Biomedical subjects

Z Schwartz

Publications and source records attributed to Z Schwartz.

At least 55 records · Page 3Linked to original sources

PubMed2002

Effect of porcine fetal enamel matrix derivative on chondrocyte proliferation, differentiation, and local factor production is dependent on cell maturation state.

Recent studies have shown that porcine fetal enamel matrix derivative (EMD) can enhance the osteoinductive ability of demineralized freeze-dried bone allograft (DFDBA) in a nude mouse muscle implantation model. This suggests that one or more components of EMD can regulate the process of endochondral ossification initiated by DFDBA. To substantiate this hypothesis, in the current study, chondrocytes in the endochondral pathway at two stages of maturation were tested for their response to EMD. Chondrocytes were isolated from the resting zone and growth zone (prehypertrophic and upper hypertrophic cell zones) of the costochondral growth plate cartilage of adolescent rats. The results showed that the relatively immature resting zone cells responded to EMD with an increase in proliferation and a decrease in differentiation as measured by alkaline-phosphatase-specific activity. In addition, EMD stimulated a fivefold increase in PGE(2) production, but was without effect on collagen synthesis, proteoglycan sulfation, and TGF-beta(1) production. The more mature growth zone cells also responded to EMD with increased proliferation, but alkaline-phosphatase-specific activity was unchanged, and there was only a modest increase in PGE(2) production. In contrast to resting zone cells, growth zone cells exhibited a decrease in collagen synthesis, proteoglycan sulfation, and TGF-beta(1) production. These observations indicate that EMD has prominent effects on cells in the endochondral pathway. In particular, EMD stimulates the production of more cells, but inhibits their maturation. This would increase the pool of cells available for subsequent differentiation in response to other factors.

Alkaline Phosphatase↗

PubMed2002

Differential regulation of growth plate chondrocytes by 1alpha,25-(OH)2D3 and 24R,25-(OH)2D3 involves cell-maturation-specific membrane-receptor-activated phospholipid metabolism.

This review discusses the regulation of growth plate chondrocytes by vitamin D(3). Over the past ten years, our understanding of how two vitamin D metabolites, 1alpha,25-(OH)(2)D(3) and 24R,25-(OH)(2)D(3), exert their effects on endochondral ossification has undergone considerable advances through the use of cell biology and signal transduction methodologies. These studies have shown that each metabolite affects a primary target cell within the endochondral developmental lineage. 1alpha,25-(OH)(2)D(3) affects primarily growth zone cells, and 24R,25-(OH)(2)D(3) affects primarily resting zone cells. In addition, 24R,25-(OH)(2)D(3) initiates a differentiation cascade that results in down-regulation of responsiveness to 24R,25-(OH)(2)D(3) and up-regulation of responsiveness to 1alpha,25-(OH)(2)D(3). 1alpha,25-(OH)(2)D(3) regulates growth zone chondrocytes both through the nuclear vitamin D receptor, and through a membrane-associated receptor that mediates its effects via a protein kinase C (PKC) signal transduction pathway. PKCalpha is increased via a phosphatidylinositol-specific phospholipase C (PLC)-dependent mechanism, as well as through the stimulation of phospholipase A(2) (PLA(2)) activity. Arachidonic acid and its downstream metabolite prostaglandin E(2) (PGE(2)) also modulate cell response to 1alpha,25-(OH)(2)D(3). In contrast, 24R,25-(OH)(2)D(3) exerts its effects on resting zone cells through a separate, membrane-associated receptor that also involves PKC pathways. PKCalpha is increased via a phospholipase D (PLD)-mediated mechanism, as well as through inhibition of the PLA(2) pathway. The target-cell-specific effects of each metabolite are also seen in the regulation of matrix vesicles by vitamin D(3). However, the PKC isoform involved is PKCzeta, and its activity is inhibited, providing a mechanism for differential autocrine regulation of the cell and events in the matrix by these two vitamin D(3) metabolites.

