Growth Zone Chondrocyte Cultures Research Articles

Lysophospholipid regulates release and activation of latent TGF-β1 from chondrocyte extracellular matrix

Transforming growth factor beta-1 (TGF-β1) 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 1α,25(OH) 2D 3 via activation of phospholipase A 2 (PLA 2), resulting in local production of lysophospholipids and matrix vesicle membrane destabilization. This effect of 1α,25(OH) 2D 3 is greater in matrix vesicles from growth zone chondrocyte cultures and PLA 2 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-β1 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-β1 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 PLA 1, and resting zone cells producing higher levels of LPC via PLA 2. LPC and LPE directly increased activation of recombinant human latent TGF-β1 in a biphasic manner with a peak at 2 μg/ml. Phosphatidylcholine, phosphatidylethanolamine, and LPE plasmalogen (LPEP), but not choline, also activated TGF-β1. Latent TGF-β1 incubated with LPC or LPE, but neither lysophospholipid alone, stimulated [ 3H]-thymidine incorporation of resting zone cells, indicating the TGF-β1 released was biologically active. LPC and LPE also released TGF-β1 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-β1 stored in the extracellular matrix.

Steroid Hormone Action in Musculoskeletal Cells Involves Membrane Receptor and Nuclear Receptor Mechanisms

Steroid hormones regulate target cells through traditional nuclear mechanisms as well as by membrane mechanisms. 1 f ,25(OH) 2 D 3 and 24R,25(OH) 2 D 3 bind membrane receptors (mVDR) and mediate their effects on the physiological responses of musculoskeletal cells via protein kinase C (PKC). In cultures of costochondral growth plate chondrocytes, 1 f ,25(OH) 2 D 3 binds the 1,25-mVDR in growth zone cells, activating phospholipase C (PLC), leading to diacylglycerol (DAG) production and PKC translocation to the plasma membrane. It also activates PLA 2 , increasing arachidonic acid release and prostaglandin synthesis. 24R,25(OH) 2 D 3 binds its membrane receptor in resting zone chondrocytes, activating phospholipase D (PLD), and increasing DAG and PKC activity, but translocation does not occur. PLA 2 activity is decreased, reducing arachidonic acid and prostaglandin production. 17 g -Estradiol (E 2 ) activates PKC in both cartilage cells, but DAG is not involved. 1 f ,25(OH) 2 D 3 and 24R,25(OH) 2 D 3 also increase PKC in osteoblasts in a cell-specific manner. Antibodies to the 1,25-mVDR block PKC activation. Membrane-mediated events influence gene expression via signaling cascades, including the ERK1/2 MAP kinases. The ability of steroid hormones to initiate events nongenomically is important for regulation of matrix vesicle (MV) function in the extracellular matrix. MVs have mVDRs, but ligand binding inhibits PKC-zeta (PKC ‘ ) via a mechanism that differs from PKC f activation in the plasma membranes. Treatment of MVs from growth zone chondrocyte cultures with 1 f ,25(OH) 2 D 3 releases stromelysin-1 (MMP-3) and increases TGF- g activation. MMP-3 is also involved in proteoglycan degradation, facilitating calcification. 24R,25(OH) 2 D 3 inhibits PKC ‘ in MV from resting zone cell cultures and inhibits MMP-3 release. Chondrocytes and osteoblasts produce 1,25(OH) 2 D 3 , 24,25(OH) 2 D 3 , and E 2 ; thus, locally produced steroids may function as autocrine regulators of matrix events, including matrix vesicle enzyme activity and matrix protein remodelling during longitudinal growth, calcification, and growth factor activation.

