CCAAT/enhancer-binding protein-β participates in oxidized LDL-enhanced proliferation in 3T3-L1 cells

Increased circulating oxidized LDL (oxLDL) have been found in obese subjects. Obesity is characterized by an excess of fat mass resulting from an increase in adipocyte number and size. The generation of new adipocytes is a tightly controlled process where multiple factors acting in a signaling cascade follow a precise temporal expression pattern; oxLDL appear to have a role in the impairment of this process. The purpose of this study was to examine the effects of oxLDL on the mechanisms involved in the proliferative stage of the differentiation process in 3T3-L1 cells. After hormonal induction, 3T3-L1 cells undergo approximately two rounds of mitotic clonal expansion (MCE), a process required for adipogenesis. CCAAT/enhancer-binding protein β (C/EBPβ) is immediately expressed after induction, and plays a crucial role in MCE, but its expression must decrease to allow preadipocytes to mature into adipocytes. We found that, in the presence of stimuli to differentiate, oxLDL induced a higher proliferation rate in this cell line, associated with a sustained up-regulation of C/EBPβ, which remained activated inside the nucleus for several days. RNAi-mediated knockdown of C/EBPβ 24 h after oxLDL treatment counteracted the increase in proliferation rate. Both C/EBPβ expression and proliferation processes appear to be influenced by cAMP/protein kinase A (PKA) and extracellular signal-regulated kinases1/2 (ERK1/2) pathways. OxLDL treatment led to increased levels of cAMP, and to a strong, prolonged phosphorylation of ERK1/2 and C/EBPβ. The addition of cAMP and PKA inhibitors, SQ22536 and H-89, respectively, reduced proliferation only in oxLDL-treated cells, whereas the addition of ERK1/2 inhibitor U0126 blocked proliferation in both control and oxLDL-treated cells. C/EBPβ nuclear expression and DNA-binding activity were reduced by U0126, under all tested conditions. These findings show that the altered expression pattern of C/EBPβ is involved in the increase in the number of proliferating cells induced by oxLDL, in hormone-stimulated 3T3-L1 cells.     

Very long-chain-fatty acids enhance adipogenesis through coregulation of Elovl3 and PPARγ in 3T3-L1 cells

Here, we show that Elovl3 (elongation of very long-chain fatty acids 3) was involved in the regulation of the progression of adipogenesis through activation of peroxisome proliferator-activated receptor (PPAR)γ in mouse adipocytic 3T3-L1 cells. The expression of the Elovl3 gene increased during adipogenesis, the expression pattern of which was similar to that of the PPARγ gene. Troglitazone, a PPARγ agonist, enhanced Elovl3 expression in adipocytes, as it did that of other PPARγ target genes. Promoter-reporter analysis demonstrated that three PPAR-responsive elements in the Elovl3 gene promoter had the potential to activate its expression in 3T3-L1 cells. Moreover, a chromatin immunoprecipitation assay revealed that PPARγ bound these PPAR-responsive elements of the Elovl3 promoter. When the Elovl3 mRNA level was suppressed by its siRNAs, the level of intracellular triglycerides was significantly decreased, and the expression levels of adipogenic, lipolytic, and lipogenic genes were also repressed. In a mammalian two-hybrid assay, C18:1 and C20:1 very long-chain fatty acids (VLCFAs), which are the products of Elovl3 and activated PPARγ function. In addition, these same VLCFAs could prevent the Elovl3 siRNA-mediated suppression of adipogenesis by enhancing the expression of adipogenic, lipolytic, and lipogenic genes in adipocytes. Moreover, this VLCFAs-mediated activation was repressed by a PPARγ antagonist. These results indicate that the expression of the Elovl3 gene was activated by PPARγ during adipogenesis. Elovl3-produced C18:1 and C20:1 VLCFAs acted as agonists of PPARγ in 3T3-L1 cells. Thus, the Elovl3-PPARγ cascade is a novel regulatory circuit for the regulation of adipogenesis through improvement of PPARγ function in adipocytes.     

Transforming growth factor β induces expression of connective tissue growth factor in hepatic progenitor cells through Smad independent signaling

Hepatic progenitor cells (HPCs) are activated in the chronic liver injury and are found to participate in the progression of liver fibrosis, while the precise role of HPCs in liver fibrosis remains largely elusive. In this study, by immunostaining of human liver sections, we confirmed that HPCs were activated in the cirrhotic liver and secreted transforming growth factor β (TGF-β) and connective tissue growth factor (CTGF), both of which were important inducers of liver fibrosis. Besides, we used HPC cell lines LE/6 and WB-F344 as in vitro models and found that TGF-β induced secretion of CTGF in HPCs. Moreover, TGF-β signaling was intracrine activated and contributed to autonomous secretion of CTGF in HPCs. Furthermore, we found that TGF-β induced expression of CTGF was not mediated by TGF-β activated Smad signaling but mediated by TGF-β activated Erk, JNK and p38 MAPK signaling. Taken together, our results provide evidence for the role of HPCs in liver fibrosis and suggest that the production of CTGF by TGF-β activated MAPK signaling in HPCs may be a therapeutic target of liver fibrosis.     

Activation of Wnt11 by transforming growth factor-β drives mesenchymal gene expression through non-canonical Wnt protein signaling in renal epithelial cells

Transforming growth factor β1 (TGF-β) promotes renal interstitial fibrosis in vivo and the expression of mesenchymal genes in vitro; however, most of its direct targets in epithelial cells are still elusive. In a screen for genes directly activated by TGF-β, we found that components of the Wnt signaling pathway, especially Wnt11, were targets of activation by TGF-β and Smad3 in primary renal epithelial cells. In gain and loss of function experiments, Wnt11 mediated the actions of TGF-β through enhanced activation of mesenchymal marker genes, such as Zeb1, Snail1, Pai1, and αSMA, without affecting Smad3 phosphorylation. Inhibition of Wnt11 by receptor knockdown or treatment with Wnt inhibitors limited the effects of TGF-β on gene expression. We found no evidence that Wnt11 activated the canonical Wnt signaling pathway in renal epithelial cells; rather, the function of Wnt11 was mediated by the c-Jun N-terminal kinase (JNK) pathway. Consistent with the in vitro results, all the TGF-β, Wnt11, and JNK targets were activated in a unilateral ureteral obstruction (UUO) model of renal fibrosis in vivo. Our findings demonstrated cooperativity among the TGF-β, Wnt11, and JNK signaling pathways and suggest new targets for anti-fibrotic therapy in renal tissue.