Evidence for metalloproteinase and metalloproteinase inhibitor imbalance in human osteoarthritic cartilage.

Cartilage specimens from tibial plateaus, obtained from 13 osteoarthritic (OA) patients and seven controls, were selected from three regions: zone A, center of fibrillated area; zone B, area adjacent to fibrillation, and zone C, remote region of plateau. Acid and neutral metalloproteinases and tissue inhibitor of metalloproteinase (TIMP) were extracted with 2 M guanidine. Methods were developed to selectively destroy either proteinases or TIMP to prevent cross-reaction during assay. Acid and neutral proteinases were elevated approximately 150% in OA; TIMP was elevated approximately 50%. A positive correlation (r = 0.50) was found between acid and neutral proteinase activities in OA, but not in controls. Both proteinases were elevated two-to threefold in zones A, B, and C. However, the self-active form of the acid metalloproteinase was elevated only in zones A and B (200%); it correlated well with the Mankin scores, whereas the total activities did not. TIMP was elevated (50%) only in zones A and B. Both the proteinase levels and the Mankin score were elevated to a greater extent in the medial, than in the lateral, compartment. Titration of TIMP against the two metalloproteinases indicates that there is a small excess of inhibitor over enzymes in normal cartilage. In OA, TIMP does not increase to the same extent as the proteinases; the resultant excess of proteinases over TIMP may contribute to cartilage breakdown.

Localization of collagenase in the growth plate of rachitic rats.

In the transition from proliferation to hypertrophic cell zones in the growth plate, there is an increase in chondrocyte volume and a corresponding decrease in collagen content to accommodate the enlarging cells. It is postulated that collagenase accounts for this collagen loss. To test this hypothesis, tibial growth plates were obtained from normal rats, rachitic rats deficient in vitamin D and phosphate, and rats after 48 and 72 h of healing from rickets. Collagenase was quantitated by a pellet assay based on the release of solubilized collagen from the endogenous insoluble collagen in the tissue homogenates. A fourfold greater collagen release and a concomitant sixfold greater hypertrophic cell volume were measured in rachitic growth plates compared with normal age-matched controls. During healing of rickets, collagenase activity and hypertrophic cell volume returned almost to control levels. Rachitic growth plates were dissected into the juxtaepiphyseal 1/3 and the juxtametaphyseal 2/3. The latter portion contained greater than 95% of the hypertrophic cells and 86% of the collagenase. The collagen-degrading activity was extracted from this region and was shown to be a true collagenase by its production of typical A fragments of tropocollagen produced by collagenase action. The enzyme was activated by aminophenylmercuric acetate and trypsin and was inhibited by EDTA, 1,10-phenanthroline, and a tissue inhibitor of metalloproteinases from human articular cartilage. Inhibitors of aspartic, cysteine, and serine proteases had no effect. Micropuncture fluids aspirated from rachitic cartilage contained latent collagenase activity, indicating an extracellular localization. Negative tests for hemoglobin in the rachitic cartilage samples indicated that there was no contamination by capillaries and that this was not a source of collagenase. It is concluded that extracellular collagenase accounts for the loss of cartilage matrix in the hypertrophic zone, and that this process may be distinct from that of capillary invasion.

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.

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.

Evidence that interleukin-1, but not interleukin-6, affects costochondral chondrocyte proliferation, differentiation, and matrix synthesis through an autocrine pathway.

