C. Onofri

Intrapituitary Expression and Regulation of the gp130 Cytokine Interleukin-6 and Its Implication in Pituitary Physiology and Pathophysiology

Interleukin (IL)‐6, a member of the gp130 cytokine family, is sometimes designated as an “endocrine” cytokine because of its strong regulatory influence on hormone production. Systemically acting IL‐6 derived from immune cells is a potent stimulator of the hypothalamus–pituitary–adrenal axis and therefore plays an important role in modulating immune–neuroendocrine interactions during inflammatory or infectious processes. However, IL‐6 is also produced within the anterior pituitary by so‐called folliculostellate (FS) cells and is also synthesized in and released by tumor cells in pituitary adenomas. Growth factors (e.g., transforming growth factor‐beta), neuropeptides (e.g., pituitary adenylate cyclase‐activating polypeptide), or hormones (e.g., glucocorticoids) regulate IL‐6 production both in FS and pituitary tumor cells. Interestingly, components of the innate immune system, such as toll‐like receptor 4 and nucleotide‐binding oligomerization domains (NODs), are expressed in FS and pituitary tumor cells. Therefore, cell‐wall components of bacteria (lipopolysaccharide, muramyl dipeptide, diamino pimelic acid) stimulate IL‐6 production in normal and tumoral pituitary. The intrinsic IL‐6 production by FS cells in normal anterior pituitary may participate in immune–neuroendocrine interactions during inflammatory processes. In pituitary adenomas, IL‐6 stimulates hormone secretion, tumor cell proliferation, and the production of angiogenic factors, such as vascular endothelial growth factor‐A, suggesting an important role of IL‐6 in the pathophysiology and progression of pituitary adenomas.

Curcumin Inhibits the Growth, Induces Apoptosis and Modulates the Function of Pituitary Folliculostellate Cells

The polyphenol curcumin (diferuloylmethane) is the active componenet of the spice plant Curcuma longa and has been shown to exert multiple actions on mammalian cells. We have studied its effect on folliculostellate (FS) TtT/GF mouse pituitary cells, representative of a multifunctional, endocrine inactive cell type of the anterior pituitary. Proliferation of TtT/GF cells was inhibited by curcumin in a monolayer cell culture and in the colony formation assay in soft agar. Fluorescence-activated cell-sorting (FACS) analysis demonstrated curcumin-induced cell cycle arrest at G2/M accompanied by inhibition of cyclin D1 protein expression. Curcumin had a small effect on necrosis of TtT/GF cells, but it mainly stimulated apoptosis as demonstrated by FACS analysis (Annexin V-fluorescein isothiocyannate/7-aminoactinomycin D staining). Curcumin-induced apoptosis involved suppression of Bcl-2, stimulation of cleaved caspase-3 and induction of DNA fragmentation. Functional studies on FS cell-derived compounds showed that curcumin inhibited mRNA synthesis and release of angiogenic vascular endothelial growth factor-A (VEGF-A). Immune-like functions of FS cells were impaired since curcumin downregulated Toll-like receptor 4, reduced nuclear factor-ĸB expression and suppressed bacterial endotoxin-induced interleukin-6 (IL-6) secretion. The inhibitory action of curcumin on VEGF-A and IL-6 production was also found in primary rat pituitary cell cultures, in which FS cells are the only source of these proteins. The observed effects of curcumin on FS cell growth, apoptosis and functions may have therapeutic consequences for the intrapituitary regulation of hormone production and release as well as for pituitary tumor pathogenesis.

Platelet-derived Growth Factor (PDGF) and PDGF Receptor Expression and Function in Folliculostellate Pituitary Cells

Homo- and heterodimers of platelet-derived growth factor-A (PDGF-A) and PDGF-B chains are involved through PDGF alpha- and beta-receptors in the growth regulation of multiple normal and tumoural cell types as well as in tumour neovascularization. Since little information is available on the impact of PDGF/PDGF receptors in normal and adenomatous pituitary, we studied the expression and action of this growth factor system in a variety of pituitary tumour cell lines and in rat anterior pituitary cell cultures. By RT-PCR, mRNA expression of PDGF-A and -B chains and of both receptors was found in rat pituitary and mouse folliculostellate TtT/GF pituitary tumour cells. Rat somatotroph MtT-S and mouse corticotroph AtT20 tumor cells expressed only a part of the PDGF/PDGF receptor components whereas mouse gonadotroph alphaT3-1 and rat lactosomatotroph GH3 pituitary tumour cells contained neither PDGF nor PDGF receptors. To further characterize the role of PDGF in TtT/GF cells, the effect of PDGF-AB and -BB on growth and vascular endothelial growth factor-A (VEGF-A) release was studied. Proliferation of TtT/GF cells was weakly but significantly stimulated by PDGF. Both in rat pituitary cell cultures and in TtT/GF cells, PDGF-AB and -BB strongly enhanced VEGF-A secretion. The PI3 kinase inhibitor LY 294002 blocked the increase in VEGF-A. Western immunoblotting confirmed the participation of key components of the PI3 kinase/Akt signal pathway (PDK1, Akt-Ser476) in PDGF-stimulated VEGF production. Thus the PDGF/PDGF receptor system is expressed in folliculostellate cells and is involved in VEGF regulation. Its role in endocrine pituitary tumour cell lines and pituitary adenomas need to be clarified in future studies.

