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

Abstract

BackgroundEts-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.ResultsWe 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.ConclusionsThis 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.

TGFβ receptor I transactivation mediates stretch-induced Pak1 activation and CTGF upregulation in mesangial cells

“…Active TbRI recruits receptor-regulated Smads (2 and 3) and phosphorylates them at a highly conserved C-terminal SSXS motif. These then dissociate from the receptor, associate with the common mediator Smad4, and move as a complex to the nucleus to bind Smad binding elements (SBE) in target gene promoter regions, usually in cooperation with other transcription factors (Geisinger et al, 2012;Wrighton and Feng, 2008). TGFb may also signal through non-Smad proteins.…”

Lentiviral Delivery of Small Hairpin RNA Targeting Connective Tissue Growth Factor Blocks Profibrotic Signaling in Tenon's Capsule Fibroblasts

“…Transforming growth factor b and endothelin-1 are two important upstream regulators of CTGF. The former induces CTGF through a complex network of transcriptional interactions requiring Smads, protein kinase C and the Ras/MEK/ERK/ Ets-1 pathway, 33,34 whereas the latter acts through JNK and ERK to induce the AP-1 components c-Fos and c-Jun. 35,36 Recently, the regulation of cell proliferation and ECM deposition by CTGF in relation to TGF-b was documented in human TCFs.…”

MicroRNAs orchestra the cellular processes driving failure of the heart

“…The transcription factor ETS-1 synergizes with TGFβ to induce CTGF expression [39] and was found to mediate the pro-fibrotic effects of angiotensin II on renal fibroblast [40]. ETS-1 expression is repressed by both miR-221 and miR-222 [13,18,26].…”

“…The ability of the eIF4F complex to interact simultaneously with eIF4E and PAB1 effectively circularizes the mRNA molecule and greatly enhances translation efficiency [39]. However, RISC can interfere with this process by deadenylation of the mRNA, limiting the circularization of the cap and PABP1-free tail of the deadenylated mRNA [40].…”

“…Decreased expression [18] and activity [27] of SERCA2a are thought to be the primary mechanisms for the impaired cardiac relaxation in the aged myocardium. In addition, age-associated alterations in proteins able to regulate SERCA2a activity, including PLB, [35][36][37][38], PKA [39], and CAMKII [40] have also been documented in the aged heart. Thus, βAR signalling plays a crucial role in maintaining a balanced calcium homeostasis and regulating cardiomyocyte contraction and relaxation, and dysregulated βAR signalling forms one of the major cellular hallmarks of the senescent myocardium.…”