Sex-specific regulation of growth plate chondrocytes by estrogen is via multiple MAP kinase signaling pathways.

Both male and female rat growth plate cartilage cells possess estrogen receptors (ERs), but 17beta-estradiol (E(2)) activates protein kinase C (PKC) and PKC-dependent biological responses to E(2) only in cells from female animals. PKC signaling can elicit genomic responses via mitogen activated protein kinase (MAPK) and E(2) has been shown to activate ERK MAPK in many cells, suggesting that MAPK may play a role in growth plate chondrocytes as well. We tested if E(2) increases MAPK activity and if so, whether the response is limited to female cells, if it is PKC-dependent, and if the mechanism involves traditional ER pathways. We also determined the contribution of MAPK to the biological response of growth plate chondrocytes and assessed the relative contributions of ERK, p38 and JNK MAPKs. Female rat costochondral cartilage cells were treated with E(2) and MAPK-specific activity determined in cell layer lysates. The mechanism of MAPK activation was determined by treating the cells with E(2) conjugated to bovine serum albumin (E(2)-BSA) to assess if membrane receptors were involved; stereospecificity was determined using 17alpha-estradiol; PKC and phospholipase C (PLC) dependence was determined using specific inhibitors; and the ER agonist diethylstilbestrol, the ER antagonist ICI 182780, and tamoxifen were used to assess the role of traditional ER pathways. E(2) regulation of ERK1/2 MAPK was assessed and the relative roles of ERK1/2, p38 and JNK MAPKs determined using specific inhibitors. E(2) caused a rapid dose-dependent activation of MAPK that was greatest in cells treated for 9 min with 10(-9) M hormone; activity remained elevated for 3 h. E(2)'s effect on MAPK was stereospecific and comparable to that of E(2)-BSA. It was insensitive to DES and ICI 182780, dependent on PKC and PLC, blocked by tamoxifen and it did not require gene transcription or translation. E(2) had no effect on ERK1 or ERK2 mRNA or protein but it caused a rapid phosphorylation of ERK1/2 at 9 min. Inhibition of ERK1/2 and p38 MAPK reduced the stimulatory effects of E(2) on alkaline phosphatase activity and [(35)S]-sulfate incorporation. These results suggest that E(2) regulates MAPK through a sex-specific membrane-mediated mechanism that does not involve cytosolic ERs in a traditional sense and that ERK1/2 and p38 mediate the downstream biological effects of the hormone.

Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption.

The effect of oxygen-based radio frequency glow discharge (rfGD) on the surface of different starch-based biomaterials (SBB) and the influence of proteins adsorption on modulating bone-cells behavior was studied. Bovine serum albumin, fibronectin and vitronectin were used in single and complex protein systems. RfGD-treated surfaces showed to increase in hydrophilicity and surface energy when compared to non-modified SBB. Biodegradable polymeric blends of cornstarch with cellulose acetate (SCA; 50/50wt%), ethylene vinyl alcohol (SEVA-C; 50/50wt%) and polycaprolactone (SPCL; 30/70wt%) were studied. SCA and SCA reinforced with 10% hydroxyapatite (HA) showed the highest degree of modification as result of the rfGD treatment. Protein and control solutions were used to incubate with the characterized SBB and, following this, MG63 osteoblast-like osteosarcoma cells were seeded over the surfaces. Cell adhesion and proliferation onto SCA was found to be enhanced for non-treated surfaces and on SCA+10%HA no alteration was brought up by the plasma modification. Onto SCA surfaces, BSA, FN and VN single solutions improved cell adhesion, and this same effect was found upscaled for ternary systems. In addition, plasma treated SEVA-C directed an increase in both adhesion and proliferation comparing to non-treated surfaces. Even though adhesion onto treated and untreated SPCL was quite similar, plasma modification clearly promoted MG63 cells proliferation. Regarding MG63 cells morphology it was shown that onto SEVA-C surfaces the variation of cell shape was primarily defined by the protein system, while onto SPCL it was mainly affected by the plasma treatment.

Lysophospholipid regulates release and activation of latent TGF-beta1 from chondrocyte extracellular matrix.

