Larry D. Swain

Matrix vesicles are enriched in metalloproteinases that degrade proteoglycans

SummaryThis study examined the presence of extracellular matrix processing enzymes in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. Optimum procedures for the extraction of each enzyme activity were determined. Enzyme activity associated with chondrocyte plasma membrane microsomes was used for comparison. There was a differential distribution of the enzyme activities related to the cartilage zone from which the cells were isolated. Acid and neutral metalloproteinase (TIMP), plasminogen activator, and betaglucuronidase were highest in the growth zone chondrocyte (GC) membrane fractions when compared with matrix vesicles and plasma membranes isolated from resting zone chondrocyte (RC) cultures. There was a threefold enrichment of total and active acid metalloproteinase in GC matrix vesicles, whereas no enrichment in enzyme activity was observed in RC matrix vesicles. Total and active neutral metalloproteinase were similarly enriched twofold in GC matrix vesicles. TIMP, plasminogen activator, and betaglucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found in any of the membrane fractions. The data indicate that matrix vesicles are selectively enriched in enzymes which degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.

Regulation of prostaglandin E2 production by vitamin D metabolites in growth zone and resting zone chondrocyte cultures is dependent on cell maturation

The production of PGE2 by chondrocytes and its regulation by vitamin D metabolites was examined in this study as a function of cell maturation. Costochondral chondrocytes, derived from the resting zone and growth zone cartilage, were grown in culture to fourth passage. At confluence, they were exposed to 10(-8)-10(-11)M 1,25-(OH)2D3 or to 10(-7)-10(-10)M 24,25-(OH)2D3 for either five minutes or 3, 6, 12, or 24 hours. Indomethacin (10(-7)M) was added to one-half of the cultures to block the production of PGE2. The amount of PGE2 released into the media was determined by radioimmunoassay. Both growth zone and resting zone cells produced PGE2 in a time-dependent manner; PGE2 concentration was greater in the resting zone cell cultures. 1,25-(OH)2D3 stimulated PGE2 production by growth zone cells in a dose-dependent manner, significant at 10(-8)-10(-10)M. This effect was observed at 3 hours and remained elevated during the 24 hours of culture. 1,25-(OH)2D3 had no effect on PGE2 production by resting zone cells. However, 24,25-(OH)2D3 (10(-7)-10(-8)M) inhibited PGE2 production from 3-24 hours. No effect was noted when 24,25-(OH)2D3 was added to growth zone cells. Indomethacin reduced PGE2 production to baseline values in all groups examined. The results indicate that chondrocytes in culture produce PGE2. Production is regulated by vitamin D3 metabolites and is cell maturation-dependent.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of arachidonic acid turnover by 1,25-(OH)2D3 and 24,25-(OH)2D3 in growth zone and resting zone chondrocyte cultures

Previous studies have shown that phospholipase A2 activity in rat costochondral chondrocyte cultures is differentially regulated by 1,25-(OH)2D3 and 24,25-(OH)2D3. 1,25-(OH)2D3 stimulates enzyme activity in growth zone chondrocytes but has no effect on the resting zone chondrocyte enzyme activity. 24,25-(OH)2D3 inhibits the resting zone enzyme but has no effect on the growth zone chondrocyte phospholipase A2. 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]arachidonic acid by exogenous phospholipases than were matrix vesicle phospholipids. The effect of 1,25-(OH)2D3 on growth zone chondrocytes was observed within 5 min. Incorporation was greatest after 60 min; release was greatest after 30 min. 24,25-(OH)2D3 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)2D3 and 24,25-(OH)2D3 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.

Nongenomic regulation of chondrocyte membrane fluidity by 1,25-(OH)2D3 and 24,25-(OH)2D3 is dependent on cell maturation

