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.
The Commonwealth Medical College, Scranton, Pennslyvania, United States of America.
Youngjin
Y
Cho
The Commonwealth Medical College, Scranton, Pennslyvania, United States of America.
Rachel
R
Silverstein
The Commonwealth Medical College, Scranton, Pennslyvania, United States of America.
Max T
MT
Geisinger
The Commonwealth Medical College, Scranton, Pennslyvania, United States of America.
Stephen
S
Martinkovich
The Commonwealth Medical College, Scranton, Pennslyvania, United States of America.
Holly
H
Corkill
The Commonwealth Medical College, Scranton, Pennslyvania, United States of America.
Jess M
JM
Cunnick
The Commonwealth Medical College, Scranton, Pennslyvania, United States of America.
Sonia L
SL
Planey
The Commonwealth Medical College, Scranton, Pennslyvania, United States of America.
John A
JA
Arnott
D000818:Animals; D000831:Animals, Newborn; D001665:Binding Sites; D002454:Cell Differentiation / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D024082:Collagen Type XII / Q000235:genetics* / Q000378:metabolism; D055513:Connective Tissue Growth Factor / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation; D008840:Microfilament Proteins / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D010006:Osteoblasts / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D061251:Primary Cell Culture; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D012886:Skull / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000378:metabolism / Q000494:pharmacology*
C000607822:Afap1 protein, rat; C526316:CCN2 protein, rat; D024082:Collagen Type XII; D008840:Microfilament Proteins; D034741:RNA, Small Interfering; D053773:Transforming Growth Factor beta1; D055513:Connective Tissue Growth Factor; D016392:Proto-Oncogene Proteins pp60(c-src)