Although the effects of interleukin-1 (IL-1) and interleukin-6 (IL-6) on articular cartilage chondrocytes have been reported, little is known concerning the effects of these cytokines on growth plate chondrocytes. In this study, we examined the effect of IL-1 alpha, IL-1 beta, and IL-6 on growth plate chondrocyte proliferation, differentiation, and matrix production as a function of cell maturation and examined the ability of these cells to produce IL-1 alpha and IL-1 beta. Confluent fourth passage cultures of rat costochondral resting zone and growth zone chondrocytes were treated with 0-100 ng/ml of IL-1 alpha, IL-1 beta, or IL-6 for 24 h and then assayed for [3H]-thymidine incorporation, alkaline phosphatase specific activity, [35S]-sulfate incorporation, and percent collagen production. Neutralizing polyclonal antibodies were used to confirm the specificity of response to each cytokine. Treatment of resting zone cells with IL-1 alpha produced a significant, dose-dependent decrease in [3H]-thymidine incorporation, while similarly treated growth zone cells were unaffected by treatment with this cytokine. IL-1 alpha also stimulated alkaline phosphatase specific activity and inhibited [35S]-sulfate incorporation by resting zone chondrocytes, but had no affect on growth zone chondrocytes. When collagen production was examined, it was observed that IL-1 alpha had a stimulatory affect on growth zone cells but no affect on resting zone cells. When the effect of IL-1 beta was examined, it was observed that this cytokine inhibited [3H]-thymidine incorporation by resting zone cells and stimulated isotope incorporation in growth zone cells. IL-1 beta also stimulated alkaline phosphatase specific activity and inhibited [35S]-sulfate incorporation by resting zone chondrocytes but had no affect on growth zone chondrocytes. In contrast to IL-1 alpha, IL-1 beta stimulated collagen production by resting zone cells but not growth zone cells. IL-6 had no affect on any of the parameters measured in either cell type. When cytokine production was measured, it was found that IL-1 alpha was produced by both cell types, while IL-1 beta was produced only by resting zone cells. Resting zone cells secreted both IL-1 alpha and IL-1 beta into the media, but 75% of the total cytokine produced by these cells was retained in the cell layer. In contrast, growth zone cells did not secrete measurable IL-1 alpha into the media. These results suggest that IL-1 alpha and IL-1 beta target resting zone cells, inducing them to differentiate and acquire a phenotype characteristic of the more mature growth zone cells. Moreover, resting zone chondrocytes produce both IL-1 alpha and IL-1 beta, suggesting the possibility of an autocrine effect of these cytokines on the cells.

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.

Activation of latent transforming growth factor beta1 by stromelysin 1 in extracts of growth plate chondrocyte-derived matrix vesicles.

Previous studies have shown that matrix vesicles isolated from cultures of costochondral growth zone chondrocytes and treated with 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] can activate recombinant human latent transforming growth factor beta1 (rhTGF-beta1). It is unknown what enzyme or other factor in the extracellular organelles is responsible for the activation. This study tested the hypothesis that enzymes present in matrix vesicles can activate latent TGF-beta1 and that this is regulated by 1alpha,25(OH)2D3. To do this, we examined the ability of matrix vesicle extracts to activate small latent rhTGF-beta1. In addition, enzymes previously determined to be present in matrix vesicles were screened for their ability to activate small latent rhTGF-beta1. Recombinant human matrix metalloproteinase 2 (rhMMP-2; 72 kDa gelatinase), rhMMP-3 (stromelysin 1), purified human plasminogen, and purified urokinase (plasminogen activator) were each tested at varying concentrations. To assess the role of cell maturation, we used a cell culture model in which chondrocytes are derived from two distinct zones of rat costochondral cartilage, the resting zone and the growth zone. Matrix vesicles were isolated from these cultures and then tested. The results showed that extracts of matrix vesicles produced by both growth zone and resting zone chondrocytes were able to activate small latent rhTGF-beta1. The effects were dose and time dependent, with greater activity being found in extracts of matrix vesicles from the growth zone chondrocyte cultures. Only rhMMP-3 was able to activate small latent rhTGF-beta1, indicating that stromelysin-1, but not MMP-2, plasminogen, or urokinase, was involved. As observed in the extracts, the effect of rhMMP-3 was time and dose dependent. When anti-MMP-3 antibody was added to matrix vesicle extracts from both cell types, activation of small latent rhTGF-beta1 was dose-dependently blocked. Neither 1alpha,25(OH)2D3 nor 24R,25(OH)2D3 had a direct effect on activation of small latent rhTGF-beta1 by the extracts. However, when intact matrix vesicles were treated with 1alpha,25(OH)2D3, their ability to activate small latent rhTGF-beta1 was increased. Inhibition of phospholipase A2 with quinacrine blocked the 1alpha,25(OH)2D3-dependent effect. These results suggest that the ability of 1alpha,25(OH)2D3-treated matrix vesicles to activate small latent TGF-beta1 is via action of the secosteroid on the matrix vesicle membrane, not on the enzymes responsible for activating latent TGF-beta1. Because matrix vesicles isolated from growth zone chondrocytes have been shown to contain increased phospholipase A2 activity after treatment with 1alpha,25(OH)2D3, it is likely that this secosteroid promotes loss of membrane integrity through phospholipase A2-dependent formation of lysophospholipids, resulting in the release of MMP-3 into the matrix, where latent TGF-beta1 is stored. Taken together, the results of the current study show that matrix vesicles produced by growth plate chondrocytes contain MMP-3, that this enzyme is at least partially responsible for activation of small latent TGF-beta1 in the matrix, and that 1alpha,25(OH)2D3 regulates MMP release from matrix vesicles.

