V. L. Sylvia

Identification of a membrane receptor for 1,25-dihydroxyvitamin D3 which mediates rapid activation of protein kinase C.

This paper is the first definitive report demonstrating a unique membrane receptor for 1,25‐dihydroxyvitamin D3(1,25(OH)2D3) which mediates the rapid and nongenomic regulation of protein kinase C (PKC). Previous studies have shown that 1,25(OH)2D3 exerts rapid effects on chondrocyte membranes which are cell maturation‐specific, do not require new gene expression, and do not appear to act via the traditional vitamin D receptor. We used antiserum generated to a [3H]1,25(OH)2D3 binding protein isolated from the basal lateral membrane of chick intestinal epithelium (Ab99) to determine if rat costochondral resting zone (RC) or growth zone (GC) cartilage cells contain a similar protein and if cell maturation‐dependent differences exist. Immunohistochemistry demonstrated that both RC and GC cells express the protein, but levels are highest in GC. The binding protein is present in both plasma membranes and matrix vesicles and has a molecular weight of 66,000 Da. The 66 kDa protein in GC matrix vesicles has a Kd of 17.2 fmol/ml and Bmax of 124 fmol/mg of protein for [3H]1,25(OH)2D3. In contrast, the 66 kDa protein in RC matrix vesicles has a Kd of 27.7 fmol/ml and a Bmax of 100 fmol/mg of protein. Ab99 blocks the 1,25(OH)2D3‐dependent increase in PKC activity in GC chondrocytes, indicating that the 1,25(OH)2D3‐binding protein is indeed a receptor, linking ligand recognition to biologic function.

Phagocytosis of wear debris by osteoblasts affects differentiation and local factor production in a manner dependent on particle composition

Wear debris is considered to be one of the main factors responsible for aseptic loosening of orthopaedic endoprostheses. Whereas the response of cells in the monocytic lineage to foreign materials has been extensively studied, little is known about cells at the bone formation site. In the present study, we examined the hypothesis that the response of osteoblasts to wear debris depends on the chemical composition of the particles. We produced particles from commercially pure titanium (cpTi), Ti-6Al-4V (Ti-A), and cobalt-chrome (CoCr) and obtained ultrahigh molecular weight polyethylene (UHMWPE; GUR 4150) particles from a commercial source. The equivalent circle diameters of the particles were comparable: 1.0 +/- 0.96 microm for UHMWPE; 0.84 +/- 0.12 microm for cpTi; 1.35 +/- 0.09 microm for Ti-A, and 1.21 +/- 0.16 microm for CoCr. Confluent primary human osteoblasts and MG63 osteoblast-like cells were incubated in the presence of particles for 24 h. Harvested cultures were examined by transmission electron microscopy to determine if the cells had phagocytosed the particles. Particles were found intracellularly, primarily in the cytosol, in both the primary osteoblasts and MG63 cells. The chemical composition of the particles inside the cells was confirmed by energy-dispersive X-ray analysis. Morphologically, both cell types had extensive ruffled cell membranes, less-developed endoplasmic reticulum, swollen mitochondria, and vacuolic inclusions compared with untreated cells. CpTi, Ti-A, and CoCr particles were also added to cultures of MG63 cells to assess their effect on proliferation (cell number) and differentiation (alkaline phosphatase activity), and PGE2 production. All three types of particles had effects on the cells. The effect on cell number was dependent on the chemical composition of the particles; Ti-A and CoCr caused a dose-dependent increase, while cpTi particles had a biphasic effect with a maximal increase in cell number observed at the 1:10 dilution. Alkaline phosphatase specific activity was also affected and cpTi was more inhibitory than Ti-A or CoCr. PGE2 production was increased by all particles, but the magnitude of the effect was particle-dependent: CoCr > cpTi > Ti-A. This study demonstrates clearly that human osteoblast-like cells and MG63 cells can phagocytose small UHMWPE, CoCr, Ti-A, and cpTi particles. Phagocytosis of the particles is correlated with changes in morphology, and analysis of MG63 response shows that cell proliferation, differentiation, and prostanoid production are affected. This may have negative effects on bone formation adjacent to an orthopaedic implant and may initiate or contribute to the cellular events that cause aseptic loosening by inhibiting bone formation. The effects on alkaline phosphatase and PGE2 release are dependent on the chemical composition of the particles, suggesting that both the type and concentration of wear debris at an implant site may be important in determining clinical outcome.

