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D D Dean

Publications and source records attributed to D D Dean.

At least 91 records · Page 5Linked to original sources
PubMed1998

The synergistic effects of vitamin D metabolites and transforming growth factor-beta on costochondral chondrocytes are mediated by increases in protein kinase C activity involving two separate pathways.

Transforming growth factor-beta (TGFbeta), as well as the vitamin D3 metabolites 1,25-dihydroxyvitamin D3 (1,25) and 24,25-dihydroxyvitamin D3 (24,25), regulate chondrocyte differentiation and maturation during endochondral bone formation. Both the growth factor and secosteroids also affect protein kinase C (PKC) activity, although each has its own unique time course of enzyme activation. Vitamin D3 metabolite effects are detected soon after addition to the media, whereas TGFbeta effects occur over a longer term. The present study examines the interrelation between the effects of 1,25, 24,25, and TGFbeta on chondrocyte differentiation, matrix production, and proliferation. We also examined whether the effect is hormone-specific and maturation-dependent and whether the effect of combining hormone and growth factor is mediated by PKC. This study used a chondrocyte culture model developed in our laboratory that allows comparison of chondrocytes at two stages of differentiation: the more mature growth zone (GC) cells and the less mature resting zone chondrocyte (RC) cells. Only the addition of 24,25 with TGFbeta showed synergistic effects on RC alkaline phosphatase-specific activity (ALPase). No similar effect was found when 24,25 plus TGFbeta was added to GC cells or when 1,25 plus TGFbeta were added to GC or RC cells. The addition of 1,25 plus TGFbeta and 24,25 plus TGFbeta to GC and RC cells, respectively, produced a synergistic increase in [35S]sulfate incorporation and had an additive effect on [3H]thymidine incorporation. To examine the signal transduction pathway involved in producing the synergistic effect of 24,25 and TGFbeta on RC cells, the level of PKC activity was examined. Addition of 24,25 and TGFbeta for 12 h produced a synergistic increase in PKC activity. Moreover, a similar effect was found when 24,25 was added for only the last 90 min of a 12-h incubation. However, a synergistic effect could not be found when 24,25 was added for the last 9 min or the first 90 min of incubation. To further understand how 24,25 and TGFbeta may mediate the observed synergistic increase in PKC activity, the pathways potentially leading to activation of PKC were examined. It was found that 24,25 affects PKC activity through production of diacylglycerol, not through activation of G protein, whereas TGFbeta only affected PKC activity through G protein. The results of the present study indicate that vitamin D metabolites and TGFbeta produced a synergistic effect that is maturation-dependent and hormone-specific. Moreover, the synergistic effect between 24,25 and TGFbeta was mediated by activation of PKC through two parallel pathways: 24,25 through diacylglycerol production and TGFbeta through G protein activation.

24,25-Dihydroxyvitamin D 3↗
PubMed1998

The effect of prostaglandin E2 on costochondral chondrocyte differentiation is mediated by cyclic adenosine 3',5'-monophosphate and protein kinase C.

Recent studies indicate that vitamin D metabolites exert rapid effects on growth plate chondrocytes via changes in PG production and protein kinase C (PKC) activity. This suggests that these two products of vitamin D action may be interrelated. To test this hypothesis, we examined the effect of PGE2 on rat costochondral resting zone and growth zone cartilage cells and determined whether the effects of PGE2 are mediated by changes in the level of cAMP and/or PKC activity, whether there is a relationship between cAMP production and PKC activity, and whether cell maturation-specific effects are involved. Confluent, fourth passage resting zone and growth zone cartilage cell cultures were incubated in DMEM containing 10% FBS, 50 microg/ml vitamin C, and 1% antibiotics. The PGE2 concentration was varied from 0.007-15 ng/ml. Low concentrations of PGE2 caused a dose-dependent increase in cell number and [3H]thymidine incorporation and stimulated alkaline phosphatase specific activity. These effects were comparable in resting zone and growth zone cartilage cells at the same PGE2 concentrations. At higher concentrations, PGE2 caused a general increase in the synthesis of collagenase-digestible protein and noncollagenase-digestible protein in resting zone cartilage cells and of collagenase-digestible protein in growth zone cartilage cells, resulting in a net increase in the percent collagen synthesis for both cell types. cAMP production was increased over the entire range of chondrocyte response. Prevention of cAMP metabolism with the protein kinase A inhibitors H-8 and H-89 blocked the PGE2-dependent inhibition of PKC in resting zone cartilage cells in a dose-dependent manner. H-8 alone had no effect on PKC in resting zone cartilage cells, but stimulated PKC activity in growth zone cartilage cells; H-89 alone stimulated PKC activity in resting zone cartilage cells. These results suggest that low levels of PGE2 promote differentiation, whereas high doses promote an anabolic response; PGE2 increases cAMP production and PKC activity in a cell maturation-dependent manner; PGE2 exerts its effects via cAMP production and PKC activity; and regulation of PGE2-dependent PKC is via cAMP.

