Peroxisome proliferator activated receptor gamma in colonic epithelial cells protects against experimental inflammatory bowel disease.

INTRODUCTION: Peroxisome proliferator activated receptor gamma (PPARgamma) is expressed in epithelial cells, macrophage, and T and B lymphocytes. Ligand induced activation of PPARgamma was reported to attenuate colitis activity but it is not clear whether this protection is mediated by epithelial or leucocyte PPARgamma. METHODS: Mice with targeted disruption of the PPARgamma gene in intestinal epithelial cells, generated using a villin-Cre transgene and floxed PPARgamma allele and designated PPARgamma(DeltaIEpC), were compared with littermate mice having only the PPARgamma floxed allele with no Cre transgene that expressed PPARgamma in the gut, designated PPARgamma(F/F). Colitis was induced by administering dextran sodium sulphate (DSS) and the two mouse lines compared for typical symptoms of disease and expression of inflammatory cytokines. RESULTS: PPARgamma(DeltaIEpC) mice displayed reduced expression of the PPARgamma target genes ADRP and FABP in the gut but were otherwise normal. Increased susceptibility to DSS induced colitis, as defined by body weight loss, colon length, diarrhoea, bleeding score, and altered histology, was found in PPARgamma(DeltaIEpC) mice in comparison with PPARgamma(F/F) mice. Interleukin (IL)-6, IL-1beta, and tumour necrosis factor alpha mRNA levels in colons of PPARgamma(DeltaIEpC) mice treated with DSS were higher than in similarly treated PPARgamma(F/F) mice. The PPARgamma ligand rosiglitazone decreased the severity of DSS induced colitis and suppressed cytokine production in both PPARgamma(F/F) and PPARgamma(DeltaIEpC) mice. CONCLUSIONS: These studies reveal that PPARgamma expressed in the colonic epithelium has an endogenous role in protection against DSS induced colitis and that rosiglitazone may act through a PPARgamma independent pathway to suppress inflammation.

Exogenous expression of Pit-1 in AtT-20 corticotropic cells induces endogenous growth hormone gene transcription.

The pituitary-specific POU-homeodomain transcription factor, Pit-1, is known to regulate the expression of the GH gene in somatotropes, prolactin (PRL) in lactotropes, and TSH in thyrotropes. It is not normally expressed in corticotropes or gonadotropes. We addressed the question of whether exogenous Pit-1 was sufficient to induce ectopic transcription of the GH gene in the corticotropic cell line, AtT-20, or the gonadotropic cell line, alpha T3-1. A fusion gene composed of enhanced green fluorescent protein gene and human Pit-1 cDNA was transfected into AtT-20 and alpha T3-1 cells. The endogenous mouse GH mRNA was induced in three of nine AtT-20 cell lines and one of three alpha T3-1 cell lines containing the fusion gene. A small amount of GH protein was also detected in these cell lines. These data indicate that transfected Pit-1 is capable of inducing transcription of the GH gene in AtT-20 cells and alpha T3-1 cells. These data also suggest that synergistic co-factors might be required to transcribe the GH gene effectively for translation into GH protein. Furthermore, our findings support the hypothesis that the function of anterior pituitary cells is determined by the combinatorial action of specific transcription factors.

Immunohistochemical analysis of RCAS1 in human pituitary adenomas.

It has been reported that RCAS1 (receptor-binding cancer antigen expressed on SiSO cells) acts as a ligand for a receptor present on normal peripheral lymphocytes and induces apoptotic cell death. It is expressed in uterine and ovarian carcinomas, especially in invasive cancers. This immunohistochemical study is aimed to elucidate the expression of RCAS1 in human pituitary adenomas in order to clarify its role in their proliferative regulation and invasiveness. Five normal pituitary glands, 50 human pituitary adenomas, and one malignant glioma were subjected to immunohistochemical studies. In normal pituitary glands, immunostaining of RCAS1 and MIB-1 was not found. In malignant glioma, large numbers of cell nuclei were positive for MIB-1 (MIB-1 index: 28%), and RCAS1 was detected both in the cytoplasm and on the membrane of the tumor cells. Expression of RCAS1 was noted in 48% of pituitary adenomas immunohistochemically (60.0% of growth hormone-secreting adenomas, 60.0% of prolactin-secreting adenomas, 42.9% of adrenocorticotrophin-secreting adenomas, 40.0% of thyroid-stimulating hormone-secreting adenomas, 33.3% of nonfunctioning adenomas, and 44.4% of gonadotropin-subunit-positive adenomas). It showed no correlation with tumor type, size, and invasiveness. The statistically significant relationship between RCAS1 and MIB-1 positivity was identified in our study. These results suggest that expression of RCAS1 as well as MIB-1 positivity predict the growth potential of individual pituitary adenomas.

Expression of interleukin-6, interleukin-6 receptor (gp80), and the receptor's signal-transducing subunit (gp130) in human normal pituitary glands and pituitary adenomas.

