cells. We treated MtT/S cells with glucocorticoid and found that they differentiated into mature GH cells with many secretory granules in their cytoplasm and they responded well to GHRH. These results suggested that MtT/E and MtT/S cells are progenitor or premature GH cells, and show different responses to differentiation factors. Our data also suggested that GH cells differentiate from their progenitor cells through multistep processes.

Serine/threonine/tyrosine phosphorylation of the LHX3 LIM‐homeodomain transcription factor

LHX3 is a LIM homeodomain transcription factor with essential roles in pituitary and motor neuron development in mammals. Patients with mutations in the LHX3 gene have combined pituitary hormone deficiency and other symptoms. In this study, we show that the LHX3 protein can be modified post-translationally by phosphorylation. LHX3 can serve as a substrate for protein kinase C and casein kinase II. Overexpression of these kinases reduces the transcriptional capacity of LHX3 to activate target genes. Following exposure of LHX3 to cellular kinases, mass spectrometry was used to map the phosphorylation of five amino acid residues within the human LHX3a isoform. Two phosphorylated residues (threonine 63 and serine 71) lie within the first LIM domain of the protein. Three other modified amino acids (tyrosine 227, serine 234, and serine 238) are located in the carboxyl terminus. Targeted replacement of these amino acids with non-modifiable residues significantly reduced the ability of LHX3 to activate both synthetic and pituitary hormone reporter genes. However, the amino acid replacements did not significantly affect the capability of LHX3 to interact with the NLI, PIT1, and MRG1 partner proteins, or its ability to bind to a high affinity DNA site. In conclusion, we have identified unique amino acids within LHX3 that are important for its transcriptional activity and are phosphorylated.

Phosphate uptake and PiT-1 protein expression in rat skeletal muscle.

Skeletal muscle fiber types differ in their contents of total phosphate, which includes inorganic phosphate (P(i)) and high-energy organic pools of ATP and phosphocreatine (PCr). At steady state, uptake of P(i) into the cell must equal the rate of efflux, which is expected to be a function of intracellular P(i) concentration. We measured (32)P-labeled P(i) uptake rates in different muscle fiber types to determine whether they are proportional to cellular P(i) content. P(i) uptake rates in isolated, perfused rat hindlimb muscles were linear over time and highest in soleus (2.42 +/- 0.17 micromol x g(-1) x h(-1)), lower in red gastrocnemius (1.31 +/- 0.11 micromol x g(-1) x h(-1)), and lowest in white gastrocnemius (0.49 +/- 0.06 micromol x g(-1) x h(-1)). Reasonably similar rates were obtained in vivo. P(i) uptake rates at plasma P(i) concentrations of 0.3-1.7 mM confirm that the P(i) uptake process is nearly saturated at normal plasma P(i) levels. P(i) uptake rate correlated with cellular P(i) content (r = 0.99) but varied inversely with total phosphate content. Sodium-phosphate cotransporter (PiT-1) protein expression in soleus and red gastrocnemius were similar to each other and seven- to eightfold greater than PiT-1 expression in white gastrocnemius. That the PiT-1 expression pattern did not match the pattern of P(i) uptake across fiber types implies that other factors are involved in regulating P(i) uptake in skeletal muscle. Furthermore, fractional turnover of the cellular P(i) pool (0.67, 0.57, and 0.33 h(-1) in soleus, red gastrocnemius, and white gastrocnemius, respectively) varies among fiber types, indicating differential management of intracellular P(i), likely due to differences in resistance to P(i) efflux from the fiber.

Glucocorticoid induction of lactotrophs and prolactin gene expression in chicken embryonic pituitary cells: a delayed response relative to stimulated growth hormone production.

