Growth Zone Chondrocytes Research Articles

The first stage of transforming growth factor beta1 activation is release of the large latent complex from the extracellular matrix of growth plate chondrocytes by matrix vesicle stromelysin-1 (MMP-3).

Transforming growth factor beta-1 (TGF-beta1) is secreted in a biologically inactive form and stored in the extracellular matrix as a 290 kDa complex consisting of the mature TGF-beta1 homodimer (Mr 25 kDa), the latency-associated peptide (LAP; Mr 75 kDa), and the latent TGF-beta1 binding protein-1 (LTBP1; Mr 190 kDa). Latent TGF-beta1, composed of these three components, is known as the "large latent TGF-beta1 complex." In contrast, latent TGF-beta1 without LTBP1 is known as "small latent TGF-beta1." For all latent forms, dissociation of the TGF-beta1 homodimer from LAP is necessary for growth factor activation and acquisition of biological activity. Matrix vesicles produced by growth plate chondrocytes contain matrix metalloproteinases that can activate small latent TGF-beta1. The enzyme responsible for this is matrix metalloproteinase-3 (MMP-3), although matrix vesicles also contain MMP-2 and plasminogen activator. The present study tested the hypothesis that matrix vesicle enzymes are also involved in the release of the large latent TGF-beta1 complex stored in the extracellular matrix. Matrix vesicles were isolated from cultures of resting zone and growth zone chondrocytes and metalloproteinases present in the matrix vesicles extracted with guanidine-HCl. Chondrocyte extracellular matrices were prepared by lysing confluent cultures and removing the lysed cells. The matrices were incubated with matrix vesicle extracts and the release of total and active TGF-beta1 was determined. To determine if MMP-2 or MMP-3 was involved in the release, matrix vesicle extracts were preincubated with anti-MMP-2 antibody or anti-MMP-3 antibody to selectively deplete the enzyme activity. Matrices were also treated with rhMMP-2 or rhMMP-3. To determine the identity of the released protein(s), digests were separated on SDS-polyacrylamide gels and Western blotting analysis was performed using a specific antibody to LTBP1. Matrix vesicle extracts released both active and total (=latent + active) TGF-beta1 in a time-dependent manner, with peak release after 1 hour of incubation. The amount of total TGF-beta1 released was 10 times higher than the release of active TGF-beta1. The effect of the matrix vesicle extracts was dose-dependent; in addition, the amount and ratio of active to total TGF-b1 released was very similar, irrespective of the source of matrix or matrix vesicle extracts. Pre-incubation of matrix vesicle extracts with anti-MMP-3 antibody blocked the release of active and total TGF-beta1, whereas pre-incubation with pre-immune IgG or anti-MMP-2 antibody had no effect. The addition of rhMMP-3, but not rhMMP-2, caused a dose-dependent increase in the release of total, but not active, TGF-beta1. Western analysis confirmed that both matrix vesicle extracts and rhMMP-3 released the large latent TGF-beta1 complex from the matrix. In addition to the expected 290, 230, and 190 kDa bands, samples run without reduction also contained proteins of molecular weights 110 and 50 kDa that reacted with the anti-LTBP1 antibody. When these same samples were electrophoresed after reduction, the high molecular weight immunoreactive bands disappeared and three bands of molecular weight 75, 32, and 25 kDa were observed. These results indicate that matrix vesicles contain enzymes, especially MMP-3, which are responsible for the release of TGF-beta1 from the matrix, most of which is in latent form. Further, the data suggest that release of the large complex occurs via cleavage at several novel sites in the 130 kDa LTBP1 molecule. Since matrix vesicle MMP-3 is also able to activate small latent TGF-beta1, these results suggest that the large latent TGF-beta1 complex protects against activation of the small latent TGF-beta1. Thus, the data suggest that release of the large latent TGF-bl complex from the matrix and activation of the latent growth factor are only two steps of what must be at least a three-step process.

1alpha,25-(OH)2D3 regulates 25-hydroxyvitamin D3 24R-hydroxylase activity in growth zone costochondral growth plate chondrocytes via protein kinase C.