Transforming growth factor beta-1 (TGF-beta1) is released from the extracellular matrix of rat growth plate chondrocytes and activated by stromelysin-1 (matrix metalloproteinase 3, MMP-3), an enzyme that is stored in matrix vesicles. MMP-3 is released from these extracellular organelles by the direct action of 1alpha,25(OH)2D3 via activation of phospholipase A2 (PLA2), resulting in local production of lysophospholipids and matrix vesicle membrane destabilization. This effect of 1alpha,25(OH)2D3 is greater in matrix vesicles from growth zone chondrocyte cultures and PLA2 activity is higher in the growth zone in vivo, suggesting that it may depend on chondrocyte maturation state in the endochondral lineage. Previous studies have shown that latent TGF-beta1 can be activated by mild detergents in vitro, suggesting that lysophospholipids may act in vivo in a similar manner. To test this hypothesis, we determined if rat costochondral growth plate cartilage cells produce lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) in a maturation state-dependent manner and if LPC or LPE could release and activate latent TGF-beta1 from the extracellular matrix produced by these cells. Rat growth plate chondrocytes produced both lysophospholipids, with growth zone cells producing higher levels of LPE via PLA1, and resting zone cells producing higher levels of LPC via PLA2. LPC and LPE directly increased activation of recombinant human latent TGF-beta1 in a biphasic manner with a peak at 2 microg/ml. Phosphatidylcholine, phosphatidylethanolamine, and LPE plasmalogen (LPEP), but not choline, also activated TGF-beta1. Latent TGF-beta1 incubated with LPC or LPE, but neither lysophospholipid alone, stimulated [3H]-thymidine incorporation of resting zone cells, indicating the TGF-beta1 released was biologically active. LPC and LPE also released TGF-beta1 in a dose- and time-dependent manner when incubated with cell-free extracellular matrices produced by the cells. These results indicate that LPC and LPE have important roles as regulators of rat growth plate chondrocytes by directly and indirectly activating TGF-beta1 stored in the extracellular matrix.

Transforming growth factor-beta1 regulation of resting zone chondrocytes is mediated by two separate but interacting pathways.

Previous studies have shown that transforming growth factor-beta1 (TGF-beta1) stimulates protein kinase C (PKC) via a mechanism that is independent of phospholipase C or tyrosine kinase, but involves a pertussis toxin-sensitive G-protein. Maximal activation occurs at 12 h and requires new gene expression. To understand the signaling pathways involved, resting zone chondrocytes were incubated with TGF-beta1 and PKC activity was inhibited with chelerythrine, staurosporine or H-7. [(35)S]Sulfate incorporation was inhibited, indicating that PKC mediates the effects of TGF-beta1 on matrix production. However, there was little, if any, effect on TGF-beta1-dependent increases in [(3)H]thymidine incorporation, and TGF-beta1-stimulated alkaline phosphatase was unaffected, indicating that these responses to the growth factor are not regulated via PKC. TGF-beta1 caused a dose-dependent increase in prostaglandin E(2) (PGE(2)) production which was further increased by PKC inhibition. The increase was regulated by TGF-beta1-dependent effects on phospholipase A(2) (PLA(2)). Activation of PLA(2) inhibited TGF-beta1 effects on PKC, and inhibition of PLA(2) activated TGF-beta1-dependent PKC. Exogenous arachidonic acid also inhibited TGF-beta1-dependent increases in PKC. The effects of TGF-beta1 on PKC involve genomic mechanisms, but not regulation of existing membrane-associated enzyme, since no direct effect of the growth factor on plasma membrane or matrix vesicle PKC was observed. These results support the hypothesis that TGF-beta1 modulates its effects on matrix production through PKC, but its effects on alkaline phosphatase are mediated by production of PGE(2) and protein kinase A (PKA). Inhibition of PKA also decreases TGF-beta1-dependent proliferation. We have previously shown that PGE(2) stimulates alkaline phosphatase through its EP2 receptor, whereas EP1 signaling causes a decrease in PKC. Thus, there is cross-talk between the two pathways.

Effect of 17 beta-estradiol on chondrocyte membrane fluidity and phospholipid metabolism is membrane-specific, sex-specific, and cell maturation-dependent.