Alkaline Phosphatase↗
PubMed1998

1,25(OH)2D3 regulates protein kinase C activity through two phospholipid-dependent pathways involving phospholipase A2 and phospholipase C in growth zone chondrocytes.

We have previously shown that 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) plays a major role in growth zone chondrocyte (GC) differentiation and that this effect is mediated by protein kinase C (PKC). The aim of the present study was to identify the signal transduction pathway used by 1,25(OH)2D3 to stimulate PKC activation. Confluent, fourth passage GC cells from costochondral cartilage were used to evaluate the mechanism of PKC activation. Treatment of GC cultures with 1,25(OH)2D3 elicited a dose-dependent increase in both inositol-1,4,5-trisphosphate and diacylglycerol (DAG) production, suggesting a role for phospholipase C and potentially for phospholipase D. Addition of dioctanoylglycerol to plasma membranes isolated from GCs increased PKC activity. Neither pertussis toxin nor choleratoxin had an inhibitory effect on PKC activity in control or 1,25(OH)2D3-treated GCs, indicating that neither Gi nor Gs proteins were involved. Phospholipase A2 inhibitors, quinacrine, OEPC (selective for secretory phospholipase A2), and AACOCF3 (selective for cytosolic phospholipase A2), and the cyclooxygenase inhibitor indomethacin decreased PKC activity, while the phospholipase A2 activators melittin and mastoparan increased PKC activity in GC cultures. Arachidonic acid and prostaglandin E2, two downstream products of phospholipase A2 action, also increased PKC activity. These results indicate that 1,25(OH)2D3-dependent stimulation of PKC activity is regulated by two distinct phospholipase-dependent mechanisms: production of DAG, primarily via phospholipase C and production of arachidonic acid via phospholipase A2.

Animals↗
PubMed1998

Identification of a membrane receptor for 1,25-dihydroxyvitamin D3 which mediates rapid activation of protein kinase C.

This paper is the first definitive report demonstrating a unique membrane receptor for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) which mediates the rapid and nongenomic regulation of protein kinase C (PKC). Previous studies have shown that 1,25(OH)2D3 exerts rapid effects on chondrocyte membranes which are cell maturation-specific, do not require new gene expression, and do not appear to act via the traditional vitamin D receptor. We used antiserum generated to a [3H]1,25(OH)2D3 binding protein isolated from the basal lateral membrane of chick intestinal epithelium (Ab99) to determine if rat costochondral resting zone (RC) or growth zone (GC) cartilage cells contain a similar protein and if cell maturation-dependent differences exist. Immunohistochemistry demonstrated that both RC and GC cells express the protein, but levels are highest in GC. The binding protein is present in both plasma membranes and matrix vesicles and has a molecular weight of 66,000 Da. The 66 kDa protein in GC matrix vesicles has a Kd of 17.2 fmol/ml and Bmax of 124 fmol/mg of protein for [3H]1,25(OH)2D3. In contrast, the 66 kDa protein in RC matrix vesicles has a Kd of 27.7 fmol/ml and a Bmax of 100 fmol/mg of protein. Ab99 blocks the 1,25(OH)2D3-dependent increase in PKC activity in GC chondrocytes, indicating that the 1,25(OH)2D3-binding protein is indeed a receptor, linking ligand recognition to biologic function.

Animals↗
PubMed1998

Treatment of resting zone chondrocytes with bone morphogenetic protein-2 induces maturation into a phenotype characteristic of growth zone chondrocytes by downregulating responsiveness to 24,25(OH)2D3 and upregulating responsiveness to 1,25-(OH)2D3.

To determine if bone morphogenetic protein-2 (BMP-2) can induce the endochondral maturation of resting zone (RC) chondrocytes, confluent fourth-passage cultures of these cells were pretreated for 24, 36, 48, 72, or 120 h with recombinant human BMP-2. At the end of pretreatment, the media were replaced with new media containing 10(-10)-10(-8) M 1,25-(OH)2D3 or 10(-9)-10(-7) M 24,25-(OH2)D3 and the cells incubated for an additional 24 h. This second treatment was chosen, because prior studies had shown that the more mature growth zone (GC) chondrocytes and RC cells respond to 1,25-(OH)2D3 and 24,25-(OH)2D3 in distinctly different ways with respect to the parameters examined. The effect of BMP-2 pretreatment on cell maturation was assessed by measuring alkaline phosphatase specific activity (ALPase). In addition, changes in matrix protein production were assessed by measuring collagen synthesis, as well as [35S]-sulfate incorporation into proteoglycans. When RC cells were pretreated for 72 or 120 h with BMP-2, treatment with 1,25-(OH)2D3 caused a dose-dependent increase in ALPase specific activity and collagen synthesis, with no effect on proteoglycan sulfation. RC cells pretreated with 1,25-(OH)2D3 responded like RC cells that had not received any pretreatment. RC cells normally respond to 24,25-(OH)2D3; however, RC cultures pretreated for 72 or 120 h with BMP-2 lost their responsiveness to 24,25-(OH)2D3. These results indicate that BMP-2 directly regulates the differentiation and maturation of RC chondrocytes into GC chondrocytes. These observations support the hypothesis that BMP-2 plays a significant role in regulating chondrocyte maturation during endochondral ossification.

