Summary The peroxisome proliferator‐activated receptor γ (PPARγ) is expressed in macrophages and plays an important role in suppressing the inflammatory response. Lipopolysaccharides (LPS), which activate Toll‐like receptor 4 (TLR4), reduced PPARγ expression and function in peritoneal macrophages and macrophage cell lines. Moreover, pretreatment with the synthetic PPARγ ligand, rosiglitazone did not prevent LPS‐mediated downregulation of PPARγ. Inhibition of PPARγ expression was not blocked by cycloheximide, indicating that de novo protein synthesis is not required for LPS‐mediated suppression of PPARγ. Destabilization of PPARγ messenger RNA (mRNA) was not observed in LPS‐stimulated macrophages, suggesting that LPS regulates the synthesis of PPARγ mRNA. LPS had no effect on PPARγ expression in macrophages from TLR4 knockout mice, whereas LPS inhibited PPARγ expression in cells that had been reconstituted to express functional TLR4. Targeting the TLR4 pathway with inhibitors of MEK1/2, p38, JNK and AP‐1 had no effect on PPARγ downregulation by LPS. However, inhibitors that target NEMO, IκB and NF‐κB abolished LPS‐mediated downregulation of PPARγ in LPS‐stimulated macrophages. Our data indicate that activation of TLR4 inhibits PPARγ mRNA synthesis by an NF‐κB‐dependent mechanism. Low‐density genomic profiling of macrophage‐specific PPARγ knockout cells indicated that PPARγ suppresses inflammation under basal conditions, and that loss of PPARγ expression is sufficient to induce a proinflammatory state. Our data reveal a regulatory feedback loop in which PPARγ represses NF‐κB‐mediated inflammatory signalling in unstimulated macrophages; however, upon activation of TLR4, NF‐κB drives down PPARγ expression and thereby obviates any potential anti‐inflammatory effects of PPARγ in LPS‐stimulated macrophages.
Suppression of colon carcinogenesis by peroxisome proliferator-activated receptor (PPAR)-gamma is likely due to some effect of PPAR-gamma on normal colonic epithelial cells. However, our understanding of the effects of PPAR-gamma in such cells is limited. We analyzed the abundance, distribution, and function of PPAR-gamma in epithelial cells isolated from the murine proximal and distal colon. Marked differences in PPAR-gamma abundance and distribution were observed, suggesting tissue-specific responses. Analysis of PPAR-gamma effects on DNA synthesis, formation of preneoplastic lesions, and activation of MAPK signaling in proximal and distal colonic epithelial cells in vivo indicates that PPAR-gamma regulates both tissue-specific and common responses within the proximal and distal colon. Three major functional cohorts of PPAR-gamma target genes were identified by genomic profiling of isolated colonic epithelial cells: genes that are involved in metabolism, in signaling, and in cellular adhesion and motility. Two subsets of PPAR-gamma target genes were differentially expressed in the proximal and distal epithelium. Proximal target genes were primarily involved in metabolic activities, whereas signal transduction, adhesion, and motility targets were more pronounced in the distal colon. Remarkably, those target genes that are differentially expressed in the proximal colon were all induced on activation of PPAR-gamma, whereas all target genes that are preferentially expressed in the distal colon were repressed. Our data indicate that PPAR-gamma exerts both common and tissue-specific effects in the colon and challenge the general conclusions that PPAR-gamma is induced on differentiation of colonic epithelial cells and that this receptor stimulates differentiated function in epithelial cells throughout the colon.
Peroxisome proliferator-activated receptor ␥ (PPAR␥) 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 PPAR␥-induced EMT, whereas constitutively active MEK1 and MEK2 induce a mesenchymal phenotype similar to that evoked by PPAR␥. PPAR␥ also stimulates ERK1/2 phosphorylation in the intestinal epithelium in vivo. PPAR␥ induces the p110␣ subunit of phosphoinositide 3-kinase (PI3K), and inhibition of PI3K blocks PPAR␥-dependent phosphorylation of MEK1 Ser 298 , activation of ERK1/2, and EMT. We conclude that PPAR␥ 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 PPAR␥ in regulating motility of intestinal epithelial cells provides fundamental new insight into the function of this receptor during renewal of the intestinal epithelium.The nuclear receptor peroxisome proliferator-activated receptor (PPAR) 2 ␥1 is expressed at high levels in many cells of epithelial origin, including those of the gastrointestinal tract (1, 2). PPAR␥ is activated by the thiazolidinedione class of drugs (3), which inhibit azoxymethane-induced colon carcinogenesis in wild type mice (4, 5). Furthermore, hemizygous knock-out of PPAR␥ exacerbates colon tumor formation in azoxymethanetreated mice (6). Paradoxically, thiazolidinediones appear to promote intestinal tumor growth in mice that harbor mutations in the adenomatous polyposis coli (APC ϩ/Min ) gene (7,8), and long term treatment with high concentrations of thiazolidinediones induces caecal tumors in mice (9, 10). These observations suggest that PPAR␥ may, under different circumstances, function as a tumor suppressor or as a tumor promoter. However, little is known about the physiological role of PPAR␥ in the gastrointestinal epithelium, which makes it difficult to construct testable hypotheses concerning those aspects of PPAR␥ signaling that may account for these paradoxical effects of thiazolidinediones.To address these questions, we carried out a series of studies to elucidate the functions of PPAR␥ in nontransformed gastrointestinal epithelial cells. Our initial studies indicate that PPAR␥ plays a critical role in a number of processes that are central to renewal of the intestinal epithelium (11). The intestinal epithelium is one of the most dynamic tissues in the adult. The entire epithelium is replaced every 3 days or so by a highly orchestrated process that involves proliferation,...
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