In this study we examined the hypothesis that 17 beta-estradiol exerts both rapid and direct, nongenomic effects of cells in the endochondral pathway. To do this, we used a cell culture model in which chondrocytes at two distinct stages of cell maturation are isolated from the costochondral cartilage of male and female rats, and examined the short-term effect of 17 alpha- and 17 beta-estradiol on [14C]arachidonic acid turnover in the cell layer and phospholipase A2 specific activity in plasma membranes and extracellular matrix vesicles isolated from similarly prepared cultures. In addition, the effect of 17 alpha- and 17 beta-estradiol on plasma membrane and matrix vesicle membrane fluidity was assessed. The effect of hormone on arachidonic acid turnover was rapid, time- and concentration-dependent, stereo-specific, and cell maturation-specific. Only resting zone cells from female rats were affected, and only 17 beta-estradiol elicited a response. Similarly, only female rat resting zone chondrocytes exhibited a change in phospholipase A2 activity after a 24 h exposure to hormone, causing an increase in enzyme activity in the matrix vesicles, but not plasma membranes. When isolated membranes were incubated directly with hormone, membrane fluidity was decreased in both plasma membranes and matrix vesicles isolated from female rat resting zone chondrocyte cultures. This nongenomic effect was dose-dependent and stereo-specific and differentially expressed in the two membrane fractions with respect to time course and magnitude of response. These results support the hypothesis that 17 beta-estradiol has a rapid action on chondrocyte membrane lipid metabolism and suggest that specific membrane components, characteristic of a particular sex and state of cell maturation, are involved in the nongenomic effects of this sex hormone on isolated matrix vesicles and plasma membranes.

Regulation of phospholipase D (PLD) in growth plate chondrocytes by 24R,25-(OH)2D3 is dependent on cell maturation state (resting zone cells) and is specific to the PLD2 isoform.

Many of the effects of 1alpha,25-(OH)2D3 and 24R,25-(OH)2D3 on costochondral chondrocytes are mediated by the protein kinase C (PKC) signal transduction pathway. 1alpha,25-(OH)2D3 activates PKC in costochondral growth zone chondrocytes through a specific membrane receptor (1alpha,25-mVDR), involving rapid increases in diacylglycerol via a phospholipase C (PLC)-dependent mechanism. 24R,25-(OH)2D3 activates PKC in resting zone chondrocytes. Although diacylglycerol is increased by 24R,25-(OH)2D3, PLC is not involved, suggesting a phospholipase D (PLD)-dependent mechanism. Here, we show that resting zone and growth zone cells express mRNAs for PLD1a, PLD1b, and PLD2. Both cell types have PLD activity, but levels are higher in resting zone cells. 24R,25-(OH)2D3, but not 24S,25-(OH)2D3 or 1alpha,25-(OH)2D3, stimulates PLD activity in resting zone cells within 3 min via nongenomic mechanisms. Neither 1alpha,25-(OH)2D3 nor 24R,25-(OH)2D3 affected PLD in growth zone cells. Basal and 24R,25-(OH)2D3-stimulated PLD were inhibited by the PLD inhibitors wortmannin and EDS. Inhibition of phosphatidylinositol 3-kinase (PI 3-kinase), PKC, phosphatidylinositol-specific PLC (PI-PLC), and phosphatidylcholine-specific PLC (PC-PLC) had no effect on PLD activity. Thus, 24R,25-(OH)2D3 stimulates PLD, and PI 3-kinase, PI-PLC and PKC are not involved, whereas PLD is required for stimulation of PKC by 24R,25-(OH)2D3. Pertussis toxin, GDPbetaS, and GTPgammaS had no effect on 24R,25-(OH)2D3-dependent PLD when added to cell cultures, indicating that G-proteins are not involved. These data show that PKC activation in resting zone cells is mediated by PLD and suggest that a functional 24R,25-(OH)2D3-mVDR is required. The results also support the conclusion that the 24R,25-(OH)2D3-responsive PLD is PLD2, since this PLD isoform is G-protein-independent.

Varying Ti-6Al-4V surface roughness induces different early morphologic and molecular responses in MG63 osteoblast-like cells.