We reported that corticosterone (CORT) induces GH cell differentiation in chicken embryonic pituitary cells in culture and in living embryos. The present study tested whether CORT could also induce prolactin (PRL) cell differentiation in vitro. CORT increased the percentage of GH cells in cultures of embryonic d (e) 13 pituitary cells after 1 d of treatment and PRL cells after 3 d of incubation. Dual immunofluorescence showed that the PRL cells induced by CORT did not contain GH, indicating they were separate cell populations. Similar PRL cell responses were also observed in cultures of e11, e15, and e17 pituitary cells. ZK98299, a glucocorticoid receptor (GR) antagonist, suppressed CORT effects, verifying involvement of GR. Northern blot analysis indicated that CORT increased GH mRNA levels after 1 d of treatment. In contrast, increases in PRL mRNA levels were delayed and observed after 3 d of treatment. Induction of a luciferase reporter driven by the PRL promoter was also delayed until 3 d of CORT treatment. Dual-labeling immunofluorescence indicated that the majority of PRL cells induced by CORT were not labeled with bromodeoxyuridine, suggesting that lactotrophs induced by CORT do not result from cell proliferation. Proportions of pituitary-specific transcription factor (Pit-1)-containing cells and the total amount of Pit-1 protein spontaneously increased with increasing culture time. However, no effect of CORT on Pit-1 levels or the number of Pit-1-containing cells were observed. We conclude that CORT can induce lactotroph differentiation in culture and that longer CORT exposure is needed for lactotroph induction compared with somatotroph induction. The effects of CORT on PRL cell induction are GR dependent and involve an increase in PRL gene expression. PRL cell induction by CORT is not associated with an increase in Pit-1-containing cells.

Immunocytochemical identification of Pit-1 containing cells in the anterior pituitary of hens.

Our goal was to identify the cells expressing Pit-1 protein in chicken anterior pituitary. The anterior pituitaries were collected from laying hens after perfusion with formalin-PBS, and fixed with Bouin's fixative followed by paraffin embedding. Sections of the anterior pituitaries were immunostained for Pit-1 in the first staining sequence followed by staining for 6 types of pituitary hormones in the second sequence. Pit-1 positive nuclei were observed in the glandular cells in both the cephalic and caudal lobes. Pit-1 immunoreaction products were colocalized in the glandular cells immunopositive for growth hormone, thyroid-stimulating hormone, follicle-stimulating hormone, luteinizing hormone, adrenocorticotropic hormone or prolactin. These results indicate that Pit-1 protein induction occurs in 6 types of glandular cells, suggesting that Pit-1 may regulate hormone synthesis in each glandular cell in the chicken pituitary.

Change in expression of basic fibroblast growth factor mRNA in a pituitary tumor clonal cell line.

To study pituitary tumor formation, we used a rat pituitary tumor cell line, MtT/E, which was derived from an estrogen-induced rat prolactinoma. MtT/E cells are known not to produce any pituitary hormone; however, they do produce the Pit-1 protein, which is known to be a common transcription factor in thyrotropes, somatotropes, and mammotropes. Although MtT/E is a clonal cell line, it exhibits two distinct phenotypes, fibroblastic (F-) and epithelial (E-) cells. We obtained subclonal cell lines from MtT/E cells with characters similar to those of F- and E-cells and called them MtT/E-G1 and MtT/E-B3, respectively. To examine tumor formation by these cells, we implanted them into female Fischer rats. One month later, typical pituitary tumors had appeared in MtT/E-B3-implanted rats; however, tumor formation by MtT/E-G1 was delayed. Interestingly, the tumors formed by MtT/E-B3 cells were intensely vascularized. To examine changes in tumor cell morphology, we performed primary culture and found that spindle-shaped cells appeared. These spindle-shaped cells were immunopositive for the Pit-1 protein, which suggests that they originated from MtT/E-B3 cells. Interestingly, reverse transcriptase polymerase chain reaction showed that both tumors and the cells obtained in primary culture expressed basic fibroblast growth factor (bFGF). By contrast, the original MtT/E-B3 cells did not express bFGF. These results suggested that MtT/E-B3 cells show a change in phenotype during tumor formation; that is, epithelial-type cells change into bFGFexpressing fibroblastic cells. These phenomena, especially the appearance of bFGFexpressing cells in tumor tissue, may explain the extensive angiogenesis in the tumors formed by MtT/E-B3 cells.

Selective repression of rat prolactin gene by stable expression of dominant-negative Ets in GH4 pituitary cells.