Maturation state determines the response of osteogenic cells to surface roughness and 1,25-dihydroxyvitamin D3

In this study we assessed whether osteogenic cells respond in a differential manner to changes in surface roughness depending on their maturation state. Previous studies using MG63 osteoblast‐like cells, hypothesized to be at a relatively immature maturation state, showed that proliferation was inhibited and differentiation (osteocalcin production) was stimulated by culture on titanium (Ti) surfaces of increasing roughness. This effect was further enhanced by 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3]. In the present study, we examined the response of three additional cell lines at three different maturation states: fetal rat calvarial (FRC) cells (a mixture of multipotent mesenchymal cells, osteoprogenitor cells, and early committed osteoblasts), OCT‐1 cells (well‐differentiated secretory osteoblast‐like cells isolated from calvaria), and MLO‐Y4 cells (osteocyte‐like cells). Both OCT‐1 and MLO‐Y4 cells were derived from transgenic mice transformed with the SV40 large T‐antigen driven by the osteocalcin promoter. Cells were cultured on Ti disks with three different average surface roughnesses (Ra): PT, 0.5 μm; SLA, 4.1 μm; and TPS, 4.9 μm. When cultures reached confluence on plastic, vehicle or 10−7 M or 10−8 M 1,25(OH)2D3 was added for 24 h to all of the cultures. At harvest, cell number, alkaline phosphatase‐specific activity, and production of osteocalcin, transforming growth factor β1 (TGF‐β1) and prostaglandin E2 (PGE2) were measured. Cell behavior was sensitive to surface roughness and depended on the maturation state of the cell line. Fetal rat calvarial (FRC) cell number and alkaline phosphatase‐specific activity were decreased, whereas production of osteocalcin, TGF‐β1, and PGE2 were increased with increasing surface roughness. Addition of 1,25(OH)2D3 to the cultures further augmented the effect of roughness for all parameters in a dose‐dependent manner; only TGF‐β1 production on plastic and PT was unaffected by 1,25(OH)2D3. OCT‐1 cell number and alkaline phosphatase (SLA > TPS) were decreased and production of PGE2, osteocalcin, and TGF‐β1 were increased on SLA and TPS. Response to 1,25(OH)2D3 varied with the parameter being measured. Addition of the hormone to the cultures had no effect on cell number or TGF‐β1 production on any surface, while alkaline phosphatase was stimulated on SLA and TPS; osteocalcin production was increased on all Ti surfaces but not on plastic; and PGE2 was decreased on plastic and PT, but unaffected on SLA and TPS. In MLO‐Y4 cultures, cell number was decreased on SLA and TPS; alkaline phosphatase was unaffected by increasing surface roughness; and production of osteocalcin, TGF‐β1, and PGE2 were increased on SLA and TPS. Although 1,25(OH)2D3 had no effect on cell number, alkaline phosphatase, or production of TGF‐β1 or PGE2 on any surface, the production of osteocalcin was stimulated by 1,25(OH)2D3 on SLA and TPS. These results indicate that surface roughness promotes osteogenic differentiation of less mature cells, enhancing their responsiveness to 1,25(OH)2D3. As cells become more mature, they exhibit a reduced sensitivity to their substrate but even the terminally differentiated osteocyte is affected by changes in surface roughness.

Surface roughness mediates its effects on osteoblasts via protein kinase A and phospholipase A2

Earlier studies have shown that implant surface roughness influences osteoblast proliferation, differentiation, matrix synthesis and local factor production. Moreover, the responsiveness of osteoblasts to systemic hormones, such as 1,25-(OH)2D3, at the implant surface is also influenced by surface roughness and this effect is mediated by changes in prostaglandins. At present, it is not known which signaling pathways are involved in mediating cell response to surface roughness and how 1,25-(OH)2D3 treatment alters the activation of these pathways. This paper reviews a series of studies that have addressed this question. MG63 osteoblast-like cells were cultured on commercially pure titanium (cpTi) surfaces of two different roughnesses (Ra 0.54 and 4.92 microm) in the presence of control media or media containing 1,25-(OH)2D3 or 1,25-(OH)2D3 plus H8 (a protein kinase A inhibitor) or quinacrine (a phospholipase A2 inhibitor). At harvest, the effect of these treatments on cell number and alkaline phosphatase specific activity was measured. Compared to cultures grown on the smooth surface, cell number was reduced on the rough surface. 1,25-(OH)2D3 inhibited cell number on both surfaces and inhibition of protein kinase A in the presence of 1,25-(OH)2D3 restored cell number to that seen in the control cultures. Inhibition of phospholipase A2 in the presence of 1,25-(OH)2D3 caused a further reduction in cell number on the smooth surface, and partially reversed the inhibitory effects of 1,25-(OH)2D3 on the rough surface. Alkaline phosphatase specific activity was increased in cultures grown on the rough surface compared with those grown on the smooth surface; 1,25-(OH)2D3 treatment increased enzyme specific activity on both surfaces. Cultures treated with H8 and 1,25-(OH)2D3 displayed enzyme specific activity that approximated that seen in control cultures. Inhibition of phospholipase A2 also inhibited the 1,25-(OH)2D3-dependent effect on the smooth surface, but on the rough surface there was an inhibition of the 1,25-(OH)2D3 effect as well as a partial inhibition of the surface roughness-dependent effect. The results indicate that surface roughness and 1,25-(OH)2 D3 mediate their effects through phospholipase A2, which catalyzes one of the rate-limiting steps in prostaglandin E2 production. Further downstream, prostaglandin E2 activates protein kinase A.

Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells

Osteocytes, the predominant cells in bone, are postulated to be responsible for sensing mechanical and electrical stimuli, transducing signals via gap junctions. Osteocytes respond to induced shear by increasing connexin 43 (Cx43) levels, suggesting that they might be sensitive to physical stimuli like low‐frequency electromagnetic fields (EMF). Immature osteoblasts exhibit decreased intercellular communication in response to EMF but no change in Cx43. Here, we examined long term effects of pulsed EMF (PEMF) on MLO‐Y4 osteocyte‐like cells and ROS 17/2.8 osteoblast‐like cells. In MLO‐Y4 cell cultures, PEMF for 8 h/day for one, two or four days increased alkaline phosphatase activity but had no effect on cell number or osteocalcin. Transforming growth factor beta‐1 (TGF‐β1) and prostaglandin E2 were increased, and NO2‐ was altered. PEMFs effect on TGF‐β1 was via a prostaglandin‐dependent mechanism involving Cox‐1 but not Cox‐2. In ROS 17/2.8 cells, PEMF for 24, 48 or 72 h did not affect cell number, osteocalcin mRNA or osteocalcin protein. PEMF reduced Cx43 protein in both cells. Longer exposures decreased Cx43 mRNA. This indicates that cells in the osteoblast lineage, including well‐differentiated osteoblast‐like ROS 17/2.8 cells and terminally differentiated osteocyte‐like MLO‐Y4 cells, respond to PEMF with changes in local factor production and reduced Cx43, suggesting decreased gap junctional signaling.

Nongenomic regulation of protein kinase C isoforms by the vitamin D metabolites 1α,25-(OH)2D3 and 24R,25-(OH)2D3

Prior studies have shown that vitamin D regulation of protein kinase C activity (PKC) in the cell layer of chondrocyte cultures is cell maturation‐dependent. In the present study, we examined the membrane distribution of PKC and whether 1α,25‐(OH)2D3 and 24R,25‐(OH)2D3 can directly regulate enzyme activity in isolated plasma membranes and extracellular matrix vesicles. Matrix vesicle PKC was activated by bryostatin‐1 and inhibited by a PKC‐specific pseudosubstrate inhibitor peptide. Depletion of membrane PKC activity using isoform‐specific anti‐PKC antibodies suggested that PKCα is the major isoform in cell layer lysates as well as in plasma membranes isolated from both cell types; PKCζ is the predominant form in matrix vesicles. This was confirmed in Western blots of immunoprecipitates as well as in studies using control peptides to block binding of the isoform specific antibody to the enzyme and using a PKCζ‐specific pseudosubstrate inhibitor peptide. The presence of PKCζ in matrix vesicles was further verified by immunoelectron microscopy. Enzyme activity in the matrix vesicle was insensitive to exogenous lipid, whereas that in the plasma membrane required lipid for full activity. 1,25‐(OH)2D3 and 24,25‐(OH)2D3 inhibited matrix vesicle PKC, but stimulated plasma membrane PKC when added directly to the isolated membrane fractions. PKC activity in the matrix vesicle was calcium‐independent, whereas that in the plasma membrane required calcium. Moreover, the vitamin D‐sensitive PKC in matrix vesicles was not dependent on calcium, whereas the vitamin D‐sensitive enzyme in plasma membranes was calcium‐dependent. It is concluded that PKC isoforms are differentially distributed between matrix vesicles and plasma membranes and that enzyme activity is regulated in a membrane‐specific manner. This suggests the existence of a nongenomic mechanism whereby the effects of 1,25‐(OH)2D3 and 24,25‐(OH)2D3 may be mediated via PKC. Further, PKCζ may be important in nongenomic, autocrine signal transduction at sites distal from the cell.