Steroid Hormone Action in Musculoskeletal Cells Involves Membrane Receptor and Nuclear Receptor Mechanisms

Steroid hormones regulate target cells through traditional nuclear mechanisms as well as by membrane mechanisms. 1 &#102 ,25(OH) 2 D 3 and 24R,25(OH) 2 D 3 bind membrane receptors (mVDR) and mediate their effects on the physiological responses of musculoskeletal cells via protein kinase C (PKC). In cultures of costochondral growth plate chondrocytes, 1 &#102 ,25(OH) 2 D 3 binds the 1,25-mVDR in growth zone cells, activating phospholipase C (PLC), leading to diacylglycerol (DAG) production and PKC translocation to the plasma membrane. It also activates PLA 2 , increasing arachidonic acid release and prostaglandin synthesis. 24R,25(OH) 2 D 3 binds its membrane receptor in resting zone chondrocytes, activating phospholipase D (PLD), and increasing DAG and PKC activity, but translocation does not occur. PLA 2 activity is decreased, reducing arachidonic acid and prostaglandin production. 17 &#103 -Estradiol (E 2 ) activates PKC in both cartilage cells, but DAG is not involved. 1 &#102 ,25(OH) 2 D 3 and 24R,25(OH) 2 D 3 also increase PKC in osteoblasts in a cell-specific manner. Antibodies to the 1,25-mVDR block PKC activation. Membrane-mediated events influence gene expression via signaling cascades, including the ERK1/2 MAP kinases. The ability of steroid hormones to initiate events nongenomically is important for regulation of matrix vesicle (MV) function in the extracellular matrix. MVs have mVDRs, but ligand binding inhibits PKC-zeta (PKC &#145 ) via a mechanism that differs from PKC &#102 activation in the plasma membranes. Treatment of MVs from growth zone chondrocyte cultures with 1 &#102 ,25(OH) 2 D 3 releases stromelysin-1 (MMP-3) and increases TGF- &#103 activation. MMP-3 is also involved in proteoglycan degradation, facilitating calcification. 24R,25(OH) 2 D 3 inhibits PKC &#145 in MV from resting zone cell cultures and inhibits MMP-3 release. Chondrocytes and osteoblasts produce 1,25(OH) 2 D 3 , 24,25(OH) 2 D 3 , and E 2 ; thus, locally produced steroids may function as autocrine regulators of matrix events, including matrix vesicle enzyme activity and matrix protein remodelling during longitudinal growth, calcification, and growth factor activation.

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.

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.

Vitamin D metabolites regulate matrix vesicle metalloproteinase content in a cell maturation-dependent manner.

Matrix vesicles are extracellular organelles produced by cells that mineralize their matrix. They contain enzymes that are associated with calcification and are regulated by vitamin D metabolites in a cell maturation-dependent manner. Matrix vesicles also contain metalloproteinases that degrade proteoglycans, macromolecules known to inhibit calcification in vitro, as well as plasminogen activator, a proteinase postulated to play a role in activation of latent TGF-beta. In the present study, we examined whether matrix vesicle metalloproteinase and plasminogen activator are regulated by 1, 25(OH)2D3 and 24,25(OH)2D3. Matrix vesicles and plasma membranes were isolated from fourth passage cultures of resting zone chondrocytes that had been incubated with 10(-10)-10(-7) M24, 25(OH)2D3 or growth zone chondrocytes incubated with 10(-11)-10(-8) M 1,25(OH)2D3, and their alkaline phosphatase, active and total neutral metalloproteinase, and plasminogen activator activities determined. 24,25(OH)2D3 increased alkaline phosphatase by 35-60%, decreased active and total metalloproteinase by 75%, and increased plasminogen activator by fivefold in matrix vesicles from resting zone chondrocyte cultures. No effect of vitamin D treatment was observed in plasma membranes isolated from these cultures. In contrast, 1,25(OH)2D3 increased alkaline phosphatase by 35-60%, but increased active and total metalloproteinase three- to fivefold and decreased plasminogen activator by as much as 75% in matrix vesicles isolated from growth zone chondrocyte cultures. Vitamin D treatment had no effect on plasma membrane alkaline phosphatase or metalloproteinase, but decreased plasminogen activator activity. The results demonstrate that neutral metalloproteinase and plasminogen activator activity in matrix vesicles are regulated by vitamin D metabolites in a cell maturation-specific manner. In addition, they support the hypothesis that 1,25(OH)2D3 regulation of matrix vesicle function facilitates calcification by increasing alkaline phosphatase and phospholipase A2 specific activities as well as metalloproteinases which degrade proteoglycans.