24,25-Dihydroxyvitamin D 3↗

PubMed2002

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

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

24,25-Dihydroxyvitamin D 3↗

PubMed2002

Coverage of previously carious roots is as predictable a procedure as coverage of intact roots.

BACKGROUND: Coverage of roots exposed by gingival recession is one of the main objectives of periodontal reconstructive surgery. A variety of highly predictable and esthetically acceptable mucogingival grafting procedures are available for treating intact root surfaces. One of the indications for root coverage procedures is prevention of root caries. However, little is known about the ability to treat previously carious roots by mucogingival grafting, and even less is known about the outcome of such treatment. METHODS: Sixty patients with gingival recession were treated with subepithelial connective tissue grafts. During initial examination, 33 intact teeth with longstanding gingival recessions of 4.09 +/- 0.16 mm (mean +/- SEM) and 27 teeth with carious roots (restored and unrestored) and longstanding gingival recessions of 3.44 +/- 0.22 mm (mean +/- SEM) were found. Prior to grafting, any carious dentin and plastic restorations were removed. The exposed roots were thoroughly planed and covered by a subepithelial connective tissue graft without any further root treatment or conditioning. Patients were evaluated periodically between 1 and 6 years. Residual recession, defect coverage, and caries incidence were assessed. RESULTS: Recession was reduced to a similar extent on all teeth whether root caries was present (0.31 +/- 0.11 mm) or not (0.15 +/- 0.04 mm). This resulted in comparable defect coverages of 92.41 +/- 2.38% for previously carious roots and 97.46 +/- 0.79% for intact roots. In 44 cases, complete coverage of the recession was achieved. The results were stable, and no further recessions or recurrent caries were found during the periodic evaluations. CONCLUSIONS: These results indicate that coverage of previously carious roots with subepithelial connective tissue grafts is a very predictable procedure with results similar to those found on intact roots. This procedure may provide a definitive biological alternative for conventional restorative treatment modalities for root caries. Moreover, the results are more esthetic, biologically acceptable, and maintainable.

Adult↗

PubMed2002

[The use of enamel matrix derivative in periodontal therapy].

Periodontal disease results in the loss of the attachment apparatus, i.e. the alveolar bone, the periodontal ligament and the cementum. In the last three decades, an increasing effort has been placed on seeking procedures and materials to promote the regeneration of these tissues. This article reviews published data concerning the enamel matrix derivative (EMD) as a regenerative mean. It encompasses in vitro and in vivo studies as well as human case reports, clinical comparative trials and histologic findings. According to the discussed studies it may be concluded that EMD possess comparable clinical abilities to initiate regeneration in previously diseased sites as guided tissue regeneration, or as the use of xenograft material. In contrast, the histologic findings are less promising regarding the amount of new bone formation. The controversy results concerning the benefit of the EMD as an additive to xenograft or alloplast materials should be attributed to the correct case selection, and particularly to the defect morphology. However, this latter statement needs further validation. Clinical examples of treatment utilizing EMD or in conjunction with allograft are presented.

Alveolar Bone Loss↗

PubMed2001

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.

Animals↗

PubMed2001

Osteoblast response to titanium surface roughness and 1alpha,25-(OH)(2)D(3) is mediated through the mitogen-activated protein kinase (MAPK) pathway.

