Regulation of estrogen receptor mRNA in normal and neoplastic rat pituitary tissues

The effects of estradiol 17β (E₂) on the regulation of estrogen receptor (ER) mRNA amounts in normal and neoplastic rat pituitary tissues were analyzed by in situ hybridization with an oligonucleotide probe. E₂ treatment produced a significant increase in ER mRNA amounts in two transplantable pituitary tumors (MtT/Wl5 and MtT/F4) and in the GH₃ cell line. ER mRNA amounts were also increased In normal pituitaries after 6 weeks of E₂ treatment, but the differences were not significant. Biochemical assay of ER proteins confirmed the presence of ER protein in MtT/Wl 5 and MtT/F4 tumors. “Cytoplasmic” ER proteins were decreased by E₂ in the MtT/Wl5 tumor.These results indicate that ER mRNA is present in normal pituitaries and in pituitary tumors and can be regulated by estrogen treatment in vivo and in vitro. The inhibitory effects of high estrogen concentration on proliferation of transplantable pituitary tumors in vivo and GH₃ cells in vitro is not explained by the absence of ER mRNAs in these tumors.     

TGF-B and Estrogen Regulate P27(Kip1) and Cyclin D(1) in Normal and Neoplastic Rat Pituitary Cells

Pituitary hyperplasia and tumor growth are regulated by various hormones and growth factors. Estrogen (E₂) stimulates pituitary cell proliferation and prolactin (PRL) production. Estrogen also regulates transforming growth factor-β (TGF-β) effects in the pituitary. TGF-β in turn regulates various cell cycle proteins including p15 and p27^Kip1 (p27). To better understand the regulatory role of growth factors and hormones in the cell cycle we analyzed cyclin D₁, cyclin E, and p27 expression in normal and neoplastic rat pituitary cells. An in vitro analysis using cultured normal pituitary cells and GH₃ tumor cells and an in vivo analysis of estrogen-treated normal pituitary and implanted GH₃ cells were performed. Semiquantitative RT-PCR was used to analyze mRNA expression for cyclin D₁, cyclin E, and p27 in cultured pituitary cells and E₂-treated pituitaries in vivo. Cyclin D₁ and p27 were localized in the nuclei of normal pituitary cells by immunocytochemistry (ICC). Very weak or absent immunostaining for cyclin D₁ and p27 was present in GH₃ cells. Both normal pituitary and GH₃ cells had strong nuclear staining for cyclin E. Normal pituitary had a 20-fold greater amount of cyclin D₁ mRNA and a 3-fold greater amount of p27 mRNA compared to GH₃ cells, whereas GH₃ cells had slightly (1.5-fold) more cyclin E than normal pituitary cells. E₂ treatment in vivo stimulated cell proliferation and decreased cyclin D₁ mRNA levels in normal pituitary. GH₃ tumor cells, implanted subcutaneously in the same animal, showed increased proliferation after E₂ treatment, but there was no change in cyclin D₁ mRNA in GH₃ cells. Cyclin E and p27 mRNA levels did not change significantly in normal pituitary or in GH₃ cells after E₂ treatment in vivo. Treatment of normal pituitary cells with 10⁻⁹ M TGF-β1 for 3 d in vitro led to significant decreases in cyclin D₁ and p27 mRNAs (p < 0.05), whereas cyclin E levels were unchanged. These results indicate that cyclin D₁ and p27 mRNAs are present at significantly higher levels in normal pituitary compared to GH₃ cells, and that both E₂ and TGF-β1 can downregulate cyclin D₁ mRNA levels in normal pituitary cells, suggesting that these factors regulate G1 to S phase transition in pituitary cells. The lower levels of specific cell cycle regulators in GH₃ cells may explain the decreased regulatory control by E₂ in GH₃ tumor cells.     

Detection of prolactin messenger RNA in rat anterior pituitary by in situ hybridization.

