Myofibroblast differentiation and activation by transforming growth factor-beta1 (TGF-beta1) is a critical event in the pathogenesis of human fibrotic diseases, but regulatory mechanisms for this effect are unclear. In this report, we demonstrate that stable expression of the myofibroblast phenotype requires both TGF-beta1 and adhesion-dependent signals. TGF-beta1-induced myofibroblast differentiation of lung fibroblasts is blocked in non-adherent cells despite the preservation of TGF-beta receptor(s)-mediated signaling of Smad2 phosphorylation. TGF-beta1 induces tyrosine phosphorylation of focal adhesion kinase (FAK) including that of its autophosphorylation site, Tyr-397, an effect that is dependent on cell adhesion and is delayed relative to early Smad signaling. Pharmacologic inhibition of FAK or expression of kinase-deficient FAK, mutated by substituting Tyr-397 with Phe, inhibit TGF-beta1-induced alpha-smooth muscle actin expression, stress fiber formation, and cellular hypertrophy. Basal expression of alpha-smooth muscle actin is elevated in cells grown on fibronectin-coated dishes but is decreased on laminin and poly-d-lysine, a non-integrin binding polypeptide. TGF-beta1 up-regulates expression of integrins and fibronectin, an effect that is associated with autophosphorylation/activation of FAK. Thus, a safer and more effective therapeutic strategy for fibrotic diseases characterized by persistent myofibroblast activation may be to target this integrin/FAK pathway while not interfering with tumor-suppressive functions of TGF-beta1/Smad signaling.
MicroRNAs are single-stranded RNA of 18 -24 nt expressed endogenously that play important roles in cancer development. Here, we show that expression of miR-378 enhances cell survival, reduces caspase-3 activity, and promotes tumor growth and angiogenesis. Proteomic analysis indicates reduced expression of suppressor of fused (Sufu), a potential target of miR-378, which was confirmed in vitro and in vivo. Expression of a luciferase construct containing the target site in Sufu was repressed when cotransfected with miR-378. Transfection of a Sufu construct reversed the effect of miR-378, suggesting an important role for miR-378 in tumor cell survival. We also discovered that miR-378 targets Fus-1. Expression of luciferase constructs harboring the target sites in Fus-1 was repressed by miR-378. Fus-1 constructs with or without its 3 UTR were also generated. Cotransfection experiments showed that the presence of miR-378 repressed Fus-1 expression. Suppression of Fus-1 expression by siRNA against Fus-1 enhanced cell survival. Transfection of the Fus-1 construct reversed the function of miR-378 in cell survival. Our results suggest that miR-378 transfection enhanced cell survival, tumor growth, and angiogenesis through repression of the expression of two tumor suppressors, Sufu and Fus-1.miRNA ͉ oncogenic miRNA ͉ tumorigenesis O ver the past few years, microRNAs (miRNAs) have emerged as a prominent class of gene regulators (1). miRNAs are single-stranded RNA of 18-24 nt in length and are generated by an RNase III-type enzyme from an endogenous transcript that contains a local hairpin structure (2, 3). miRNA functions as a guide molecule in posttranscriptional gene silencing by partially complementing with the 3Ј untranslated region (UTR) of the target mRNAs, leading to translational repression (4). In animals, miRNA genes are transcribed to generate long primary transcripts, which are processed by the RNase III-type enzyme Drosha to produce precursor miRNAs (premiRNAs) in the nucleus (5). PremiRNAs are then exported to the cytoplasm by exportin-5 (6). After arrival in the cytoplasm, premiRNAs are subjected to secondary processing by Dicer, a cytoplasmic RNase III-type enzyme (7,8). miRNAs normally repress gene expression, opposing the activity of transcription factors, which initiate gene expression (9). By silencing various target mRNAs, miRNAs have key roles in diverse regulatory pathways, including control of development (10), cell differentiation (11), apoptosis (12-14), cell proliferation (15), division (16), protein secretion (17), and viral infection (18,19). Most importantly, miRNAs have been known to play roles in cancer development (20)(21)(22)(23). Previous studies have shown that miR-378 is expressed in a number of cancer cell lines (24) and is involved in the expression of vascular endothelial growth factor (25). To understand the biological functions of miR-378, we have generated a miR-378 expression construct for functional studies. Here, we show that stable expression of miR-378 plays a role in cell sur...
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