Local factor production by MG63 osteoblast-like cells in response to surface roughness and 1,25-(OH)2D3 is mediated via protein kinase C- and protein kinase A-dependent pathways

Titanium (Ti) surface roughness affects bone formation in vivo and osteoblast attachment, proliferation and differentiation in vitro. MG63 cells exhibit decreased proliferation and increased differentiation when cultured on rough Ti surfaces (Ra>2 μm) and response to 1,25-(OH)2D3 is enhanced, resulting in synergistic increases in TGF-β1 and PGE2. To examine the hypothesis that surface roughness and 1,25-(OH)2D3 exert their effects on local factor production through independent, but convergent, signaling pathways, MG63 cells were cultured on tissue culture plastic or on smooth (PT, Ra=0.60 μm) and rough (SLA, Ra=3.97 μm; TPS, Ra=5.21 μm) Ti disks. At confluence (5 days), cultures were treated for 24 h with 10−8 1α,25-(OH)2D3 and active and latent TGF-β1 in the conditioned media measured by ELISA. Cell layers were digested with plasmin and released TGF-β1 was also measured. 1,25-(OH)2D3 regulated the distribution of TGF-β1 between the media and the matrix in a surface-dependent manner; the effect was greatest in the matrix of cells cultured on SLA and TPS. Inhibition of PKA with H8 for the last 24 h of culture increased PGE2 on SLA and TPS, but when present throughout the entire culture period H8 caused an increase in PGE2 on all surfaces. 1,25-(OH)2D3 reduced the effect of H8 an PGE2 production in cultures treated for 24 h. H8 had no effect on TGF-β1 in the media by itself but caused a complete inhibition of the 1,25-(OH)2D3 dependent increase. Inhibition of PKC with chelerythrine increased PGE2 in a surface-dependent manner and 1,25-(OH)2D3 reduced the effect of the PKC inhibitor. Chelerythrine also increased TGF-β1 but the effect was not surface dependent; however, 1,25-(OH)2D3 reduced the effects of chelerythrine with the greatest effects on the smooth surface. Thus, the distribution of TGF-β1 between the media and the matrix is regulated by 1,25-(OH)2D3 in a surface-dependent manner. Surface roughness exerts its effects on TGF-β1 production via PKC but not PKA. The effect of 1,25-(OH)2D3 on TGF-β1 production is not via PKC. PKA is involved in the surface-dependent regulation of PGE2 but not in the regulation of PGE2 by 1,25-(OH)2D3 on rough surfaces. Regulation of PKC affects PGE2 production but it is not involved in the surface roughness-dependent response to 1,25-(OH)2D3. These results suggest two independent but interconnected pathways are involved.