Src is a major signaling component for CTGF induction by TGF-beta1 in osteoblasts.
Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor beta1 (TGF-beta1) where it acts as a downstream mediator of TGF-beta1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk, and Smad signaling for CTGF induction by TGF-beta1 in osteoblasts; however, the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-beta1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-beta1. Additionally, inhibiting Src activation prevented Erk activation, Smads 2 and 3 activation and nuclear translocation by TGF-beta1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway directly by mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059, it inhibited TGF-beta1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) of the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. These data demonstrate that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-beta1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts.
Zhang
X
X
Department of Anatomy and Cell Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Arnott
J A
JA
Rehman
S
S
Delong
W G
WG
Sanjay
A
A
Safadi
F F
FF
Popoff
S N
SN
D000818:Animals; D002460:Cell Line; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005419:Flavonoids / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000502:physiology*; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology*; D051785:Smad Proteins / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism*; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005419:Flavonoids; D034741:RNA, Small Interfering; D051785:Smad Proteins; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D019061:src-Family Kinases; D048049:Extracellular Signal-Regulated MAP Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one