Search

Activation of peroxisome proliferator-activated receptor gamma (PPARgamma) down-regulates the expression of follistatin mRNA in intestinal epithelial cells in vivo. The mechanism of PPARgamma-mediated down-regulation of follistatin was investigated using non-transformed, rat intestinal epithelial cells (RIE-1). RIE cells expressed activin A, the activin receptors ActRI and ActRII, and the follistatin-315 mRNA. RIE-1 cells responded to endogenous activin A, and this response was antagonized by follistatin, as evidenced by changes in cell growth and regulation of an activin-responsive reporter. Using RIE-1 cells, we show that activation of PPARgamma by rosiglitazone reduced follistatin mRNA levels in a dose- and concentration-dependent manner. Down-regulation of follistatin by rosiglitazone required the DNA binding domain of PPARgamma and was dependent upon dimerization with the retinoid X receptor. Inhibition of follistatin expression by rosiglitazone was not associated with decreased follistatin mRNA stability, suggesting that regulation may be at the promoter level. Analysis of the follistatin promoter revealed consensus binding sites for AP-1, AP-2, and Sp1. Targeting the AP-1 pathway with SP600125, an inhibitor of JNK, and TAM67, a dominant negative c-Jun, had no effect on PPARgamma-mediated down-regulation of follistatin. However, the follistatin promoter was dramatically regulated by Sp1, and this regulation was inhibited by PPARgamma expression. Knockdown of Sp1 expression relieved repression of follistatin levels by rosiglitazone. Moreover, PPARgamma was found to interact with Sp1 and repress its transcriptional activation function. Collectively, our data indicate that repression of Sp1 transcriptional activity by PPARgamma is the underlying mechanism responsible for PPARgamma-mediated regulation of follistatin expression.

Peroxisome proliferator-activated receptor gamma (PPARgamma) causes epithelial to mesenchymal transformation (EMT) in intestinal epithelial cells, as evidenced by reorganization of the actin cytoskeleton, acquisition of a polarized, mesenchymal cellular morphology, increased cellular motility, and colony scattering. This response is due to activation of Cdc42, resulting in p21-activated kinase-dependent phosphorylation and activation of MEK1 Ser(298) and activation of ERK1/2. Dominant negative MEK1, MEK2, and ERK2 block PPARgamma-induced EMT, whereas constitutively active MEK1 and MEK2 induce a mesenchymal phenotype similar to that evoked by PPARgamma. PPARgamma also stimulates ERK1/2 phosphorylation in the intestinal epithelium in vivo. PPARgamma induces the p110alpha subunit of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K blocks PPARgamma-dependent phosphorylation of MEK1 Ser(298), activation of ERK1/2, and EMT. We conclude that PPARgamma regulates the motility of intestinal epithelial cells through a mitogen-activated protein kinase cascade that involves PI3K, Cdc42, p21-activated kinase, MEK1, and ERK1/2. Regulation of cellular motility through Rho family GTPases has not been previously reported for nuclear receptors, and elucidation of the mechanism that accounts for the role of PPARgamma in regulating motility of intestinal epithelial cells provides fundamental new insight into the function of this receptor during renewal of the intestinal epithelium.

