Local factor production by MG63 osteoblast-like cells in response to surface roughness and 1,25-(OH)(2)D-3 is mediated via protein kinase C- and protein kinase A-dependent pathways

Titanium (Ti) surface roughness affects bone formation in vivo and osteobla st attachment, proliferation and differentiation in vitro. MG63 cells exhib it decreased proliferation and increased differentiation when cultured on r ough Ti surfaces (R-a > 2 mum) and response to 1,25-(OH)(2)D-3 is enhanced, resulting in synergistic increases in TGF-beta1 and PGE(2). To examine the hypothesis that surface roughness and 1,25-(OH)(2)D-3 exert their effects on local factor production through independent, but convergent, signaling p athways, MG63 cells were cultured on tissue culture plastic or on smooth (P T, R-a = 0.60 mum) and rough (SLA, R-a = 3.97 mum; TPS, R-a = 5.21 mum) Ti disks. At confluence (5 days), cultures were treated for 24h with 10(-8)M 1 alpha ,25-(OH)(2)D-3 and active and latent TGF-beta1 in the conditioned me dia measured by ELISA. Cell layers were digested with plasmin and released TGF-beta1 was also measured. 1,25-(OH)(2)D-3 regulated the distribution of TGF-beta1 between the media and the matrix in a surface-dependent manner; t he effect was greatest in the matrix of cells cultured on SLA and TPS. Inhi bition of PKA with H8 for the last 24h of culture increased PGE(2) on SLA a nd TPS, but when present throughout the entire culture period H8 caused an increase in PGE(2) on all surfaces. 1,25-(OH)(2)D-3 reduced the effect of H 8 on PGE(2) production in cultures treated for 24 h. H8 had no effect on TG F-beta1 in the media by itself but caused a complete inhibition of the 1,25 -(OH)(2)D-3 dependent increase. Inhibition of PKC with chelerythrine increa sed PGE(2) in a surface-dependent manner and 1,25-(OH)(2)D-3 reduced the ef fect of the PKC inhibitor. Chelerythrine also increased TGF-beta1 but the e ffect was not surface dependent; however, 1,25-(OH)(2)D-3 reduced the effec ts of chelerythrine with the greatest effects on the smooth surface. Thus, the distribution of TGF-beta1 between the media and the matrix is regulated by 1,25-(OH)(2)D-3 in a surface-dependent manner. Surface roughness exerts its effects on TGF-beta1 production via PKC but not PKA. The effect of 1,2 5-(OH)(2)D-3 on TGF-beta1 production is not via PKC. PKA is involved in the surface-dependent regulation of PGE(2) but not in the regulation of PGE(2) by 1,25-(OH)(2)D-3 on rough surfaces. Regulation of PKC affects PGE(2) pro duction but it is not involved in the surface roughness dependent response to 1,25-(OH)(2)D-3. These results suggest two independent but interconnecte d pathways are involved. (C) 2001 Elsevier Science Ltd. All rights reserved .