Rat costochondral chondrocytes possess 25-hydroxyvitamin D3 1alpha- and 24R-hydroxylase activities and metabolize 25-(OH)D3 to 1,25-(OH)2D3 and 24,25-(OH)2D3 in a cell maturation-specific and time-dependent manner. This study examined the hypothesis that 1alpha,25-(OH)2D3 and 24R,25-(OH)2D3 regulate the activities of both hydroxylases in prehypertrophic/upper hypertrophic (growth zone) chondrocytes, and 1alpha,25-(OH)2D3 exerts its effects via mechanisms involving protein kinase C (PKC) mediated pathways. Rat costochondral growth zone chondrocytes were treated with 10(-9) - 10(-7) M 1alpha,25-(OH)2D3 or 24R,25-(OH)2D, for 24 hours, and 1alpha- and 24R-hydroxylase activities in cell homogenates determined by measuring the conversion of [3H]-25-(OH)D3 to [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3. Metabolite production by intact cells was determined at 6 and 24 hours. Involvement of PKC was assessed using chelerythrine, and the requirement for protein synthesis was assessed using cycloheximide. In addition, the effect of 10(-10) - l0(-8) M 1alpha,25-(OH)2D3 on 24-hydroxylase expression was assessed by semiquantitative measurement of mRNA levels using RT-PCR. Involvement of the membrane receptor for 1alpha,25-(OH)2D3 (1,25-mVDR), which exerts its effects via PKC, was assessed by blocking the 1,25-mVDR with an antibody (Ab99) generated against the 1,25-mVDR in chick enterocyte membranes. Specificity of the 1,25-(OH)2D3-dependent effect on 24,25-(OH)2D3 production was determined by comparing the response to 1alpha,25-(OH)2D3 to the response to 1beta,25-(OH)2D3. 1alpha,25-(OH)2D3 increased 24R-hydroxylase specific activity in a dose-dependent manner, 24,25-(OH)2D3 production by intact cells was also increased. The effect of 1alpha,25-(OH)2D3 on 24,25-(OH)2D3 production was stereospecific. Only 1alpha,25-(OH)2D3 caused an increase; 1beta,25-(OH)2D3 was without effect. 24R,25-(OH)2D3 had no effect on 24R-hydroxylase activity at 24 hours. 1alpha-hydroxylase activity was unaffected by either metabolite at 24 hours. 1alpha,25-(OH)2D3 affected 24R-hydroxylase activity via a PKC-dependent mechanism requiring new protein synthesis. In addition, 1alpha,25-(OH)2D3 caused a dose-dependent increase in 24-hydroxylase mRNA levels. The 1,25-mVDR was involved in the 1alpha,25(OH)2D3-dependent stimulation of 24R-hydroxylase. These results suggest an interrelationship between the 1,25-mVDR and gene expression via the nuclear VDR (nVDR) and/or a PKC-mediated mechanism in modulating 24R-hydroxylase in growth zone chondrocytes.

Characterization of PGE 2 receptors (EP) and their role as mediators of 1α,25-(OH) 2D 3 effects on growth zone chondrocytes

Growth plate chondrocyte function is modulated by the vitamin D metabolite 1α,25-(OH) 2D 3 via activation of protein kinase C (PKC). In previous studies with cells derived from prehypertrophic and upper hypertrophic zones of rat costochondral cartilage (growth zone cells), inhibition of prostaglandin production with indomethacin caused a decrease in the stimulation of PKC activity, suggesting that changes in prostaglandin levels mediate the 1α,25-(OH) 2D 3-dependent response in these cells. Growth zone cells also respond to PGE 2 directly, indicating that prostaglandins act as autocrine or paracrine regulators of chondrocyte metabolism in the growth plate. The aim of the present study was to identify which PGE 2 receptor subtypes (EP) mediate the effects of PGE 2 on growth zone cells. Using primers specific for EP1–EP4, reverse transcription-polymerase chain reaction (RT-PCR) amplified EP1 and EP2 cDNA in a RT-dependent manner. In parallel experiments, we used EP subtype-specific agonists to examine the role of EP receptors in 1α,25-(OH) 2D 3-mediated cell proliferation and differentiation. 17-Phenyl-trinor-PGE 2 (PTPGE 2), an EP1 agonist, decreased [ 3H]-thymidine incorporation in a dose-dependent manner and augmented the 1α,25-(OH) 2D 2-induced inhibition of [ 3H]-thymidine incorporation. PTPGE 2 also caused significant increases in proteoglycan production, as measured by [ 35S]-sulfate incorporation, and alkaline phosphatase specific activity. 1α,25-(OH) 2D 3-induced alkaline phosphatase activity was only slightly stimulated by PTPGE 2. In contrast, 1α,25-(OH) 2D 3-induced PKC activity was synergistically increased by PTPGE 2, whereas EP1 antagonists SC-19220 and AH6809 inhibited PKC activity in a dose-dependent manner. The EP2, EP3 and EP4 agonists had no effect on the various cell-induced responses measured. EP1 receptor-induced responses were blocked by the phospholipase C inhibitor U73122, and reduced by PKA inhibitors. EP1 receptor-induced PKC activity was insensitive to pertussis toxin or choleratoxin but blocked by the G-protein inhibitor GDPβS, suggesting the involvement of G q. These results suggest that the EP1 receptor subtype mediates various PGE 2-induced cellular responses in growth zone chondrocytes leading to decreased proliferation and enhanced differentiation, as well as the effect of 1α,25-(OH) 2D 3 on cellular maturation.

The effect of 24R,25-(OH)(2)D(3) on protein kinase C activity in chondrocytes is mediated by phospholipase D whereas the effect of 1alpha,25-(OH)(2)D(3) is mediated by phospholipase C.