Dexamethasone promotes von kossa-positive nodule formation and increased alkaline phosphatase activity in costochondral chondrocyte cultures

This study examined the effect of dexamethasone on von Kossa-positive nodule formation and alkaline phosphate specific activity of costochondral chondrocytes at two distinct stages of maturation. The nodules formed by the more mature growth zone chondrocyte cultures contained von Kossa-positive deposits in the extracellular matrix that had a punctate morphology. The nodules formed by the less mature resting zone cells also contained von Kossa-positive deposits, but differentiation was delayed by three-to-five days compared to the growth zone cell cultures. Dexamethasone stimulated the number of nodules formed and shortened the length of time required for von Kossa-positive nodule formation in both types of cultures. During the first 48 h of exposure to dexamethasone, alkaline phosphatase specific activity in the cell layer of both resting zone and growth zone cultures was increased in a dose-dependent manner. At 12 days post-confluence and thereafter, enzyme activity was inhibited in the dexamethasone-treated cultures. Changes in matrix vesicle alkaline phosphatase specific activity reflected those changes seen in the cell layer after dexamethasone treatment, but with higher magnitude, suggesting that one effect of dexamethasone might be to regulate matrix vesicle function. With the exception of one culture, the chondrocytes did not synthesize type X collagen under any of the experimental conditions used. Fourier transform infrared spectroscopy (FTIR) failed to detect the presence of calcium phosphates in any of the cultures exposed to dexamethasone except one. These results demonstrate that dexamethasone promotes early differentiation events, including nodule formation and increased alkaline phosphatase activity, in costochondral chondrocyte cultures. The failure to detect type X collagen synthesis and mineralization in both dexamamethasone-treated and control cultures suggests that these cultures lack the factors necessary for terminal differentiation and mineralization.

Maturation‐dependent regulation of protein kinase C activity by vitamin D3 metabolites in chondrocyte cultures

Vitamin D3 metabolites regulate the differentiation of chondrocytes isolated from the growth zone or resting zone of rat costochondral cartilage. Since some of the direct membrane effects of vitamin D metabolites are nongenomic, we hypothesized that protein kinase C (PKC) plays a role in signal transduction for these chondrocyte differentiation factors and that the regulation of PKC by the vitamin D metabolites is cell maturation dependent. Confluent, fourth passage cultures of growth zone and resting zone chondrocytes were treated with vitamin D3 metabolites for up to 24 h, lysed, and cell extracts assayed for kinase activity using a specific PKC substrate peptide. The addition of 1,25-(OH)2D3 to growth zone cell cultures resulted in a rapid dose-dependent stimulation of PKC, significant at 10(-9)-10(-7) M, beginning at 3 min and sustained until 90 min; 1,25-(OH)2D3 had no effect on PKC activity in resting zone chondrocyte cultures. The addition of 24,25-(OH)2D3 to resting zone cultures showed a slower PKC activation, with significant stimulation seen at 90-360 min for 10(-8)-10(-7) M 24,25-(OH)2D3. However, 24,25-(OH)2D3 had no effect on PKC activity in growth zone cell cultures at all times and concentrations examined. The specificity of PKC stimulation by the vitamin D3 metabolites was verified using a specific pseudosubstrate region peptide inhibitor, which reduced PKC activity when included in the reaction mixture. Pretreatment of the cultures with U73, 122, a phospholipase C inhibitor, decreased 1,25-(OH)2D3-stimulated PKC activity but had no effect upon 24,25-(OH)2D3-induced activity. The tyrosine kinase inhibitor, genistein, did not inhibit the PKC response in either vitamin D3 metabolites-treated culture. Neither actinomycin D nor cycloheximide affected 1,25-(OH)2D3-induced PKC activity in growth zone chondrocyte cultures, while both compounds inhibited 24,25-(OH)2D3-induced activity in resting zone chondrocyte cultures. The results of this study indicate that vitamin D metabolites stimulate PKC activity in a metabolite- and cell-maturation-specific manner. Effects of 1,25-(OH)2D3 appear to be nongenomic, whereas the effects of 24,25-(OH)2D3 probably involve a genomic mechanism.

