Ets-1 Is Essential for Connective Tissue Growth Factor (CTGF/CCN2) Induction by TGF-β1 in Osteoblasts

Ets-1 controls osteoblast differentiation and bone development; however, its downstream mechanism of action in osteoblasts remains largely undetermined. 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; however, the molecular mechanisms that control CCN2 induction are poorly understood. In this study, we investigated the role of Ets-1 for CCN2 induction by TGF-β1 in primary osteoblasts.We demonstrated that Ets-1 is expressed and induced by TGF-β1 treatment in osteoblasts, and that Ets-1 over-expression induces CCN2 protein expression and promoter activity at a level similar to TGF-β1 treatment alone. Additionally, we found that simultaneous Ets-1 over-expression and TGF-β1 treatment synergize to enhance CCN2 induction, and that CCN2 induction by TGF-β1 treatment was impaired using Ets-1 siRNA, demonstrating the requirement of Ets-1 for CCN2 induction by TGF-β1. Site-directed mutagenesis of eight putative Ets-1 motifs (EBE) in the CCN2 promoter demonstrated that specific EBE sites are required for CCN2 induction, and that mutation of EBE sites in closer proximity to TRE or SBE (two sites previously shown to regulate CCN2 induction by TGF-β1) had a greater effect on CCN2 induction, suggesting potential synergetic interaction among these sites for CCN2 induction. In addition, mutation of EBE sites prevented protein complex binding, and this protein complex formation was also inhibited by addition of Ets-1 antibody or Smad 3 antibody, demonstrating that protein binding to EBE motifs as a result of TGF-β1 treatment require synergy between Ets-1 and Smad 3.This study demonstrates that Ets-1 is an essential downstream signaling component for CCN2 induction by TGF-β1 in osteoblasts, and that specific EBE sites in the CCN2 promoter are required for CCN2 promoter transactivation in osteoblasts

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

Ets-1 synergizes with TGF-β1 for CCN2 promoter induction in osteoblasts.

<p>(<b>A</b>) Osteoblasts were plated in 96 well tissue culture plates and transfected with either 0.4 µg of an empty vector control (−) or the Ets-1 expression construct (+). All samples were co-transfected with 0.4 µg of our previously described CCN2 promoter luciferase reporter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone.0035258-Arnott2" target="_blank">[22]</a> and 0.2 µg of a renilla luciferase expression vector as an internal control. The cells were serum starved for 24 hrs and then treated with TGF-β1 (5 ng/ml) (+) or mock treated (−) with TGF-β1 diluent for 24 hrs. Luciferase activity was then assessed and expressed as a ratio of firefly/renilla luciferase (+SEM, n = 6). A = p<0.05 compared to +TGF-β1 only or +Ets-1 only. (<b>B</b>) Osteoblasts were plated in 96 well tissue culture plates and transfected with either 100 nM of Ets-1 siRNA (Ets-1) or control siRNA (C) for 48 hrs. All samples were co-transfected with 0.4 µg of our previously described CCN2 promoter luciferase reporter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone.0035258-Arnott2" target="_blank">[22]</a> and 0.2 µg of a renilla luciferase expression vector as an internal control. The cells were serum starved for 24 hrs and then treated with 5 ng/ml of TGF-β1 (+) or mock treated (−) with TGF-β1 diluent for 24 hrs. Luciferase activity was then assessed and expressed as a ratio of firefly/renilla luciferase (+SEM, n = 6). Star symbol indicates p<0.05 compared to control siRNA.</p

EBE sites are required for CCN2 promoter activation by TGF-β1 in osteoblasts.

<p>Osteoblasts were plated in 96-well tissue culture plates and transfected with 0.4 µg of either EBE mutation construct 1–8, pGL3-Basic (negative control) or W787 (positive control) and all were co-transfected with 0.2 µg of a renilla luciferase expression vector (internal control) for 24 hrs. The cells were serum starved for 24 hrs and then treated with TGF-β1 (5 ng/ml) for 24 hrs. Luciferase activity was assessed, and expressed as a % of activity obtained using the full length W787 construct. (+SEM, n = 6). A = p<0.05 compared to W787.</p

Mutation of EBE sites prevents protein complex binding.

<p>(<b>A</b>) Electro-mobility shift assays (EMSA) probes were created that were homologues to the CCN2 promoter and contained either mutated or unmutated EBE sites (#5-8) as indicated. Each probe was dsDNA and 5′ biotinylated. (<b>B</b>) Electro-mobility shift assays (EMSA) from nuclear lysates were generated from osteoblasts that were treated with TGF-β1 (5 ng/ml) for 2 hrs. Nuclear protein binding to the wild type and mutated EBE sites in the CTGF promoter was assessed using 5 µg of nuclear lysates. The lane number above each well corresponds to the probe used for that reaction. The experiment was repeated four times with similar results.</p

Ets-1 binds to EBE sites in the CCN2 promoter in osteoblasts.

<p>Electro-mobility shift assays (EMSA) from nuclear lysates were generated from osteoblasts that were treated with TGF-β1 (5 ng/ml) for 2 hrs. (<b>A</b>) Nuclear protein binding to the wild type E-E-E (lanes 1–6) (this probe contains EBE # 6-8; for probe design see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone-0035258-g006" target="_blank">Figure 6</a>) probe was assessed using 5 µg of nuclear lysates for each reaction. In some reactions, Ets-1 antibody was added at increasing concentrations (1 ug of antibody in lane 4; Two micrograms of antibody in lane 5) to test for Ets-1/probe interaction. Control antibody (2 ug) was also used (lane 6; C). In some cases, probe only (lane 1) or a molar excess of unlabeled probe (lane 3) was also used to demonstrate specificity. (<b>B</b>) Nuclear protein binding to the wild type S-E-T (lanes 7–14) (this probe contains EBE#5; for probe design see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035258#pone-0035258-g006" target="_blank">Figure 6</a>) probe was assessed using 5 µg of nuclear lysates for each reaction. In some reactions, Ets-1 antibody was added at increasing concentrations (1 ug of antibody in lane 10; 2 ug of antibody in lane 11) to test for Ets-1 protein/probe interaction or Smad 3 antibody (1 ug of antibody in lane 12; 2 ug of antibody in lane 13) to test for Smad 3 protein/probe interaction. Control antibody (2 ug) was also used (lane 14). In some cases, probe only (lane 7) or a molar excess of unlabeled probe (lane 9) was also used to demonstrate specificity.</p

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