Members of the Ets family of transcription factors are key regulators controlling prolactin (PRL) gene expression. Utilizing a transient transfection approach and the GH4 rat pituitary cell line, we have shown that Ets- 1 acts synergistically with the pituitary-specific POU homeodomain transcription factor, Pit-1, to mediate basal and Ras-induced regulation of the proximal (-425) rat PRL (rPRL) promoter. Although the transient transfection approach has provided important information regarding rPRL proximal promoter regulation, the role of Ets factors in the regulation of the intact, endogenous PRL promoter has not been explored. To address this area of question, we created several clonal GH4 cell lines that stably express either dominant-negative Ets (dn-EtsZ) or dominant-active Ets (VP16 Ets) constructs and used these cell lines as a model system to analyze the role of Ets factors on endogenous PRL gene expression. Northern blot analysis of these cells showed that PRL mRNA levels were dramatically reduced, by an average of 80%, in the cell lines expressing dn-Ets compared to vector-only controls. Conversely, stable expression of the dominant-active VP16 Ets led to an average threefold increase in PRL mRNA. GH4 cells expressing dn-EtsZ displayed significantly lower levels of intracellular PRL protein content and greatly diminished secretion of PRL into the cell culture medium, compared to vector-only controls. Consistent with our previous observations, the mRNA levels for growth hormone were unaffected by either dn-EtsZ or VP16 Ets expression. Expression of dn-EtsZ reduced Pit-1 mRNA levels by about 30%; however, the intracellular levels of Pit-1 protein were unchanged. Taken together, these results verify and strengthen the view that Ets factors play a critical role in the regulation of endogenous PRL gene expression and PRL protein production.

IFN-γ Increases the hGH Gene Promoter Activity in Rat GH3 Cells

Aim: To study the effect(s) of interferon gamma (IFN-γ) on the activity of human growth hormone (hGH) gene promoter in rat pituitary GH3 cells and the molecular mechanism underlying the effect(s). Methods: Cell transfection and luciferase reporter gene were used. Results: IFN-γ (10² and 10³ U/ml) increased the activity of hGH in GH3 cells. The addition of the mitogen-activated protein kinase inhibitor PD98059 (40 µmol/l) to the cells blocked the stimulatory effect of IFN-γ. Neither overexpression of Pit-1 nor inhibiting Pit-1 expression affected IFN-γ induction of hGH promoter activity. To identify the DNA sequence that mediated the effect of IFN-γ, four deletion constructs of hGH gene promoter were created. The stimulatory effect of IFN-γ was abolished following deletion of the –250 to –132 fragment. Conclusions: IFN-γ increases the activity of hGH gene promoter in rat pituitary GH3 cells. This stimulatory effect of IFN-γ appears to require the intracellular mitogen-activated protein kinase-dependent signaling pathway. The effect of IFN-γ requires the promoter sequence that spans the –250 to –132 fragment of the gene, but is unrelated to Pit-1 protein.

Reassessing the role of region A in Pit1-mediated viral entry.

The mammalian gammaretroviruses gibbon ape leukemia virus (GALV) and feline leukemia virus subgroup B (FeLV-B) can use the same receptor, Pit1, to infect human cells. A highly polymorphic nine-residue sequence within Pit1, designated region A, has been proposed as the virus binding site, because mutations in this region abolish Pit1-mediated cellular infection by GALV and FeLV-B. However, a direct correlation between region A mutations deleterious for infection and loss of virus binding has not been established. We report that cells expressing a Pit1 protein harboring mutations in region A that abolish receptor function retain the ability to bind virus, indicating that Pit1 region A is not the virus binding site. Furthermore, we have now identified a second region in Pit1, comprising residues 232 to 260 (region B), that is required for both viral entry and virus binding. Epitope-tagged Pit1 proteins were used to demonstrate that mutations in region B result in improper orientation of Pit1 in the cell membrane. Compensatory mutations in region A can restore proper orientation and full receptor function to these region B mutants. Based on these results, we propose that region A of Pit1 confers competence for viral entry by influencing the topology of the authentic binding site in the membrane and hence its accessibility to a viral envelope protein. Based on glycosylation studies and results obtained by using N- and C-terminal epitope-tagged Pit1, region A and region B mutants, and the transmembrane helices predicted with the PHD PredictProtein algorithm, we propose a new Pit1 topology model.