The effects of chronic diethylstilbestrol treatment on rat prolactin mRNA was analyzed by in situ hybridization histochemistry. Forty-day-old female rats were treated with 10 mg diethylstilbestrol in Silastic tubes for 3, 6, and 9 weeks. Estrogen treatment for 9 weeks increased pituitary wet weight (51.6 +/- 2.4 versus 7.9 +/- 0.31 mg for controls), serum prolactin (4155 +/- 571 versus 47.1 +/- 8.9 ng/ml for controls), and the percentage of immunoreactive prolactin cells (69% +/- 3% versus 34% +/- 2% for controls). In situ hybridization studies showed an increase in rat prolactin mRNA with increasing duration of estrogen treatment. After 9 weeks of estrogen treatment, there was a 2.3-fold increase in rat prolactin mRNA. 3H-cDNA was distributed diffusely throughout the anterior pituitary in both normal and hyperplastic pituitaries. There were no separate foci of adenomatous pituitary with increased labeling or with increased immunoreactive PRL cells. Although transplantable pituitary MtT/W15 tumors secreted very large amounts of PRL, compared with pituitaries from DES-treated rats, rat prolactin mRNA as evaluated by mean grain counts was considerably less in the MtT/W15 tumor than in DES-treated pituitary cells. These results show that in situ hybridization histochemistry can be used to detect changes in rat prolactin mRNA in tissue sections from the anterior pituitary with chronic estrogen treatment and that these pituitaries show a diffuse increase in immunoreactive prolactin cells and cellular prolactin mRNA, rather than distinct adenomatous areas within the glands.     

Regulation of prolactin gene expression in a DMBA-estrogen-induced transplantable rat pituitary tumor.

A new transplantable rat pituitary tumor was induced in F344 female rats with dimethylbenz(a)anthracene and estrogen (MtT/F-DMBA) and studied for 20 serial transplant generations. The tumor grew without estrogen supplements in female rats by the second transplant generation. Sensitivity to estrogens, as indicated by a prolonged latency period for tumor development, was seen at the 20th, but not the 5th transplant generation. MtT/F-DMBA tumors expressed prolactin (PRL), growth hormone (GH), and adrenocorticotropin (ACTH) mRNAs. A decrease in the percentage of cells expressing PRL mRNA, PRL protein, and in the number of secretory granules per cell occurred with serial transplantation. S-100 protein-positive folliculostellate cells were present in the hyperplastic pituitary but not in the transplantable tumors. Estrogen treatment at the 20th transplant generation prolonged the tumor latency period, increased the number of cells expressing PRL mRNA greater than 5-fold by in situ hybridization analysis (14 +/- 2% versus 77 +/- 5%), increased PRL secretion (132 +/- 40 ng/ml versus 3762 +/- 890 ng/ml), and increased the number of cytoplasmic secretory granules per cell. These results indicate that hyperplastic pituitary and true pituitary neoplasms differ in their ability to grow readily after transplantation. The presence of S-100 protein-positive folliculostellate cells, which are present in hyperplastic but not in neoplastic pituitary tissues, may serve as a morphologic marker to separate hyperplastic and neoplastic rat pituitary tissues. Transplantable tumors remained responsive to estrogen with expression of a more differentiated phenotype, including an increased number of cells expressing PRL mRNA and increased numbers of PRL secretory granules.     

The effects of estrogens on tumor growth and on prolactin and growth hormone mRNA expression in rat pituitary tissues.

Estrogens inhibit tumor growth and modify PRL and GH expression in the MtT/W15 transplantable rat pituitary tumor. The effects of estradiol (E2) and diethylstilbestrol (DES) on PRL and GH mRNA levels were investigated. Estrogens increased GH mRNA levels and decreased PRL mRNA levels as detected by in situ hybridization and Northern blot hybridization with oligonucleotide probes, while inhibiting tumor growth. Similar changes in immunoreactive GH and PRL were seen in the tumor cells. The pituitary glands of tumor-bearing rats treated with estrogen for 3 weeks were increased in weight with a concurrent increase in pituitary PRL mRNA when analyzed by dot blot hybridization. These results indicate that estrogens have an inhibitory effect on the growth of the MtT/W15 tumor and increase GH protein and mRNA levels, while causing PRL protein and mRNA levels to decrease. The pituitaries of tumor-bearing rats concomitantly undergo PRL cell hyperplasia with an increase in PRL mRNA. These results also demonstrate a paradoxical effect of estrogens on different pituitary tissues.     

The effects of estrogen on prolactin gene methylation in normal and neoplastic rat pituitary tissues.

