TGF-beta1 calcium signaling increases alpha5 integrin expression in osteoblasts.
TGF-beta1 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-beta1 intracellular Ca2+ signaling pathway in osteoblasts, and found that this signaling is required for the TGF-beta1 mediated enhancement of osteoblast adhesion to substrate. Given that interaction between the extracellular matrix protein fibronectin and alpha5beta1 integrin on the cell surface is principally responsible for osteoblast substrate adhesion, we examined here whether the TGF-beta1 stimulated Ca2+ signal is involved in this pathway. Our results show that, in primary human osteoblasts, the TGF-beta1 induced intracellular Ca2+ signal is responsible, in part, for the stimulation of expression of alpha5 integrin, but not of beta1 integrin or fibronectin. Increased levels of alpha5 integrin protein and mRNA were seen as early as 12 h after TGF-beta1 treatment, but were inhibited by co-treatment of cells with nifedipine, a selective L-type Ca2+ channel blocker. TGF-beta1 treatment increased both fibronectin and beta1 integrin protein production within 48 h, in a manner unaffected by co-treatment with nifedipine. Immunofluorescence observations revealed that TGF-beta1 treatment resulted in increased alpha5 integrin staining, and more prominent alpha5 integrin clustering, with increased co-localization with the actin cytoskeleton, effects that were blocked by co-treatment with nifedipine. The TGF-beta1 induced intracellular Ca2+ signal in human osteoblasts is thus an important mechanistic step in the regulation of alpha5 integrin expression, later contributing to enhanced cell adhesion.
Nesti
Leon J
LJ
Department of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Caterson
E J
EJ
Wang
Mark
M
Chang
Richard
R
Chapovsky
Felix
F
Hoek
Jan B
JB
Tuan
Rocky S
RS
D002121:Calcium Channel Blockers / Q000494:pharmacology; D020013:Calcium Signaling; D002448:Cell Adhesion / Q000187:drug effects; D002452:Cell Count; D005353:Fibronectins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D015151:Immunoblotting; D039081:Integrin alpha5beta1 / Q000235:genetics / Q000378:metabolism*; D009543:Nifedipine / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D012313:RNA / Q000032:analysis; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology; D053773:Transforming Growth Factor beta1
D002121:Calcium Channel Blockers; D005353:Fibronectins; D039081:Integrin alpha5beta1; D012333:RNA, Messenger; C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D012313:RNA; D009543:Nifedipine