Osteoblast response to Ti implants depends not only on the chemistry of the implant but also on the physical properties of the implant surface, such as microtopography and roughness. This study was undertaken to examine early changes in cell morphology and gene expression during the early phase of osteoblast interaction with titanium alloy (Ti-6Al-4V) surfaces of two different roughnesses. MG63 osteoblast-like cells were cultured for 2, 6, 24, and 72 h on smooth (Ra=0.18+/-0.03 microm) and rough (Ra=2.95+/-0.23 microm) Ti-6Al-4V surfaces. Changes in cell proliferation were assessed by measuring cell number after 72 h in culture. Morphological characteristics were observed by scanning electron microscopy after 2, 6, and 24 h of culture. Changes in gene expression for extracellular signal-regulated kinase 2 (Erk2), type I collagen (alpha2[I] collagen), phospholipase C-gamma2 (Plc-gamma2), and beta-actin were measured by RT-PCR after 6 and 24 h in culture. Cell number was significantly higher on the smooth surface. In scanning electron micrographs, cells on smooth Ti-6Al-4V were spherical and raised up from the surface after 2 h in culture. In contrast, cells on the rough surface adopted an irregular, elongated shape that spanned across pits in the surface. At 24 h, cells on the smooth surface had flattened, become elongate, and covered the surface. In contrast, cells on the rough surface appeared more differentiated in shape and the margins of the cells were irregular, with many processes extending out, following the contour of the surface. Of the genes examined, only Erk2 and beta-actin showed a change in expression with surface roughness. Both genes were upregulated (p<0.05) on the rough surface at 6 h. These results indicate that Ti-6Al-4V surface roughness affects osteoblast proliferation, morphology, and gene expression, and that these effects can be measured after periods as short as 2-6 h.

Regulation of protein kinase C by transforming growth factor beta 1 in rat costochondral chondrocyte cultures.

Transforming growth factor beta (TGF-beta) regulates the proliferation and differentiation of chondrocytes; however, the mechanism of TGF-beta signal transduction remains unclear. We examined whether the response to TGF-beta is mediated by protein kinase C activity in chondrocytes at different stages of maturation. The aims were to examine the effect of recombinant human TGF-beta 1 (rhTGF-beta 1) on protein kinase C in rat costochondral chondrocyte cultures; determine the major isoform present; assess the involvement of phospholipase C or tyrosine kinases; determine whether genomic or nongenomic pathways are involved; and test whether these mechanisms differ as a function of the stage of cell maturation. Dose-dependent increases in protein kinase C activity were observed in confluent, fourth-passage cultures of rat costochondral growth zone and resting zone chondrocytes treated with rhTGF-beta 1. In growth zone cells, elevated activity was observed at 12 h and decreased markedly by 24 h. In resting zone cells, elevated activity was observed at 9 h, maximum stimulation occurred at 12 h, and activity returned to baseline levels after 48 h. Immunoprecipitation studies showed protein kinase C alpha is the major isoform present in both untreated and treated cells. Neither the phospholipase C inhibitor, U73122, nor the tyrosine kinase inhibitor, genistein, significantly reduced the protein kinase C response to rhTGF-beta 1. Actinomycin D and cycloheximide, inhibitors of transcription and translation, produced dose-dependent inhibition of rhTGF-beta 1 stimulated protein kinase C activity in both resting zone and growth zone chondrocytes. The time course of activation and insensitivity to U73122 suggest that phospholipase C-mediated events are not involved in rhTGF-beta 1 stimulation of protein kinase C in costochondral chondrocytes. Similarly, because genistein had no effect, tyrosine kinases are not implicated. Rather, the reduction in protein kinase C activity observed when rhTGF-beta 1 is administered along with actinomycin D or cycloheximide indicates that new gene expression and protein synthesis are required for the response. These results indicate that the effect of rhTGF-beta 1 is mediated by protein kinase C; however, it is very slow and may require new protein kinase C production, perhaps via a cytokine cascade. Moreover, the classic mechanism of activation of protein kinase C by phospholipase C was not found, suggesting a novel mechanism of activation. Finally, the effects of rhTGF-beta 1 on protein kinase C are dependent on the state of cell maturation with respect to onset and duration of response.

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.

1,25(OH)2D3 regulates protein kinase C activity through two phospholipid-dependent pathways involving phospholipase A2 and phospholipase C in growth zone chondrocytes.

We have previously shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 1,25(OH)2D3 to stimulate PKC activation. Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of GC cultures with 1,25(OH)2D3 elicited a dose-dependent increase in both inositol-1,4,5-trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D. Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity. Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH)2D3-treated GCs, indicating that neither Gi nor Gs proteins were involved. Phospholipase A2 inhibitors, quinacrine, OEPC (selective for secretory phospholipase A2), and AACOCF3 (selective for cytosolic phospholipase A2), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A2 activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E2, two downstream products of phospholipase A2 action, also increased PKC activity. These results indicate that 1,25(OH)2D3-dependent stimulation of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A2.