1alpha,25-(OH)(2)D(3) regulates protein kinase C (PKC) activity in growth zone chondrocytes by stimulating increased phosphatidylinositol-specific phospholipase C (PI-PLC) activity and subsequent production of diacylglycerol (DAG). In contrast, 24R,25-(OH)(2)D(3) regulates PKC activity in resting zone (RC) cells, but PLC does not appear to be involved, suggesting that phospholipase D (PLD) may play a role in DAG production. In the present study, we examined the role of PLD in the physiological response of RC cells to 24R,25-(OH)(2)D(3) and determined the role of phospholipases D, C, and A(2) as well as G-proteins in mediating the effects of vitamin D(3) metabolites on PKC activity in RC and GC cells. Inhibition of PLD with wortmannin or EDS caused a dose-dependent inhibition of basal [3H]-thymidine incorporation by RC cells and further increased the inhibitory effect of 24R,25-(OH)(2)D(3). Wortmannin also inhibited basal alkaline phosphatase activity and [35]-sulfate incorporation and decreased the stimulatory effect of 24R,25-(OH)(2)D(3). This inhibitory effect of wortmannin was not seen in cultures treated with the PI-3-kinase inhibitor LY294002, verifying that wortmannin affected PLD. Wortmannin also inhibited basal PKC activity and partially blocked the stimulatory effect of 24R,25-(OH)(2)D(3) on this enzyme activity. Neither inhibition of PI-PLC with U73122, nor PC-PLC with D609, modulated PKC activity. Wortmannin had no effect on basal PLD in GC cells, nor on 1alpha,25-(OH)(2)D(3)-dependent PKC. Inhibition of PI-PLC blocked the 1alpha,25-(OH)(2)D(3)-dependent increase in PKC activity but inhibition of PC-PLC had no effect. Activation of PLA(2) with melittin inhibited basal and 24R,25-(OH)(2)D(3)-stimulated PKC in RC cells and stimulated basal and 1alpha,25-(OH)(2)D(3)-stimulated PKC in GC cells, but wortmannin had no effect on the melittin-induced changes in either cell type. Pertussis toxin modestly increased the effect of 24R,25-(OH)(2)D(3) on PKC, whereas GDPbetaS had no effect, suggesting that PLD2 is the isoform responsible. This indicates that 1alpha,25-(OH)(2)D(3) regulates PKC in GC cells via PI-PLC and PLA(2), but not PC-PLC or PLD, whereas 24R,25-(OH)(2)D(3) regulates PKC in RC cells via PLD2.

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.

24,25-(OH) 2D 3 regulates cartilage and bone via autocrine and endocrine mechanisms

The purpose of this paper is to summarize recent advances in our understanding of the physiological role of 24(R),25(OH) 2D 3 in bone and cartilage and its mechanism of action. With the identification of a target cell, the growth plate resting zone (RC) chondrocyte, we have been able to use cell biology methodology to investigate specific functions of 24(R),25(OH) 2D 3 and to determine how 24(R),25(OH) 2D 3 elicits its effects. These studies indicate that there are specific membrane-associated signal transduction pathways that mediate both rapid, nongenomic and genomic responses of RC cells to 24(R),25(OH) 2D 3. 24(R),25(OH) 2D 3 binds RC chondrocyte membranes with high specificity, resulting in an increase in protein kinase C (PKC) activity. The effect is stereospecific; 24R,25(OH) 2D 3, but not 24S,25-(OH) 2D 3, causes the increase, indicating a receptor-mediated response. Phospholipase D-2 (PLD2) activity is increased, resulting in increased production of diacylglycerol (DAG), which in turn activates PKC. 24(R),25(OH) 2D 3 does not cause translocation of PKC to the plasma membrane, but activates existing PKCα. There is a rapid decrease in Ca 2+ efflux, and influx is stimulated. 24(R),25(OH) 2D 3 also reduces arachidonic acid release by decreasing phospholipase A 2 (PLA 2) activity, thereby decreasing available substrate for prostaglandin production via the action of cyclooxygenase-1. PGE 2 that is produced acts on the EP1 and EP2 receptors expressed by RC cells to downregulate PKC via protein kinase A, but the reduction in PGE 2 decreases this negative feedback mechanism. Both pathways converge on MAP kinase, leading to new gene expression. One consequence of this is production of new matrix vesicles containing PKCα and PKCζ and an increase in PKC activity. The chondrocytes also produce 24(R),25(OH) 2D 3, and the secreted metabolite acts directly on the matrix vesicle membrane. Only PKCζ is directly affected by 24(R),25(OH) 2D 3 in the matrix vesicles, and activity of this isoform is inhibited. This effect may be involved in the control of matrix maturation and turnover. 24(R),25(OH) 2D 3 causes RC cells to mature along the endochondral developmental pathway, where they become responsive to 1α,25(OH) 2D 3 and lose responsiveness to 24(R),25(OH) 2D 3, a characteristic of more mature growth zone (GC) chondrocytes. 1α,25(OH) 2D 3 elicits its effects on GC through different signal transduction pathways than those used by 24(R),25(OH) 2D 3. These studies indicate that 24(R),25(OH) 2D 3 plays an important role in endochondral ossification by regulating less mature chondrocytes and promoting their maturation in the endochondral lineage.

Effects of 1alpha,25-(OH)(2)D(3) on rat growth zone chondrocytes are mediated via cyclooxygenase-1 and phospholipase A(2).

