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Biomedical subjects

Zvi Schwartz

Publications and source records attributed to Zvi Schwartz.

At least 19 recordsLinked to original sources
PubMed2007

1alpha,25(OH)2D3 is an autocrine regulator of extracellular matrix turnover and growth factor release via ERp60 activated matrix vesicle metalloproteinases.

Growth plate chondrocytes produce proteoglycan-rich type II collagen extracellular matrix (ECM). During cell maturation and hypertrophy, ECM is reorganized via a process regulated by 1alpha,25(OH)(2)D(3) and involving matrix metalloproteinases (MMPs), including MMP-3 and MMP-2. 1alpha,25(OH)(2)D(3) regulates MMP incorporation into matrix vesicles (MVs), where they are stored until released. Like plasma membranes (PM), MVs contain the 1alpha,25(OH)(2)D(3)-binding protein ERp60, phospholipase A(2) (PLA(2)), and caveolin-1, but appear to lack nuclear Vitamin D receptors (VDRs). Chondrocytes produce 1alpha,25(OH)(2)D(3) (10(-8)M), which binds ERp60, activating PLA(2), and resulting lysophospholipids lead to MV membrane disorganization, releasing active MMPs. MV MMP-3 activates TGF-beta1 stored in the ECM as large latent TGF-beta1 complexes, consisting of latent TGF-beta1 binding protein, latency associated peptide, and latent TGF-beta1. Others have shown that MMP-2 specifically activates TGF-beta2. TGF-beta1 regulates 1alpha,25(OH)(2)D(3)-production, providing a mechanism for local control of growth factor activation. 1alpha,25(OH)(2)D(3) activates PKCalpha in the PM via ERp60-signaling through PLA(2), lysophospholipid production, and PLCbeta. It also regulates distribution of phospholipids and PKC isoforms between MVs and PMs, enriching the MVs in PKCzeta. Direct activation of MMP-3 in MVs requires ERp60. However, when MVs are treated with 1alpha,25(OH)(2)D(3), PKCzeta activity is decreased and PKCalpha is unaffected, suggesting a more complex feedback mechanism, potentially involving MV lipid signaling.

Animals↗
PubMed2007

Platelet-rich plasma inhibits demineralized bone matrix-induced bone formation in nude mice.

BACKGROUND: It is unclear whether platelet-rich plasma is a clinically effective adjunct to osteoinductive agents such as demineralized bone matrix. It contains platelet-derived growth factor (PDGF), which decreases osteoinduction by human demineralized bone matrix in nude-mouse muscle, suggesting that platelet-rich plasma may also have a negative impact. This study tested the hypothesis that platelet-rich plasma reduces demineralized bone matrix-induced bone formation and that this effect varies with donor-dependent differences in platelet-rich plasma and demineralized bone matrix. METHODS: Human platelet-rich plasma was prepared from blood from six men (average age [and standard error of the mean], 29.2 +/- 2.4 years). Platelet numbers were determined, and growth factors were quantified before and after platelet activation. Human demineralized bone matrix from two donors (demineralized bone matrix-1 and demineralized bone matrix-2) was mixed with activated platelet-rich plasma and was implanted bilaterally in the gastrocnemius muscle in eighty male nude mice (eight implants per variable). Fifty-six days after implantation, the hindlimb calf muscles were harvested for histological analysis. Osteoinduction was evaluated with use of a qualitative score and morphometric measurements of ossicle size, new bone formation, and residual demineralized bone matrix. RESULTS: Compared with platelet-poor plasma, platelet-rich plasma preparations exhibited a fourfold increase in the platelet count, a fifteenfold increase in the amount of transforming growth factor-beta, a sixfold increase in the amount of PDGF-BB, a fivefold increase in the amount of PDGF-AA, and a twofold increase in the amount of PDGF-AB. Demineralized bone matrix-1 was more osteoinductive than demineralized bone matrix-2, as determined on the basis of a greater ossicle area. The effect of platelet-rich plasma was either neutral or inhibitory depending on the demineralized bone matrix batch. When used with demineralized bone matrix-1, platelet-rich plasma did not alter the qualitative score or overall ossicle size, but it decreased the new bone area. When used with demineralized bone matrix-2, platelet-rich plasma reduced the qualitative score, ossicle area, and new bone area and increased the amount of residual demineralized bone matrix. The effects on osteoinduction also varied with the donor of the platelet-rich plasma. CONCLUSIONS: Platelet-rich plasma decreased the osteoinductivity of demineralized bone matrix implanted in immunocom-promised mice, and the activities of both demineralized bone matrix and platelet-rich plasma were donor-dependent.

