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.

Are folliculo-stellate cells in the anterior pituitary gland supportive cells or organ-specific stem cells?

Folliculo-stellate cells (FS-cells) in the anterior pituitary gland are star-shaped cells and form tiny follicles. FS-cells are positive for S-100 protein and produce many cytokines or growth factors, such as interleukin-6 (IL-6), leukemia inhibitory factor (LIF), basic fibroblastic growth factor (bFGF) and vascular endothelial cell growth factor (VEGF). Therefore, it is generally accepted that FS-cells regulate endocrine cells through these growth factors. FS-cells also exhibit a phagocytotic activity and are known to work as scavenger cells. In addition to these functions, FS-cells are considered to have some unknown functions. In order to reveal the biological significance of FS-cells in the anterior pituitary gland, we performed a morphological study and obtained some new findings. First, we were interested in the colloid formation in the senescent porcine pituitary gland. We analyzed the colloids and found that clusterin is a major protein in them. We also found that the accumulation of clusterin in the colloids is related to the phagocytotic activity of FS-cells. In our next study, we found that FS-cells have the potential to differentiate into striated muscle cells. From FS-cells show multi-potent cell character and other cytological evidence, we propose that FS-cells are candidate of organ-specific stem cells in the anterior pituitary gland.

Leukaemia inhibitory factor and interleukin 6 inhibit secretion of prolactin and growth hormone by rat pituitary MtT/SM cells.

The rat pituitary cell line, MtT/SM, has the characteristics of somatomammotrophs. The cells secrete both prolactin (PRL) and growth hormone (GH). We examined the effects of cytokines such as leukaemia inhibitory factor (LIF), interleukin 6 (IL-6), oncostatin M and interleukin 11 on the secretion of these hormones by the cells. These cytokines stimulate proliferation of the cells and inhibit the secretion of PRL by 70-80% and that of GH by 50%. They induce tyrosine phosphorylation of STAT3 in the cells. The cells containing PRL or GH decreased at 48 h after treatment of the cells with LIF or IL-6. These results suggest that the LIF/IL-6 family of cytokines inhibits the functions of mammotrophs and somatotrophs in the pituitary gland.

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.