Surface roughness modulates the response of MG63 osteoblast-like cells to 1,25-(OH)₂D₃ through regulation of phospholipase A₂ activity and activation of protein kinase A

Implant surface roughness influences osteoblast proliferation, differentiation, and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones such as 1,25-(OH)₂D₃ is altered by the effects of surface roughness; on the roughest Ti surfaces the effects of roughness and 1,25-(OH)₂D₃ are synergistic. Prostaglandin E₂ (PGE₂) appears to be involved in mediating the effects of surface roughness on the cells, as well as in the response to 1,25-(OH)₂D₃. However, it is not yet known through which signaling pathways surface roughness exerts its effects on the response of osteoblasts to 1,25-(OH)₂D₃. The present study examined the potential role of protein kinase A (PKA), phospholipase A₂(PLA₂), and protein kinase C (PKC) in this process. MG63 osteoblast-like human osteosarcoma cells were cultured on cpTi disks with R(a) values of 0.54 μm (PT), 4.14 μm (SLA), or 4.92 μm (TPS). PKA was inhibited by adding H8 to the cultures; similarly, PLA₂ was inhibited with quinacrine or activated with melittin, and PKC was inhibited with chelerythrine. Inhibitors or activators were included in the culture media through the entire culture period or for 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)₂D₃. The effects on cell number and alkaline phosphatase specific activity were determined after 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 increased. 1,25-(OH)₂D₃ had a synergistic effect with surface roughness on alkaline phosphatase. However, neither surface roughness nor 1,25- (OH)₂D₃ had an effect on PKC. H8 treatment for 24 h inhibited cell number and alkaline phosphatase on all surfaces; however, when it was present throughout the culture period, the PKA inhibitor had no effect on cell number, but decreased alkaline phosphatase-specific activity. H8 reduced the 1,25(OH)₂D₃-mediated effect on cell number and alkaline phosphatase. Quinacrine inhibited cell proliferation and alkaline phosphatase on all surfaces and further reduced the 1,25(OH)₂D₃-dependent decreases in both parameters. Melittin had no effect when applied for 24 h and did not modify the 1,25-(OH)₂D₃ effect; however, when present throughout the culture period, it caused a decrease in proliferation and an increase in enzyme activity. Chelerythrine, the PKC inhibitor, only inhibited cell proliferation when it was present throughout the entire culture period. However, it decreased alkaline phosphatase in cultures treated for 24 h, but increased enzyme activity when it was present for the entire culture period. The results indicate that surface roughness and 1,25-(OH)₂D₃ both mediate their effects through PLA₂ which catalyzes the rate-limiting step in PGE₂ production. Further downstream, PGE₂ activates PKA. Surface roughness- dependent effects are also mediated through PKC, but only after the cells have reached confluence and are undergoing phenotypic maturation. The effect of surface roughness on responsiveness to 1,25-(OH)₂D₃ is mediated through PLA₂/PKA and not through PKC.