Adult↗
PubMed2006

Integrin beta1 silencing in osteoblasts alters substrate-dependent responses to 1,25-dihydroxy vitamin D3.

Surface microroughness increases osteoblast differentiation and enhances responses of osteoblasts to 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. The observations that beta1 integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and that it is regulated by 1alpha,25(OH)2D3 in a surface-dependent manner, suggest that beta1 may play a role in mediating osteoblast response. To test this hypothesis, we silenced beta1 expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA) and examined the responses of the beta1-silenced osteoblasts to surface microtopography and 1alpha,25(OH)2D3. To better understand the role of beta1, MG63 cells were also treated with two different monoclonal antibodies to human beta1 to block ligand binding. beta1-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor beta1, prostaglandin E2, and osteoprotegerin in comparison with control cells. Moreover, beta1-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1alpha,25(OH)2D3. Anti beta1 antibody AIIB2 had no significant effect on cell number and osteocalcin, but decreased alkaline phosphatase; MAB2253Z caused dose-dependent decreases in cell number and alkaline phosphatase and an increase in osteocalcin. Effects of 1alpha,25(OH)2D3 on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that beta1 plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1alpha,25(OH)2D3. The results also show that beta1 mediates, in part, the synergistic effects of surface roughness and 1alpha,25(OH)2D3.

Animals↗
PubMed2006

Decreased response of osteoblasts obtained from aged Cohen diabetic sensitive rats to sex steroid hormones and 1,25OH2D3 in culture.

Patients with diabetes mellitus are known to develop osteopenia and osteoporosis, apparently as a reduction in the process of bone formation. In order to evaluate whether bone-modulating hormones--estradiol, testosterone, and 1,25(OH)(2)D(3)--have different effects on osteoblasts derived from diabetic and from normal non-diabetic rats, we studied the specific effects of these hormones on the differentiation and function of cultured osteoblasts derived from 1-year-old Cohen diabetic rats. (The Cohen diabetic model consists of a diabetic-sensitive strain [CDs; diabetic] and a diabetic-resistant strain [CDr; normal]). The CDs and CDr male and female rats were fed on a regular diet (RD) or a high-sucrose low-copper diet (HSD; diabetogenic). On the HSD diet, only CD rats develop type 2 diabetes, while CDr do not. Bones were removed for primary osteoblast cultures, and osteoblastic responses to the bone-modulation hormones--estradiol, testosterone, and 1,25(OH)(2)D(3)--were studied. In male rats fed RD, primary cultures of osteoblasts without hormone addition to the culture medium showed that alkaline phosphatase (ALP) activity was similar in the Cohen diabetic rats (both CDr and CDs) to that of the original Sabra strain. However, collagen synthesis was reduced in the CDr and CDs compared to the Sabra strain. The addition of the hormones to the culture medium did not change ALP activity or collagen synthesis in the male-derived osteoblasts, but increased mineralization in all strains. In female rats (studied only in CDs and CDr animals) there were no differences between animals fed the RD. HSD increased the basal activity of ALP in the CDr but not in the CDs rats, and decreased the rate of collagen synthesis in both CDr and CDs (diabetic) animals. The addition of the bone-modulation hormones to the culture medium further increased ALP activity in the osteoblasts derived from the CDr animals, while decreasing ALP activity in the CDs. These hormones also decreased collagen synthesis in both strains and increased mineralization in all osteoblasts. In conclusion, the metabolic status (HSD and diabetes) in rats prior to culture affected the phenotype of cultured osteoblasts, decreasing their response to bone-modulation hormones. This decreased response, especially to estradiol, may be a major cause of the osteopenia observed in diabetes.

Aging↗
PubMed2006

Use of growth factors to modify osteoinductivity of demineralized bone allografts: lessons for tissue engineering of bone.

Biologically active bone graft substitute materials are needed for repair and regeneration of skeletal tissues. Current approaches are focused on the use of osteoinductive agents, including bone morphogenetic proteins (BMP) in combination with biodegradable carriers. Demineralized freeze-dried bone allograft (DFDBA) can provide an osteoconductive surface and, at the same time, function as a time-release carrier for BMP. Donor variability, however, limits the predictability of DFDBA as an osteoinductive material. This article examines the use of growth factors, including platelet-rich plasma, platelet-derived growth factor, enamel matrix derivatives, and BMP-2, to enhance the osteoinductive properties of human DFDBA.

