The present studies demonstrate for the first time in vivo antagonism between a collaborative oncogene, cyclin D1, and a nuclear receptor, PPARγ. Cyclin D1 inhibited PPARγ-dependent reporter activity and repressed the trans
activity of PPARγ when linked to a heterologous DNA binding domain. Consistent with previous findings that PPARγ expression is induced by PPARγ (12
), cyclin D1 also inhibited PPARγ expression and promoter activity. The reduction in cyclin D1 abundance in cyclin D1−/−
3T3 cells, cyclin D1−/−
MEFs, and cyclin D1 antisense transgenic mice correlated with the induction of PPARγ expression (Fig. ). The induction of adipocyte differentiation by PPARγ-specific ligands was substantially enhanced in cyclin D1
-deficient cells. The reintroduction of cyclin D1 into cyclin D1-deficient cells abolished the adipogenic phenotype, consistent with a key role for cyclin D1 as an inhibitor of PPARγ-specific functional activity. Since cyclin D1 abundance is regulated by diverse oncogenic and mitogenic stimuli, the inhibition of PPARγ transactivation by cyclin D1 may have important implications for signal transduction and tumorigenesis.
Repression of PPARγ transactivation by cyclin D1 was independent of the cdk and pRb binding functions and required a C-terminal region of cyclin D1 that is predicted to form a helix-loop-helix structure. The mechanism by which cyclin D1 regulated PPARγ activity is thus distinct from that regulating the ERα through recruiting a p160 coactivator SRC-1 in vitro (66
), as an SRC-1 binding point mutation of cyclin D1 maintained wt repression of liganded PPARγ activity. Herein, genetic deletion of cyclin D1
enhanced PPARγ ligand-mediated differentiation of MEFs into adipocytes. Although interactions between cyclin D1 and nuclear receptors have been previously described, the present studies provide strong genetic evidence for a functional interaction between cyclin D1 and a nuclear receptor. Ectopic expression of PPARγ induced differentiation of NIH-3T3 fibroblast cells into fat-laden adipocyte cells (51
). In the present studies, the cyclin D1−/−
MEF adipogenic phenotype induced by PPARγ ligands was reversed by cyclin D1 overexpression. ACRP30 was increased 50-fold in the cyclin D1−/−
MEFs, consistent with the enhanced induction of the adipogenic phenotype. The relatively modest increase in PPARγ abundance, together with the dramatic enhancement of PPARγ activity in the cyclin D1−/−
MEFs, is consistent with the reporter gene studies in which cyclin D1 inhibited PPARγ trans
These studies raise the possibility that reduced PPARγ expression, together with increased cyclin D1, may be a genetic feature of the transition from normal breast epithelium, to benign breast disease and adenocarcinoma. PPARγ immunopositivity was decreased in benign breast disease compared with normal mammary epithelium and was reduced further in adenocarcinomas. Cyclin D1 immunopositivity increased from normal epithelium to benign disease and adenocarcinomas. The reduction in PPARγ expression in the cyclin D1-infected MEFs, together with the finding of increased levels of PPARγ mRNA and protein in cyclin D1−/− livers by microarray (not shown) and Western blotting, suggest that cyclin D1 inhibits PPARγ expression. The overexpression of cyclin D1 with ERα reflects poor prognosis in human breast cancer. Given the repression of PPARγ function and expression by cyclin D1 and the cytoinhibitory role of PPARγ in breast epithelium, these studies raise the question of whether reduced PPARγ may contribute to poor prognosis in a subset of patients. The reduction in PPARγ staining in proliferative breast disease suggests that further studies of PPARγ as a prognostic indicator and candidate target for prevention or therapy of human breast cancer warrants consideration.
The present studies are important in demonstrating a functional antagonism between a collaborative oncogene, cyclin D1, and a candidate tumor suppressor, PPARγ. Several lines of evidence suggest that PPARγ may function as a tumor suppressor (42
). Consistent with a role for PPARγ as an inhibitor of tumorigenesis, heterozygous mutations of PPARγ were detected in 4 of 55 patients with colon cancer (45
). In follicular thyroid cancer, a fusion oncoprotein has been described, formed by a chromosomal translocation between PPARγ1 and PAX8 with a deletion in its C-terminal activation domain. The PPARγ fusion protein functioned as a powerful dominant-negative of wt PPARγ and was not observed in benign follicular adenomas (24
). The addition of PPARγ ligands (TZD or 15d-PGJ2
) inhibited breast and colonic cellular proliferation (7
). In contrast, cyclin D1 abundance is induced by diverse oncogenic and mitogenic signals in breast and colonic epithelial cells and functions as a collaborative oncogene (18
). Cyclin D1 antisense inhibits the growth of murine mammary tumors derived from MMTV-ErbB2 mice (25
), and cyclin D1−/−
mice are resistant to the induction of tumor formation by ErbB2 (64
). Since PPARγ inhibits the expression of several genes promoting tumor invasion (those encoding iNOS, gelatinase B, matrix metalloproteinases [10
], and UPA) (data not shown), cyclin D1 antagonism of PPARγ function may enhance expression of tumor invasion genes.
The functional antagonism between PPARγ and cyclin D1 may also have implications for signal transduction cross talk. Cyclin D1 is induced by diverse mitogenic signaling pathways, including those of Src, Rac mutants, Dbl proteins, and β-catenin, and the NF-κB signaling pathway (1
). PPARγ activity is also induced by a large number of synthetic and natural ligands, including prostaglandins and fatty acids (42
). The inhibition of PPARγ function by cyclin D1 may contribute to altered metabolism and altered inflammatory responses and remains to be further explored.