AFAP1 Is a Novel Downstream Mediator of TGF-β1 for CCN2 Induction in Osteoblasts.

BACKGROUND:CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-β1 and acts as a mediator of TGF-β1 induced matrix production in osteoblasts and Src is required for CCN2 induction by TGF-β1; however, the molecular mechanisms that control CCN2 induction in osteoblasts are poorly understood. AFAP1 binds activated forms of Src and can direct the activation of Src in certain cell types, however a role for AFAP1 downstream of TGF-β1 or in osteoblats is undefined. In this study, we investigated the role of AFAP1 for CCN2 induction by TGF-β1 in primary osteoblasts. RESULTS:We demonstrated that AFAP1 expression in osteoblasts occurs in a biphasic pattern with maximal expression levels occurring during osteoblast proliferation (~day 3), reduced expression during matrix production/maturation (~day 14-21), an a further increase in expression during mineralization (~day 21). AFAP1 expression is induced by TGF-β1 treatment in osteoblasts during days 7, 14 and 21. In osteoblasts, AFAP1 binds to Src and is required for Src activation by TGF-β1 and CCN2 promoter activity and protein induction by TGF-β1 treatment was impaired using AFAP1 siRNA, indicating the requirement of AFAP1 for CCN2 induction by TGF-β1. We also demonstrated that TGF-β1 induction of extracellular matrix protein collagen XIIa occurs in an AFAP1 dependent fashion. CONCLUSIONS:This study demonstrates that AFAP1 is an essential downstream signaling component of TGF-β1 for Src activation, CCN2 induction and collagen XIIa in osteoblasts

AFAP1 signaling is necessary for TGF-β1 induced Src activation in osteoblast.

<p>The data show that AFAP1 is required for Src activation by TGF-β1 (A) and that AFAP1 binds Src in osteoblasts in a TGF-β1 inducible manner (B). <u>Methods:</u> (A) Primary osteoblasts were pretreated with AFAP1 siRNA (siRNA) or non-targeting control siRNA (control) and the treated with TGF-β1 (5ng/ml; 2hrs) (+) or mock treated (-) and AFAP1, pTyr416-Src (pSrc) and Actin were assessed by Western blot. (B) Primary osteoblasts were treated with TGF-β1 (5ng/ml; 24hrs) (+) or mock treated (-) and IP with AFAP1 or IgG and blotted with Src or AFAP1 The data show that AFAP1 is required for Src activation by TGF-β1 and that AFAP1 binds Src in osteoblasts in a TGF-β1 inducible manner.</p

AFAP1 is required for CCN2 induction by TGF-β1.

<p>The data show that blocking AFAP1 expression with siRNA impairs CCN2 protein and promoter induction by TGF-β1 in primary osteoblasts. Additionally, CCN2 induction by TGF-β1 is impaired in osteoblasts derived from AFAP1^-/- mice. <u>Methods:</u><b>(A)</b> Primary osteoblasts were pretreated with AFAP1 siRNA (siRNA) or non-targeting control siRNA (control) or transfection reagent only (transfectant only) and then treated with TGF-β1 (5ng/ml; 24hrs) (+) or mock treated (-) and AFAP1, CCN2 and Actin were assessed by Western blot. (<b>B</b>) Primary Osteoblasts were treated as in A, co-transfected with a full length CCN2 proximal promoter reporter, serum starved and then treated with TGF-β1 (5ng/ml) for 24hrs. Luciferase is expressed as a ratio of firefly/renilla. (SEM, n = 6) star symbol = p<0.05 compared to TGF-B1 +, control siRNA sample.</p

AFAP1 is necessary for TGF-β1 induced Col XIIa.

<p>The data show that blocking AFAP1 expression with siRNA impairs Col XIIa protein expression. Primary osteoblasts were pretreated with AFAP1 siRNA (siRNA) and then treated with TGF-β1 (5ng/ml; 24hrs) (+) or mock treated (-) and Col XIIa, AFAP1, CCN2 and Actin were assessed by Western Blot. Western Blots are representative of triplicate determinations.</p