When osteoblasts are cultured on surfaces of increasing microroughness, they exhibit decreases in proliferation, increases in differentiation and local factor production, and enhanced response to 1alpha,25(OH)(2)D(3). The cells interact with surfaces through integrins, which signal by the same pathways used by 1alpha,25(OH)(2)D(3), including protein kinase C via phospholipase C and protein kinase A via phospholipase A(2). This provides opportunities for crosstalk that may contribute to the synergistic effects of surface roughness and the vitamin D metabolite. Because these pathways converge at mitogen-activated protein kinase (MAPK), we tested the hypothesis that the extracellular signal-regulated kinase (ERK1/2) subclass of MAPKs mediates the effects of surface roughness and 1alpha,25(OH)(2)D(3). MG63 osteoblast-like osteosarcoma cells were cultured on commercially pure Ti disks with various surface roughnesses: pretreatment (PT; 0.6 microm average roughness [Ra]), coarse grit-blasted and acid-etched (SLA; 4 microm RA), and titanium plasma-sprayed (TPS; 5.2-microm R(a)). At confluence, cells were treated for 24 h with control media or media containing 10(-7) M 1alpha,25(OH)(2)D(3). One-half of the cultures received 1 microm or 10 microm PD98059, a specific inhibitor of the ERK family of MAPKs. PD98059 alone did not affect proliferation, osteocalcin production, or production of transforming growth factor-beta1 or nitric oxide, regardless of the surface roughness. Alkaline phosphatase was reduced by the inhibition of the ERK family kinases on all surfaces to a comparable extent. However, when PD98059 was added to the cultures with 1alpha,25(OH)(2)D(3), the effects of the seco-steroid were blocked, including the synergistic increases seen in MG63 cells cultured on SLA or TPS. These results indicate that ERK1/2 MAPK is required for the maintenance of alkaline phosphatase at control levels and that the effects of 1alpha,25(OH)(2)D(3) are mediated by ERK1/2. However, the effects of surface roughness are not due to the ERK family of MAPKs. This suggests that alternative pathways may be used, including those mediated by other MAPK subclasses.

Cell Adhesion↗

PubMed2001

Both cyclooxygenase-1 and cyclooxygenase-2 mediate osteoblast response to titanium surface roughness.

Previous studies suggest that the enhanced expression of the osteoblastic phenotype exhibited by MG63 osteoblast-like cells on rough Ti surfaces (R(a) 4-5 microm) involves increased production of prostaglandin. Inhibition of prostaglandin synthesis by indomethacin blocks surface-roughness-dependent decreases in cell proliferation and increases in alkaline phosphatase activity and the production of osteocalcin and TGF-beta1. This study examined the hypothesis that the increase in expression of the osteoblastic phenotype noted in MG63 cells cultured on rough Ti surfaces is mediated by inducible cyclooxygenase-2 (Cox-2) whereas Cox-1 modulates prostaglandin production and phenotypic expression of the cells under standard conditions and on smooth Ti surfaces. MG63 cells were cultured on tissue culture plastic, smooth Ti (PT, R(a) = 0.60 microm), and two rough Ti surfaces with differing morphologies (SLA, R(a) = 3.97 microm and TPS, R(a) = 5.21 microm). At 24 h after plating, media were replaced with media containing the general Cox inhibitor indomethacin (10(-7)M), the Cox-1 inhibitor resveratrol (1 or 10 microM), or the Cox-2 inhibitor NS-398 (1 or 10 microM). Media were changed again after 48 h. Five days after plating, osteocalcin, PGE(2), and TGF-beta1 content of the conditioned media were determined. Cell numbers were assessed in the same cultures used for determination of osteocalcin production. Cell layer protein and alkaline phosphatase specific activity were assessed in cultures used to measure PGE(2) and TGF-beta1. Indomethacin, resveratrol, and NS-398 had no effect on cell number. Indomethacin blocked the surface-roughness-dependent increase in PGE(2) production by up to 80%. Similarly, resveratrol inhibited up to 50% of the PGE(2) production on smooth surfaces and up to 80% on rough surfaces. In contrast, NS-398 had no effect on PGE(2) production by cells on smooth surfaces but caused a 60% reduction in cultures on rough surfaces. Indomethacin reduced alkaline phosphatase on all surfaces below basal levels. However, neither resveratrol nor NS-398 had an effect. Indomethacin blocked the stimulatory effect of surface roughness on osteocalcin production while resveratrol only partially reduced osteocalcin production, and NS398 completely blocked the surface-dependent increase. TGF-beta1 production on rough surfaces was blocked by indomethacin. The effects of resveratrol and NS-398 were dose dependent, but neither agent caused total inhibition of the increase noted on SLA, and only resveratrol blocked the increase on TPS. These results indicate that both Cox-1 and Cox-2 are involved in the response of osteoblasts to surface roughness with respect to production of PGE(2), TGF-beta1, and osteocalcin. While prostaglandin mediates the effects of surface roughness on alkaline phosphatase, neither Cox-1 nor Cox-2 appears to be involved, at least with respect to the two inhibitors used.