Production of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 by growth zone and resting zone chondrocytes is dependent on cell maturation and is regulated by hormones and growth factors.

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 have been shown to promote chondrocyte proliferation and differentiation; resting zone chondrocytes respond primarily to 24,25-(OH)2D3, whereas growth zone chondrocytes respond primarily to 1,25-(OH)2D3. This study determined whether resting zone and growth zone cells produce 24,25-(OH)2D3 or 1,25-(OH)2D3; whether this production is regulated by 1,25-(OH)2D3 (10(-8) M), 24,25-(OH)2D3 (10(-7) M), dexamethasone (10(-7) M), or recombinant human transforming growth factor-beta 1 (11 ng/ml); and whether the metabolites produced are biologically active. Confluent fourth passage rat costochondral growth zone or resting zone chondrocytes were cultured in Dulbecco's Modified Eagle's Medium containing [3H]25-hydroxyvitamin D3 ([3H]25OHD3), 2% fetal bovine serum, and antibiotics. Metabolism of [3H]25OHD3 was measured by analyzing the lipid extracts of the conditioned medium and the cell layer for [3H]1,25OHD3, [3H]1,25-(OH)2D3, and [3H]24,25-(OH)2D3 using flow-through scintillation spectroscopy of HPLC eluates. Chemically synthesized radioinert vitamin D3 metabolites were used as standards, and their migration was determined by absorbance at 254 nm. To ensure that the radioactive peaks were 1,25-(OH)2D3 and 24,25-(OH)2D3, the fractions were rechromatographed into three other HPLC solvent systems. Biological activity was confirmed; the addition of HPLC-purified 1,25-(OH)2D3 produced by growth zone chondrocytes elicited a dose-dependent stimulation of alkaline phosphatase specific activity in growth zone cell cultures, but had no effect on the resting zone cells. There was a time-dependent increase in both [3H]1,25-(OH)2D3 and [3H]24,25-(OH)2D3 in the conditioned medium of both types of cultures. At 24 h, the percent conversion of [3H]25OHD3 to [3H]1,25-(OH)2D3 was 5.3 +/- 1.2, and the percent conversion to [3H]24,25-(OH)2D3 was 1.8 +/- 0.4 in growth zone chondrocyte cultures. No such effect was found in cultures freeze-thawed five times or without cells. When resting zone cells were cultured with [3H]25OHD3, the percent conversion to 1,25-(OH)2D3 and 24,25-(OH)2D3 was 4.5 +/- 1.0 and 1.7 +/- 0.4, respectively. The addition of dexamethasone significantly increased the percent production of 1,25-(OH)2D3 at 6 and 24 h and at 6 h by resting zone and growth zone cells, respectively, compared to the control values. Recombinant human transforming growth factor-beta 1 increased the percent production of 1,25-(OH)2D3 after 1 h in resting zone cells and, after 24 h, the production of 24,25-(OH)2D3 in growth zone cells. Radiolabeled 1,25-(OH)2D3 and 24,25-(OH)2D3 were not detected in the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)

Production of 1,25-dihydroxyvitamin D3 and 24,25-dihydroxyvitamin D3 by growth zone and resting zone chondrocytes is dependent on cell maturation and is regulated by hormones and growth factors