Characterization of transactivation domain and developmental expression of pituitary specific transcription factor, Pit-1 of ayu ( Plecoglossus altivelis)

Pit-1 is a pituitary-specific transcription factor, which regulates the expression of growth hormone, prolactin, and thyroid stimulating hormone-β genes. We previously reported the expression of a Pit-1 gene from ayu ( Plecoglossus altivelis), which is an important cultivated food fish in Taiwan and Japan. Comparison of ayu Pit-1 with that of salmon, turkey, and rodent, revealed that the Pit-1 structure is highly conserved through vertebrates, especially in POU-specific and POU-homeo domains. The variation among fish, bird, and mammal are mainly found in transactivation domain by alternative splicing and initiation. Three insertions were found. The γ-insert in fish Pit-1 is homologous to the exon 2a of avian Pit-1, which is not found in mammals. The β-insert of fish Pit-1 is homologous to the 28 amino acids (a.a.) and 26 a.a. insert of avian Pit- 1 β ∗ and mammalian Pit-1β, respectively. An additional similarity was noticed between fish and bird, as both of them contain 7 a.a. insert that is not present in mammalian Pit-1. By site directed mutagenesis, we demonstrated that the β, γ, and the 7 a.a. inserts of ayu Pit-1 are critical for activation of zebrafish growth hormone promoter. The ayu Pit-1 protein was found to be expressed specifically in pituitary gland, and its mRNA was first detected at embryonic day 4, significantly increased at embryonic day 5, then sustained to time of hatching at day 8.

Differential conservation of transcriptional domains of mammalian Prophet of Pit-1 proteins revealed by structural studies of the bovine gene and comparative functional analysis of the protein

The Prophet of Pit-1 (PROP1) gene encodes a paired class homeodomain transcription factor that is exclusively expressed in the developing mammalian pituitary gland. PROP1 function is essential for anterior pituitary organogenesis, and heritable mutations in the gene are associated with combined pituitary hormone deficiency in human patients and animals. By cloning the bovine PROP1 gene and by comparative analysis, we demonstrate that the homeodomains and carboxyl termini of mammalian PROP1 proteins are highly conserved while the amino termini are diverged. Whereas the carboxyl termini of the human and bovine PROP1 proteins contain potent transcriptional activation domains, the amino termini and homeodomains have repressive activities. The bovine PROP1 gene has four exons and three introns and maps to a region of chromosome seven carrying a quantitative trait locus affecting ovulation rate. Two alleles of the bovine gene were found that encode distinct protein products with different DNA binding and transcriptional activities. These experiments demonstrate that mammalian PROP1 genes encode proteins with complex regulatory capacities and that modest changes in protein sequence can significantly alter the activity of this pituitary developmental transcription factor.

Pit-1/GHF-1 and GH expression in the MCF-7 human breast adenocarcinoma cell line.

GH expression in mammary tumors has been related to the increase and spreading of cell proliferation. Using the MCF-7 human breast adenocarcinoma cell line, it has been demonstrated that autocrine GH-stimulated mammary carcinoma cell proliferation decreased the apoptosis rate and enhanced cell spreading. Surprisingly, no data are available about the presence of Pit-1 (the main pituitary regulator of GH) or GH expression in this cell line. Using RT-PCR, Western blot and immunohistochemistry, we have demonstrated the presence of both mRNA coding Pit-1 and GH as well as Pit-1 and GH protein in the MCF-7 cell line. These data could imply that Pit-1 may be an adequate target to inhibit breast cell proliferation.

Expression of GATA-2 in Human Pituitary Adenomas

Transcription factor GATA-2 contains two copies of a highly conserved zinc finger domain and plays unique roles at an early stage of hematopoietic differentiation. In the mouse pituitary gland, Pit-1-GATA-2 protein–protein interaction has been shown to lead to gene-specific actions to obtain cell-specific roles. In this study, we investigated the expression of GATA-2 and Pit-1 in human pituitary adenomas using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical techniques. By immunohistochemical analysis, GATA-2 was detected in all of the gonadotropin-subunit (Gn-su)-positive adenomas (n = 8) and in four of five thyroid-stimulating hormone (TSH)-secreting adenomas, but its incidence was low in the other types of adenomas. Pit-1 protein was detected in 4 of 5 TSH-secreting adenomas and in 10 of 10 growth hormone (GH)-secreting adenomas. By RT-PCR analysis, GATA-2 was detected in all Gn-su-positive adenomas and TSH-secreting adenomas, and Pit-1 was detected in all TSH-secreting adenomas and GH-secreting adenomas. These results suggested that GATA-2 contributes to the functional expression and the differentiation of Gn-su-positive adenomas and the TSH-secreting adenomas and that the interaction between GATA-2 and Pit-1 can lead to gene-specific action and differentiation of TSH-secreting adenomas. It is further speculated that GATA-2 and transcriptional interaction with Pit-1 play roles in the functional differentiation of specific pituitary adenomas.