24,25-Dihydroxyvitamin D 3↗
PubMed1998

Addition of human recombinant bone morphogenetic protein-2 to inactive commercial human demineralized freeze-dried bone allograft makes an effective composite bone inductive implant material.

COMMERCIAL PREPARATIONS OF HUMAN DEMINERALIZED freeze-dried bone allograft (DFDBA) vary in their ability to induce new bone formation. This study tested the hypothesis that inactive DFDBA can be used as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2). Two batches of active DFDBA were used as controls. Two batches of DFDBA, previously shown to be inactive, were treated with vehicle or with 5 or 20 microg rhBMP-2 and implanted into the calf muscle of male Nu/Nu (nude) mice. Each mouse received one implant in each hind limb, both of which were of the same formulation, resulting in 8 groups of 4 mice per group: active DFDBA batch A, active DFDBA batch B, inactive DFDBA batch A, inactive DFDBA batch B, inactive DFDBA batch A plus 5 microg rhBMP-2, inactive DFDBA batch A plus 20 microg rhBMP-2, inactive DFDBA batch B plus 5 microg rhBMP-2, and inactive DFDBA batch B plus 20 microg rhBMP-2. After 56 days, the implants were removed and histologically examined. A semiquantitative bone induction index was calculated based on the amount of new bone covering each histological section. Histomorphometry was also used to evaluate the area of new bone formed and the area of residual implant material. The results showed that active DFDBA induces new bone formation, whereas inactive DFDBA does not. Addition of rhBMP-2 to inactive DFDBA results in new bone formation with a bone induction index comparable to that of active DFDBA. Histomorphometric analysis, however, revealed that the rhBMP-2-containing implants caused a dose-dependent increase in new bone area that exceeded that induced by active DFDBA. At the highest concentration of rhBMP-2, bone formation was exuberant. rhBMP-2 also caused the resorption of residual implant material to levels comparable to that seen in sites treated with active DFDBA, suggesting that this growth factor may regulate resorptive cells either directly or indirectly. This study shows that addition of rhBMP-2 to inactive DFDBA provides reproducible, consistent bone induction, and suggests that inactive commercial preparations may contain inadequate amounts of BMP to cause bone induction compared to active preparations.

Animals↗
PubMed1998

Ability of commercial demineralized freeze-dried bone allograft to induce new bone formation is dependent on donor age but not gender.

Demineralized freeze-dried bone allografts (DFDBA) have been used extensively in periodontal therapy. DFDBA is used because it contains bone morphogenetic protein (BMP), which induces new bone formation during the healing process. Most commercial bone banks do not verify the presence or activity of BMP in DFDBA nor the ability of DFDBA to induce new bone. Recently, we showed that different bone bank preparations of DFDBA, even from the same bank, varied considerably in their ability to induce new bone, suggesting inherent differences in the quality of the material. Therefore, we examined whether donor age or gender contributed to the variability seen with these preparations. Twenty-seven batches of DFDBA from different donors were donated by one bone bank which had been shown previously to supply DFDBA that was consistently able to induce new bone formation. Each batch was implanted bilaterally in the thigh muscle of nude mice. After 56 days, the implants were excised and examined by light microscopy and histomorphometry. Seventy percent of the preparations tested induced new bone formation. Most of these preparations produced ossicles containing cortical bone surrounding bone marrow-like tissue. The ability to induce bone appears to be age-dependent, with DFDBA from older donors being less likely to have strong bone-inducing activity. By contrast, no difference in ability to induce new bone was noticed between male or female donors. The results of this study confirm that commercial preparations of DFDBA differ in their ability to induce new bone formation. In fact, some of the batches had no activity at all. The ability of DFDBA to induce new bone formation is suggested to be age-dependent, but not gender-dependent by our study. These results indicate that commercial bone banks need to verify the ability of DFDBA to induce new bone formation and should reconsider the advisability of using bone from older donors.