Alkaline Phosphatase↗

PubMed2001

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

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

24,25-Dihydroxyvitamin D 3↗

PubMed2001

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.

Animals↗

PubMed2001

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.

Alkaline Phosphatase↗

PubMed2001

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.

24,25-Dihydroxyvitamin D 3↗

PubMed2001

Tissue response and osteoinduction of human bone grafts in vivo.

Freeze-dried human bone allograft is used clinically as an adjunct to autologous bone graft. When freeze-dried human bone allograft is demineralized, the allograft is osteoinductive, since it causes bone to form heterotopically. Both types of allograft are also used alone, such as in spinal fusions, critical size defects, and periodontal therapy. The purpose of this study was to determine the effect of demineralization on the osteoinductive potential of human bone grafts obtained from two different groups of patients. One group consisted of six patients younger than 42 years of age, while the other group consisted of six patients who were older than 70 years of age. The harvested material was lyophilized and divided into two portions, one of which was used directly while the other was demineralized. Osteoinductive ability was established using an in vivo assay for heterotopic bone formation. Activity in these bone grafts was compared with a batch of commercially prepared demineralized, freeze-dried human bone grafts that had been previously shown to be active and another batch that had been shown to display low ('inactive') osteoinductive ability. A bone induction score was determined for each group of grafts based on the number and size of any ossicles formed. In addition, the area of new bone formation and area of residual particles were determined histomorphometrically. Tissue response to the bone grafts varied with donor age and whether the samples had been demineralized or not. Only demineralized, freeze-dried bone graft from patients younger than 42 years of age was osteoinductive; all other batches displayed little or no osteoinductive activity. In the demineralized, freeze-dried bone from donors younger than 42 years of age, the bone induction score and new bone area were significantly higher than in the other batches of bone graft, and the area of residual particles was reduced. Both demineralized and nondemineralized bone graft from patients older than 70 years of age were encapsulated in dense, fibrous connective tissue. These results may help explain the observed differences in clinical outcome when demineralized, freeze-dried bone graft or nondemineralized, freeze-dried bone graft from different donors is used in bone regeneration applications.

Adult↗

PubMed2001

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.

Androstadienes↗

PubMed2001

Local factor production by MG63 osteoblast-like cells in response to surface roughness and 1,25-(OH)2D3 is mediated via protein kinase C- and protein kinase A-dependent pathways.

Titanium (Ti) surface roughness affects bone formation in vivo and osteoblast attachment, proliferation and differentiation in vitro. MG63 cells exhibit decreased proliferation and increased differentiation when cultured on rough Ti surfaces (Ra > 2 microm) and response to 1,25-(OH)2D3 is enhanced, resulting in synergistic increases in TGF-beta1 and PGE2. To examine the hypothesis that surface roughness and 1,25-(OH)2D3 exert their effects on local factor production through independent, but convergent, signaling pathways, MG63 cells were cultured on tissue culture plastic or on smooth (PT, Ra = 0.60 microm) and rough (SLA, Ra = 3.97 microm; TPS, Ra = 5.21 microm) Ti disks. At confluence (5 days), cultures were treated for 24h with 10(-8) M 1alpha,25-(OH)2D3 and active and latent TGF-beta1 in the conditioned media measured by ELISA. Cell layers were digested with plasmin and released TGF-beta1 was also measured. 1,25-(OH)2D3 regulated the distribution of TGF-beta1 between the media and the matrix in a surface-dependent manner; the effect was greatest in the matrix of cells cultured on SLA and TPS. Inhibition of PKA with H8 for the last 24 h of culture increased PGE2 on SLA and TPS, but when present throughout the entire culture period H8 caused an increase in PGE2 on all surfaces. 1,25-(OH)2D3 reduced the effect of H8 on PGE2 production in cultures treated for 24 h. H8 had no effect on TGF-beta1 in the media by itself but caused a complete inhibition of the 1,25-(OH)2D3 dependent increase. Inhibition of PKC with chelerythrine increased PGE2 in a surface-dependent manner and 1,25-(OH)2D3 reduced the effect of the PKC inhibitor. Chelerythrine also increased TGF-beta1 but the effect was not surface dependent; however, 1,25-(OH)2D3 reduced the effects of chelerythrine with the greatest effects on the smooth surface. Thus, the distribution of TGF-beta1 between the media and the matrix is regulated by 1,25-(OH)2D3 in a surface-dependent manner. Surface roughness exerts its effects on TGF-beta1 production via PKC but not PKA. The effect of 1,25-(OH)2D3 on TGF-beta1 production is not via PKC. PKA is involved in the surface-dependent regulation of PGE2 but not in the regulation of PGE2 by 1,25-(OH)2D3 on rough surfaces. Regulation of PKC affects PGE2 production but it is not involved in the surface roughness-dependent response to 1,25-(OH)2D3. These results suggest two independent but interconnected pathways are involved.