The effects of estrogen treatment on rat prolactin (PRL) gene methylation were analyzed in normal pituitaries and in three transplantable rat pituitary tumors. Northern analysis showed increased PRL mRNA expression in estrogen-treated pituitary and in MtT/F4 and MtT/F-DMBA tumors. Prolactin mRNA amounts in MtT/W15 tumor were decreased by estrogen treatment. There was an inverse relationship between changes in PRL mRNA expression and changes in gene methylation in the coding regions of the PRL gene after estrogen treatment. The amounts of the 4.6-Kb and 1.8-Kb restriction fragments generated by HpaII digestion in pituitary tissues were influenced by estrogen, with an increase in these fragments in normal pituitary, MtT/F4, and MtT/F-DMBA tumors after estrogen treatment. In contrast, the amounts of the 4.6-Kb and 1.8-Kb fragments were decreased in MtT/W15 tumors after estrogen treatment. Most of the internal -CCGG- sites in the PRL gene were methylated in the liver, and the PRL gene was not expressed in the liver. These data suggest that there is a tissue-specific pattern of DNA methylation of the PRL gene and that PRL gene methylation is influenced by estrogen in vivo in normal and tumorous pituitary tissues. These results also suggest that estrogen may influence PRL expression by multiple mechanisms, including changes in the level of DNA methylation.     

Human growth hormone and prolactin secreting pituitary adenomas analyzed by in situ hybridization

Acidophilic pituitary adenomas commonly produce growth hormone (GH) or prolactin (PRL), according to studies employing immunohistochemical and ultrastructural methods. To examine this question, in situ hybridization with oligonucleotide probes was done on routinely processed tissues received in the pathology laboratory to analyze for the presence of GH and PRL messenger RNA (mRNA) in 4 normal pituitaries, 10 prolactinomas, and 16 GH-secreting adenomas. Most acidophilic cells in normal pituitaries expressed either GH or PRL hormone and the respective mRNAs, but GH mRNA and PRL hormone were also detected in some of the same cells. Patients with a clinical diagnosis of prolactinoma had cells with only PRL mRNA in their tumors, while most (14 of 16) patients with a clinical diagnosis of acromegaly or gigantism had both GH and PRL mRNAs in their tumors. The GH adenomas varied in these studies. In situ hybridization was helpful in characterizing the adenoma from a patient with acromegaly who had immunoreactive PRL, but no immunoreactive GH in the resected tumor; in situ hybridization analysis revealed mRNAs for both GH and PRL in the same tumor cells. Our findings indicate that pituitary adenomas from patients with acromegaly commonly express PRL mRNA. It is concluded that in situ hybridization provides new information about the clinical biology and the histopathologic classification of pituitary adenomas.     

Evidence That Folliculo-Stellate Cells Mediate the Inhibitory Effect of Japanese Kampo Medicine,Unkei-To,on Growth Hormone Secretion in Rat Anterior Pituitary Cell Cultures

PROBLEM: Cytokine-induced neutrophil chemoattractant (CINC) was reported to influence anterior pituitary hormone release. We recently found that Unkei-to, one of the Japanese Kampo medicines, stimulated CINC secretion by rat anterior pituitary cells and the pituitary folliculo-stellate (FS)-like cell line (TtT/GF). Therefore, the effect of Unkei-to on growth hormone (GH) secretion by rat anterior pituitary cells was investigated. METHOD OF STUDY: Dispersed normal anterior pituitary cells, the folliculo-stellate-like cell line TtT/GF, and the GH₃ cell were used to test the effect of Unkei-to on GH secretion. In reconstitutive coculture experiments, TtT/GF cells were mixed with GH₃ cells at a ratio (TtT/GF cells: GH₃ cells) of 1:99. From this mixture, cells were seeded onto plates at a density of 10⁴ cells/well and were cultured for 5 days. The cells were then used in the experiments. RESULTS: Unkei-to at 20 μg/ml significantly inhibited GH secretion by normal anterior ptuitary cells within 12 hr of incubation. In contrast Unkei-to stimulated GH secretion by GH₃ cells in a time- and dose-dependent manner, suggesting that an accessory cell type was involved. To assess the contribution of CINC as a paracrine factor, an experiment using a reconstitutive coculture system was performed, and Unkei-to was found to inhibit GH secretion when GH₃ cells were cocultured with TtT/GF cells. The addition of anti-CINC antibody to the reconstitutive coculture system antagonized Unkei-to-inhibited GH secretion. CONCLUSION: CINC, which was secreted from FS cells by Unkei-to, may be responsible for mediating the inhibitory effect of Unkei-to on GH secretion by rat anterior pituitary cells.     