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 microm; SLA, 4.1 microm; and TPS, 4.9 microm. 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 beta1 (TGF-beta1) 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-beta1, 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-beta1 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-beta1 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-beta1 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-beta1, 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-beta1 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.

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, 1alpha-CH2OH-3beta-25D3; 3a, 1alpha-CH2OH-3beta-20-epi-22-oxa-25D3; and 3b, 1beta-CH2OH-3alpha-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.

The synergistic effects of vitamin D metabolites and transforming growth factor-beta on costochondral chondrocytes are mediated by increases in protein kinase C activity involving two separate pathways.

Transforming growth factor-beta (TGFbeta), as well as the vitamin D3 metabolites 1,25-dihydroxyvitamin D3 (1,25) and 24,25-dihydroxyvitamin D3 (24,25), regulate chondrocyte differentiation and maturation during endochondral bone formation. Both the growth factor and secosteroids also affect protein kinase C (PKC) activity, although each has its own unique time course of enzyme activation. Vitamin D3 metabolite effects are detected soon after addition to the media, whereas TGFbeta effects occur over a longer term. The present study examines the interrelation between the effects of 1,25, 24,25, and TGFbeta on chondrocyte differentiation, matrix production, and proliferation. We also examined whether the effect is hormone-specific and maturation-dependent and whether the effect of combining hormone and growth factor is mediated by PKC. This study used a chondrocyte culture model developed in our laboratory that allows comparison of chondrocytes at two stages of differentiation: the more mature growth zone (GC) cells and the less mature resting zone chondrocyte (RC) cells. Only the addition of 24,25 with TGFbeta showed synergistic effects on RC alkaline phosphatase-specific activity (ALPase). No similar effect was found when 24,25 plus TGFbeta was added to GC cells or when 1,25 plus TGFbeta were added to GC or RC cells. The addition of 1,25 plus TGFbeta and 24,25 plus TGFbeta to GC and RC cells, respectively, produced a synergistic increase in [35S]sulfate incorporation and had an additive effect on [3H]thymidine incorporation. To examine the signal transduction pathway involved in producing the synergistic effect of 24,25 and TGFbeta on RC cells, the level of PKC activity was examined. Addition of 24,25 and TGFbeta for 12 h produced a synergistic increase in PKC activity. Moreover, a similar effect was found when 24,25 was added for only the last 90 min of a 12-h incubation. However, a synergistic effect could not be found when 24,25 was added for the last 9 min or the first 90 min of incubation. To further understand how 24,25 and TGFbeta may mediate the observed synergistic increase in PKC activity, the pathways potentially leading to activation of PKC were examined. It was found that 24,25 affects PKC activity through production of diacylglycerol, not through activation of G protein, whereas TGFbeta only affected PKC activity through G protein. The results of the present study indicate that vitamin D metabolites and TGFbeta produced a synergistic effect that is maturation-dependent and hormone-specific. Moreover, the synergistic effect between 24,25 and TGFbeta was mediated by activation of PKC through two parallel pathways: 24,25 through diacylglycerol production and TGFbeta through G protein activation.

Arachidonic acid directly mediates the rapid effects of 24,25-dihydroxyvitamin D3 via protein kinase C and indirectly through prostaglandin production in resting zone chondrocytes.