Adolescent↗
PubMed1997

Bacterial lipopolysaccharide induces early and late activation of protein kinase C in inflammatory macrophages by selective activation of PKC-epsilon.

Experiments from our and other laboratories have shown that specific inhibitors of protein kinase C (PKC) inhibited the secretion of nitric oxide, TNF alpha, and IL-1 beta from lipopolysaccharide (LPS)-stimulated macrophages, suggesting an important role for PKC in the inflammatory response. The present study was designed to investigate the mechanism whereby LPS stimulates PKC activity in inflammatory macrophages. Mouse macrophages were stimulated with 0-1 microgram/ml LPS for 0-18 hours, and PKC activity was detected in cell lysates. PKC isoform specificity was determined by blocking PKC activity with isoform-specific antibodies. Treatment of macrophages with 1 microgram/ml LPS induced a two-fold increase in PKC activity within 15 minutes and an additional more significant peak of PKC activity appeared 3 hours post-LPS stimulation. A lower dose of LPS (10 ng/ml) induced the later peak only. The enhancement in PKC activity induced by LPS occurred in both the cytosol and membrane fractions, but the enhancement in the membrane fraction was significantly greater than in the cytosol. The increase in PKC activity in both peaks was abolished only by the addition of anti-PKC-epsilon antibody. The present experiments suggest that PKC activation is an important pathway in the LPS-induced secretory response of macrophages and that PKC-epsilon is the major isoform involved.

Animals↗
PubMed1997

Hybrid structural analogues of 1,25-(OH)2D3 regulate chondrocyte proliferation and proteoglycan production as well as protein kinase C through a nongenomic pathway.

1,25-(OH)2D3 and 24,25-(OH)2D3 mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two hybrid analogues of 1,25-(OH)2D3 which have been modified on the A-ring and C,D-ring side chain (1 alpha-(hydroxymethyl)-3 beta-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D3 (analogue MCW-YA = 3a) and 1 beta-(hydroxymethyl)-3 alpha-hydroxy-20-epi-22-oxa-26,27-dihomo vitamin D3 (analogue MCW-YB = 3b) to examine the role of the VDR in response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25-(OH)2D3 and 24,25-(OH)2D3. These hybrid analogues are only 0.1% as effective in binding to the VDR from calf thymus as 1,25-(OH)2D3. Chondrocyte proliferation ([3H]-thymidine incorporation), proteoglycan production ([35S]-sulfate incorporation), and activity of protein kinase C (PKC) were measured after treatment with 1,25-(OH)2D3, 24,25-(OH)2D3, or the analogues. Both analogues inhibited proliferation of both cell types, as did 1,25-(OH)2D3 and 24,25-(OH)2D3. Analogue 3a had no effect on proteoglycan production by GCs but increased that by RCs. Analogue 3b increased proteoglycan production in both GC and RC cultures. Both analogues stimulated PKC in GC cells; however, neither 3a nor 3b had an effect on PKC activity in RC cells. 1,25-(OH)2D3 and 3a decreased PKC in matrix vesicles from GC cultures, whereas plasma membrane PKC activity was increased, with 1,25-(OH)2D3 having a greater effect. 24,25-(OH)2D3 caused a significant decrease in PKC activity in matrix vesicles from RC cultures; 24,25-(OH)2D3, 3a, and 3b increased PKC activity in the plasma membrane fraction, however. Thus, with little or no binding to calf thymus VDR, 3a and 3b can affect cell proliferation, proteoglycan production, and PKC activity. The direct membrane effect is analogue-specific and cell maturation-dependent. By studying analogues with greatly reduced affinity for the VDR, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites.

24,25-Dihydroxyvitamin D 3↗
PubMed1997

A-ring analogues of 1, 25-(OH)2D3 with low affinity for the vitamin D receptor modulate chondrocytes via membrane effects that are dependent on cell maturation.