Effects of estrogens on pituitary cell and pituitary tumor growth

Estrogens have been known to induce PRL cell hyperplasia in the anterior pituitary of some species for many decades. Recent studies have shown variable susceptibility to estrogen-induced hyperplasia in different strains of rats. The distinction between hyperplastic pituitaries and adenomas is usually not made by most investigators in this field, although true neoplasms can usually be propagated by serial transplantation. The growth of transplantable tumors is usually inhibited by estrogen in vivo. Estrogens have a biphasic effect on pituitary cell proliferation in vitro with higher concentrations of estradiol inhibit cell growth, and lower concentrations stimulating PRL secretion. Estrogens can regulate PRL gene methylation in vivo thus affecting PRL mRNA expression. Recent studies have suggested that estrogen regulates signal transduction by stimulating protein kinase C. Estrogens also regulate specific proto-oncogenes such as c-myc and c-fos. These observations may help to explain some of the regulatory effects of estrogens on cell proliferation and tumor development.     

DNA Methylation Regulates p27Kip1 Expression in Rodent Pituitary Cell Lines

We previously reported loss of expression of p27^Kip1 (p27) protein in rat GH₃ and mouse GHRH-CL1 pituitary tumor cells compared with normal pituitary (NP). The molecular basis for the loss of expression of p27 protein in GH₃ and GHRH-CL1 cells is unknown. To determine the role of p27 gene methylation in the regulation of the expression of this cell cycle protein, the methylation patterns of p27 in normal and neoplastic pituitary cells was analyzed. Inhibition of DNA methyltransferase (DNA-MTase) with 5-aza-2′-deoxycytidine (AZAdC) induced expression of both p27 protein and mRNA when GH₃ and GHRH-CL1 cells were treated for 7 days in vitro. DNA methylation correlated inversely with the expression of p27 gene products in NP and pituitary tumor cell lines. Bisulfite genomic sequencing analysis showed that the normally unmethylated cytosines in exon 1 in NP and AtT20 cells were extensively methylated in GH₃ and GHRH-CL1 cells. After treatment of GH₃ and GHRH-CL1 cells with 10 μmol/L AZAdC, there were decreased numbers of methylated cytosines (by 60% to 90%) with variable methylation patterns observed by bisulfite genomic sequencing. Analysis of genomic DNA with methylation-sensitive enzymes showed that all SmaI, HhaI, and AvaI enzyme sites of the p27 gene in exon 1 were methylated in GH₃ cells but not in NP, confirming the bisulfite genomic sequencing results. AtT20 cells and a human pituitary null cell adenoma cell line (HP75), which expressed abundant p27, had a methylation pattern similar to the NP. DNA-MTase activity was elevated fourfold in GH₃ cells and twofold in GHRH-CL1 cells compared with DNA-MTase activity in NP and AtT20 cells. These results suggest that increased DNA methylation is another mechanism of silencing of the p27 gene in some pituitary tumors and possibly in other types of neoplasms.     

Neuropeptides of anterior pituitary origin. Autocrine or paracrine functions?

Several neuropeptides classically associated with the hypothalamus have been found in the anterior pituitary. The question arises whether they are locally synthesized and if they play a paracrine or autocrine role on pituitary cell functions. Using normal and tumoral human pituitaries we found neuropeptides (TRH, SRIH, GHRH) and dopamine in variable quantities according to the nature of the tissue. They were all present in normal pituitaries, while stimulatory hormones (TRH and GHRH) were predominantly found in tumoral tissue, implying an imbalance of pathophysiological importance between the stimulatory and inhibitory control of hypophyseal hormones (PRL and GH) in pituitary adenomas. Both normal and tumoral pituitaries released TRH, SRIH and GHRH in large amounts suggesting their local synthesis. The in situ synthesis was demonstrated for SRIH by the evidence of SRIH mRNA, the detection of SRIH immunoreactivity in peculiar cells and the presence of SRIH precursor. The possible role of these pituitary neuropeptides was suggested for instance by the negative correlation found in vitro between SRIH and GH secretions. Moreover, neuropeptides could interact with each other. Indeed DA stimulated TRH release while PRL secretion decrease at the same time. Pulses of TRH had differential effects on SRIH release according to the nature of the tissue as TRH inhibited SRIH release from adenoma while it stimulated SRIH release from normal pituitary. Concerning the effects of SRIH and GHRH on GH secretion, there was an endogenous regulatory pattern comparable to that observed in rat portal blood vessels. Pulses of GHRH induced GH secretion only when endogenous SRIH release was not stimulated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coexpression of galanin and adrenocorticotropic hormone in human pituitary and pituitary adenomas.