1alpha,25-(OH)(2)D(3) mediates its effects on growth zone chondrocytes via rapid membrane-associated events as well as through traditional nuclear receptor mechanisms. The membrane-associated signaling pathways include rapid production of diacylglycerol and activation of protein kinase C (PKC), as well as activation of phospholipase A(2) (PLA(2)), increased production of arachidonic acid, and increased production of prostaglandins. This study examined the roles of PLA(2) and cyclooxygenase (Cox) in the mechanism of action of 1alpha,25-(OH)(2)D(3) in these cells to determine whether one or both enzymes catalyze the rate limiting step and whether constitutive or inducible Cox is involved. Cultures were incubated with 1alpha,25-(OH)(2)D(3) for 9 min to measure PKC or for 24 h to measure physiological responses ([(3)H]-thymidine incorporation, alkaline phosphatase specific activity, [(35)S]-sulfate incorporation). Based on RT-PCR and Northern blot analysis, growth zone chondrocytes expressed mRNAs for both Cox-1 and Cox-2 and neither Cox was modulated by 1alpha,25-(OH)(2)D(3). To examine the role of Cox, the cultures were also treated with resveratrol (a specific inhibitor of Cox-1), NS-398 (a specific inhibitor of Cox-2), or indomethacin (a general Cox inhibitor). The results showed that Cox-1 inhibition reduced the 1alpha,25-(OH)(2)D(3)-dependent effects on proliferation, differentiation, and matrix production, whereas inhibition of Cox-2 only had an effect on proliferation. The effects of Cox inhibition were not rate limiting, based on experiments in which PLA(2) was activated with melittin or inhibited with quinacrine. However, at least part of the action of 1alpha,25-(OH)(2)D(3) was regulated by metabolism of arachidonic acid to prostaglandins. This supports the hypothesis that 1alpha,25-(OH)(2)D(3) exerts its effects via more than one signaling pathway and that these pathways are interrelated via the modulation of PLA(2) as a rate-limiting step. PKC regulation may occur at multiple stages in the signal transduction cascade. J. Cell. Biochem. Suppl. 36: 32-45, 2001.

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

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

Regulation of phospholipase D (PLD) in growth plate chondrocytes by 24 R,25-(OH) 2D 3 is dependent on cell maturation state (resting zone cells) and is specific to the PLD2 isoform

Many of the effects of 1α,25-(OH) 2D 3 and 24 R,25-(OH) 2D 3 on costochondral chondrocytes are mediated by the protein kinase C (PKC) signal transduction pathway. 1α,25-(OH) 2D 3 activates PKC in costochondral growth zone chondrocytes through a specific membrane receptor (1α,25-mVDR), involving rapid increases in diacylglycerol via a phospholipase C (PLC)-dependent mechanism. 24 R,25-(OH) 2D 3 activates PKC in resting zone chondrocytes. Although diacylglycerol is increased by 24 R,25-(OH) 2D 3, 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. 24 R,25-(OH) 2D 3, but not 24 S,25-(OH) 2D 3 or 1α,25-(OH) 2D 3, stimulates PLD activity in resting zone cells within 3 min via nongenomic mechanisms. Neither 1α,25-(OH) 2D 3 nor 24 R,25-(OH) 2D 3 affected PLD in growth zone cells. Basal and 24 R,25-(OH) 2D 3-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, 24 R,25-(OH) 2D 3 stimulates PLD, and PI 3-kinase, PI-PLC and PKC are not involved, whereas PLD is required for stimulation of PKC by 24 R,25-(OH) 2D 3. Pertussis toxin, GDPβS, and GTPγS had no effect on 24 R,25-(OH) 2D 3-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 24 R,25-(OH) 2D 3-mVDR is required. The results also support the conclusion that the 24 R,25-(OH) 2D 3-responsive PLD is PLD2, since this PLD isoform is G-protein-independent.

Expression and production of stathmin in growth plate chondrocytes is cell‐maturation dependent

Growth plate cartilage is comprised of linear columns of chondrocytes with the least differentiated cells at one end and the terminally differentiated cells at the other end. Rat costochondral chondrocytes can be divided into the resting cell zone (reserve cell zone), which contains relatively immature chondrocytes (RC cells), and the phenotypically more mature prehypertrophic and upper hypertrophic cell zones, which together may be termed the growth zone chondrocytes (GC cells). When grown separately in monolayer culture, they continue to express their zone-specific phenotype, providing a model for assessing cell-maturation–dependent expression of molecules associated with differentiation. Stathmin (also called prosolin, Op18, p19, 19K, and others) is a highly conserved, phosphorylated cytosolic protein with apparent ubiquitous expression. Although its exact function is unknown, stathmin is considered to be a messenger phosphorylated protein, it plays a role in tubulin stability, and it may participate in both general and specific regulatory pathways. One uniform observation is that the expression of stathmin protein decreases in all cells as they become more terminally differentiated in culture. There have been no published data regarding stathmin expression and production in chondrocytes. This study was based on the hypothesis that stathmin exists in chondrocytes and that the mRNA and protein levels decline in the GC cell with respect to the RC cell. Stathmin mRNA levels were determined and quantitated by reverse transcription-polymerase chain reaction (RT-PCR) and northern blots. Protein levels were determined using immunoblots. It was found that stathmin exists in chondrocytes and that RC cells express approximately twice the level of mRNA and protein to that found in GC cells. The results support the hypothesis and suggest that the level of stathmin expression and production in culture is related to the level of differentiation of RC and GC cells in vivo. J. Cell. Biochem. 79:150–163, 2000. © 2000 Wiley-Liss, Inc.

