SummaryNuclear receptor signaling plays an important role in energy metabolism. In this study we demonstrate that the nuclear receptor corepressor RIP140 is a key regulator of metabolism in skeletal muscle. RIP140 is expressed in a fiber type-specific manner, and manipulation of its levels in null, heterozygous, and transgenic mice demonstrate that low levels promote while increased expression suppresses the formation of oxidative fibers. Expression profiling reveals global changes in the expression of genes implicated in both myofiber phenotype and metabolic functions. Genes involved in fatty-acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis are upregulated in the absence of RIP140. Analysis of cultured myofibers demonstrates that the changes in expression are intrinsic to muscle cells and that nuclear receptor-regulated genes are direct targets for repression by RIP140. Therefore RIP140 is an important signaling factor in the regulation of skeletal muscle function and physiology.

WISP-2/CCN5 is an estrogen-regulated member of the "connective tissue growth factor/cysteine-rich 61/ nephroblastoma overexpressed" (CCN) family of the cell growth and differentiation regulators. The WISP-2/ CCN5 mRNA transcript is undetectable in normal human mammary cells, as well as in highly aggressive breast cancer cell lines, in contrast with its higher level in the breast cancer cell lines characterized by a more differentiated phenotype. We report here that knockdown of WISP-2/CCN5 by RNA interference in estrogen receptor alpha (ER␣)-positive MCF-7 breast cancer cells induced an estradiol-independent growth linked to a loss of ER␣ expression and promoted epithelial-to-mesenchymal transdifferentiation. In contrast, forced expression of WISP-2/CCN5 directed MCF-7 cells toward a more differentiated phenotype. When introduced into the poorly differentiated, estrogen-independent, and invasive MDA-MB-231 breast cancer cells, WISP-2/ CCN5 was able to reduce their proliferative and invasive phenotypes. In a series of ER␣-positive tumor biopsies, we found a positive correlation between the expression of WISP-2/CCN5 and ID2, a transcriptional regulator of differentiation in normal and transformed breast cells. We propose that WISP-2/CCN5 is an important regulator involved in the maintenance of a differentiated phenotype in breast tumor epithelial cells and may play a role in tumor cell invasion and metastasis.

Estrogen receptors (ER) are ligand-dependent transcription factors that regulate growth, differentiation, and maintenance of cellular functions in a wide variety of tissues. We report here that p21 WAF1/CIP1 , a cyclin-dependent kinase (Cdk) inhibitor, cooperates with CBP to regulate the ER␣-mediated transcription of endogenous target genes in a promoter-specific manner. The estrogen-induced expression of the progesterone receptor and WISP-2 mRNA transcripts in MCF-7 cells was enhanced by p21 WAF1/CIP1 , whereas that of the cyclin D1 mRNA was reduced and the pS2 mRNA was not affected. Chromatin immunoprecipitation assays revealed that p21 WAF1/CIP1 was recruited simultaneously with ER␣ and CBP to the endogenous progesterone receptor gene promoter in an estrogen-dependent manner. Experiments in which the p21 WAF1/CIP1 protein was knocked down by RNA interference showed that the induction of the expression of the gene encoding the progesterone receptor required p21 WAF1/CIP1 , in contrast with that of the cyclin D1 and pS2 genes. p21 WAF1/CIP1induced not only cell cycle arrest in breast cancer cells but also milk fat globule protein and lipid droplets, indicators of the differentiated phenotype, as well as cell flattening and increase of the volume of the cytoplasm. These results indicate that p21 WAF1/CIP1 , in addition to its Cdk-regulatory role, behaves as a transcriptional coactivator in a gene-specific manner implicated in cell differentiation.