Animals↗
PubMed2006

Osteoblast-like cells are sensitive to submicron-scale surface structure.

OBJECTIVES: Studies showing that osteoblasts exhibit a more differentiated phenotype on rough titanium (Ti) surfaces and osteoclast-resorbed bone surfaces used materials characterized by average peak to valley distance (Ra). Other surface features impacting the cells include distance between peaks, curvature of the valleys, and relative distribution of flat and smooth regions. We used novel Ti surfaces prepared by electrochemical micromachining as models to examine specific contributions of individual design features to osteoblast response. Results show that micron-scale topography modulates cell number, cell morphology and prostaglandin E2 (PGE2). In the presence of the appropriate microtopography, submicron-scale rugosity modulates differentiation and transforming growth factor-beta1 (TGF-beta1) levels. In this study, we examined the role of different types of submicron-scale structures. MATERIAL AND METHODS: Thirty micrometer diameter craters on Ti disks were produced by photolithography resulting in an electropolished smooth surface, and arranged so that inside crater area vs. outside flat area was 6 (30/6). Submicron-scale structures were superposed by acid etching and porous anodization. Ra's were 700, 400, 60 nm on acid-etched, porous anodized and smooth 30/6 surfaces, respectively. RESULTS: MG63 osteoblast-like cells were sensitive to submicron-scale architecture. Cell morphology on anodized surfaces was similar to morphology on smooth surfaces, whereas on etched surfaces, cells had a more elongated differentiated shape. Cell number was greatest on smooth surfaces > anodized > etched. Osteocalcin and PGE2 were affected in a reverse manner. Active TGF-beta1 was greatest on etched 30/6 surfaces > anodized > smooth; latent TGF-beta1 was elevated on all rough surfaces. CONCLUSIONS: These results support our previous observations that submicron-scale structures modulate osteoblastic phenotype and show that the physical properties of the submicron-scale structures are important variables in determining osteoblast response to substrate topography.

Acid Etching, Dental↗
PubMed2006

Regulation of growth plate chondrocytes by 1,25-dihydroxyvitamin D3 requires caveolae and caveolin-1.

UNLABELLED: We examined the role of caveolae and caveolin-1 in the mechanism of 1alpha,25(OH)(2)D(3) action in growth plate chondrocytes. We found that caveolae are required for rapid 1alpha,25(OH)(2)D(3)-dependent PKC signaling, and caveolin-1 must be present based on studies using chondrocytes from Cav-1(-/-) mice. INTRODUCTION: 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] regulates endochondral ossification in part through membrane-associated mechanisms, including protein kinase C (PKC) signaling activated by a membrane-associated 1alpha,25(OH)(2)D(3)-binding protein, ERp60. We tested the hypothesis that caveolae are required for 1alpha,25(OH)(2)D(3) action and play an important role in regulating chondrocyte biology and growth plate physiology. MATERIALS AND METHODS: Rat costochondral chondrocytes were examined for caveolae by transmission electron microscopy of cultured cells and of cells in situ. Western blots and confocal microscopy were used to detect caveolae proteins including caveolin-1 (Cav-1) and 1alpha,25(OH)(2)D(3) receptors. Caveolae cholesterol was depleted with beta-cyclodextrin (CD) and effects of 1alpha,25(OH)(2)D(3) on PKC, DNA synthesis, alkaline phosphatase, and proteoglycan production determined. Chondrocytes from Cav-1(-/-) and C57BL/6 wildtype mice were also treated with 1alpha,25(OH)(2)D(3). Epiphyses and costochondral junctions of 8-week-old male Cav-1(-/-) and wildtype mice (N = 8) were compared by histomorphometry and microCT. Data were analyzed by ANOVA and Bonferroni for posthoc comparisons. RESULTS: Growth zone chondrocytes had caveolae and Cav-1, -2, and -3. Resting zone chondrocytes, which do not exhibit a rapid 1alpha,25(OH)(2)D(3)-dependent increase in PKC activity, also had these caveolins, but caveolae were larger and fewer in number. ERp60 but not VDR co-localized with Cav-1 in plasma membranes and in lipid rafts. CD-treatment blocked 1alpha,25(OH)(2)D(3) effects on all parameters tested. The Cav-1(-/-) cells did not respond to 1alpha,25(OH)(2)D(3), although 1alpha,25(OH)(2)D(3) increased PKC, alkaline phosphatase, and [(35)S]-sulfate incorporation in wildtype C57BL/6 cells. Histology and microCT showed that Cav-1(-/-) growth plates were longer and had more hypertrophic cells in each column. Growth plate changes were reflected in the metaphysis. CONCLUSIONS: The membrane-mediated effects of 1alpha,25(OH)(2)D(3) require caveolae and Cav-1, and Cav-1 deficiency results in altered growth plate physiology.