The membrane effects of 17 β-estradiol on chondrocyte phenotypic expression are mediated by activation of protein kinase C through phospholipase C and G-proteins

Growth plate chondrocytes from both male and female rats have nuclear receptors for 17β-estradiol (E 2); however, recent studies indicate that an alternative pathway involving a membrane receptor may also be involved in the female cell response. E 2 directly affects the fluidity of chondrocyte membranes derived from female, but not male, rats. In addition, E 2 activates PKC in a nongenomic manner in female cells, and chelerythrine, a specific inhibitor of PKC, inhibits E 2-dependent alkaline phosphatase activity in these cells, indicating PKC is involved in the signal transduction mechanism. The aims of this study were: (1) to examine if PKC mediates the effect of E 2 on chondrocyte proliferation, differentiation, and matrix synthesis; and (2) to determine the pathway that mediates the membrane effect of E 2 on PKC. Confluent, fourth passage resting zone (RC) and growth zone (GC) chondrocytes from female rat costochondral cartilage were treated with 10 −10 to 10 −7 M E 2 in the presence or absence of the PKC inhibitor chelerythrine, and changes in alkaline phosphatase specific activity, proteoglycan sulfation, and [ 3H]thymidine incorporation were measured. To examine the pathway of PKC activation, chondrocyte cultures were treated with E 2 in the presence or absence of genistein (an inhibitor of tyrosine kinases), U73122 or D609 (inhibitors of phospholipase C [PLC]), quinacrine (an inhibitor of phospholipase A 2 [PLA 2]), and melittin (an activator of PLA 2). Alkaline phosphatase specific activity and proteoglycan sulfation were increased and [ 3H]thymidine incorporation was decreased by E 2. The effects of E 2 on all parameters were blocked by chelerythrine. Treatment of the cultures with E 2 produced a significant dose-dependent increase in PKC. U73122 dose-dependently inhibited the activation of PKC in E 2-stimulated female chondrocyte cultures. However, the classical receptor antagonist ICI 182780 was unable to block the stimulatory effect of E 2 on PKC. Moreover, the classical receptor agonist diethylstilbestrol (DES) had no effect on PKC, nor did it alter the stimulatory effect of E 2. Inhibition of tyrosine kinase and PLA 2 had no effect on the activation of PKC by E 2. The PLA 2 activator also had no effect on PKC activation by E 2. E 2 stimulated PKC activity in membranes isolated from the chondrocytes, demonstrating a direct membrane effect for this steroid hormone. These data indicate that the rapid nongenomic effect of E 2 on PKC activity in chondrocytes from female rats is sex-specific and dependent upon a G-protein-coupled phospholipase C.

Parathyroid hormone and transforming growth factor-beta1 coregulate chondrocyte differentiation in vitro.

Parathyroid hormone (1-34) (PTH(1-34) and transforming growth factor-beta1 (TGF-beta1) regulate chondrocyte proliferation, differentiation, and matrix synthesis. Both proteins mediate their effects in a dose- and time-dependent manner, and the effects are cell maturation specific. Moreover, similar signaling pathways are used, suggesting that there may be cross talk leading to coregulated cell response. To test this hypothesis, confluent cultures of rat costochondral resting zone and growth zone chondrocytes were treated with 0.22, 0.44, or 0.88 ng/mL of rhTGF-beta1 for 24 h, followed by treatment with 10(-11) to 10(-8) M PTH(1-34) for 10 min or 24 h. [3H]-Thymidine incorporation, specific activity of alkaline phosphatase (AP), and [35S]-sulfate incorporation were measured. PTH(1-34) had no effect on [3H]-thymidine incorporation by growth zone cells pretreated with 0.22 or 0.44 ng/mL of TGF-beta1, but in cultures treated with 0.88 ng/mL, PTH(1-34) caused a dose-dependent decrease that was maximal at the lowest concentration tested. By contrast, PTH(1-34) stimulated [3H]-thymidine incorporation by resting zone cells, and this effect was additive with the stimulation caused by 0.22 ng/mL of TGF-beta1. PTH(1-34) caused a synergistic increase in AP in growth zone cells treated with 0.44 or 0.88 ng/mL of TGF-beta1, but not in cells treated with 0.22 ng/mL of TGF-beta1. It had no effect on AP in resting zone cells pretreated with any concentration of TGF-beta1. PTH(1-34) increased [35S]-sulfate incorporation in growth zone and resting zone cell cultures treated with 0.22 ng/mL of TGF-beta1 to levels seen in cultures treated with 0.88 ng/mL of TGF-beta1 alone. These results support the hypothesis that PTH(1-34) and TGF-beta1 coregulate growth plate chondrocytes and that the effects are cell maturation dependent.

Expression and production of stathmin in growth plate chondrocytes is cell-maturation dependent

Growth plate cartilage is comprised of linear columns of chondrocytes with the least differentiated cells at one end and the terminally differentiated cells at the other end. Rat costochondral chondrocytes can be divided into the resting cell zone (reserve cell zone), which contains relatively immature chondrocytes (RC cells), and the phenotypically more mature prehypertrophic and upper hypertrophic cell zones, which together may be termed the growth zone chondrocytes (GC cells). When grown separately in monolayer culture, they continue to express their zone-specific phenotype, providing a model for assessing cell-maturation-dependent expression of molecules associated with differentiation. Stathmin (also called prosolin, Op18, p19, 19K, and others) is a highly conserved, phosphorylated cytosolic protein with apparent ubiquitous expression. Although its exact function is unknown, stathmin is considered to be a messenger phosphorylated protein, it plays a role in tubulin stability, and it may participate in both general and specific regulatory pathways. One uniform observation is that the expression of stathmin protein decreases in all cells as they become more terminally differentiated in culture. There have been no published data regarding stathmin expression and production in chondrocytes. This study was based on the hypothesis that stathmin exists in chondrocytes and that the mRNA and protein levels decline in the GC cell with respect to the RC cell. Stathmin mRNA levels were determined and quantitated by reverse transcription-polymerase chain reaction (RT-PCR) and northern blots. Protein levels were determined using immunoblots. It was found that stathmin exists in chondrocytes and that RC cells express approximately twice the level of mRNA and protein to that found in GC cells. The results support the hypothesis and suggest that the level of stathmin expression and production in culture is related to the level of differentiation of RC and GC cells in vivo.