Interleukin-6 (IL-6) is an important cytokine in cell proliferation and differentiation in several organs. It has also been reported that IL-6 plays a role in secretion or release of pituitary hormones in pituitary hormone-secreting cells and pituitary adenomas, but convincing data in situ have not yet been reported. In this study, we examined the participation of IL-6 in the production of pituitary hormones and the differences between human normal pituitary glands and pituitary adenomas by determination of the localization or expression of IL-6, IL-6 receptor (IL-6R, gp80), and the signal-transducing subunit (gp130) of the receptor using immunohistochemical staining and RT-PCR. IL-6 was mainly expressed in ACTH- and FSH/LH-secreting cells in normal pituitary glands, as shown by double staining. gp 80 and gp130 were coexpressed in almost all GH- and PRL-secreting cells and in approximately 30% of FSH/LH-secreting cells. RT-PCR showed that IL-6 mRNA was expressed in only one of all the pituitary adenomas examined, whereas gp 80 and gp 130 mRNAs were detected in all these pituitary adenomas. In conclusion, IL-6 was mainly expressed in ACTH- and FSH/LH-secreting cells, and the receptors were expressed in GH-, PRL- and FSH/LH-secreting cells in human normal pituitary glands. Furthermore, our data emphasized that the mechanism of IL-6 function in human pituitary adenoma cells is distinct from that in normal pituitary cells.

Expression of prolactin gene in spontaneous mammary tumors in aging Fischer 344 rats.

Expression of extra pituitary prolactin (PRL) has been recently reported in the mammary gland. However, spontaneous mammary tumors occurring in aging rats have not been investigated for PRL production. The present study was undertaken to examine the expression of PRL gene in rat mammary tumors spontaneously arisen in rats with pituitary prolactinomas among 130 female Fischer-344 (F-344) rats. The tumors examined were fibroadenoma (adenomatous type) in the 18-month old rat and adenocarcinoma (alveolar/tubular type) in the 21-month old rat. PRL mRNA was examined by solution and in situ reverse transcription-polymerase chain reaction (RT-PCR) method. The predicted amplified products for PRL mRNA were identified in both tumors, and its expression was confirmed to be in the cytoplasm of epithelial cells. The results of the present study showed that PRL gene is expressed in spontaneously arising mammary tumors.

Cytochemical and molecular biological aspects of the pituitary and pituitary adenomas--cell differentiation and transcription factors.

The anterior pituitary is composed of several cell types, each responsible for the production of specific hormones. Each hormone secreting cells is defined by the activation of its respective hormone genes in a temporally and spatially regulated manner. Recent development in cytochemistry and molecular biology have provided various aspects of human pituitary adenomas, i.e., functional differentiation and classification. The molecular factors that determine hormone production have now been identified as transcription factors. Many novel transcription factors that play a role in anterior pituitary development are implicated. In this review, we focus on the transcriptional factors roles on functional differentiation of the pituitary cells and adenomas and the contribution of cytochemistry and recent development in molecular biological techniques.

Cytological characterization of a pituitary folliculo-stellate-like cell line, Tpit/F1, with special reference to adenosine triphosphate-mediated neuronal nitric oxide synthase expression and nitric oxide secretion.

An immortal nonhormone-producing cell line with a characteristic star-shaped morphology, named Tpit/F1, was derived from an anterior pituitary gland of a temperature-sensitive large T antigen transgenic mouse. To characterize Tpit/F1 cells, we performed cytological studies, which revealed that Tpit/F1 cells express the messenger RNAs of neruonal nitric oxide (NO) synthase, S-100 protein, basic fibroblast growth factor, and pituitary-restricted transcription factor. The Tpit/F1 cells response to pituitary adenylate cyclase-activating peptide comprised the stimulated secretion of interleukin-6. Furthermore, glucocorticoids stimulate glutamine synthase production by Tpit/F1 cells. Considering these cytological characteristics together with their morphology, we deduced that Tpit/F1 cells are derived from pituitary folliculo-stellate (FS) cells. Our cytophysiological analyses of Tpit/F1 cells revealed that intracellular Ca2+ increased dose dependently on ATP administration (0-100 microM), and that this effect did not require the presence of extracellular Ca2+ and was not abolished by treatment with gadolinium, a Ca2+ channel blocker. The ATP-induced increase in intracellular Ca2+ ([Ca2+]i) was completely abolished by treatment with the Ca2+-adenosine triphosphatase (Ca2+-ATPase) inhibitor thapsigargin, which suggests that ATP increases [Ca2+]i by mobilizing internally stored Ca2+ followed by an influx of Ca2+. Moreover, UTP was equipotent with ATP in causing the [Ca2+]i increase in Tpit/F1 cells. Also, the Ca2+ response was prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. From these results we therefore concluded that ATP acts on Tpit/F1 cells via P2Y2-purinoceptors. Interestingly, both neuronal nitric oxide synthase messenger RNA and NO secretion were increased by ATP administration (10 and 100 microM). These results suggest the biological significance of the topological colocalization of FS cells and endocrine cells. Namely, ATP is cosecreted with hormones from endocrine cells and stimulates NO production by FS cells, and the released NO may regulate neighboring endocrine cell and blood vessels.