1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] and 24,25-(OH)2D3 have been shown to promote chondrocyte proliferation and differentiation; resting zone chondrocytes respond primarily to 24,25-(OH)2D3, whereas growth zone chondrocytes respond primarily to 1,25-(OH)2D3. This study determined whether resting zone and growth zone cells produce 24,25-(OH)2D3 or 1,25-(OH)2D3; whether this production is regulated by 1,25-(OH)2D3 (10(-8) M), 24,25-(OH)2D3 (10(-7) M), dexamethasone (10(-7) M), or recombinant human transforming growth factor-beta 1 (11 ng/ml); and whether the metabolites produced are biologically active. Confluent fourth passage rat costochondral growth zone or resting zone chondrocytes were cultured in Dulbecco's Modified Eagle's Medium containing [3H]25-hydroxyvitamin D3 ([3H]25OHD3), 2% fetal bovine serum, and antibiotics. Metabolism of [3H]25OHD3 was measured by analyzing the lipid extracts of the conditioned medium and the cell layer for [3H]1,25OHD3, [3H]1,25-(OH)2D3, and [3H]24,25-(OH)2D3 using flow-through scintillation spectroscopy of HPLC eluates. Chemically synthesized radioinert vitamin D3 metabolites were used as standards, and their migration was determined by absorbance at 254 nm. To ensure that the radioactive peaks were 1,25-(OH)2D3 and 24,25-(OH)2D3, the fractions were rechromatographed into three other HPLC solvent systems. Biological activity was confirmed; the addition of HPLC-purified 1,25-(OH)2D3 produced by growth zone chondrocytes elicited a dose-dependent stimulation of alkaline phosphatase specific activity in growth zone cell cultures, but had no effect on the resting zone cells. There was a time-dependent increase in both [3H]1,25-(OH)2D3 and [3H]24,25-(OH)2D3 in the conditioned medium of both types of cultures. At 24 h, the percent conversion of [3H]25OHD3 to [3H]1,25-(OH)2D3 was 5.3 +/- 1.2, and the percent conversion to [3H]24,25-(OH)2D3 was 1.8 +/- 0.4 in growth zone chondrocyte cultures. No such effect was found in cultures freeze-thawed five times or without cells. When resting zone cells were cultured with [3H]25OHD3, the percent conversion to 1,25-(OH)2D3 and 24,25-(OH)2D3 was 4.5 +/- 1.0 and 1.7 +/- 0.4, respectively. The addition of dexamethasone significantly increased the percent production of 1,25-(OH)2D3 at 6 and 24 h and at 6 h by resting zone and growth zone cells, respectively, compared to the control values. Recombinant human transforming growth factor-beta 1 increased the percent production of 1,25-(OH)2D3 after 1 h in resting zone cells and, after 24 h, the production of 24,25-(OH)2D3 in growth zone cells. Radiolabeled 1,25-(OH)2D3 and 24,25-(OH)2D3 were not detected in the cell layer.(ABSTRACT TRUNCATED AT 400 WORDS)

α2-HS-glycoprotein: Expression in chondrocytes and augmentation of alkaline phosphatase and phospholipase A 2 activity

The α 2-HS-glycoprotein is a plasma protein synthesized in liver and enriched in bone. The concentration of α 2-HS-glycoprotein dynamically changes in various physiological conditions and is highest in bone during growth, suggesting that it is involved in regulation of endochondral ossification. Northern blot analysis demonstrated that mRNA transcripts from growth zone and resting zone costochondral chondrocyte cultures hybridized with α 2-HS-glycoprotein cDNA. However, a difference of mRNA transcript size was observed, with chondrocyte mRNA transcripts being 2.2 kb, while mRNA isolated from liver was 1.6 kb. Presence of α 2-HS-glycoprotein in cartilage cells was found by immuno-histochemical staining of human fetal epiphyses using anti-human α 2-HS-glycoprotein antibody. To understand the role of α 2-HS-glycoprotein in cartilage growth, the effects of exogenous α 2-HS-glycoprotein were correlated with alkaline phosphatase (ALPase) and phospholipase A 2 (PA 2) activity in the chondrocyte cultures. Alkaline phosphatase specific activity was stimulated by α 2-HS-glycoprotein at concentrations between 0.25 and 1.25μ/mL in the growth zone and resting zone cultures 2.7 and 2.0-fold, respectively. Matrix vesicle PA 2 activity was increased only in the growth zone chondrocyte cultures. These results suggested that α 2-HS-glycoprotein may contribute to the regulation of the expression of the chondrocyte phenotype. Steady state mRNA levels of ALPase were analyzed in chondrocytes after additions of α 2-HS-glycoprotein. The ALPase mRNA levels remained stationary during the stimulation of enzymatic activity, indicating that the effect of α 2-HS-glycoprotein upon alkaline phosphatase activity is not at the transcriptional level.