Animals↗
PubMed2006

Osteoinductive ability of human allograft formulations.

BACKGROUND: Bone graft materials are needed in periodontics that are osteoinductive, have good handling characteristics, and have physical properties that provide appropriate stiffness for the treatment site. Demineralized freeze-dried bone allograft (DFDBA), also called demineralized bone matrix (DBM), is osteoinductive but requires a carrier to meet the other clinical objectives, thereby decreasing the DBM content per volume of the bone graft material. The present study determined whether the DBM content of a carrier formulation is an important variable with respect to its effectiveness as an osteoinductive material. METHODS: The immunocompromised Nu/Nu mouse-muscle implantation assay of osteoinductivity was used to test human DBM formulated with hyaluronic acid (HY) and cancellous and cortical bone granules from the same donor: DBM alone (11 mg); DBM (11 mg):HY, 55:45, weight/weight (wt/wt); DBM (6.4 mg):HY, 32:68, wt/wt; DBM mixed with cortical and cancellous bone chips 1:4 (DBMC) (11 mg total, of which 2.2 mg was DBM); DBMC (11 mg):HY, 55:45, wt/wt; heat-treated DBM (11 mg); HY alone; and positive-control DBM (11 mg). Osteoinduction was scored using a qualitative scale and by histomorphometry. RESULTS: Results showed that all DBM was osteoinductive and the addition of HY did not change this as long as the amount of DBM used was held constant. The reduction in the absolute amount of DBM resulted in a reduced osteoinduction score, reduced ossicle area, and reduced new bone formation. The addition of HY also caused a decrease in the amount of residual non-vital bone particles, particularly when DBMC was implanted. Results were donor dependent. CONCLUSION: This study showed the importance of DBM content and donor variability in osteoinductivity of DBM formulations with improved handling and stiffness characteristics.

Adult↗
PubMed2006

Response of musculoskeletal cells to biomaterials.

Many of our current therapies are based on information obtained in cell cultures using substrates that have little in common with the substrates the cells will encounter in vivo. To produce materials that are clinically valuable, we must analyze more deeply how musculoskeletal cells interact with the physical features of their environments. An increasing body of information has examined the mechanisms by which osteoblasts interact with their substrate. The underlying substrate, particularly in bone, also has structural features that can alter the mechanical environment experienced by the cells. These structural features modulate the nature of cell attachment and the resulting cell shape, affecting cell proliferation and differentiation. The chemistry, surface energy, and microarchitecture of a material all influence the kinds of proteins that adsorb onto the surface, which in turn affects integrin-mediated attachment. Signaling via integrins initiates the transfer of information to the cell about the microenvironment. Cells can differentiate between crystallinities of the same chemistry and distinguish complex differences in surface structure. These differences in the in vitro response correspond to differences in clinical effectiveness. By designing biomaterials that maximally enhance mesenchymal cell attachment, migration, proliferation, and differentiation, the value of these materials for tissue repair will be markedly increased. The goal is to provide materials that are capable of supporting tissue regeneration in vivo, often at sites compromised by infection and loss of structure.

Biocompatible Materials↗
PubMed2005

Plasma membrane requirements for 1alpha,25(OH)2D3 dependent PKC signaling in chondrocytes and osteoblasts.