Expression of pituitary homeo box 1 (Ptx1) in human non-neoplastic pituitaries and pituitary adenomas.

We investigated the localization of pituitary homeo box 1 (Ptx1) protein in five human non-neoplastic pituitaries and 73 of all types of pituitary adenomas using immunohistochemistry, and the expression of Ptx1 messenger RNA (mRNA) in 18 representative pituitary adenomas using the reverse transcriptase polymerase chain reaction (RT-PCR) technique. By immunohistochemical analysis, Ptx1 protein was extensively detected in the nuclei of normal human pituitary cells. Ptx1 was detected in 10/14 (71.4%) of growth hormone (GH)-secreting adenomas, 12/12 (100%) of prolactin (PRL)-secreting adenomas, 18/20 (90%) of adrenocorticotropic hormone (ACTH)-secreting adenomas, 6/7 (85.7%) of thyroid-stimulating hormone (TSH)-secreting adenomas, and 17/20 (85%) of clinically non-functioning adenomas, including 9/10 (90%) of gonadotropin-subunit-positive adenomas. Thus, there was no relationship between Ptx1 expression and a particular type of pituitary adenomas. By RT-PCR analysis, Ptx1 mRNA was expressed in all 18 cases of pituitary adenomas, including two cases negative for Ptx1 protein by immunohistochemistry. These results suggested that Ptx1 may be an universal transcription factor in both neoplastic and non-neoplastic conditions in human pituitaries. The synergistic action with other transcription factors may be speculated to determine the specific production of the anterior pituitary hormones.

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.

Contributions of immunohistochemistry and in situ hybridization to the functional analysis of pituitary adenomas.

Immunohistochemistry (IHC) and recently in situ hybridization (ISH) have elucidated various aspects of human pituitary adenomas, i.e., functional differentiation and classification, transcription factors and mechanism of hormone production, regulation of hormone secretion, and processing of prohormones. Recently, the use of tyramide (catalyzed signal amplification; TSA or CSA) and RT-PCR has been effective for detection of trivial amount of proteins (peptides) and mRNA, respectively. Immunomolecular histochemistry is expected to further clarify the function and biology of human pituitary adenomas.

Expression of Ptx1 in the adult rat pituitary glands and pituitary cell lines: hormone-secreting cells and folliculo-stellate cells.

The pituitary homeobox1 gene (Ptx1) was initially identified as encoding a pituitary-restricted transcription factor for the proopiomelanocortin (POMC) gene. In order to elucidate the expression pattern of the Ptx1 protein, we investigated the localization of the protein in adult rat pituitary gland and in various pituitary cell lines. We produced an antibody specific for Ptxl protein, and confirmed its specificity by Western blot analysis. Immunohistochemically, many nuclei in the anterior pituitary cells as well as in the intermediate cells were positive for Ptxl staining with this specific antibody. Immunohistochemical double staining revealed the presence of Ptx1 not only in all types of hormone-secreting cells but also in some folliculo-stellate (FS) cells. Furthermore, the expression of Ptx1 mRNA was confirmed in various pituitary cell lines and in the FS cell line by using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Our studies indicated that Ptxl may not only play a role as a basic transcriptional factor for production of various hormones, but may also play some important role(s) in FS cells. Possible synergistic actions with other factors remain to be investigated. The novel finding of Ptx1 in FS cells is of particular interest, and may suggest that FS cells and hormone-secreting cells are derived from a common cellular ancestor.

Pit-1 positive alpha-subunit positive nonfunctioning human pituitary adenomas: a dedifferentiated GH cell lineage?

Pit-1 is a transcription factor which has been reported to regulate differentiation toward GH, PRL and TSH in the anterior pituitary glands. In the human pituitary adenomas, Pit-1 is highly expressed in GH secreting and TSH secreting adenomas as it can well be anticipated. Interestingly, human non-functioning pituitary adenomas also express Pit-1, especially it was expressed in all alpha SU positive nonfunctioning adenomas. The human anterior pituitary cells are special in comparison with rodents in a finding that alpha SU is frequently colocalized with GH. As alpha SU is the first hormone appearing during fetal development in the rodent pituitary glands, it may be postulated that alpha SU Pit-1 positive cells undergo differentiation in the GH cell lineage. From this background, this paper proposes that "alpha SU positive Pit-1 Positive" cells are the ones in the GH cell lineage, more specifically a dedifferentiated cell lineage toward alpha SU/GH/Pit-1.