Physiological importance of the 1,25(OH)2D3 membrane receptor and evidence for a membrane receptor specific for 24,25(OH)2D3

We have recently identified a membrane vitamin D receptor (mVDR) specific for 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) and shown that it mediates the rapid activation of protein kinase C (PKC) in growth zone chondrocytes (GCs). In this study, we examine the role of the 1,25(OH)2D3‐mVDR in chondrocyte physiology and provide evidence for the existence of a specific membrane receptor for 24,25‐dihydroxyvitamin D3 (24,25(OH)2D3‐mVDR). Fourth‐passage cultures of growth plate chondrocytes at two distinct stages of endochondral development, resting zone (RC) and growth zone (GC) cells, were used to assess the role of the mVDR in cell proliferation, PKC activation, and proteoglycan sulfation. To preclude the involvement of the nuclear vitamin D receptor (nVDR), we used hybrid analogs of 1,25(OH)2D3 with <0.1% affinity for the nVDR (2a, 1α‐CH2OH‐3β‐25D3; 3a, 1α‐CH2OH‐3β‐20‐epi‐22‐oxa‐25D3; and 3b, 1β‐CH2OH‐3α‐20‐epi‐22‐oxa‐25D3). To determine the involvement of the mVDR, we used an antibody generated against the highly purified 1,25(OH)2D3 binding protein from chick intestinal basolateral membranes (Ab99). Analog binding to the mVDR was demonstrated by competition with [3H]1,25(OH)2D3 using matrix vesicles (MVs) isolated from cultures of RC and GC cells. Specific recognition sites for 24,25(OH)2D3 in RC MVs were demonstrated by saturation binding analysis. Specific binding of 24,25(OH)2D3 was also investigated in plasma membranes (PMs) from RC and GC cells and GC MVs. In addition, we examined the ability of Ab99 to block the stimulation of PKC by analog 2a in isolated RC PMs as well as the inhibition of PKC by analog 2a in GC MVs. Like 1,25(OH)2D3, analogs 2a, 3a, and 3b inhibit RC and GC cell proliferation. The effect was dose dependent and could be blocked by Ab99. In GC cells, PKC activity was stimulated maximally by analogs 2a and 3a and very modestly by 3b. The effect of 2a and 3a was similar to that of 1,25(OH)2D3 and was blocked by Ab99, whereas the effect of 3b was unaffected by antibody. In contrast, 2a was the only analog that increased PKC activity in RC cells, and this effect was unaffected by Ab99. Analog 2a had no effect on proteoglycan sulfation in RC cells, whereas analogs 3a and 3b stimulated it and this was not blocked by Ab99. Binding of [3H]1,25(OH)2D3 to GC MVs was displaced completely with 1,25(OH)2D3 and analogs 2a, 3a, and 3b, but 24,25(OH)2D3 only displaced 51% of the bound ligand. 24,25(OH)2D3 displaced 50% of [3H]1,25(OH)2D3 bound to RC MVs, but 2a, 3a, and 3b displaced <50%. Scatchard analysis indicated specific binding of 24,25(OH)2D3 to recognition sites in RC MVs with a Kd of 69.2 fmol/ml and a Bmax of 52.6 fmol/mg of protein. Specific binding for 24,25(OH)2D3 was also found in RC and GC PMs and GC MVs. GC membranes exhibited lower specific binding than RC membranes; MVs had greater specific binding than PMs in both cell types. 2a caused a dose‐dependent increase in PKC activity of RC PMs that was unaffected by Ab99; it inhibited PKC activity in GC MVs, and this effect was blocked by Ab99. The results indicate that the 1,25(OH)2D3 mVDR mediates the antiproliferative effect of 1,25(OH)2D3 on chondrocytes. It also mediates the 1,25(OH)2D3‐dependent stimulation of PKC in GC cells, but not the 2a‐dependent increase in RC PKC activity, indicating that 24,25(OH)2D3 mediates its effects through a separate receptor. This is supported by the failure of Ab99 to block 2a‐dependent stimulation of PKC in isolated PMs. The data demonstrate for the first time the presence of a specific 24,25(OH)2D3 mVDR in endochondral chondrocytes and show that, although both cell types express mVDRs for 1,25(OH)2D3 and 24,25(OH)2D3, their relative distribution is cell maturation–dependent.