CLONING OF THE FULL LENGTH PIG PIT1 (POU1F1) CDNA AND A NOVEL ALTERNATIVE PIT1 TRANSCRIPT, AND FUNCTIONAL STUDIES OF THEIR ENCODED PROTEINS

PIT1 is an essential regulatory gene of growth hormone (GH), prolactin (PRL) and thyrotropin β subunit (TSHβ). Previously, a partial pig PIT1 cDNA and a genomic clone of the entire 3′ end of the PIT1 gene was isolated, and polymorphisms at PIT1 were associated with several performance traits in the pig. In order to understand the biological function of the pig PIT1 gene and its possible application in swine genetics, reverse transcriptase-polymerase chain reaction (RT-PCR) was used to complete the cloning of the full length cDNA for pig PIT1. The pig PIT1 cDNA and its deduced protein sequence have approximately 90% and 95% identity, respectively, with the PIT1 cDNA and protein of other mammals (human, bovine, sheep and rodents). Surprisingly, sequence comparison to other pig PIT1 sequences indicated only approximately 93% identity. Additional sequencing confirmed our sequence, and identified a new polymorphism in exon 4. Phylogenetic analysis of several mammalian PIT1 sequences indicates sequencing errors may account for the discrepancies observed in the other pig sequences reported. Several PIT1 alternative spliced forms were also identified by RT-PCR. They were the Δ3PIT1 (missing entire exon 3), Δ4PIT1 (missing entire exon 4) and PIT1β (additional 26 amino acids inserted in front of exon 2) transcripts. The Δ4PIT1 and PIT1β transcripts have been found to encode functionally different proteins in rodents. The Δ3PIT1 transcript is a novel isoform of PIT1. Potentially different functions between pig Δ3PIT1 and PIT1 were analyzed by expressing these proteins in bacteria. The E. coli-expressed PIT1 and Δ3PIT1 proteins were used with rat growth hormone (rGH) and rat prolactin (rPRL) promoter DNA in DNA mobility shift assays. The results showed that pig PIT1 can specifically bind rGH and rPRL promoter regions, but that the pig Δ3PIT1 cannot, even at very high protein concentrations. Possible protein-protein interactions between Δ3PIT1 and PIT1 were tested by mixing protein extracts before the gel shift assay, and the results showed that Δ3PIT1 protein did not affect PIT1 binding to its target DNA. These data demonstrate the functionality of the PIT1 cDNA cloned in this study, and identify a novel Δ3PIT1 transcript which encodes a protein that cannot bind rGH/rPRL target sequences.

Molecular Cloning of the Chicken Prolactin Gene and Activation by Pit-1 and cAMP-Induced Factor in GH3 Cells

Transcription of the prolactin (PRL) gene has been reported to be activated by a nuclear factor, Pit-1. However, the precise molecular mechanisms of the Pit-1-mediated PRL gene activation are still unclear. We have cloned the chicken PRL (cPRL) gene and its 5′-flanking region to analyze their structure and transcription-initiating mechanism. In luciferase assay, forskolin activated the proximal promoter region between −248 and −76 to transcribe the cPRL gene in GH3 cells, although there is no canonical cyclic AMP-responsive element in the promoter region. In gel mobility shift assay, a DNA fragment between −104 and −76 containing a putative Pit-1 binding site was bound by nuclear factors from the GH3 cells. Furthermore, it was observed that Pit-1 protein specifically bound to the DNA fragment in the supershift assay. These results indicate that both Pit-1 and cAMP-induced factor(s) associated with the cis element on the proximal promoter region to activate cPRL gene expression in GH3 cells.

