4C). This up-regulation of HuR was confirmed by western blotting in 5-day cultured HSCs, compared to quiescent HSCs (Fig. 4D). HuR silencing in primary HSCs, as confirmed by immunocytochemsitry (Fig. 4E), induced morphological changes selleck compound (F-actin immunostaining) (Fig. 4E), significantly reduced levels of activation (α-SMA, col1a1, and TGF-β) and proliferation markers (cyclin D1), and markedly increased expression of the quiescent marker, GFAP15 (Fig. 4F). Taken together, our data show

that HuR could play a role during HSC activation. We next examined whether HuR activity controlled the functions of two principal mediators of HSC activation (i.e., PDGF and TGF-β). PDGF potently promotes HSC migration and proliferation during fibrosis.16 HuR silencing in primary HSCs isolated from BDL mice (Supporting Fig. 2A) significantly reduced their migratory rate, both basally (Supporting Fig. 2C) and after PDGF treatment (Supporting Fig. 2D), and Selleck BAY 57-1293 decreased bromodeoxyuridine (BrdU) incorporation after PDGF stimulation (Supporting Fig. 2E). HuR silencing in a cell line of activated HSCs (cirrhotic liver fat-storing cells-8B [CFSC-8B] cells)12 (Supporting Fig. 2B) also blocked PDGF-induced migration and proliferation (Fig. 5A–C).

In CFSC-8B cells, HuR silencing prevented PDGF-induced increase in mRNA levels of genes regulating proliferation (cyclin D1 and B1), migration (MMP9 and Actin17), and infiltration (MCP-118) (Fig. 5D) as well as cyclin D1 protein (Supporting

Fig. 2B). RNA immunoprecipitation 上海皓元医药股份有限公司 of ribonucleaotide complexes coupled to qPCR (RIP-qPCR) analyses revealed a significantly increased binding of HuR to these mRNAs after PDGF stimuli (Fig. 5E). These data demonstrate the importance of HuR in PDGF-mediated HSC proliferation and migration. The abundance and subcellular localization of HuR are important determinants of its activity.19, 20 PDGF treatment increased the expression of HuR mRNA (Fig. 6A) and protein (Fig. 6B,C) levels in CFSC-8B cells as well as its cytoplasmic localization (Fig. 6D). Inhibition of both extracellular signal-related kinase (ERK) and phosphatidylinositol 3-kinase (PI3K) blocked PDGF-induced up-regulation of HuR mRNA and protein (Fig. 6A-C), thus controlling HuR abundance. Recently, it was reported that HuR transcription is controlled by nuclear factor kappa-light-chain enhancer of activated B cells (NFκB)/p65.21 We found that both ERK and PI3K induced nuclear translocation of the NFκB subunit (p65) in response to PDGF (Supporting Fig. 3A,C), and inhibition of this translocation by BAY 11-7802 treatment prevented PDGF-mediated up-regulation of HuR protein expression (Supporting Fig. 3B,D).