The essential role for c-Ski in mediating TGF-β1-induced bi-directional effects on skin fibroblast proliferation through a feedback loop

Abstract

The bi-directional regulation of TGF-beta1 (transforming growth factor-beta1) on fibroblast proliferation with stimulation at low concentration, but inhibition at high concentration, has important significance during tissue repair. The mechanism has not been defined. c-Ski is a major co-repressor of TGF-beta1/Smad3 signalling; however, the exact role of c-Ski in the bi-directional regulation of fibroblast proliferation remains to be determined. In the present study, we established a dose-effect relationship of bi-directional regulation of TGF-beta1-mediated proliferation in rat skin fibroblasts, and found that c-Ski overexpression promoted fibroblast proliferation by inhibiting Smad3 activity. Importantly, c-Ski expression was decreased at the high concentration of TGF-beta1, but increased at the low concentration of TGF-beta1. This dose-dependent change in TGF-beta1 action did not affect Smad3 phosphorylation or nuclear translocation, but altered Smad3 DNA-binding activity, transcriptional activity and expression of the downstream gene p21 that both increased at the high concentration and decreased at the low concentration. Furthermore, c-Ski overexpression exerted synergistic stimulation with TGF-beta1 at the low concentration, but reversed the inhibitory effect of TGF-beta1 at high concentrations, while knockdown of c-Ski by RNA interference abrogated bi-directional role of TGF-beta1 on fibroblast proliferation. Thus our data reveal a new mechanism for this bi-directional regulation, i.e. c-Ski expression change induced by low or high TGF-beta1 concentration in turn determines the promoting or inhibiting effects of TGF-beta1 on fibroblast proliferation, and suggests an important role of c-Ski that modulates the local availability of TGF-beta1 within the wound repair microenvironment.

Upregulation of Ski in Fibroblast is Implicated in the Peroxisome Proliferator- Activated Receptor d-Mediated Wound Healing

“…Briefly, rat skin fibroblasts were synchronized with DMEM/0.4% FBS for 24 h and subsequently treated with GW501516 (50 nmol/L) or DMSO. To investigate whether Ski was implicated in the effect of PPARδ activation on fibroblasts proliferation, 200 ng pSuper-Ski-RNAi was transfected into fibroblasts for 6 hours and its effect on Ski protein expression was detected by western blot as previously described, which has been confirmed to efficiently RNAi Ski expression in fibroblasts in our recent study [11], then followed by GW501516 treatment. And 24 h, 48 h and 72 h after GW501516 or DMSO treatment respectively, the cells were incubated for 4 h at 37°C with WST-8 and the absorbance was finally determined at 450 nm using a microplate reader.…”

“…Fibroblast culture was established as described previously [11,25]. Briefly, fibroblasts were incubated at 5% CO 2 in a humidified 37°C incubator, cultured with Dulbecco's minimum essential medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) three times a week.…”

“…scxk (Yu) 2002-0002, grade II). pSuper-c-Ski-RNAi was made previously [11], which has been confirmed to successfully knockdown Ski expression both in rat fibroblasts and wound tissues in our recent study [10,11]. Antibodies specific to Ski (sc-9140), PPARδ (sc-7197), β-actin (sc-1616), PPARδ agonist GW501516 (sc-202642) and antagonist GSK0660 (sc-203985) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).…”

“…However, the transcriptional or translational modulation of Ski gene in these conditions is not clear. Our recent study has demonstrated that Ski is a new tissue repair-related gene, which is mainly expressed in fibroblasts and modulates wound healing and scar formation by regulating TGF-β-dependent or -independent pathway [10][11][12]. Accordingly, it is essential to investigate the endogenous or exogenous factors to regulate Ski expression or function, which has the potential to provide a novel strategy to accelerate wound healing via modulating Ski-mediated signaling and effects.…”

The Glucocorticoid Dexamethasone Inhibits U937 Cell Adhesion and Neutrophil Release via RhoA/ROCK1-Dependent and Independent Pathways

“…The proteins were visualized using an enhanced chemiluminescence (ECL) detection system and imaged. The intensity of each protein band was determined using Labworks 4.6 image analysis software, and β-actin was used as the internal reference [10]. …”