1,25-(OH)2D3 (1,25) and 24,25-(OH)2D3(24,25) mediate their effects on chondrocytes through the classic vitamin D receptor (VDR) as well as through rapid membrane-mediated mechanisms, which result in both nongenomic and genomic effects. In intact cells, it is difficult to distinguish between genomic responses via the VDR and genomic and nongenomic responses via membrane-mediated pathways. In this study, we used two analogues of 1,25 that have been modified on the A-ring (2a, 2b) and are only 0.1% as effective in binding to the VDR as 1,25, to examine the role of the VDR in the response of rat costochondral resting zone (RC) and growth zone (GC) chondrocytes to 1,25 and 24,25. Chondrocyte proliferation ([3H]-thymidine incorporation), proteoglycan production ([35S]-sulfate incorporation), and second messenger activation (activity of protein kinase C) were measured after treatment with 10(-8) M 1,25, 10(-7) M 24,25, or the analogues at 10(-9)-10(-6) M. Both analogues inhibited proliferation of both cell types, as did 1,25 and 24,25. Neither 2a nor 2b had an effect on proteoglycan production by GCs or RCs. 2a caused a dose-dependent stimulation of protein kinase C (PKC) that was not inhibited by cycloheximide or actinomycin D in either GC or RC cells. 2b, on the other hand, had no effect on PKC activity in RCs and only a slight stimulatory effect in GCs. Both cells produce matrix vesicles, extracellular organelles associated with the initial stages of calcification, in culture that are regulated by vitamin D metabolites. Since these organelles contain no DNA or RNA, they provide an excellent model for studying the mechanisms used by vitamin D metabolites to mediate their nongenomic effects. When matrix vesicles were isolated from naive cultures of growth zone cells and treated with 2a, a dose-dependent inhibition of PKC activity was observed that was similar to that found with 1,25-(OH)2D3. Plasma membranes contained increased PKC activity after treatment with 2a, but the magnitude of the effect was less than that seen with 1,25-(OH)2D3. Analogue 2b had no affect on PKC activity in either membrane fraction. When matrix vesicles from resting zone chondrocyte cultures were treated with 24,25-(OH)2D3, a significant decrease in PKC activity was observed. No change in enzyme activity was found for either 1,25-(OH)2D3 or the analogues. PKC activity in the plasma membrane fraction, however, was increased by 24,25-(OH)2D3 as well as by analogue 2a. This study shows that these analogues, with little or no binding to the vitamin D receptor, can affect cell proliferation and PKC activity, but not proteoglycan production. The direct membrane effect is analogue specific and cell maturation dependent. Further, by eliminating the VDR-mediated component of the cellular response, we have provided further evidence for the existence of a membrane receptor(s) involved in mediating nongenomic effects of vitamin D metabolites.

Animals↗
PubMed1997

Recombinant bone morphogenetic protein (BMP)-2 regulates costochondral growth plate chondrocytes and induces expression of BMP-2 and BMP-4 in a cell maturation-dependent manner.

This study examined the effect of recombinant human bone morphogenetic protein-2 on several parameters of growth, differentiation, and matrix synthesis and on the endogenous production of mRNA of bone morphogenetic proteins 2 and 4 by growth plate chondrocytes in culture. Chondrocytes from resting and growth zones were obtained from rat costochondral cartilage and cultured for 24 or 48 hours in medium containing 0.05-100 ng/ml recombinant human bone morphogenetic protein-2 and 10% fetal bovine serum. Incorporation of [3H]thymidine, cell number, alkaline phosphatase specific activity, incorporation of [3H]proline into collagenase-digestible protein and noncollagenase-digestible protein, and incorporation of [35S]sulfate were assayed as indicators of cell proliferation, differentiation, and extracellular matrix synthesis. mRNA levels for bone morphogenetic proteins 2 and 4 were determined by Northern blot analysis. Recombinant human bone morphogenetic protein-2 increased the incorporation of [3H]thymidine by quiescent resting-zone and growth-zone cells in a similar manner, whereas it had a differential effect on nonquiescent cultures. At 24 and 48 hours, 12.5-100 ng/ml recombinant human bone morphogenetic protein-2 caused a dose-dependent increase in cell number and DNA synthesis in resting-zone chondrocytes. No effect was seen in growth-zone cells. Recombinant human bone morphogenetic protein-2 stimulated alkaline phosphatase specific activity in resting-zone chondrocytes in a bimodal manner, causing significant increases between 0.2 and 0.8 ng/ml and again between 25 and 100 ng/ml. In contrast, alkaline phosphatase specific activity in growth-zone chondrocytes was significantly increased only between 12.5 and 100 ng/ml. Recombinant human bone morphogenetic protein-2 increased the production of both collagenase-digestible protein and noncollagenase-digestible protein by resting-zone and growth-zone cells, but incorporation of [35S]sulfate was unaffected. Administration of recombinant human bone morphogenetic protein-2 also increased incorporation of [3H]uridine in both resting-zone and growth-zone chondrocytes; these cells produced mRNA for bone morphogenetic proteins 2 and 4. Bone morphogenetic protein-2 mRNA levels in both resting-zone and growth-zone chondrocytes increased in the presence of recombinant human bone morphogenetic protein-2; however, bone morphogenetic protein-4 mRNA levels in growth-zone cells decreased under its influence, and those in resting-zone cells were upregulated only with a dose of 10 ng/ml. This indicates that recombinant human bone morphogenetic protein-2 regulates chondrocyte proliferation, differentiation, and matrix production, and the effects are dependent on the stage of cell maturation. Resting-zone chondrocytes were more sensitive, suggesting that they are targeted by bone morphogenetic protein-2 and that this growth factor may have autocrine effects on these cells.

