PPAR-y Expression in Pituitary Tumours and the Functional Activity of the Glitazones

It has been reported that both normal pituitary and pituitary tumours express PPAR-γ, a nuclear hormone receptor, the expression being more abundant in pituitary tumours, and that this is the basis for the reported antiproliferative effects of the thiazolidinedione, rosiglitazone, in animal models. However, the mechanisms for the responsivity to rosiglitazone have remained unclear.

To investigate this further, 'real-time' PCR was used to assess PPAR-γ mRNA expression, and Western blotting and immunohistochemistry to study its protein expression, in 46 human pituitary tumours and normal pituitary tissue. Cell proliferation of the GH3 pituitary cell line was assessed by [³H]-thymidine-incorporation after 48 h rosiglitazone and pioglitazone (10^-4 m- 10^-10 m) treatment alone, or rosiglitazone in combination with the PPAR-γ antagonist GW9662.

PPAR-γ mRNA and protein was found to be expressed in normal pituitary and was variably expressed in pituitary tumours, but were increased specifically in nonfunctioning pituitary adenomas. However, very little staining was observed with immunohistochemistry, with only occasional cell nuclei stained, and no difference was detectable between controls and tumours. Rosiglitazone at 10^-4 m and 10^-5 m concentrations inhibited cell proliferation (10^-4 m 14·0% ± 1·5% and 10^-5 m 67% ± 4% [mean ± SEM] vs Control 100% ± 3%, P < 0·0001) while lower concentrations showed no significant effect. Following withdrawal of rosiglitazone 10^-5 m, the cells fully recovered at a further 48 h, while lower doses showed a 'rebound' of stimulation. Pioglitazone was of similar potency to rosiglitazone in inhibiting proliferation. The PPAR-γ antagonist did not show a significant reversal of the antiproliferative effect of rosiglitazone, and indeed suppressed proliferation on its own.

Peroxisome proliferator activated receptor-gamma (PPAR-γ) belongs to the family of nuclear hormone receptors and is related to thyroid hormone, retinoic acid, androgen and oestrogen receptors. PPAR-γ is expressed abundantly in adipose tissue, the colon and haemopoetic cells, moderately in kidney, liver and small intestine, and in small amounts in skeletal muscle. PPAR-γ was originally described as playing a primary role in adipocyte differentiation, but more recent studies have implicated it in processes of inflammation, atherosclerosis, cell cycle control, apoptosis and carcinogenesis. It acts as a transcription factor when heterodimerized with the retinoid X receptor (RXR) and ligand-bound: natural ligands include prostaglandin-J2 and polyunsaturated fatty acids, while synthetic ligands include thiazolidinedione (TZD) and nonsteroidal anti-inflammatory drugs.

Some studies have implicated PPAR-γ in the regulation of cell proliferation. PPAR-ligand activation has been shown to have an antiproliferative effect in a variety of cancers, including thyroid, pancreatic, breast, prostate and testicular cancer, and it is assumed that these operate via the PPAR-γ receptor. Mutations of PPAR-γ have been found in colon cancer,while chromosomal translocation of PPAR-γ and the PAX8 gene induces follicular thyroid carcinoma. Mice which are heterozygous for PPAR-γ loss have increased rates of colon, ovarian and mammary cancer. However, the evidence is not conclusive regarding PPAR-γ's role in tumorigenesis. Mice which are PPAR-γ^-/- in their mammary epithelium do not have an increased incidence of mammary tumours. Furthermore, some studies have shown that PPAR-γ can actually stimulate tumour formation: min mice, which are a model for familial adenomatous polyposis, show increased levels of polyposis when treated with TZDs.

Recently, PPAR-γ protein expression was shown to occur in corticotroph, and subsequently other types, of pituitary tumour to a greater extent than in the normal pituitary, while the administration of the TZD rosiglitazone caused changes in cell-cycle and apoptotic proteins compatible with an antiproliferative effect; this was also shown to occur in vivo. We have now sought to investigate whether the increased expression of PPAR-γ occurs at the level of mRNA, to explore whether the proposed antiproliferative effect of rosiglitazone on the somato-mammotroph rat pituitary tumour cell line GH3 can be shown in terms of tritiated thymidine incorporation, and finally to determine whether any effects of rosiglitazone are indeed likely to be mediated by the PPAR-γ receptor.