SIGNAL-TRANSDUCTION OF MECHANICAL STIMULI IS DEPENDENT ON MICROFILAMENT INTEGRITY - IDENTIFICATION OF OSTEOPONTIN AS A MECHANICALLY INDUCEDGENE IN OSTEOBLASTS

Mechanical perturbation has been shown to modulate a wide variety of c hanges in second message signals and patterns of gene expression in os teoblasts. Embryonic chick osteoblasts were subjected to a dynamic spa tially uniform biaxial strain (1.3% applied strain) at 0.25 Hz for a s ingle 2-h period, and osteopontin (OPN), an Arg-Gly-Asp (RGD)-containi ng protein, was shown to be a mechanoresponsive gene. Expression of op n mRNA reached a maximal 4-fold increase 9 h after the end of the mech anical perturbation that was not inhibited by cycloheximide, thus demo nstrating that mechanoinduction of opn expression is a primary respons e through the activation of pre-existing transcriptional factors, The signal transduction pathways, which mediated the increased expression of opn in response to mechanical stimuli, were shown to be dependent o n the activation of a tyrosine kinase(s) and protein kinase A (PKA) or a PKA-like kinase, Selective inhibition of protein kinase C (PKC) had no effect on the mechanoinduction of osteopontin even though opn has been demonstrated to be an early response gene to phorbol 12-myristate 13-acetate (PMA) stimulation, Mechanotransduction was dependent on mi crofilament integrity since cytochalasin-D blocked the up-regulation o f the opn expression; however, microfilament disruption had no effect on the PMA induction of the gene, The microtubule component of the cyt oskeleton was not related to the mechanism of signal transduction invo lved in controlling opn expression in response to mechanical stimulati on since colchicine did not block opn expression. Mechanical stimulus, vas shown to activate focal adhesion kinase (FAK), which specifically became associated with the cytoskeleton after mechanical perturbation, and its association with the cytoskeleton was dependent on tyrosine k inase activity, In conclusion, these results demonstrate that the sign al transduction pathway for mechanical activation of opn is uniquely d ependent on the structural integrity of the microfilament component of the cytoskeleton, In contrast, the PKC pathway, which also activates this gene in osteoblasts, acts independently of the cytoskeleton in th e transduction of its activity.