Mechanisms Of TNF Receptor-associated Factor (TRAF6) On The Differentiation Of C-kit~+ Cardiac Stem Cells

Objective Cardiac stem cells(CSCs)can differentiate into cardiac muscle-like cells upon stimulation by angiotensin?(Ang?).TNF receptor-associated factor 6 TRAF6)has been shown to promote JNK-and p38-induced myogenic differentiation and mediate Smad-independent activation of TGF-?.However,the detailed echanisms underlying the activation of these signaling pathways are not entirely known.Herein,we hypothesized that Ang?could promote the differentiation of -kit~+CSCs into cardiac muscle-like cells by non-canonical TGF-?/TRAF6 signaling pathway,and sought to test the hypothesis.Methods C-kit~+CSCs were isolated from neonatal Sprague Dawley(SD)rats,and their c-kit status was confirmed with immunofluorescence staining.A TGF-?type?receptor inhibitor(SB431542)was used to inhibit SMAD2/3 phosphorylation.The small interfering RNA(siRNA)-mediated knockdown of TRAF6 was used to nvestigate the role of TRAF6 in TGF-?signaling.Rescue of TRAF6 siRNA ransfected cells with a 3'UTR-deleted siRNA insensitive construct was performed to rule out any off-target effects of the siRNA.TRAF6 dominant-negative(TRAF6DN)vector was constructed and used to infect c-kit~+CSCs.After transfected by RAF6-siRNA or Ad-TRAF6,cardiac specific markers and Wnt signaling proteins were tested by Western blotting.Physical interactions between TRAF6 and TGF-?receptors were studied by co-immunoprecipitation.The transfecting effect RAF6-siRNA or Ad-TRAF6 into c-kit~+CSCs were assessed after treatment with or without Ang II.The levels of Wnt3a,Wnt5a,and cTnT in cell lysates were analyzed by Western blotting.Results After culture for 48 h,the c-kit-labeled cell population routinely constituted~95.51%of the total gated cells.The expression of the stem cell markers CD117/c-kit,CD34,CD45,and CD133 in these cells.There was no detectable signal of CD34~+, D45~+and CD133~+cells in c-kit~+cells.Cardiac stem cells have been shown to sponta-neously differentiate into a cardiomyocyte phenotype under standard culturing onditions,which can be enhanced by Ang II stimulation.The Ang?-stimulated phosphorylation of p38 and JNK was elevated in c-kit~+CSCs in a time-dependent manner,and was not suppressed by SB431542.The increase in JNK and p38 ctivation by Ang?was blocked by siRNA.The re-introduction of a FLAG-tagged siRNA-insensitive TRAF6 construct lacking its 3'UTR into c-kit~+CSCs reversed the effect of TRAF6-specific siRNA on the ability of Ang?to activate TAK1,JNK,and P38.A ladder of high molecular weight TRAF6 bands was observed in the nti-TRAF6 immunocomplex,suggesting that 30 min of Ang?treatment promoted TRAF6 ubiquitination.Forced expression of TRAF6 enhanced the expression of TnT and Cx-43 but inhibited the expression of Wnt3a.Conclusion TRAF6 functions as an important mediator for Ang?-induced P38 and JNK activation,and cardiac-specific proteins cTnT and Cx-43 expression in c-kit~+CSCs.To achieve this function,an intact RING domain of TRAF6 as well as oly-ubiquitination and activation of TAK1 are required.