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.

Prostaglandins mediate the effects of 1,25-(OH) 2D 3 and 24,25-(OH) 2D 3 on growth plate chondrocytes in a metabolite-specific and cell maturation-dependent manner

Prior studies have shown that 1,25-(OH) 2D 3 stimulates alkaline phosphatase, phospholipase A 2 (PLA 2), and protein kinase C (PKC)-specific activities, and production of prostaglandin E 2 (PGE 2) in growth zone chondrocytes. In contrast, 24,25-(OH) 2D 3 stimulates alkaline phosphatase and PKC-specific activities but inhibits PLA 2-specific activity and PGE 2 production in resting zone cells. This indicates that different mechanisms are involved in the action of 1,25-(OH) 2D 3 and 24,25-(OH) 2D 3 on their respective target cells. In this study, we examined the hypothesis that differential regulation of prostaglandin production modulates the activity of PKC and alkaline phosphatase. To do this, we examined the effect of the cyclooxygenase inhibitor indomethacin (Indo) on alkaline phosphatase, PLA 2, and PKC-specific activities in growth plate chondrocytes treated with these two vitamin D metabolites. In addition, we examined whether inhibition of PKC altered PGE 2 production. In growth zone cells, Indo inhibited basal alkaline phosphatase and blocked the 1,25-(OH) 2D 3-dependent increase in alkaline phosphatase. This effect was due to inhibition of both plasma membrane and matrix vesicle alkaline phosphatase. In resting zone cells, Indo increased basal alkaline phosphatase activity in a dose-dependent manner, but it did not further enhance the 24,25-(OH) 2D 3-dependent stimulation of this enzyme. The effect of Indo was found in both plasma membranes and matrix vesicles. These data indicate that 1,25-(OH) 2D 3-dependent increases in alkaline phosphatase-specific activity in growth zone cells are mediated through increased prostaglandin production, whereas 24,25-(OH) 2D 3-mediated changes in enzyme activity in resting zone cells are mediated through decreased prostaglandin production. Regulation of PLA 2 by either 1,25-(OH) 2D 3 or 24,25-(OH) 2D 3 in their target cells was unaffected by Indo, indicating that the effect of the vitamin D metabolites on this enzyme is not dependent on changes in PGE 2 production. The rapid increase in 1,25-(OH) 2D 3-dependent PKC-specific activity in growth zone cells was inhibited by Indo, whereas there was a potentiation of the effect of 24,25-(OH) 2D 3 on PKC activity in resting zone cells. In addition, inhibition of PKC blocked the 1,25-(OH) 2D 3-dependent increase in PGE 2 production in growth zone cells and the 24,25-(OH) 2D 3-dependent decrease in PGE 2 production by resting zone cells. These data indicate that prostaglandins are involved in mediating the rapid effects of 1,25-(OH) 2D 3 on growth zone cells, and contribute to the effects of 24,25-(OH) 2D 3 on resting zone cells; in both instances, the vitamin D metabolites exert their effects on PKC through changes in arachidonic acid via the action of PLA 2. In addition, PKC by itself may mediate the production of PGE 2.

TGFbeta1 regulates 25-hydroxyvitamin D3 1alpha- and 24-hydroxylase activity in cultured growth plate chondrocytes in a maturation-dependent manner.

Chondrocytes metabolize 25-(OH)D3 to the two active dihydroxylated forms of the secosteroid, 1,25-(OH)2D3 and 24,25-(OH)2D3. The aim of the present study was to examine the activity of the enzymes responsible for this metabolism, 1alpha-hydroxylase and 24R-hydroxylase, and their regulation by TGFbeta1. Basal 1alpha- and 24R-hydroxylase activities were measured in homogenates of confluent, fourth passage rat costochondral resting zone and growth zone chondrocytes and mouse cortico-tubular cells (MCT) were used as a positive control. The cells were harvested and homogenized in buffer optimized to maintain the activity and stability of the hydroxylases. Homogenates were incubated for 90 minutes and 1alpha- and 24R-hydroxylase activities determined by measuring the conversion of [3H]-25-(OH)D3 to [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3 using an HPLC with an inline radioisotope detector. Resting zone cells were also treated with various concentrations of recombinant human TGFbeta1 for 24 hours, and enzyme activity in total cell homogenates as well as 24-hydroxylase mRNA levels were determined. In addition, [3H]-1,25-(OH)2D3 and [3H]-24,25-(OH)2D3 released into the conditioned media by resting zone chondrocyte cultures in response to TGFbeta1 were measured. In culture, all three cell types were found to contain 1alpha- and 24R-hydroxylase activities. Basal 1alpha-hydroxylase specific activity was significantly higher than 24R-hydroxylase specific activity in all cells. RT-PCR confirmed that resting zone and growth zone cells expressed mRNA for 24R-hydroxylase. Treatment of resting zone cells with TGFbeta1 increased 24R-hydroxylase mRNA levels in a dose-dependent manner. TGFbeta1 also increased 24R-hydroxylase activity 2- to 5-fold and decreased 1alpha-hydroxylase activity by 20-30%. Similar changes were observed with MCT cells, but not growth zone cells. Production of [3H]-24,25-(OH)2D3 by resting zone cells increased with TGFbeta1 treatment, while [3H]-1,25-(OH)2D3 production decreased. The effect was time- and dose-dependent, correlating with hydroxylase activity and 24-hydroxylase gene expression. These results demonstrate that growth plate chondrocytes contain the necessary enzymes to produce 1, 25-(OH)2D3 and 24,25-(OH)2D3 from 25-(OH)D3. In addition, the activity of these enzymes in resting zone cells, but not growth zone cells, is regulated by TGFbeta1 by increasing gene transcription, indicating that cell maturation-dependent autocrine/paracrine pathways exist for regulating vitamin D metabolite production.

