Curcumin Suppresses Fibrosis Of TGF-β Signaling In Scleroderma

Background and ObjectivesSystemic scleroderma (SSc) is a generalized disease which typically results in fibrosis of the skin and various other organs. The hallmark of Scleroderma is the excessive accumulation of extracellular matrix (ECM) proteins. Although the fundamental mechanism of scleroderma is still unclear, excessive fibrosis might be the result of a failure to down-regulate the normal tissue repair program.Several fibrogenic cytokines are involved in the process of tissue repair and fibrosis. Among them, TGF-βis the most extensively studied. TGF-βis a major profibrotic cytokine, which regulates multiple cellular functions including differentiation, proliferation and induction of synthesis of extracellular matrix proteins. Recently, TGIF, an exclusively nuclear protein, raised much attention duo to its potent activity of suppression of TGF-β. TGIF also can attenuate TGF-βsignaling by suppression of Smad2 phosphorylation through sequestering cPML in the nucleus.Curcumin, a yellow curry powder from Turmeric (Curcuma longa) in Indian and other Asian cuisines, shows potent biological effects in vitro and in vivo. It activates multiple pathways to exert antioxidant, anti-inflammatory, antifibrotic and anticancer activities. It can exert its antioxidant activity via stimulation of Nrf2, which binds and regulates several genes coding for cytoprotective, antioxidants, and detoxification proteins, such as oxygenase-1, ferritin, etc. Curcumin also exerts potent anti-inflammatory effects through inhibition of NF-κB, AP-1, STAT and MAP kinases.Previous studies have demonstrated that curcumin can restrict Smad signal transduction. Considering the critical role of TGIF in TGF-βsignaling and the potent antifibrotic action of curcumin, in this study, we hypothesized curcumin might regulate TGF-βsignaling by targeting its downstream regulator TGIF in SSc fibroblasts. To test this hypothesis, we primarily cultured SSc fibroblasts and studied the impact of curcumin on TGF-βsignaling and its molecular mechanism. Objects and Methods1.Cell culture. Skin biopsies were obtained during diagnostic procedures from five female SSc patients (age 58±8 years), after informed consent and Ethical Committee approval. Fibroblasts obtained from the above biopsies were plated on tissue culture dishes in DMEM. SSc fibroblasts in 100 mm-diameter culture dishes were grown to confluency and rested in medium without FBS overnight. After treated with the indicated doses of curcumin, cells were treated with TGF-? for appropriate time in the absence of FBS.2.Western blot analysis. Cells were dissolved in lysis buffer containing 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 0.1 % SDS, 100μg/mL PMSF, 2μg/mL aprotinin, 2μg/mL leupeptin, 1% NP40. After centrifugation, the supernatant was collected for immunoblotting analysis.3.Real-time qPCR. Cellular total RNA was extracted using TRIzol according to the manufacturer's instructions. After DNase I treatment, 2μg of total RNA was reverse-transcribed into cDNA by using Superscript? First-Strand Synthesis System. Real-time quantitative PCR was performed using ABI PRISM 7300 and QuantiFast SYBR Green PCR Kit.4.Luciferase activity analysis. Cells were transfected with the luciferase reporter construct (CAGA)9-Lux in which luciferase expression is under control of the Smad3/4, or with FAST1and the ARE-Lux construct, which contains Smad2/4-responsive cis-elements. Luciferase activity was measured using Dual luciferase reporter assay system according to the manufacturer's recommendations.5.Gene silencing. RNA interference experiments were performed using TGIF-specific siRNA. siRNA transfection was performed using Lipofectamine 2000 according to the manufacturer's recommendation. Scramble siRNA was used as an off-target control.Results1.Curcumin represses TGF-β-Smad2/3 signaling in SSc fibroblasts.TGF-βtreatment significantly increased the expression of several profibrotic genes, such as type I collagen, fibronectin and plasminogen activator inhibitor-1 (PAI-1). After treatment of curcumin, the induction of these genes by TGF-βwas remarkably inhibited.TGF-βtreatment significantly induced the luciferase activity of the Smad3/4-responsive reporter construct (CAGA) 9-Lux. The induced luciferase activity was strongly inhibited by curcumin addition in a dose dependent way.2.TGF-β-induced Smad2 phosphorylation is suppressed by curcumin concomitant with upregulation of TGIF. TGF-βtreatment significantly induced Smad2 phosphorylation. However, this induction was dramatically inhibited by curcumin treatment.we did not observe significant changes of Smad3 phosphorylation by TGF-βafter treatment of curcumin. This result indicates TGIF might be involved in curcumin-mediated suppression of TGF-βsignaling in SSc fibroblasts.3.TGIF is required for the inhibition of Smad2 phosphorylation by curcumin. TGIF specific siRNA, but not scramble siRNA significantly decreased TGIF levels. Knockdown of TGIF dramatically enhanced TGF-β-induced phosphorylation of Smad2 and upregulation of profibrotic genes, which confirmed the negative regulatory role of TGIF in TGF-β/Smad2 signaling.4.Curcumin-induced TGIF upregulation is via decreased proteasome-mediated TGIF degradation.We examined the mRNA levels of TGIF after curcumin treatment in SSc fibroblasts. And found that curcumin treatment alone did not significantly increase TGIF mRNA expression. We next examined the degradation process of TGIF protein after curcumin treatment.And observed that curcumin significantly increased the amount of TGIF protein. To show more directly that curcumin inhibits TGIF degradation, cells were incubated with curcumin in the presence of cycloheximide (CHX) which blocks new protein synthesis. In cells that did not receive curcumin, TGIF protein level decreased after CHX treatment.ConclusionsThe present study once more confirmed the anti-fibrotic activity of curcumin by suppression of TGF-βsignaling pathway. Moreover, our findings revealed a novel mechanism that links curcumin to TGIF, a novel negative modulator of TGF-βsignaling. Further studies should be conducted to more fully elucidate the molecular mechanism by which curcumin suppresses TGF-βsignaling in scleroderma. In view of the negligible toxicity of curcumin, our results reassure the promising clinical use of curcumin for scleroderma therapy.