Alkaline Phosphatase↗
PubMed1997

The effects of 17 beta-estradiol on chondrocyte differentiation are modulated by vitamin D3 metabolites.

Both 17 beta-estradiol (17 beta) and the vitamin D metabolites, 1,25-(OH)2D3(1,25) and 24,25-(OH)2D3(24,25), regulate endochondral bone formation in vivo and in vitro. The effects of 17 beta are sex-specific and cell maturation-dependent. Similarly, the effects of 1,25 and 24,25 are cell maturation-dependent, with 1,25 affecting growth zone chondrocytes (GC) and 24,25 affecting resting zone chondrocytes (RC). This study examined whether the response of chondrocytes to 17 beta is altered after pretreatment with 1,25 or 24,25. Cells were isolated from the costochondral cartilage of male or female rats. Confluent, fourth-passage GC and RC cultures were pretreated with 1,25 or 24,25, respectively, for 24 or 48 h followed by treatment with 17 beta for an additional 24 h. At harvest, cell proliferation ([3H]-thymidine incorporation), differentiation (alkaline phosphatase specific activity [ALPase]), general metabolism ([3H]-uridine incorporation), and proteoglycan production ([35S]-sulfate incorporation) were determined. 1,25 enhanced the inhibitory effect of 17 beta on [3H]-thymidine incorporation by female GC cells; in contrast, no effect was observed in GC cells obtained from male rats. When male RC cells were treated with 17 beta, [3H]-thymidine incorporation was inhibited; however, when these cells were pretreated with 24,25 for 48 h, 17 beta stimulated [3H]-thymidine incorporation 24,25 had no effect on 17 beta-dependent [3H]-thymidine incorporation by female RC cells. 17 beta stimulated ALPase in female GC cells, but had no effect on male GC cells. 1,25 pretreatment of female GC cells inhibited the stimulatory effect of 17 beta on ALPase, but had no effect on ALPase in male GC cultures. 17 beta had no effect on male RC cell ALPase and stimulated ALPase in female RC cells. This was not affected by pretreatment with 24,25. Pretreatment with 1,25 increased the basal level of sulfate incorporation only in female GC. No effect was found in RC cells. These results indicate that pretreatment of rat costochondral chondrocytes with vitamin D metabolites modulate the effect of 17 beta. Although the effect of vitamin D metabolites alone on these chondrocytes is maturation-dependent and not sex-specific, the influence of preincubation with vitamin D metabolites on the effect of 17 beta is hormone-specific, sex-specific, and maturation-dependent.

24,25-Dihydroxyvitamin D 3↗
PubMed1997

Platelet derived growth factor stimulates chondrocyte proliferation but prevents endochondral maturation.

Platelet-derived growth factor (PDGF) is a cytokine released by platelets at sites of injury to promote mesenchymal cell proliferation. Since many bone wounds heal by endochondral bone formation, we examined the response of chondrocytes in the endochondral lineage to PDGF. Confluent cultures of rat costochondral resting zone cartilage cells were incubated with 0-300 ng/mL PDGF-BB for 24 h to determine whether dose-dependent changes in cell proliferation (cell number and [3H]-thymidine incorporation), alkaline phosphatase specific activity, [35S]-sulfate incorporation, or [3H]-proline incorporation into collagenase-digestible protein (CDP) or noncollagenase-digestible protein (NCP), could be observed. Long-term effects of PDGF were assessed in confluent cultures treated for 1, 2, 4, 6, 8, or 10 d with 37.5 or 150 ng/mL PDGF-BB. To determine whether PDGF-BB could induce resting zone chondrocytes to change maturation state to a growth zone chondrocyte phenotype, confluent resting zone cell cultures were treated for 1, 2, 3, or 5 d with 37.5 or 150 ng/ml PDGF-BB and then challenged for an additional 24 h with 1,25-(OH)2D3. PDGF-BB caused a dose-dependent increase in cell number and [3H]-thymidine incorporation at 24 h. The proliferative effect of the cytokine decreased with time. PDGF-BB had no effect on alkaline phosphatase at 24 h, but at later times, the cytokine prevented the normal increase in enzyme activity seen in post-confluent cultures. This effect was primarily on the cells and not on the matrix. PDGF-BB stimulated [35S]-sulfate incorporation at all times examined, but had no effect on [3H]-proline incorporation into either the CDP or NCP pools. Thus, percent collagen production was not changed. Treatment of the cells for up to 5 d with PDGF-BB failed to elicit a 1,25-(OH)2D3 responsive phenotype typical of rat costochondral growth zone cartilage cells. These results show that committed chondrocytes can respond to PDGF-BB with increased proliferation. The effect of the cytokine is to enhance cartilage matrix production, but at the same time to prevent progression of the cells along the endochondral maturation pathway.