Vitamin D3 metabolites regulate LTBP1 and latent TGF‐β1 expression and latent TGF‐β1 incorporation in the extracellular matrix of chondrocytes

Growth plate chondrocytes make TGF-β1 in latent form (LTGF-β1) and store it in the extracellular matrix via LTGF-β1 binding protein (LTBP1). 1,25-(OH)2D3 (1,25) regulates matrix protein production in growth zone (GC) chondrocyte cultures, whereas 24,25-(OH)2D3 (24,25) does so in resting zone (RC) cell cultures. The aim of this study was to determine if 24,25 and 1,25 regulate LTBP1 expression as well as the LTBP1-mediated storage of TGF-β1 in the extracellular matrix of RC and GC cells. Expression of LTBP1 and TGF-β1 in the growth plate and in cultured RC and GC cells was determined by in situ hybridization using sense and antisense oligonucleotide probes based on the published rat LTBP1 and TGF-β1 cDNA sequences. Fourth passage male rat costochondral RC and GC chondrocytes were treated for 24 h with 10-7-10-9 M 24,25 and 10-8-10-10 M 1,25, respectively. LTBP1 and TGF-β1 mRNA levels were measured by in situ hybridization; production of LTGF-β1, LTGF-β2, and LTBP1 protein in the conditioned media was verified by immunoassays of FPLC-purified fractions. In addition, ELISA assays were used to measure the effect of 1,25 and 24,25 on the level of TGF-β1 in the media and matrix of the cultures. Matrix-bound LTGF-β1 was released by digesting isolated matrices with 1 U/ml plasmin for 3 h at 37°C. LTBP1 and TGF-β1 mRNAs are co-expressed throughout the growth plate, except in the lower hypertrophic area. Cultured GC cells express more LTBP1 and TGF-β1 mRNAs than RC cells. FPLC purification of the conditioned media confirmed that RC cells produce LTGF-β1, LTGF-β2, and LTBP1. GC cells also produce LTGF-β2, but at lower concentrations. 1,25 dose-dependently increased the number of GC cells with high LTBP1 expression, as seen by in situ hybridization. 24,25 had a similar, but less pronounced, effect on RC cells. 1,25 also caused a dose-dependent increase in the amount of TGF-β1 protein found in the matrix, significant at 10-8 and 10-9 M, and a corresponding decrease in TGF-β1 in the media. 24,25 had no effect on the level of TGF-β1 in the matrix or media produced by RC cells. This indicates that 1,25 induces the production of LTBP1 by GC cells and suggests that the TGF-β1 content of the media is reduced through the formation of latent TGF-β1-LTBP1 complexes which mediates storage in the matrix. Although 24,25 induced the expression of LTBP1 by RCs, TGF-β1 incorporation into the matrix is not regulated by this vitamin D3 metabolite. Thus, vitamin D3 metabolites may play a role in regulating the availability of TGF-β1 by modulating LTBP1 production. J. Cell. Biochem. 72:151–165, 1999. © 1999 Wiley-Liss, Inc.

Vitamin D3 metabolites regulate LTBP1 and latent TGF-?1 expression and latent TGF-?1 incorporation in the extracellular matrix of chondrocytes