Estrogen-modulated Estrogen Receptor·Pit-1 Protein Complex Formation and Prolactin Gene Activation Require Novel Protein Synthesis

Both estrogen receptor (ER) and Pit-1 proteins are essential for the estrogen-activated expression of the rat prolactin gene. Our results show that ER.Pit-1 protein complex formation is reduced by estrogen in GH3 and PR1 rat pituitary tumor cells. In the latter, this decrease was blocked by cycloheximide, a protein synthesis inhibitor. On the other hand, the direct addition of estrogen to PR1 cell lysates had no effect on the formation of ER.Pit-1 complexes. Estrogen-activated prolactin gene expression was also inhibited by cycloheximide, suggesting that some form of protein synthesis is involved in ER.Pit-1 complex formation and subsequent prolactin gene activation. In support of this notion, we showed that estrogen-induced regulation of ER.Pit-1 complex formation could be transferred from cell lysates prepared from estrogen-treated PR1 cells to control cell lysates. This is not true for GH3 cells; instead, direct administration of estrogen to GH3 cell lysates readily abolished ER.Pit-1 protein complex formation in a dose-dependent manner, and such estrogen-induced regulation was blocked by the antiestrogen ICI 182,780. These findings thus indicate that 1) interaction between ER and Pit-1 proteins is estrogen-regulated in ways specific to different cell types, and 2) auxiliary protein factor synthesis may be involved in this process.

Immunoreactive pit-1 protein in hyperplastic pars intermedia induced by calcitonin of the rat pituitary gland.

To elucidate the effects of synthetic salmon calcitonin (sCT) on the cells in the rat pituitary gland, we histopathologically and immunohistochemically examined the early changes after 4 or 13 weeks treatment with sCT 120 IU/kg. Focal proliferative lesions of the anterior pituitary glands were consistently found after treatment with sCT for 13 weeks. Histologically, the cells with the focal proliferative lesions were classified into the following three groups: 1) enlarged basophilic cell focus, 2) vacuolated cell focus and 3) chromophobe cell focus. These focal proliferative lesions had positive staining only for the alpha-subunit and failed to show Pit-1 protein immunoreactivity. The sCT treatment also increased the thickness of the pars intermedia. Hypertrophy of the pars intermediate cells was characteristically seen. Furthermore, Pit-1 protein immunoreactivity was clearly detected in the nuclei of the hyperplastic pars intermediate cells. All pars intermediate cells were equally stained by alpha- or beta-MSH and beta-endorphin in both vehicle- and sCT-treatment. No difference was seen. These findings strongly suggest a very close relationship between Pit-1 protein immunoreactivity and cellular proliferation induced by sCT.

Retinoid X receptors (RXRs) mRNA expression in human pituitary adenomas

Retinoid-X receptors (RXRs) are transcriptional factors that belong to the steroid/thyroid hormone receptor (TR) superfamily. It has been demonstrated that those nuclear receptors act as ligand-activated transcription factors in pituitary cells. To determine whether RXRs play roles in the cell differentiation of pituitary adenomas, we have investigated the expression of RXRγ mRNA in various types of pituitary adenomas usingin situ reverse transcriptase-polymerase chain reaction (RT-PCR). The synergistic function on promoters of specific hormones between these nuclear receptors and pituitary specific transcription factor, Pit-1, has been noticed in in vitro experiments. The colocalization between RXRγ mRNA and Pit-1 protein was examined by combinedin situ RT-PCR and immunohistochemistry. RXRγ mRNA was detected in normal pituitary gland as well as all five growth hormone-(GH)-secreting adenomas and five thyroid stimulating hormone (TSH) secreting adenomas, two of four prolactin- (PRL) secreting adenomas, one of two adrenocorticotropin-(ACTH) secreting adenomas, one of four nonfunctioning adenomas. Byin situ hybridization andin situ RT-PCR followed by immunohistochemistry, the colocalization of Pit-1 mRNA with RXRγ as well as RXRγ mRNA with Pit-1 was observed in adenoma cells of GH-secreting adenomas and TSH-secreting adenomas. We suggest that RXRγ may play a role in cell differentiation and hormonal transcription synergistically with Pit-1 in normal and neoplastic human pituitaries.