1,25-(OH)2D3 and 24,25-(OH)2D3 regulate rat costochondral chondrocyte cultures in a metabolite-specific manner; 1,25-(OH)2D3 targets primarily growth zone cells (GC) and 24,25-(OH)2D3 targets primarily resting zone cells (RC). Some of the effects are nongenomic, since incubation of isolated membrane fractions with the metabolites results in regulation of enzyme activities comparable to that seen in culture. This study examined whether changes in membrane fluidity might be one mechanism involved in the nongenomic regulatory pathway. Chondrocyte cultures were incubated with the vitamin D metabolites and changes in plasma membrane fluidity monitored using the fluorophore, TMA-DPH, which is specific for membranes exposed to external fluids. Isolated matrix vesicles were also incubated directly with the metabolites and anisotropy of the membrane, as well as alkaline phosphatase-specific activity, determined. 1,25-(OH)2D3 caused a rapid and constant increase in alkaline phosphatase-specific activity in GC matrix vesicles; 24,25-(OH)2D3 caused an increase in RC matrix vesicle enzyme activity that was both dose- and time-dependent. Matrix vesicles produced by GC had a lower degree of fluidity than their parent plasma membranes or RC plasma membranes and matrix vesicles. Fluidity of the GC membrane fractions was increased by 1,25-(OH)2D3 in a dose- and time-dependent manner. 1,25-(OH)2D3 had no effect on the fluidity of the RC membranes. 24,25-(OH)2D3 caused a decrease in fluidity in GC at later time points. This metabolite caused an increase in fluidity of RC plasma membranes that returned to normal levels by 6 h; however, the increase induced in the matrix vesicles remained elevated throughout the 24-h experimental period.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential regulation of prostaglandin E2 synthesis and phospholipase A2 activity by 1,25-(OH)2D3 in three osteoblast-like cell lines (MC-3T3-E1, ROS 17/2.8, and MG-63)

Both 1,25-(OH)2D3 and prostaglandin E2 (PGE2) stimulate alkaline phosphatase activity in MC-3T3-E1 cells. Previous studies, demonstrating a correlation between 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activities in matrix vesicles isolated from growth cartilage chondrocyte cultures, suggest that one mechanism of vitamin D action may be via autocrine or paracrine action of PGE2. Since most PGE2 is derived from arachidonic acid released by the action of phospholipase A2, we examined whether 1,25-(OH)2D3 stimulates phospholipase A2 activity in three osteoblastic cell lines: ROS 17/2.8 cells, MC-3T3-E1 cells, and MG-63 cells. 1,25-(OH)2D3-dependent alkaline phosphatase and phospholipase A2 activity were correlated with production of PGE2 and PGE1 in the MC-3T3-E1 cells. Alkaline phosphatase specific activity was enriched in the matrix vesicles produced by all three cell types and was stimulated by 1,25-(OH)2D3 at 10(-8) to 10(-7) M. Although phospholipase A2 specific activity was enriched in the matrix vesicles produced only by the ROS 17/2.8 cell cultures, stimulation of this enzyme activity was observed only in the MC-3T3-E1 cell cultures. The effects of 1,25-(OH)2D3 on phospholipase A2 were dose-dependent and were significant at 10(-8) to 10(-7) M. There was a significant increase in PGE2 production in the MC-3T3-E1 cell cultures only. Indomethacin reduced PGE2 production to base line values. Even at baseline, MC-3T3-E1 cells produced ten times more PGE2 than did the ROS 17/2.8 or MG-63 cell cultures. The effects of 1,25-(OH)2D3 on PGE1 were comparable to those on PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

1,25-(OH)2D3 and 24,25-(OH)2D3 regulation of arachidonic acid turnover in chondrocyte cultures is cell maturation-specific and may involve direct effects on phospholipase A2

Previous studies have shown that 1,25-(OH)2D3 stimulates phospholipase A2 (PA2) activity in growth zone chondrocytes (GC), but has no effect on the resting zone chondrocyte (RC) enzyme activity. 24,25-(OH)2D3 inhibits the RC enzyme but has no effect on the GC. This study examined whether the vitamin D metabolites affect arachidonic acid turnover in their contra-target cell populations. Incorporation and release of [14C]arachidonate was measured at various times following addition of hormone. Acylation and reacylation were measured independently by incubating with p-chloromercuribenzoate. The results demonstrated that 1,25-(OH)2D3 has no effect on arachidonic acid turnover in RC, but stimulates turnover in GC. In contrast, 24,25-(OH)2D3 stimulates arachidonic acid turnover in RC, but inhibits both incorporation and release in GC. To determine whether direct interaction with PA2 is one mechanism by which 1,25-(OH)2D3 and 24,25-(OH)2D3 regulate arachidonic acid release, snake venom (Niger niger) PA2 was incubated with the vitamin D metabolites. Enzyme specific activity was inhibited by 24,25-(OH)2D3 and stimulated by 1,25-(OH)2D3 in a time- and dose-dependent manner. These results suggest that at least part of the direct effect of vitamin D3 metabolites on cell membranes may be related to changes in PA2 activity. The regulation is related to the stage of differentiation of the target cell population. Changes in fatty acid acylation and reacylation may be one mode of vitamin D3 action in cartilage.