Immunohistochemical analysis of VEGF-C/VEGFR-3 system and lymphatic vessel extent in normal and adenomatous human pituitary tissues.

The angiogenic growth factor Vascular Endothelial Growth Factor-C (VEGF-C) and its receptor VEGFR-3 are also known to be implicated in the development of lymphatic vessels. We assessed the expression of VEGF-C and VEGFR-3, together with blood and lymphatic vessel extents and proliferation index (PI) values, by immunohistochemistry (IHC) in 6 normal human pituitary glands and 53 pituitary adenomas of different tumour grade, on consecutive tissue sections. VEGF-C was detected in around 10% of the endocrine cells in normal pituitary tissue, while this gland was devoid of lymphatic vascularization and showed very few vessels positive for VEGFR-3. Concerning tumour tissue, most of the adenomas showing VEGF-C immunoreactivity (21/47) were positive in 60% of the tumour cells and the ones positive for VEGFR-3 showed a number of immunostained vessels higher than those observed in the normal pituitary. Most of the tumours positive for VEGFR-3 did not show any LYVE-1 positive vessels (18/53), suggesting that at least in these cases, VEGFR-3 is expressed on blood vessels. Nevertheless, we observed a significant association between low expression of VEGFR-3 and low lymphatic vessel number, suggesting that VEGFR-3 might be involved in the starting of DE NOVO lymphangiogenesis in this tumour type. Moreover, tumours bearing lymphatic vessels showed the tendency to shift towards a more aggressive behaviour (high tumour grade and high PI). In conclusion, the VEGF-C/VEGFR-3 system might be involved in controlling tumour angiogenesis in the pituitary adenomas lacking lymphatic vessels, but may also play a role in starting the process of tumour lymphangiogenesis.

Expression of VEGF receptors in normal and adenomatous human pituitary

Vascular endothelial growth factor (VEGF) is a potent endothelial cell mitogen and exerts its action through different tyrosine kinase receptors: VEGFR-1 (Flt-1), VEGFR-2 (Flk-1/KDR) and neuropilin-1, a co-receptor of VEGFR-2, which are almost specifically expressed in endothelial cells and VEGFR-3 (Flt-4), which is detected mainly in lymphatic vessels. However, it has been shown that VEGF receptors may also be expressed by non-endothelial cells, especially by tumor cells. We have observed that pituitary adenoma cells produce variable amounts of VEGF and have studied the expression of VEGF receptors in normal human pituitary and pituitary adenomas by RT-PCR and immunohistochemistry. Protein expression of VEGFR-1, VEGFR-2, VEGFR-3 and neuropilin-1 was found in normal pituitary and in the pituitary adenomas studied so far. The expression was variable and no correlation between VEGFR-1 and VEGFR-2 expression and different parameters as tumor grade, vessels number and proliferation index (PI) was found. In normal pituitary, VEGFR-1 immunoreactivity was observed to co-localize with ACTH-, FSH-, GH- and LH- secreting cells, but not with endothelial cells, suggesting that VEGF may affect function of endocrine cell types by paracrine mechanisms. Moreover, VEGFR-1 immunostaining was observed mainly in pituitary adenoma cells. In contrast, VEGFR-2, VEGFR-3 and neuropilin-1 immunoreactivity was detected only in vascular formations, in both normal pituitary and pituitary adenomas and interestingly, we observed a correlation between neuropilin-1 expression and vessel count and proliferation index of the tumors analyzed. This finding could be consistent with the observation that neuropilin-1 acts as a co-receptor for VEGFR-2, enhancing the activity of VEGF. According on its receptors localization, VEGF seems to have a role both on tumor cells and on tumor vessels, even if not directly on vessel proliferation. Further studies are anyway necessary to better understand the possible connection between VEGF and its receptors and pituitary adenomas development.