Galanin is a neuropeptide that regulates the secretion of several pituitary hormones, including prolactin (PRL) and growth hormone (GH). Galaninlike immunoreactivity (Gal-IR) and galanin mRNA in the rat anterior pituitary is cell lineage specific, with predominant expression in lactotrophs and somatotrophs. The authors examined the cellular distribution of human Gal-IR in seven normal postmortem pituitaries and 62 pituitary tumors by immunoperoxidase staining. In contrast to the rat, Gal-IR in human anterior pituitaries was present in corticotrophs scattered throughout the gland, but not in lactotrophs, somatotrophs, thyrotrophs, or gonadotrophs. Distinct Gal-IR also was present in hyperplastic and neoplastic corticotrophs in 19 of 22 patients with Cushing's disease. In noncorticotroph cell tumors, unequivocal Gal-IR was present in 5 of 11 GH-secreting tumors associated with clinical acromegaly, 9 of 18 nonfunctioning pituitary adenomas, and 2 of 14 prolactinomas. Of these galanin-positive tumors, four of the five GH-secreting adenomas, six of the nine nonfunctioning adenomas, and both of the prolactinomas also contained adrenocorticotropic hormone immunoreactivity (ACTH-IR). Immunostaining and in situ hybridization on adjacent sections using an 35S-labeled probe complementary to human galanin mRNA demonstrated predominant galanin expression in normal corticotrophs. Immunoelectron microscopy confirmed the presence of Gal-IR in pituitary cells characteristic of corticotrophs in both normal and neoplastic pituitaries. Thus, as in the rat, galanin gene expression in the human pituitary is cell-type specific. Unlike the rat, however, human galanin gene expression is restricted to the corticotroph lineage. Studies of tumors confirmed the observed coexpression of galanin and adrenocorticotropic hormone. The divergent cell type specificity of galanin production in human and rat pituitaries reflects different patterns of gene activation in these two species. In addition, these results suggest that galanin in the human pituitary may participate locally in the regulation of the hypothalamic-pituitary-adrenal axis.     

Percoll density gradient-enriched populations of rat pituitary cells: Interleukin 6 secretion, proliferative activity, and nitric oxide synthase expression

We used a discontinuous Percoll density gradient centrifugation to prepare enriched populations of prolactin (PRL), growth hormone (GH), and folliculo-stellate (FS) cells from rat anterior pituitaries in order to characterize these various cell populations. After cell dissociation and centrifugation, enriched PRL cells (55% of total cells as determined by immunocytochemistry [ICC]) were present in Fraction 1 (Fr1) (density ([d])=1.059). Fr2 (d=1.071) had enriched S100-positive FS cells (31% of total cells), but enriched GH cell (60% of total cells) were present in Fr3 (d=1.094). Interleukin 6 (IL-6) was secreted mainly by enriched PRL cells in Fr1 (350 pg/mL/10⁶ cells) and Fr2 (194 pg/mL/10⁶ cells), and much less by the enriched GH cells in Fr3 (16 pg/mL/10⁶). Proliferation studies with combined³H-thymidine and ICC for pituitary hormones showed that only the PRL cell had significant proliferative activity. Immunostaining showed that immediately after separation, all three isoforms of nitric oxide synthase (NOS) were expressed in anterior pituitary cells. After 3 d of culture, there was a marked increase in nuclear staining for neuronal NOS (nNOS) in all three fractions, whereas inducible NOS (iNOS) and endothelial NOS (eNOS) expression did not change significantly. These results indicate that:

Effects of propylthiouracil on growth hormone and prolactin messenger ribonucleic acids in the rat pituitary

The effects of hypothyroidism induced by propylthiouracil (PTU) treatment on growth hormone (GH) and prolactin (PRL) messenger ribonucleic acid (mRNA) levels were analyzed in adult female rat adenohypophyses by in situ hybridization histochemistry and Northern hybridization analyses. Twenty-eight days of PTU treatment produced a significant decrease in GH mRNA levels and a smaller decrease in PRL mRNA determined by both in situ hybridization histochemistry and Northern hybridization analyses. A combined procedure of in situ hybridization histochemistry followed by immunochemistry on the same sections revealed mammosomatotropic cells expressing GH mRNA and PRL protein in the same pituitary cells from all treatment groups. Cells expressing GH mRNA and thyroid-stimulating hormone protein were not detected by this method. Immunochemical staining revealed a decrease in GH cells and an increase in thyroid-stimulating hormone cells in hypothyroid rats. Cells expressing both GH and thyroid-stimulating hormone protein were not detected by immunostaining. These results indicate that hypothyroidism produces significant decreases in GH mRNA and also decreases PRL mRNA and that mammosomatotropic cells can be detected in pituitaries from normal and hypothyroid rats.