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

SummaryVitamin 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. Influex was measured as incorporation of45Ca. Efflux was measured as release of45Ca from prelabeled cultures into fresh media. The pattern of45Ca 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 of45Ca 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 in45Ca 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. These studies demonstrate that changes in Ca influx and efflux are metabolite specific and may be a mechanism by which vitamin D metabolites directly regulated chondrocytes in culture.

α2-HS-glycoprotein: Expression in chondrocytes and augmentation of alkaline phosphatase and phospholipase A2 activity

Abstract 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 glass ceramic and titanium implants on primary calcification during rat tibial bone healing

SummaryThe effect of bone bonding (KG Cera, Mina 13, and titanium) and nonbone bonding (KGy-213, M 8/1) implants on primary calcification in endosteal bone was examined by comparing changes in the morphometry of matrix vesicles to those occurring during normal bone healing following ablation of rat tibial marrow. The concentration of matrix vesicles, their diameter, and their distance from the calcification front were determined using computerized cytomorphometry at the transmission electron microscopic level. The results demonstrated that bone bonding materials supported an increase in matrix vesicle concentration when compared with control bone at 6 and 14 days postimplantation. At 14 days, there were fewer matrix vesicles in the bone adjacent to the nonbonding implants. Though matrix vesicle diameter decreased in the control bone between 6 and 14 days, it increased in all of the experimental samples. Diameters were significantly greater in the bone bonding samples at 14 days and significantly lower in the nonbonding samples at 6 days. Distance from the calcification front decreased between 6 and 14 days in all groups except in bone adjacent to the KGy-213 implants. In bone adjacent to the bone bonding implants, distance from the calcification front was comparable to or further than that of control bone; in the nonbonding samples it was closer to the calcification front. These results demonstrate that production and maturation of matrix vesicles is influenced in a differential manner by the presence of implant materials.

Stimulation of matrix vesicle enzyme activity in osteoblast-like cells by 1,25(OH)2D3 and transforming growth factor β (TGFβ)

Abstract After demonstrating the presence of matrix vesicles in three osteosarcoma cell lines, MG-63, ROS 17/2.8 and MC-3T3-E1, we sought to determine whether two major enzymes localized to matrix vesicles, alkaline phosphatase and phospholipase A 2 , could be regulated by 1,25(OH) 2 D 3 and/or TGFβ. Intravesicular calcification is probably dependent on these two enzymes. Alkaline phosphatase is essential for hydrolysis of phosphate-containing substrates and phospholipase A 2 hydrolyzes diacylphosphatides in a calcium-mediated manner at lipid-aqueous interfaces leading to changes in membrane fluidity and possibly breakdown of the matrix vesicle. The 1,25(OH) 2 D 3 induced increase of alkaline phosphatase in bone cells is localized to the matrix vesicle. TGFβ also increased alkaline phosphatase activity in two of the cell lines, MG-63 and ROS 17/2.8 but to a greater degree than 1,25(OH) 2 D 3 . Matrix vesicle alkaline phosphatase activity exhibited a greater response than that in the plasma membrane. TGFβ increased phospholipase A 2 activity in both matrix vesicles and plasma membranes, therefore, no targeting was observed with respect to this enzyme. When TGFβ was combined with 1,25(OH) 2 D 3 , 1,25(OH) 2 D 3 had no effect on phospholipase A 2 and did not interfere with TGFβ stimulation of phospholipase A 2 activity. When 1,25(OH) 2 D 3 and TGFβ were combined, a tremendous synergy was observed in alkaline phosphatase specific activity in both plasma membranes and matrix vesicles with targeting to matrix vesicles. Therefore, TGFβ not only plays an important role in matrix formation and differentiation, but works in conjunction with 1,25(OH) 2 D 3 to greatly potentiate the effects seen with 1,25(OH) 2 D 3 alone.