Prior studies have shown that 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] plays a major role in resting zone chondrocyte differentiation and that this vitamin D metabolite regulates both phospholipase A2 and protein kinase C (PKC) specific activities. Arachidonic acid is the product of phospholipase A2 action and has been shown in other systems to affect a variety of cellular functions, including PKC activity. The aim of the present study was to examine the interrelationship between arachidonic acid and 24,25-(OH)2D3 on markers of proliferation, differentiation, and matrix production in resting zone chondrocytes and to characterize the mechanisms by which arachidonic acid regulates PKC, which was shown previously to mediate the rapid effects of 24,25-(OH)2D3 and arachidonic acid on these cells. Confluent, fourth passage resting zone cells from rat costochondral cartilage were used to evaluate these mechanisms. The addition of arachidonic acid to resting zone cultures stimulated [3H]thymidine incorporation and inhibited the activity of alkaline phosphatase and PKC, but had no effect on proteoglycan sulfation. In contrast, 24,25-(OH)2D3 inhibited [3H]thymidine incorporation and stimulated alkaline phosphatase, proteoglycan sulfation, and PKC activity. In cultures treated with both agents, the effects of 24,25-(OH)2D3 were reversed by arachidonic acid. The PKC isoform affected by arachidonic acid was PKCalpha; cytosolic levels were decreased, but membrane levels were unaffected, indicating that translocation did not occur. Arachidonic acid had a direct effect on PKC in isolated plasma membranes and matrix vesicles, indicating a nongenomic mechanism. Plasma membrane PKCalpha was inhibited, and matrix vesicle PKCzeta was stimulated; these effects were blocked by 24,25-(OH)2D3. Studies using cyclooxygenase and lipoxygenase inhibitors indicate that the effects of arachidonic acid are due in part to PG production, but not to leukotriene production. This is supported by the fact that H8-dependent inhibition of protein kinase A, which mediates the effects of PGE2, had no effect on the direct action of arachidonic acid but did mediate the role of arachidonic acid in the cell response to 24,25-(OH)2D3. Diacylglycerol does not appear to be involved, indicating that phospholipase C and/or D do not play a role. Gamma-linolenic acid, an unsaturated precursor of arachidonic acid, elicited a similar response in matrix vesicles but not plasma membranes, whereas palmitic acid, a saturated fatty acid, had no effect. These data suggest that arachidonic acid may act as a negative regulator of 24,25-(OH)2D3 action in resting zone chondrocytes.

The effect of prostaglandin E2 on costochondral chondrocyte differentiation is mediated by cyclic adenosine 3',5'-monophosphate and protein kinase C.

Recent studies indicate that vitamin D metabolites exert rapid effects on growth plate chondrocytes via changes in PG production and protein kinase C (PKC) activity. This suggests that these two products of vitamin D action may be interrelated. To test this hypothesis, we examined the effect of PGE2 on rat costochondral resting zone and growth zone cartilage cells and determined whether the effects of PGE2 are mediated by changes in the level of cAMP and/or PKC activity, whether there is a relationship between cAMP production and PKC activity, and whether cell maturation-specific effects are involved. Confluent, fourth passage resting zone and growth zone cartilage cell cultures were incubated in DMEM containing 10% FBS, 50 microg/ml vitamin C, and 1% antibiotics. The PGE2 concentration was varied from 0.007-15 ng/ml. Low concentrations of PGE2 caused a dose-dependent increase in cell number and [3H]thymidine incorporation and stimulated alkaline phosphatase specific activity. These effects were comparable in resting zone and growth zone cartilage cells at the same PGE2 concentrations. At higher concentrations, PGE2 caused a general increase in the synthesis of collagenase-digestible protein and noncollagenase-digestible protein in resting zone cartilage cells and of collagenase-digestible protein in growth zone cartilage cells, resulting in a net increase in the percent collagen synthesis for both cell types. cAMP production was increased over the entire range of chondrocyte response. Prevention of cAMP metabolism with the protein kinase A inhibitors H-8 and H-89 blocked the PGE2-dependent inhibition of PKC in resting zone cartilage cells in a dose-dependent manner. H-8 alone had no effect on PKC in resting zone cartilage cells, but stimulated PKC activity in growth zone cartilage cells; H-89 alone stimulated PKC activity in resting zone cartilage cells. These results suggest that low levels of PGE2 promote differentiation, whereas high doses promote an anabolic response; PGE2 increases cAMP production and PKC activity in a cell maturation-dependent manner; PGE2 exerts its effects via cAMP production and PKC activity; and regulation of PGE2-dependent PKC is via cAMP.

Estrogen-dependent rapid activation of protein kinase C in estrogen receptor-positive MCF-7 breast cancer cells and estrogen receptor-negative HCC38 cells is membrane-mediated and inhibited by tamoxifen.