Enhancement of osteoinduction by vitamin D metabolites in rachitic host rats.

Diaphyseal bone from normal Sprague-Dawley rats was delipidated in chloroform-methanol and demineralized in 0.6 N HCl at 4 degrees C. The bones were then implanted for 7-28 days into rats made rachitic by a low-phosphate, vitamin D-deficient diet (VDP-) for 3 weeks. Bones from VDP- and normal rats were also implanted into normal hosts. When normal rats were used as the host environment, a consistent sequence of cartilage induction and bone formation was observed. Demineralized rachitic bone (RB) implanted into normal host rats resulted in cartilage and bone induction similar to that seen for normal bone (NB) implants. Transmission electron microscopy of RB in normal hosts revealed morphologically normal chondrocytes and cartilage matrix with normal mineralization. In contrast, implantation of NB in VDP- hosts resulted in delayed chondrogenesis and lack of calcification. Furthermore, similar results were observed when RB was implanted into VDP- hosts. Treatment of VDP- hosts with either 1 alpha-hydroxyvitamin D3 or 24,25-dihydroxyvitamin D3 did not accelerate the sequential appearance of precartilage or cartilage. However, 24,25-(OH)2D3 administered alone or in combination with 1 alpha-OHD3 significantly increased the amount of calcified cartilage observed at 2 weeks postimplantation compared to implants from either untreated VDP-hosts or those treated only with 1 alpha-OHD3. New bone formation was observed at 4 weeks postimplantation in all vitamin D-treated groups as determined by von Kossa staining or direct electron microscope examination. There was no apparent difference in the quantitative or qualitative bone formed within the various vitamin D-treated groups. Serum calcium and phosphorus levels were lower and alkaline phosphatase levels were higher in VDP- hosts compared with normal animals or those treated with vitamin D metabolites. The results of this study show a reduction in the capacity of progenitor cells in VDP- rat hosts to respond to osteoinductive factor(s). This impaired response appears to be corrected by vitamin D metabolites.

Osteosarcoma hybrids can preferentially target alkaline phosphatase activity to matrix vesicles: evidence for independent membrane biogenesis.

Alkaline phosphatase is the marker enzyme for matrix vesicles, extracellular organelles that play a major role in primary bone formation and calcification. Recently, we developed osteosarcoma x fibrosarcoma hybrids in which alkaline phosphatase expression was greatly reduced, a phenomenon known as extinction. In the present study, we used to cell hybrids, LTA-1 and LTA-5, constructed from a human osteoblast-like osteosarcoma. TE85, and a mouse fibrosarcoma, La-t-, to examine the differential distribution of alkaline phosphatase between matrix vesicles and the plasma membrane, postulated to be the parent membrane from which matrix vesicles are derived. While alkaline phosphatase in plasma membranes was extinguished, enzyme activity in matrix vesicles from LTA-1 hybrid cells was 34.2% of that present in matrix vesicles from the TE85 parent cells and 200 times that found in La-t- matrix vesicles. Matrix vesicles from LTA-5 had alkaline phosphatase levels similar to La-t-. When other membrane enzymes (phospholipase A2, 5'-nucleotidase, and Na+/K+ ATPase) were examined, hybrid matrix vesicle and plasma membrane levels were similar to those of TE85 and significantly higher than in La-t- membrane fractions. Northern analysis detected mRNA for alkaline phosphatase in TE85 cells, but not in the hybrids or La-t- cells. In contrast, reverse transcription-polymerase chain reaction (RT-PCR) revealed alkaline phosphatase mRNA in the hybrid cells, but at very low levels. Taken together, the data indicate that regulation of plasma membrane and matrix vesicle alkaline phosphatase is independent and suggest that matrix vesicle biogenesis is independent and distinct from that of plasma membrane biogenesis. Analysis of 1B- and 1L-type alkaline phosphatase mRNA by RT-PCR showed that alternate promoter usage of the alkaline phosphatase gene was not responsible for the differential localization of this enzyme in matrix vesicle. Thus, it is likely that matrix vesicle and plasma membrane alkaline phosphatase are regulated differently at a post-transcriptional level.