Fusion genes and fusion gene products are widely employed as biomarkers and therapeutic targets in hematopoietic cancers, but their applications have yet to be appreciated in solid tumors. Here, we report the use of SnowShoes-FTD, a powerful new analytic pipeline that can identify fusion transcripts and assess their redundancy and tumor subtype-specific distribution in primary tumors. In a study of primary breast tumors, SnowShoes-FTD was used to analyze paired-end mRNA-Seq data from a panel of estrogen receptor (ER)+, HER2+, and triple-negative primary breast tumors, identifying tumor-specific fusion transcripts by comparison with mRNA-Seq data from nontransformed human mammary epihelial cell cultures plus the Illumina Body Map data from normal tissues. We found that every primary breast tumor that was analyzed expressed one or more fusion transcripts. Of the 131 tumor-specific fusion transcripts identified, 86 were "private" (restricted to a single tumor) and 45 were "redundant" (distributed among multiple tumors). Among the redundant fusion transcripts, 7 were unique to ER+ tumors and 8 were unique to triple-negative tumors. In contrast, none of the redundant fusion transcripts were unique to HER2+ tumors. Both private and redundant fusion transcripts were widely expressed in primary breast tumors, with many mapping to genomic loci implicated in breast carcinogenesis and/or risk. Our finding that some fusion transcripts are tumor subtype-specific suggests that these entities may be critical determinants in the etiology of breast cancer subtypes, useful as biomarkers for tumor stratification, or exploitable as cancer-specific therapeutic targets. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor γ (PPARγ) causes epithelial to mesenchymal transformation (EMT) in intestinal epithelial cells, as evidenced by reorganization of the actin cytoskeleton, acquisition of a polarized, mesenchymal cellular morphology, increased cellular motility, and colony scattering. This response is due to activation of Cdc42, resulting in p21-activated kinase-dependent phosphorylation and activation of MEK1 Ser298 and activation of ERK1/2. Dominant negative MEK1, MEK2, and ERK2 block PPARγ-induced EMT, whereas constitutively active MEK1 and MEK2 induce a mesenchymal phenotype similar to that evoked by P PARγ. PPARγ also stimulates ERK1/2 phosphorylation in the intestinal epithelium in vivo. PPARγ induces the p110α subunit of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K blocks PPARγ-dependent phosphorylation of MEK1 Ser298, activation of ERK1/2, and EMT. We conclude that PPARγ regulates the motility of intestinal epithelial cells through a mitogen-activated protein kinase cascade that involves PI3K, Cdc42, p21-activated kinase, MEK1, and ERK1/2. Regulation of cellular motility through Rho family GTPases has not been previously reported for nuclear receptors, and elucidation of the mechanism that accounts for the role of PPARγ in regulating motility of intestinal epithelial cells provides fundamental new insight into the function of this receptor during renewal of the intestinal epithelium. [ABSTRACT FROM AUTHOR]

Abstract: Peroxisome proliferator-activated receptor-gamma (PPARγ) is expressed in the intestinal epithelium, yet little is known about the physiological role of PPARγ in the small bowel or the effects of PPARγ on small intestinal epithelial cells. The present studies investigate cellular and genomic effects of PPARγ in nontransformed rat intestinal epithelial cells (RIE). These cells were engineered to express mouse PPARγ1, and thereby to model the molecular phenotype that obtains upon induction of PPARγ at the crypt/villus junction in the small intestine. In these studies, we have used a novel third generation thiazolidinedione derivative, RS5444, which activates PPARγ with an EC50 about 1/50th that of rosiglitazone and has no effect on RIE cells that do not express PPARγ. We used Affymetrix oligonucleotide microarrays to identify potential PPARγ-regulated processes in RIE cells, including lipid metabolism, cell proliferation and differentiation, remodeling of the extracellular matrix, cell morphology, cell–cell adhesion, and motility. The genomic profile reflects cellular events that occur following PPARγ activation: RS5444 inhibited culture growth and caused irreversible G1 arrest, but did not induce apoptosis. In addition, RS5444 caused dramatic changes in cellular morphology which were associated with increased motility and diminished cellular adherence, but no increase in the ability of such cells to digest and invade Matrigel. Inhibition of proliferation, cell cycle arrest, increased motility, and altered adherence are aspects of the differentiated phenotype of villus epithelial cells, which withdraw from the cell cycle at the crypt/villus interface, migrate to the villus tips, and are subsequently shed by loss of contact with the epithelium and the underlying extracellular matrix. Our results are consistent with the hypothesis that PPARγ regulates critical aspects of differentiation in the small intestinal epithelium. Many nuclear receptors regulate differentiation. However, our results point to novel effects of PPARγ on cell–cell and cell–matrix interactions, which are not typical of other nuclear receptors. [Copyright &y& Elsevier]