Surface roughness modulates the response of MG63 osteoblast-like cells to 1,25-(OH)(2)D-3 through regulation of phospholipase A(2) activity and activation of protein kinase A

Implant surface roughness influences osteoblast proliferation, differentiat ion, and local factor production. Moreover, the responsiveness of osteoblas ts to systemic hormones such as 1,25-(OH)(2)D-3 is altered by the effects o f surface roughness; on the roughest Ti surfaces the effects of roughness a nd 1,25-(OH)(2)D-3 are synergistic. Prostaglandin E-2 (PGE(2)) appears to b e involved in mediating the effects of surface roughness on the cells, as w ell as in the response to 1,25-(OH)(2)D-3. However, it is not yet known thr ough which signaling pathways surface roughness exerts its effects on the r esponse of osteoblasts to 1,25-(OH)(2)D-3. The present study examined the p otential role of protein kinase A (PKA), phospholipase A(2)(PLA(2)), and pr otein kinase C (PKC) in this process. MG63 osteoblast-like human osteosarco ma cells were cultured on cpTi disks with R-a values of 0.54 mu m (PT), 4.1 4 mu m (SLA), or 4.92 mu m (TPS). PKA was inhibited by adding H8 to the cul tures; similarly, PLA(2) was inhibited with quinacrine or activated with me littin, and PKC was inhibited with chelerythrine. Inhibitors or activators were included in the culture media through the entire culture period or fur the last 24 h of culture. In addition, cultures were treated for 24 h with inhibitors or activators in the presence of 1,25-(OH)(2)D-3. The effects o n cell number and alkaline phosphatase specific activity were determined af ter 24 h; PKC activity was determined after 9 min and at 24 h. Cell number was reduced on rough surfaces, and alkaline phosphatase activity was increa sed. 1,25-(OH)(2)D-3 had a synergistic effect with surface roughness on alk aline phosphatase. However, neither surface roughness nor 1,25-(OH)(2)D-3 h ad an effect on PKC. H8 treatment for 24 h inhibited cell. number and alkal ine phosphatase on all surfaces; however, when it was present throughout th e culture period, the PKA inhibitor had no effect on cell number, but decre ased alkaline phosphatase-specific activity. H8 reduced the 1,25-(OH)(2)D-3 -mediated effect on cell number and alkaline phosphatase. Quinacrine inhibi ted cell proliferation and alkaline phosphatase on all surfaces and further reduced the 1,25-(OH)(2)D-3-dependent decreases in both parameters. Melitt in had no effect when applied for 24 h and did not modify the 1,25-(OH)(2)D -3 effect; however, when present throughout the culture period, it caused a decrease in proliferation and an increase in enzyme activity. Chelerythrin e, the PKC inhibitor, only inhibited cell proliferation when it was present throughout the entire culture period. However, it decreased alkaline phosp hatase in cultures treated for 24 h, but increased enzyme activity when it was present for the entire culture period. The results indicate that surfac e roughness and 1,25-(OH)(2)D-3 both mediate their effects through PLA(2) w hich catalyzes the rate-limiting step in PGE(2) production. Further downstr eam, PGE(2) activates PKA. Surface rough ness-dependent effects are also me diated through PKC, but only after the cells have reached confluence and ar e undergoing phenotypic maturation. The effect of surface roughness on resp onsiveness to 1,25-(OH)(2)D-3 is mediated through PLA(2)/PKA and not throug h PKC. (C) 1999 John Wiley & Sons, Inc.