Alkaline Phosphatase↗
PubMed1997

Evidence for the involvement of carbonic anhydrase and urease in calcium carbonate formation in the gravity-sensing organ of Aplysia californica.

To better understand the mechanisms that could modulate the formation of otoconia, calcium carbonate granules in the inner ear of vertebrate species, we examined statoconia formation in the gravity-sensing organ, the statocyst, of the gastropod mollusk Aplysia californica using an in vitro organ culture model. We determined the type of calcium carbonate present in the statoconia and investigated the role of carbonic anhydrase (CA) and urease in regulating statocyst pH as well as the role of protein synthesis and urease in statoconia production and homeostasis in vitro. The type of mineral present in statoconia was found to be aragonitic calcium carbonate. When the CA inhibitor, acetazolamide (AZ), was added to cultures of statocysts, the pH initially (30 min) increased and then decreased. The urease inhibitor, acetohydroxamic acid (AHA), decreased statocyst pH. Simultaneous addition of AZ and AHA caused a decrease in pH. Inhibition of urease activity also reduced total statoconia number, but had no effect on statoconia volume. Inhibition of protein synthesis reduced statoconia production and increased statoconia volume. In a previous study, inhibition of CA was shown to decrease statoconia production. Taken together, these data show that urease and CA play a role in regulating statocyst pH and the formation and maintenance of statoconia. CA produces carbonate ion for calcium carbonate formation and urease neutralizes the acid formed due to CA action, by production of ammonia.

Animals↗
PubMed1997

24,25-(OH)2D3 regulation of matrix vesicle protein kinase C occurs both during biosynthesis and in the extracellular matrix.

Plasma membranes and matrix vesicles isolated from rat costochondral resting zone chondrocyte cultures contain predominantly protein kinase C alpha (PKCalpha) and PKCzeta, respectively, and the level of PKC specific activity in these membrane fractions is regulated by 24,25-(OH)2D3 [14]. In the present study, we examined whether the effect of 24,25-(OH)2D3 on membrane PKC is via genomic mechanisms during biogenesis and through a nongenomic mechanism after the matrix vesicles are resident in the matrix. There was a dose-dependent decrease in matrix vesicle PKC specific activity and a significant increase in plasma membrane enzyme activity in cultures treated for 90 minutes with 10(-9)-10(-7) M 24,25-(OH)2D3. However, at 12 hours, matrix vesicle PKC was stimulated, but no effect was seen in the plasma membranes, suggesting that the effect seen at 90 minutes was due to a direct action of the hormone on PKC activity in the membrane, and that the effect seen at 12 hours was due to new matrix vesicle production with altered PKC content. Neither actinomycin D nor cycloheximide inhibited matrix vesicle PKC at 30, 60, or 90 minutes, but by 12 hours, these inhibitors blocked the effect of the hormone. 24,25-(OH)2D3-dependent plasma membrane PKC was sensitive to both actinomycin D and cycloheximide at early time points, but by 12 hours, no effect of the inhibitors was seen. Monensin did not alter basal plasma membrane PKC activity or the 24, 25-(OH)2D3-dependent increase, suggesting that this increase was due to translocation of cytosolic PKC rather than new membrane synthesis. Monensin did not affect matrix vesicle PKC at early time points, but it decreased 24,25-(OH)2D3-dependent enzyme activity at later times, indicating that new matrix vesicle production was blocked. At least part of the effect of 24,25-(OH)2D3 on PKC involved phospholipase A2 (PA2). Quinacrine (a PA2 inhibitor) alone had no effect on matrix vesicle PKC, but in cultures treated for 12 hours with quinacrine and 24,25-(OH)2D3, a synergistic increase in matrix vesicle PKC was observed. Quinacrine caused a time-dependent decrease in matrix vesicle PKC and a dose- and time-dependent increase in plasma membrane PKC when incubated directly with the membranes, supporting the hypothesis that PA2 plays a role in the nongenomic regulation of PKC by 24,25-(OH)2D3. Experiments using anti-isoform specific antibodies showed that 24,25-(OH)2D3 modulated the distribution of PKCalpha, beta, and zeta between the plasma membrane and matrix vesicle compartments via translocation and new PKC synthesis. Thus, the data support the hypothesis that 24, 25-(OH)2D3 regulates matrix vesicles through two pathways: a genomic one at the stage of biosynthesis and packaging, and a second nongenomic mechanism acting directly upon matrix vesicles in the matrix. These data also indicate that matrix vesicle regulation consists of complex events with several different points of regulation.

24,25-Dihydroxyvitamin D 3↗
PubMed1997

Rapid and long-term effects of PTH(1-34) on growth plate chondrocytes are mediated through two different pathways in a cell-maturation-dependent manner.