Growth plate chondrocytes make TGF-beta1 in latent form (LTGF-beta1) and store it in the extracellular matrix via LTGF-beta1 binding protein (LTBP1). 1,25-(OH)2D3 (1,25) regulates matrix protein production in growth zone (GC) chondrocyte cultures, whereas 24,25-(OH)2D3 (24,25) does so in resting zone (RC) cell cultures. The aim of this study was to determine if 24,25 and 1,25 regulate LTBP1 expression as well as the LTBP1 -mediated storage of TGF-beta1 in the extracellular matrix of RC and GC cells. Expression of LTBP1 and TGF-beta1 in the growth plate and in cultured RC and GC cells was determined by in situ hybridization using sense and antisense oligonucleotide probes based on the published rat LTBP1 and TGF-beta1 cDNA sequences. Fourth passage male rat costochondral RC and GC chondrocytes were treated for 24 h with 10(-7)-10(-9) M 24,25 and 10(-8)-10(-10) M 1,25, respectively. LTBP1 and TGF-beta1 mRNA levels were measured by in situ hybridization; production of LTGF-beta1, LTGF-beta2, and LTBP1 protein in the conditioned media was verified by immunoassays of FPLC-purified fractions. In addition, ELISA assays were used to measure the effect of 1,25 and 24,25 on the level of TGF-beta1 in the media and matrix of the cultures. Matrix-bound LTGF-beta1 was released by digesting isolated matrices with 1 U/ml plasmin for 3 h at 37 degrees C. LTBP1 and TGF-beta1 mRNAs are co-expressed throughout the growth plate, except in the lower hypertrophic area. Cultured GC cells express more LTBP1 and TGF-beta1 mRNAs than RC cells. FPLC purification of the conditioned media confirmed that RC cells produce LTGF-beta1, LTGF-beta2, and LTBP1. GC cells also produce LTGF-beta2, but at lower concentrations. 1,25 dose-dependently increased the number of GC cells with high LTBP1 expression, as seen by in situ hybridization. 24,25 had a similar, but less pronounced, effect on RC cells. 1,25 also caused a dose-dependent increase in the amount of TGF-beta1 protein found in the matrix, significant at 10(-8) and 10(-9) M, and a corresponding decrease in TGF-beta1 in the media. 24,25 had no effect on the level of TGF-beta1 in the matrix or media produced by RC cells. This indicates that 1,25 induces the production of LTBP1 by GC cells and suggests that the TGF-beta1 content of the media is reduced through the formation of latent TGF-beta1 -LTBP1 complexes which mediates storage in the matrix. Although 24,25 induced the expression of LTBP1 by RCs, TGF-beta1 incorporation into the matrix is not regulated by this vitamin D3 metabolite. Thus, vitamin D3 metabolites may play a role in regulating the availability of TGF-beta1 by modulating LTBP1 production.

Chondrocyte cultures express matrix metalloproteinase mRNA and immunoreactive protein; stromelysin-1 and 72 kDa gelatinase are localized in extracellular matrix vesicles.

Previous studies have shown that costochondral cartilage cell cultures produce extracellular matrix vesicles which contain metalloproteinase activity. In the present study, we examined whether two matrix metalloproteinases (MMPs) known to be present in cartilage, stromelysin-1 and 72 kDa gelatinase, are expressed by fourth passage resting zone and growth zone costochondral chondrocytes and whether they are specifically incorporated into matrix vesicles produced by the cells. We also examined whether the cells synthesize tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1 and TIMP-2). Oligonucleotide primers for stromelysin-1, 72 kDa gelatinase, tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2), and GAPDH were synthesized and optimized for use in the reverse transcription-polymerase chain reaction (RT-PCR). It was found that both resting zone and growth zone chondrocytes produced mRNA for both MMPs and the two TIMPs. Further, immunostaining of cell layers with antibodies to 72 kDa gelatinase and stromelysin-1 showed that both cell types produced these MMPs in culture. Substrate gel electrophoresis and Western analysis were used to characterize MMP activity in matrix vesicles, media vesicles, or plasma membranes as well as in conditioned media produced by the chondrocyte cultures. It was found that matrix vesicles but not plasma membranes or media vesicles were selectively enriched in stromelysin-1. Also, 72 kDa gelatinase was found in matrix vesicles, but to a lesser extent than seen in media vesicles. The relative activity of each enzyme detected was cell maturation-dependent. No MMP activity was detected in conditioned media produced by either cell type. The results of this study show that MMPs are expressed by resting zone and growth zone chondrocytes in culture and differentially distributed among three different membrane compartments. This suggests that, in addition to the well-known activators and inhibitors of MMP activity in the matrix, differential membrane distribution may enable more precise control over the site, rate, and extent of matrix degradation by the cell.

Growth plate chondrocytes store latent transforming growth factor (TGF)-beta 1 in their matrix through latent TGF-beta 1 binding protein-1.

Osteoblasts produce a 100 kDa soluble form of latent transforming growth factor beta (TGF-beta) as well as a 290 kDa form containing latent TGF-beta binding protein-1 (LTBP1), which targets the latent complex to the matrix for storage. The nature of the soluble and stored forms of latent TGF-beta in chondrocytes, however, is not known. In the present study, resting zone and growth zone chondrocytes from rat costochondral cartilage were cultured to fourth passage and then examined for the presence of mRNA coding for LTBP1 protein. In addition, the matrix and media were examined for LTBP1 protein and latent TGF-beta. Northern blots, RT-PCR, and in situ hybridization showed that growth zone cells expressed higher levels of LTBP1 mRNA in vitro than resting zone cells. Immunohistochemical staining for LTBP1 revealed fine fibrillar structures around the cells and in the cell matrix. When the extracellular matrix of these cultures was digested with plasmin, LTBP1 was released, as determined by immunoprecipitation. Both active and latent TGF-beta1 were found in these digests by TGF-beta1 ELISA and Western blotting. Immunoprecipitation demonstrated that the cells also secrete LTBP1 which is not associated with latent TGF-beta, in addition to LTBP1 that is associated with the 100 kDa latent TGF-beta complex. These studies show for the first time that latent TGF-beta is present in the matrix of costochondral chondrocytes and that LTBP1 is responsible for storage of this complex in the matrix. The data suggest that chondrocytes are able to regulate both the temporal and spatial activation of latent TGF-beta, even at sites distant from the cell, in a relatively avascular environment.