Animals↗

PubMed2001

Effect of polymer molecular weight and addition of calcium stearate on response of MG63 osteoblast-like cells to UHMWPE particles.

Periprosthetic osteolysis and implant loosening is associated with the presence of ultrahigh molecular weight polyethylene (UHMWPE) wear debris particles. Osteoblast phenotypic expression in vitro is affected by UHMWPE particles, suggesting that bone formation may also be affected by wear debris. Here we tested the hypothesis that the response of osteoblasts to UHMWPE can be modified by changes in UHMWPE particle chemistry. We used four different commercially available preparations of GUR UHMWPE particles to determine if chemical composition (+/- Ca-stearate) or polymer molecular weight (3.1-4.2 million or 5.4-6.5 million g/mol) modulates osteoblast response. Particles were characterized by size distribution, morphology, and number of particles added to the culture medium. They had an average equivalent circle diameter ranging from 0.46-1.26 microm. MG63 cell response was assessed by measuring cell number, cellular and cell layer alkaline phosphatase, and prostaglandin E2 (PGE2) production. There were dose-dependent effects of the particles on cell response. Cell number and PGE, production were increased, while alkaline phosphatase specific activity was decreased. In addition, there was a marked difference between cultures treated with particles containing Ca-stearate and as a function of polymer molecular weight. Particles of higher molecular weight caused a greater stimulation of proliferation and inhibition of alkaline phosphatase than particles of lower molecular weight. The presence of Castearate exerted a more pronounced depression of osteoblast phenotype as well as a significantly greater increase in PGE2 release by the cells. The present study shows that chemical composition and polymer molecular weight of UHMWPE are capable of modulating osteoblast response to particles. The results suggest that osteoblast differentiation is inhibited by UHMWPE particles, whereas cell proliferation and PGE2 production are stimulated. This may have direct effects on osteoblasts and bone formation, but also paracrine effects on cells of the monocytic lineage inducing bone resorption and promoting inflammation which may lead to aseptic loosening. The present results suggest that the cellular events in aseptic loosening may be modulated or even accelerated by changes in the composition of the UHMWPE used to fabricate implants.

Alkaline Phosphatase↗

PubMed2001

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

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

24,25-Dihydroxyvitamin D 3↗

PubMed2001

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

Growth plate chondrocyte function is modulated by the vitamin D metabolite 1alpha,25-(OH)(2)D(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 1alpha,25-(OH)(2)D(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 1alpha,25-(OH)(2)D(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 1alpha,25-(OH)(2)D(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. 1alpha,25-(OH)(2)D(3)-induced alkaline phosphatase activity was only slightly stimulated by PTPGE(2). In contrast, 1alpha,25-(OH)(2)D(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 GDPbetaS, 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 1alpha,25-(OH)(2)D(3) on cellular maturation.

Alkaline Phosphatase↗