The aims of this study were to clarify the role of cell maturation stage on chondrocyte response to parathyroid hormone (PTH) by examining the effect of PTH(1-34) on alkaline-phosphatase-specific activity (ALPase) of chondrocyte cultures at two distinct stages of maturation, and to determine the signaling pathways used by the cells to mediate this effect. Confluent, fourth passage rat costochondral resting zone (RC) and growth zone (GC) chondrocytes were used. ALPase was measured in the cell layer, as well as in matrix vesicles (MV) and plasma membranes (PM), after the addition of 10(-7) 10(-11) mol/L bovine PTH(1-34), the active peptide, or bovine PTH(3-34), the inactive peptide, to the cultures. PTH(1-34) increased ALPase in the GC cultures at two separate times: between 5 and 180 min, with maximal stimulation at 10 min, and 36 to 48 h. In contrast, PTH(3-34) had no effect. At 10 min and 48 h, PTH(1-34) produced a dose-dependent increase in ALPase of both MV and PM isolated from GC cultures. Addition of forskolin and IBMX to increase cAMP increased ALPase in GC cultures to a level similar to that seen after addition of PTH(1-34). In contrast, the addition of PTH(1-34) to RC cells only increased ALPase between 5 and 60 min, with peak activity at 10 min. As with GC, PTH increased ALPase in both MV and PM. Moreover, the addition of PTH(3-34) or forskolin and IBMX had no effect on ALPase in RC. PTH(1-34) had no effect on GC protein kinase C (PKC) activity; however, the addition of PTH(1-34) to RC caused a dose-dependent increase in PKC activity. H8, an inhibitor of PKA, had no effect on PTH-stimulated ALPase in RC cells, but inhibited the PTH-dependent response in GC cells. In contrast, chelerythrine, an inhibitor of PKC activity, inhibited PTH-stimulated ALPase in RC cells, but had no effect on PTH-stimulated ALPase in GC cells. This study shows that the effect of PTH(1-34) on RC and GC cells is maturation dependent in terms of time course and mechanism. Whereas both cell types exhibit a rapid response to PTH, only GC cells show a long-term response. In GC, the effects of PTH are associated with changes in cAMP and may also involve at least one other pathway, whereas, in RC, the PTH effects appear to be associated with changes in PKC.

1-Methyl-3-isobutylxanthine↗
PubMed1997

Underlying mechanisms at the bone-surface interface during regeneration.

The goal of regenerative therapy around teeth and implants is to create a suitable environment in which the natural biological potential for functional regeneration of periodontal ligament and/or bone can be maximized. In order for the regenerative process to be successful, the following factors must be addressed: prevention of acute inflammation from bacteria, mechanical stability of the wound, creation and maintenance of blood clot-filled space, isolation of the regenerative space from undesirable competing tissue types, and the creation of a desirable surface chemistry, energy, roughness and microtopography that can directly influence cellular response, ultimately affecting the rate and quality of new tissue formation and, therefore, the regeneration process. This paper will review how surface characteristics (chemistry and roughness) can affect cell response and local factor production. To evaluate the effect of surface chemistry on cell proliferation and differentiation costochondral chondrocytes were grown on standard tissue culture plastic dishes sputter-coated with different materials. The results indicate that surface materials can elicit differential responses in cell metabolism and phenotypic expression in vitro. In a second study, the effect of varying titanium surface roughnesses on osteoblast-like cell behavior was examined. Surface roughness was found to alter osteoblast proliferation, differentiation and matrix production in vitro. In addition, production of PGE2 and TGF beta by these cells was also shown to increase with increasing surface roughness, indicating that substrate surface roughness also affects cytokine and growth factor production. The role of surface roughness in determining cellular response was further explored by comparing the response of osteoblasts grown on new and previously used surfaces. The results of these latter studies showed that cell proliferation, expression of differentiation markers and overall matrix production are not altered when cells are grown on used vs. virgin surfaces. This suggests the possibility that implants may be re-used, especially in the same patient, if they are appropriately treated. In this context, it should also be noted that rougher titanium surfaces may require more extensive cleaning procedures. From a global perspective, these studies provide some insight into how bone regeneration can be optimized in the presence of an implant or tooth root residing at the site of a bony defect. Since the new bone being produced, during regeneration, grows from a distal area toward the implant or tooth root surface, it is hypothesized that the osteoblasts growing on the surface of the implant may produce local factors that can affect the bone healing process distally. In short, it appears that the surface characteristics of an implant, particularly roughness, may direct tissue healing and, therefore, subsequent implant success in sites of regeneration by modulating osteoblast phenotypic expression.

Alkaline Phosphatase↗