Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2

Studies in several cell types indicate that the actions of integrin receptors for extracellular matrix and receptors for growth factors are synergistic in regulating cellular differentiation and function. We studied the roles of the alpha 1 beta 1 and alpha 2 beta 1 integrin collagen receptors in regulating the differentiation of 2T3 osteoblastic cells in response to bone morphogenetic protein (BMP)-2 The immortalized 2T3 cell line was established from the calvaria of mice transgenic for a BMP-2 promoter driving SV40 T-antigen, These cells require exogenous BMP-2, as well as ascorbic acid and beta-glycerolphosphate, for expression of a mature osteoblast phenotype and formation of a mineralized matrix. To determine how integrin receptors for collagen-I affect BMP-2 signaling, function-perturbing anti-rat alpha 1 and/or alpha 2 integrin subunit, or anti-type I collagen (Col-I), antibodies mere added to human recombinant (hr)BMP-2-treated 2T3 cultures at confluence (CO) or at 4 or 8 days postconfluenee (C4, C8), After 4 days of exposure to the antibodies, cultures mere assayed for alkaline phosphatase (ALP) mRNA levels and enzyme activity and for cAMP production in response to parathyroid hormone. Addition of anti-collagen-I or both anti-integrin-alpha 1 and -alpha 2 antibodies to CO cultures blocked expression of these early osteoblast markers by more than 90%, and also blocked mineralization (0.5-1.8% control) of these cells. In all cases, adding anti-alpha 1 or anti-alpha 2 antibodies separately produced partial effects, while their combined effect approached that of anti-collagen-I. When antibodies were added to more differentiated 2T3 cells, the inhibitory effects decreased. 2T3 cells carrying constitutively active BMP receptor (caBMPR-IB) showed elevated ALP activity without hrBMP-2; this constitutive activity was also suppressed by alpha 1 and alpha 2 integrin antibodies and by anti-Col-I antibody. Together, our data suggest that a signal(s) from collagen integrin receptors regulates the response to BMP downstream of BMPR-IB and upstream of the regulation of ALP mRNA and other early markers of osteoblast differentiation.