17β-estradiol-BSA conjugates and 17β-estradiol regulate growth plate chondrocytes by common membrane associated mechanisms involving PKC dependent and independent signal transduction

Nuclear receptors for 17β‐estradiol (E2) are present in growth plate chondrocytes from both male and female rats and regulation of chondrocytes through these receptors has been studied for many years; however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the cell response. E2 was found to directly affect the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E2 activates protein kinase C (PKC) in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E2‐dependent alkaline phosphatase activity and proteoglycan sulfation in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of the present study were: (1) to examine the effect of a cell membrane‐impermeable 17β‐estradiol‐bovine serum albumin conjugate (E2‐BSA) on chondrocyte proliferation, differentiation, and matrix synthesis; (2) to determine the pathway that mediates the membrane effect of E2‐BSA on PKC; and (3) to compare the action of E2‐BSA to that of E2. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10−9 to 10−7 M E2 or E2‐BSA and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [3H]‐thymidine incorporation measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E2‐BSA in the presence or absence of GDPβS (inhibitor of G‐proteins), GTPγS (activator of G‐proteins), U73122 or D609 (inhibitors of phospholipase C [PLC]), wortmannin (inhibitor of phospholipase D [PLD]) or LY294002 (inhibitor of phosphatidylinositol 3‐kinase). E2‐BSA mimicked the effects of E2 on alkaline phosphatase specific activity and proteoglycan sulfation, causing dose‐dependent increases in both RC and GC cell cultures. Both forms of estradiol inhibited [3H]‐thymidine incorporation, and the effect was dose‐dependent. E2‐BSA caused time‐dependent increases in PKC in RC and GC cells; effects were observed within three minutes in RC cells and within one minute in GC cells. Response to E2 was more robust in RC cells, whereas in GC cells, E2 and E2‐BSA caused a comparable increase in PKC. GDPβS inhibited the activation of PKC in E2‐BSA‐stimulated RC and GC cells. GTPγS increased PKC in E2‐BSA‐stimulated GC cells, but had no effect in E2‐BSA‐stimulated RC cells. The phosphatidylinositol‐specific PLC inhibitor U73122 blocked E2‐BSA‐stimulated PKC activity in both RC and GC cells, whereas the phosphatidylcholine‐specific PLC inhibitor D609 had no effect. Neither the PLD inhibitor wortmannin nor the phosphatidylinositol 3‐kinase inhibitor LY294022 had any effect on E2‐BSA‐stimulated PKC activity in either RC or GC cells. The classical estrogen receptor antagonist ICI 182780 was unable to block the stimulatory effect of E2‐BSA on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E2‐BSA. The specificity of the membrane response to E2 was also demonstrated by showing that the membrane receptor for 1α,25‐(OH)2D3 was not involved. These data indicate that the rapid nongenomic effect of E2‐BSA on PKC activity in RC and GC cells is dependent on G‐protein‐coupled PLC and support the hypothesis that many of the effects of E2 involve membrane‐associated mechanisms independent of classical estrogen receptors. J. Cell. Biochem. 81:413–429, 2001.

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)(2)D(3) is altered by the effects of surface roughness; on the roughest Ti surfaces the effects of roughness and 1, 25-(OH)(2)D(3) are synergistic. Prostaglandin E(2) (PGE(2)) appears to be involved in mediating the effects of surface roughness on the cells, as well as in the response to 1,25-(OH)(2)D(3). However, it is not yet known through which signaling pathways surface roughness exerts its effects on the response of osteoblasts to 1, 25-(OH)(2)D(3). The present study examined the potential role of protein kinase A (PKA), phospholipase A(2)(PLA(2)), 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 microm (PT), 4.14 microm (SLA), or 4.92 microm (TPS). PKA was inhibited by adding H8 to the cultures; similarly, PLA(2) 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)(2)D(3). 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)(2)D(3) had a synergistic effect with surface roughness on alkaline phosphatase. However, neither surface roughness nor 1,25-(OH)(2)D(3) 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)(2)D(3)-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)(2)D(3)-dependent decreases in both parameters. Melittin 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. 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)(2)D(3) both mediate their effects through PLA(2) which catalyzes the rate-limiting step in PGE(2) production. Further downstream, PGE(2) 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)(2)D(3) is mediated through PLA(2)/PKA and not through PKC.

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 microm) and response to 1,25-(OH)2D3 is enhanced, resulting in synergistic increases in TGF-beta1 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 microm) and rough (SLA, Ra = 3.97 microm; TPS, Ra = 5.21 microm) Ti disks. At confluence (5 days), cultures were treated for 24h with 10(-8) M 1alpha,25-(OH)2D3 and active and latent TGF-beta1 in the conditioned media measured by ELISA. Cell layers were digested with plasmin and released TGF-beta1 was also measured. 1,25-(OH)2D3 regulated the distribution of TGF-beta1 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 on PGE2 production in cultures treated for 24 h. H8 had no effect on TGF-beta1 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-beta1 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-beta1 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-beta1 production via PKC but not PKA. The effect of 1,25-(OH)2D3 on TGF-beta1 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.