We examined protein kinase C (PKC) in the regulation of breast cancer cells by estrogen. Estrogen receptor (ER)- positive (+) MCF-7 and ER-negative (-) HCC38 cells were treated with 17 beta-estradiol (E(2)) or E(2)-BSA, which cannot enter the cell. E(2) and E(2)-BSA rapidly increased PKC-alpha in both cells via phosphatidylinositol-dependent phospholipase C and G protein, but not phospholipase A(2) or arachidonic acid. In MCF-7 cells, E(2) and E(2)-BSA had comparable effects, maximal at 90 min. In HCC38 cells, PKC was maximal at 9 min, with E(2)-BSA more than E(2). Tamoxifen blocked estrogen-dependent PKC in MCF-7 cells and reduced it in HCC38 cells. ER-antagonist ICI 182780, ER-agonist diethylstilbestrol, and antibodies to ER alpha and ER beta had no effect. E(2) stimulated [(3)H]thymidine incorporation in MCF-7 only; E(2)-BSA had no effect. Tamoxifen did not alter E(2)-dependent increases in MCF-7 cells, whereas ICI 182780 reduced DNA synthesis in control and E(2)-treated cultures. PKC activity was positively correlated with tumor severity in 133 breast cancer specimens and was greater in ER(-) tumors. Tamoxifen treatment reduced recurrence, and recurrent tumors had higher PKC activity. This indicates that E(2) rapidly increases PKC activity via membrane pathways not involving ER alpha or ER beta and suggests that tamoxifen works by reducing PKC activity through non-ER alpha/ER beta-dependent mechanisms.

1alpha,25-dihydroxyvitamin D(3) and 24R,25-dihydroxyvitamin D(3) modulate growth plate chondrocyte physiology via protein kinase C-dependent phosphorylation of extracellular signal-regulated kinase 1/2 mitogen-activated protein kinase.

Membrane-mediated increases in protein kinase C (PKC) activity and PKC-dependent physiological responses of growth plate chondrocytes to vitamin D metabolites depend on the state of endochondral maturation; 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] regulates growth zone (GC) cells, whereas 24R,25-(OH)(2)D(3) regulates resting zone (RC) cells. Different mechanisms, including protein kinase A signaling, mediate the effects of 1alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) on PKC, suggesting that different mechanisms may also regulate any MAPK involvement in the physiological responses. This study used confluent cultures of rat costochondral chondrocytes as a model. 1alpha,25-(OH)(2)D(3) stimulated MAPK specific activity in GC in a time- and dose-dependent manner, evident within 9 min. 24R,25-(OH)(2)D(3) stimulated MAPK in RC; increases were dose dependent, occurred after 9 min, and were greatest at 90 min. In both cells the effect was due to ERK1/2 activation (p42 > p44 in GC; p42 = p44 in RC). MAPK activation was dependent on PKC, but not protein kinase A. The effect of 1alpha,25-(OH)(2)D(3) required phospholipase C, and the effect of 24R,25-(OH)(2)D(3) required phospholipase D. Inhibition of cyclooxygenase activity reduced the effect of 1alpha,25-(OH)(2)D(3) on MAPK in GC and enhanced the effect of 24R,25-(OH)(2)D(3) in RC. Based on MAPK inhibition with PD98059, ERK1/2 MAPK mediated the effect of 24R,25-(OH)(2)D(3) on [(3)H]thymidine incorporation and [(35)S]sulfate incorporation by RC, but only partially mediated the effect of 1alpha,25-(OH)(2)D(3) on GC. ERK1/2 was not involved in the regulation of alkaline phosphatase specific activity by either metabolite. This paper supports the hypothesis that 1alpha,25-(OH)(2)D(3) regulates the physiology of GC via rapid membrane-mediated signaling pathways, and some, but not all, of the response to 1alpha,25-(OH)(2)D(3) is via the ERK family of MAPKs. In contrast, 24R,25-(OH)(2)D(3) exerts its effects on RC via PKC-dependent MAPK. Whereas 1alpha,25-(OH)(2)D(3) increases MAPK activity via phospholipase C and increased prostaglandin production, 24R,25-(OH)(2)D(3) increases MAPK via phospholipase D and decreased prostaglandin production. The cell specificity, metabolite stereospecificity, and the dependence on PKC argue for the participation of membrane receptors for 1alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3) in the regulation of ERK1/2 in the growth plate.

Metalloproteinase activity in growth plate chondrocyte cultures is regulated by 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) and mediated through protein kinase C.

