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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.

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. 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. This study demonstrates that AFAP1 is an essential downstream signaling component of TGF-β1 for Src activation, CCN2 induction and collagen XIIa in osteoblasts.

CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-[beta]1 and acts as a mediator of TGF-[beta]1 induced matrix production in osteoblasts and Src is required for CCN2 induction by TGF-[beta]1; 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-[beta]1 or in osteoblats is undefined. In this study, we investigated the role of AFAP1 for CCN2 induction by TGF-[beta]1 in primary osteoblasts. 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-[beta]1 treatment in osteoblasts during days 7, 14 and 21. In osteoblasts, AFAP1 binds to Src and is required for Src activation by TGF-[beta]1 and CCN2 promoter activity and protein induction by TGF-[beta]1 treatment was impaired using AFAP1 siRNA, indicating the requirement of AFAP1 for CCN2 induction by TGF-[beta]1. We also demonstrated that TGF-[beta]1 induction of extracellular matrix protein collagen XIIa occurs in an AFAP1 dependent fashion. This study demonstrates that AFAP1 is an essential downstream signaling component of TGF-[beta]1 for Src activation, CCN2 induction and collagen XIIa 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-[beta]1 and acts as a mediator of TGF-[beta]1 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-[beta]1 in primary osteoblasts. We demonstrated that Ets-1 is expressed and induced by TGF-[beta]1 treatment in osteoblasts, and that Ets-1 over-expression induces CCN2 protein expression and promoter activity at a level similar to TGF-[beta]1 treatment alone. Additionally, we found that simultaneous Ets-1 over-expression and TGF-[beta]1 treatment synergize to enhance CCN2 induction, and that CCN2 induction by TGF-[beta]1 treatment was impaired using Ets-1 siRNA, demonstrating the requirement of Ets-1 for CCN2 induction by TGF-[beta]1. 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-[beta]1) 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-[beta]1 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-[beta]1 in osteoblasts, and that specific EBE sites in the CCN2 promoter are required for CCN2 promoter transactivation in osteoblasts.

Background 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-[beta]1 and acts as a mediator of TGF-[beta]1 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-[beta]1 in primary osteoblasts. Results We demonstrated that Ets-1 is expressed and induced by TGF-[beta]1 treatment in osteoblasts, and that Ets-1 over-expression induces CCN2 protein expression and promoter activity at a level similar to TGF-[beta]1 treatment alone. Additionally, we found that simultaneous Ets-1 over-expression and TGF-[beta]1 treatment synergize to enhance CCN2 induction, and that CCN2 induction by TGF-[beta]1 treatment was impaired using Ets-1 siRNA, demonstrating the requirement of Ets-1 for CCN2 induction by TGF-[beta]1. 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-[beta]1) 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-[beta]1 treatment require synergy between Ets-1 and Smad 3. Conclusions This study demonstrates that Ets-1 is an essential downstream signaling component for CCN2 induction by TGF-[beta]1 in osteoblasts, and that specific EBE sites in the CCN2 promoter are required for CCN2 promoter transactivation in osteoblasts.

Background CCN2 acts as an anabolic growth factor to regulate osteoblast differentiation and function. CCN2 is induced by TGF-[Beta]1 and acts as a mediator of TGF-[Beta]1 induced matrix production in osteoblasts and Src is required for CCN2 induction by TGF-[Beta]1; 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-[Beta]1 or in osteoblats is undefined. In this study, we investigated the role of AFAP1 for CCN2 induction by TGF-[Beta]1 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-[Beta]1 treatment in osteoblasts during days 7, 14 and 21. In osteoblasts, AFAP1 binds to Src and is required for Src activation by TGF-[Beta]1 and CCN2 promoter activity and protein induction by TGF-[Beta]1 treatment was impaired using AFAP1 siRNA, indicating the requirement of AFAP1 for CCN2 induction by TGF-[Beta]1. We also demonstrated that TGF-[Beta]1 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-[Beta]1 for Src activation, CCN2 induction and collagen XIIa in osteoblasts.

Understanding mechanisms of hepatocellular damage may lead to new treatments for liver disease, and genome-wide association studies (GWAS) of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum activities have proven useful for investigating liver biology. Here we report 100 loci associating with both enzymes, using GWAS across 411,048 subjects in the UK Biobank. The rare missense variant SLC30A10 Thr95Ile (rs188273166) associates with the largest elevation of both enzymes, and this association replicates in the DiscovEHR study. SLC30A10 excretes manganese from the liver to the bile duct, and rare homozygous loss of function causes the syndrome hypermanganesemia with dystonia-1 (HMNDYT1) which involves cirrhosis. Consistent with hematological symptoms of hypermanganesemia, SLC30A10 Thr95Ile carriers have increased hematocrit and risk of iron deficiency anemia. Carriers also have increased risk of extrahepatic bile duct cancer. These results suggest that genetic variation in SLC30A10 adversely affects more individuals than patients with diagnosed HMNDYT1. Circulating liver enzymes, like alanine aminotransferase (ALT) and aspartate aminotransferase (AST), are highly heritable and predictive of disease. Here, the authors perform a genome-wide association study on ALT and AST, revealing a rare variant in SLC30A10 associated with elevated ALT and AST.