title: TNF receptor-associated factor 6 (TRAF6) mediates the angiotensin-induced non-canonical TGF-β pathway activation of c-kit(+) cardiac stem cells.
abstract: Cardiac stem cells (CSCs) can differentiate into cardiac muscle-like cells upon stimulation by angiotensin II (Ang II). TNF receptor-associated factor 6 (TRAF6) has been shown to promote JNK- and p38-induced myogenic differentiation and mediate Smad-independent activation of TGF-β. However, the detailed mechanisms underlying the activation of these signaling pathways are not entirely known. Herein, we hypothesized that Ang II could promote the differentiation of CSCs into cardiac muscle-like cells by non-canonical TGF-β/TRAF6 signaling pathway, and sought to test the hypothesis. C-kit(+) CSCs were isolated from neonatal Sprague Dawley (SD) rats, and their c-kit status was confirmed with immunofluorescence staining. A TGF-β type I receptor inhibitor (SB431542) was used to inhibit SMAD2/3 phosphorylation. The small interfering RNA (siRNA)-mediated knockdown of TRAF6 was used to investigate the role of TRAF6 in TGF-β signaling. Rescue of TRAF6 siRNA transfected 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. Our results showed that the increase in JNK and p38 activation by Ang-II was blocked by siRNA. After transfection by TRAF6-siRNA or Ad-TRAF6, the cardiac specific markers and Wnt signaling proteins were tested by Western blotting. Physical interactions between TRAF6 and TGF-β receptors were studied by co-immunoprecipitation. Forced expression of TRAF6 enhanced the expression of cTnT and Cx-43 but inhibited the expression of Wnt3a.Our data suggested that TRAF6 mediated Ang II-induced differential responses in c-kit(+) CSCs via the non-canonical TGF-β signaling pathway.
authors.affiliation: Xinhua Hospital, Shanghai Jiao Tong University School of Medicine 1665 Kongjiang Road, Shanghai, 200092, China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Cao
authors.affiliation: Xinhua Hospital, Shanghai Jiao Tong University School of Medicine 1665 Kongjiang Road, Shanghai, 200092, China.
authors.forename: Yuqiang
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Xinhua Hospital, Shanghai Jiao Tong University School of Medicine 1665 Kongjiang Road, Shanghai, 200092, China.
authors.forename: Liya
authors.initials: L
authors.lastname: Huang
authors.affiliation: Xinhua Hospital, Shanghai Jiao Tong University School of Medicine 1665 Kongjiang Road, Shanghai, 200092, China.
authors.forename: Fei
authors.initials: F
authors.lastname: Wang
authors.affiliation: Xinhua Hospital, Shanghai Jiao Tong University School of Medicine 1665 Kongjiang Road, Shanghai, 200092, China.
authors.forename: Shuyan
authors.initials: S
authors.lastname: Chen
keywords: Cardiac stem cells; TGF-β; TRAF6; differentiation; ubiquitination