Effect of 1,25(OH)2D3 and 24,25(OH)2D3 on calcium ion fluxes in costochondral chondrocyte cultures

Vitamin D3 metabolites have been shown to affect proliferation, differentiation, and maturation of cartilage cells. Previous studies have shown that growth zone chondrocytes respond primarily to 1,25(OH)2D3 whereas resting zone chondrocytes respond primarily to 24,25(OH)2D3. To examine the role of calcium in the mechanism of hormone action, this study examined the effects of the Ca ionophore A23187, 1,25(OH)2D3, and 24,25(OH)2D3 on Ca influx and efflux in growth zone chondrocytes and resting zone chondrocytes derived from the costochondral junction of 125 g rats. Influx was measured as incorporation of 45Ca. Efflux was measured as release of 45Ca from prelabeled cultures into fresh media. The pattern of 45Ca influx in unstimulated (control) cells over the incubation period was different in the two chondrocyte populations, whereas the pattern of efflux was comparable. A23187 induced a rapid influx of 45Ca in both types of chondrocytes which peaked by 3 minutes and was over by 6 minutes. Influx was greatest in the growth zone chondrocytes. Addition of 10(-8)-10(-9) M 1,25(OH)2D3 to growth zone chondrocyte cultures results in a dose-dependent increase in 45Ca influx after 15 minutes. Efflux was stimulated by these concentrations of hormone throughout the incubation period. Addition of 10(-6)-10(-7) M 24,25(OH)2D3 to resting zone chondrocytes resulted in an inhibition in ion efflux between 1 and 6 minutes, with no effect on influx during this period. Efflux returned to control values between 6 and 15 minutes. 45Ca influx was inhibited by these concentrations of hormone from 15 to 30 minutes.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of arachidonic acid turnover by 1,25-(OH) 2D 3 and 24,25-(OH) 2D 3 in growth zone and resting zone chondrocyte cultures

Previous studies have shown that phospholipase A 2 activity in rat costochondral chondrocyte cultures is differentially regulated by 1,25-(OH) 2D 3 and 24,25-(OH) 2D 3. 1,25-(OH) 2D 3 stimulates enzyme activity in growth zone chondrocytes but has no effect on the resting zone chondrocyte enzyme activity. 24,25-(OH) 2D 3 inhibits the resting zone enzyme but has no effect on the growth zone chondrocyte phospholipase A 2. This study examined whether the metabolites affect arachidonic acid turnover in their target cell populations. Incorporation and release of [ 14C]arachidonate was measured at various times following addition of hormone to the cultures. Acylation and reacylation were measured independently by incubating half of the [ 14C]arachidonate-labeled cultures with p-chloromercuribenzoate. The results demonstrated that the distribution of [ 14C]arachidonate in membrane phospholipids differed between growth zone and resting zone chondrocytes and between the plasma membranes and matrix vesicles isolated from the growth zone chondrocyte cultures. Plasma membrane phospholipids were more susceptible to the release of [ 14C]arachidomic acid by exogenous phospholipases than were matrix vesicles phospholipids. The effect of 1,25-(OH) 2D 3 on growth zone chondrocytes was observed within 5 min. Incorporation was greatest after 60 min; release was greatest after 30 min. 24,25-(OH) 2D 3 stimulated consistently elevated incorporation throughout the incubation period, peaking at 15 min. Peak release was at 60 min. The results confirm that resting zone chondrocytes and growth zone chondrocytes retain a differential phenotype in culture and demonstrate that matrix vesicles are distinct from the plasma membrane in terms of lipid composition and arachidonic acid incorporation. 1,25-(OH) 2D 3 and 24,25-(OH) 2D 3 appear to stimulate arachidonic acid turnover in their target cells by different mechanisms. Changes in fatty acid acylation and reacylation may be one mode of vitamin D-3 action in cartilage.