Interleukin-1alpha and beta in growth plate cartilage are regulated by vitamin D metabolites in vivo.

Matrix remodeling plays a prominent role in growth plate calcification. Since interleukin-1 (IL-1) has been implicated in stimulating proteinase production and inhibiting matrix synthesis in articular cartilage, we examined whether IL-1 was present in growth plate and whether the vitamin D metabolites, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 1,25) and 24,25(OH)2D3 (24,25), regulate the level of IL-1 found in this tissue. Sprague-Dawley rats were placed on normal (Normal rats) or rachitogenic diet (-VDP rats). The -VDP rats were either left untreated, injected 24 h prior to euthanasia with 24,25 (-VDP+24,25 rats) or 1,25 (-VDP+1,25 rats), or were given ergocalciferol (Ergo rats) orally, 48 h prior to euthanasia. Growth plates were harvested and extracted in buffer containing 1 M guanidine. IL-1 activity was measured by adding authentic cytokine or growth plate extracts to cultures of lapine articular cartilage and assaying release of glycosaminoglycans (GAGs) and changes in collagenase and neutral metalloproteinase activity. Neutralization of activity in the extracts was performed using polyclonal antisera to IL-1alpha or IL-1beta. An ELISA was used to determine levels of IL-1alpha and beta in the extracts. All extracts contained IL-1alpha and beta, as determined by ELISA. Levels of IL-1beta, but not IL-1alpha, were affected by the vitamin D status of the animal. Extracts from -VDP+24,25 animals contained significantly more IL-1beta than any of the other treatment groups, with the level found in these animals being 3-fold higher than normal and 2-fold higher than -VDP. Extracts were also tested in the bioassay to determine the level of active cytokine present. All growth plate extracts contained activity which altered GAG and proteinase release by lapine articular cartilage. Extracts from -VDP-, -VDP+1,25-, and -VDP+Ergo-treated rats stimulated a 40% increase in glycosaminoglycan release compared with extracts from normal rats. In contrast, extracts from -VDP+24,25-treated rats stimulated a 300% increase in glycosaminoglycan release. Both collagenase and neutral metalloproteinase activity of lapine cartilage were increased after incubation with the growth plate extracts. Collagenase activity was significantly increased 8- to 13-fold by the addition of extracts from -VDP-, -VDP+24,25-, or -VDP+1,25-treated animals. Neutral metalloproteinase activity was similarly increased by 4- to 10-fold. To characterize this activity further, growth plate extracts were incubated with neutralizing antibody to IL-1alpha or beta prior to addition to the lapine articular cartilage cultures. When antibodies were used separately, only partial inhibition was observed; incubation with both antibodies blocked 25% of the glycosaminoglycan release observed without antibody and greater than 80% of the enzyme activity released by the articular cartilage cultures. The results of this study show that growth plate cartilage contains both IL-1alpha and beta and indicate that vitamin D regulates the level of IL-1 in this tissue.

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.

The preovulatory increase in ovarian collagenase activity in the rat is independent of prostaglandin production.