During endochondral development, growth plate chondrocytes must remodel their matrix in a number of ways as they differentiate and mature. In previous studies, we have shown that matrix metalloproteinases (MMPs) extracted from matrix vesicles can extensively degrade aggrecan and that this is modulated by vitamin D metabolites in a manner involving protein kinase C (PKC). Matrix vesicles represent only a small component of the extracellular matrix, however, and it is unknown if the total metalloproteinase complement, including the MMPs and aggrecanases in the culture, is also regulated in a similar way. This study tested the hypothesis that vitamin D metabolites regulate the level of metalloproteinase activity in growth plate chondrocytes via a PKC-dependent mechanism and play a role in partitioning this proteinase activity between the media and cell layer (cells+matrix) in these cultures. To do this, resting zone cells (RC) were treated with 10(-9)-10(-7) M 24R,25-(OH)(2)D(3), while growth zone cells (GC) were treated with 10(-10)-10(-8) M 1alpha,25-(OH)(2)D(3). Cultures of both cell types were also treated with the PKC inhibitor chelerythrine in the presence and absence of vitamin D metabolites. At harvest, the media were either left untreated or treated to destroy metalloproteinase inhibitors, while enzyme activity in the cell layers was extracted with buffered guanidine and then treated like the media to destroy metalloproteinase inhibitors. Neutral metalloproteinase (aggrecan-degrading activity) activity was assayed on aggrecan-containing polyacrylamide gel beads and collagenase activity was measured on telopeptide-free type I collagen. Neutral metalloproteinase activity was found primarily in the cell layer of both cell types; however, activity was greater in extracts of GC cell layers. No collagenase activity could be detected in RC extracts until the metalloproteinase inhibitors were destroyed. In contrast, extracts of GC cell layers contained measurable activity without removing the inhibitors, and destroying the inhibitors resulted in a greater than two-fold increase in activity. No collagenase activity was found in the media of either cell type. 24,25-(OH)(2)D(3) caused a dose-dependent increase in neutral metalloproteinase activity in extracts of RC cells, but had no effect on collagenase activity. In contrast, 1,25-(OH)(2)D(3) caused a dose-dependent decrease in collagenase activity in extracts of GC cells, but had no effect on neutral metalloproteinase activity. In both cases, the effect of the vitamin D metabolite was mediated through the activation of PKC. These results support the hypothesis that metalloproteinases are involved in regulating the bulk turnover of collagen and aggrecan in growth plate chondrocytes and that the amount of metalloproteinase activity found is a function of the cell maturation state. Furthermore, 83-93% of neutral metalloproteinase activity and 100% of collagenase activity is localized to the cell layer. Moreover, the regulation of metalloproteinase activity by 1,25-(OH)(2)D(3) and 24,25-(OH)(2)D(3) involves a PKC-dependent pathway that is controlled by the target cell-specific vitamin D metabolite.

Decline of signal transduction by phospholipase C gamma 1 in IMR 90 human diploid fibroblasts at high population doubling levels.

During cellular senescence in vitro, the cells do not respond mitogenically to serum growth factors at high population doubling levels. Phospholipase C activity in low PDL IMR 90 cells showed a 4.7-fold stimulation in response to 10% serum compared to 3.3-fold in high PDL cells when measured in whole cell extracts. Immunoaffinity purified tyrosine phosphorylated protein fraction showed a greater increase (5.2-fold) in phospholipase C activity in low PDL than high PDL cells (2.1-fold) in response to serum. Serum stimulated PLC gamma 1 activity was diminished in high PDL cells. Immunokinase assay of PLC gamma 1 immunoprecipitates from serum stimulated IMR 90 fibroblasts suggested that diminished enzymatic activity in high PDL cells is not due to less receptor coupled tyrosine phosphorylated PLC gamma 1 enzyme. Serum stimulated [3H]thymidine incorporation into DNA declined in parallel with the activity of PLC gamma 1, suggesting that its activation might play significant roles in this in vitro model for cellular senescence.

The kinase insert domain of colony stimulating factor-1 receptor is dispensable for CSF-1 induced phosphatidylcholine hydrolysis.

Mouse NIH 3T3 fibroblasts transfected with human colony stimulating factor-1 receptor produced diacylglycerol in response to CSF1 and this correlated with elevated phosphatidylcholine hydrolyzing activity measured in an in vitro assay. Treatment of cells with the isoflavone derivative genistein attenuated PC hydrolysis in vitro suggesting a role for CSF1R tyrosine kinase activity. A CSF1R mutant lacking 67 amino acids of the kinase insert domain, which may affect the association of receptor with certain substrates, stimulated PC hydrolysis in response to CSF1. Coupling to PC hydrolysis is likely a general property of CSF1R and the kinase insert domain is dispensable for this activity.