TGF- beta 1 calcium signaling increases alpha 5 integrin expression in osteoblasts

TGF- beta 1 is a potent osteoactive factor and exhibits a wide variety of effects on osteoblasts, most of which are mediated through receptor associated Smad proteins. We have recently reported a novel TGF- beta 1 intracellular Ca super(2+) signaling pathway in osteoblasts, and found that this signaling is required for the TGF- beta 1 mediated enhancement of osteoblast adhesion to substrate. Given that interaction between the extracellular matrix protein fibronectin and alpha 5 beta 1 integrin on the cell surface is principally responsible for osteoblast substrate adhesion, we examined here whether the TGF- beta 1 stimulated Ca super(2+) signal is involved in this pathway. Our results show that, in primary human osteoblasts, the TGF- beta 1 induced intracellular Ca super(2+) signal is responsible, in part, for the stimulation of expression of alpha 5 integrin, but not of beta 1 integrin or fibronectin. Increased levels of alpha 5 integrin protein and mRNA were seen as early as 12 h after TGF- beta 1 treatment, but were inhibited by co-treatment of cells with nifedipine, a selective L-type Ca super(2+) channel blocker. TGF- beta 1 treatment increased both fibronectin and beta 1 integrin protein production within 48 h, in a manner unaffected by co-treatment with nifedipine. Immunofluorescence observations revealed that TGF- beta 1 treatment resulted in increased alpha 5 integrin staining, and more prominent alpha 5 integrin clustering, with increased co-localization with the actin cytoskeleton, effects that were blocked by co-treatment with nifedipine. The TGF- beta 1 induced intracellular Ca super(2+) signal in human osteoblasts is thus an important mechanistic step in the regulation of alpha 5 integrin expression, later contributing to enhanced cell adhesion.