Protein Kinase C (PKC)‐mediated actin disruption regulates [Ca2+]i responses to mechanical load in osteoblasts

Abstract

Osteoblasts respond to mechanical load with a rapid and transient increase in intracellular free Ca concentration ([Ca2+]i) that is essential for load-induced bone formation. PKC is also rapidly increased during mechanical stimulation and we postulate that PKC regulates the [Ca2+]i response through alteration of the actin cytoskeleton. Using fura-2 loaded MC3T3-E1 preosteoblastic cells, we examined the [Ca2+]i response to hypotonic swelling (HS) during phorbol ester (PMA)-induced PKC activation. PKC activation potentiated the peak [Ca2+]i response to HS by 57% compared to HS alone. A significant increase in the rising slope of the transient was also observed. PKC inhibition with 1 μM GF109203X reduced the peak [Ca2+]i response to 32% below HS alone peak levels. Voltage sensitive calcium or mechanosensitive channel block with nifedipine (10−6M) or gadolinium (5x10−6M), respectively, abrogated the effects of PKC on the HS-induced [Ca2+]i increase. HS alone increased actin stress fibers in MC3T3-E1 cells. However, PMA (10−6M) disrupted of the HS-induced actin organization. Osteoblasts pretreated with actin cytoskeleton stabilizer, phalloidin, failed to exhibit the PKC potentiation of HS-induced [Ca2+]i. These studies suggest that PKC regulates the [Ca2+]i response to mechanical load in osteoblasts by reorganization of actin and through control of channel activity. Supported by NIH/NIAMS AR43222