In the present study, we have examined the role of gonadotropins and prostaglandins in the preovulatory increase of ovarian collagenase activity in the rat. Whole ovaries of immature PMSG-primed rats (20 IU) were removed before and 8 h after the rats were treated with human (h) CG, Nembutal, and/or indomethacin. The ovaries were homogenized in a solution containing Triton X-100 (0.25%) and centrifuged. Collagenase was extracted by resuspending the pellets in buffer containing 100 mM CaCl2, heating to 60 C for 6 min, and centrifuging. The supernatants were treated with dithiothreitol (2 mM) and iodoacetamide (5 mM) to inactivate collagenase inhibitors. Collagenase activity was measured as the percent digestion of 3H-type I collagen/100 microliters aliquot of ovarian sample. At zero time (52 h after PMSG), ovarian collagenase activity was 4.2 +/- 1.2% digestion (mean +/- SEM, n = 3). In ovaries collected 8 h after the endogenous LH surge or 8 h after the administration of 10 IU hCG at time zero, collagenase activity rose to 19.6 +/- 2.1 (n = 6) and 22.5 +/- 1.7% digestion (n = 11), respectively. Indomethacin (1.5 mg/100 g BW) administered 30 min after hCG, produced no change in collagenase activity (24.8 +/- 2.5% digestion, n = 7) although the expected increase in ovarian prostaglandin E after hCG treatment was blocked. When the endogenous LH surge was blocked with Nembutal (3 mg/100 g BW), collagenase activity in 8-h ovaries was 6.8 +/- 1.1% digestion (n = 10). The Nembutal block of the preovulatory collagenase increase was overcome by administration of hCG (8-h ovarian enzyme activity = 22.7 +/- 3.2% digestion, n = 8). These observations demonstrate that hCG stimulates ovarian collagenase activity and that this stimulation is not dependent on prostaglandin synthesis.

Treatment of resting zone chondrocytes with 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3] induces differentiation into a 1,25-(OH)2D3-responsive phenotype characteristic of growth zone chondrocytes.

Vitamin D3 metabolites affect the proliferation and differentiation of cartilage cells. Previous reports have shown that rat costochondral cartilage chondrocytes isolated from the growth zone (GC) respond to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], whereas those from the resting zone (RC) respond to 24,25-(OH)2D3. The aim of the present study was to determine whether 24,25-(OH)2D3 induces differentiation of RC cells into a 1,25-(OH)2D3-responsive GC phenotype. To do this, confluent, fourth passage RC chondrocytes were pretreated for 24, 36, 48, 72, and 120 h with 10(-7) M 24,25-(OH)2D3. The medium was then replaced with new medium containing 10(-10) to 10(-8) M 1,25-(OH)2D3, and the cells were incubated for an additional 24 h. At harvest, DNA synthesis was measured as a function of [3H]thymidine incorporation; cell maturation was assessed by measuring alkaline phosphatase (ALPase) specific activity. Incorporation of [3H]uridine was used as a general indicator of RNA synthesis. Matrix protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and collagenase-nondigestible protein (NCP) as well as 35SO4 incorporation into proteoglycans. When RC cells were pretreated for 24 h with 24,25-(OH)2D3, they responded like RC cells that had received no pretreatment; further treatment of these cells with 1,25-(OH)2D3 had no effect on ALPase, proteoglycan, or NCP production, but CDP production was inhibited. However, when RC cells were pretreated for 36-120 h with 24,25-(OH)2D3, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase, CDP, and proteoglycan synthesis, with no effect on NCP production. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. To determine whether these responses were specific to chondrocytes in the endochondral pathway, cells were isolated from the xiphoid process, a hyaline cartilage. In these cells, 1,25-(OH)2D3 inhibited ALPase, whereas 36 h of pretreatment with 24,25-(OH)2D3 caused these cells to lose their response to 1,25-(OH)2D3. These results indicate that 24,25-(OH)2D3 can directly regulate the differentiation and maturation of RC chondrocytes into GC chondrocytes, as evidenced by increased responsiveness to 1,25-(OH)2D3. 24,25-(OH)2D3 also promotes differentiation of cells derived from xiphoid cartilage, resulting in the loss of 1,25-(OH)2D3 responsiveness. These observations support the hypothesis that 24,25-(OH)2D3 plays a significant role in cartilage development.

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.