1,25-Dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] acts on chondrocytes and osteoblasts through traditional nuclear Vitamin D receptor (VDR) mechanisms as well as through rapid actions on plasma membranes that initiate intracellular signaling pathways. We have investigated the mechanisms involved in activation of protein kinase C (PKC) and downstream biological responses that depend on the latter pathway. These studies show that PKC activation depends on presence of a membrane receptor ERp60 and rapid increases in phospholipase A(2) (PLA(2)) activity. Cells that are responsive to 1alpha,25(OH)(2)D(3) express PLA(2) activating protein (PLAA), suggesting a link between ERp60 and PLA(2). Increased PLA(2) results in increased arachidonic acid release and formation of lysophospholipid, which then activates phospholipase C beta (PLCbeta), leading to rapid formation of inositol-trisphosphate (IP3) and diacylglycerol (DAG). PLA(2), PLC, and DAG are all associated with lipid rafts including caveolae in many cells, suggesting that the caveolar environment may be an important mediator of PKC activation by 1alpha,25(OH)(2)D(3). Here, we use the VDR(-/-) mouse costochondral cartilage growth plate to examine the expression of ERp60 and PLAA in vivo in 1alpha,25(OH)(2)D(3)-responsive hypertrophic chondrocytes (growth zone cells) and in resting zone cells that do not respond to this Vitamin D metabolite in vitro. In addition, we determined if intact lipid rafts are required for the response of rat costochondral cartilage growth zone cells to 1alpha,25(OH)(2)D(3). The results show that ERp60 and PLAA are localized to 1alpha,25(OH)(2)D(3)-responsive growth zone cells and metaphyseal osteoblasts, even in VDR(-/-) mice. Disruption of lipid rafts using beta-cyclodextrin blocks the activation of PKC by 1alpha,25(OH)(2)D(3) and reduces the ability of 1alpha,25(OH)(2)D(3) to regulate [(35)S]-sulfate incorporation.

Animals↗
PubMed2005

The response to sex steroid hormones and vitamin D of cultured osteoblasts derived from ovariectomized mice with and without 17beta-estradiol pretreatment.

This study investigated whether 17beta-estradiol (E2) may have different effects on osteoblasts derived from estrogen-deficient ovariectomized (OVX) mice compared to sham-operated normal animals. We studied the specific effects of 17beta-estradiol on the differentiation and function of cultured osteoblasts derived from these groups of animals, with or without estrogen replacement treatment. One-month-old mice were ovariectomized or sham-operated, and treated (every second day) for 4 weeks with 0.5 mg/kg 17beta-estradiol or with vehicle alone. At the end of the experiment, bones were removed for primary osteoblast cultures or for morphological and chemical evaluation. In cells from untreated OVX animals, alkaline phosphatase (ALP) specific activity was reduced, while collagen production and mineralization were unchanged when compared to cells from controls. In vivo estrogen pretreatment of the OVX mice elevated ALP activity and mineralization of the cells, while collagen production was reduced. The addition of 17beta-estradiol to the culture medium increased ALP activity, collagen production, and mineralization by all cultured osteoblasts, except in those derived from sham-operated, estrogen-pretreated mice, where these features remained unchanged. Osteocalcin production was unchanged. Addition of testosterone or 1,25(OH)2D3 to the culture medium induced changes that differed among the groups depending on the source of the cultured cells. It seems that ovariectomy in mice prior to culture affected the phenotype of the cultured osteoblasts and their response to estradiol, testosterone, and 1,25(OH)2D3, depending on whether animals were pretreated with estradiol or not. These results imply that the animal's estrogen status prior to culture can influence the response to estrogens; this finding may have important implications for hormone replacement therapy (HRT) in postmenopausal women.

Alkaline Phosphatase↗
PubMed2005

Platelet-derived growth factor inhibits demineralized bone matrix-induced intramuscular cartilage and bone formation. A study of immunocompromised mice.

BACKGROUND: Platelet-derived growth factor (PDGF) has been proposed as a therapeutic agent to promote bone-healing. The purpose of this study was to examine the effect of PDGF on the ability of human demineralized bone matrix to induce bone formation in a nude-mouse muscle-implantation model. We also examined whether platelet-rich plasma, which contains PDGF, also modulates osteoinduction in this model. METHODS: Human demineralized bone matrix, previously shown to be osteoinductive in the calf muscles of nude mice, was mixed with PDGF-BB (0, 0.1, 1, and 10 microg/10 mg of demineralized bone matrix) and was implanted bilaterally in the calf muscles of immunocompromised (nu/nu) mice (six mice in each group). Heat-inactivated demineralized bone matrix was used as a control. Tissue was harvested at fourteen, twenty-eight, and fifty-six days after implantation. Platelet-rich plasma was prepared from the blood of a healthy donor with use of the Harvest PRP preparation device, activated with thrombin, and mixed with active and inactive demineralized bone matrix. Fifty-six days post-implantation, tissues were harvested. Osteoinduction was assessed with use of a qualitative scoring system and with quantitative histomorphometry. RESULTS: Cartilage was present at fourteen days in all tissues that had received an implant, but the amount decreased as the PDGF concentration increased. PDGF reduced bone formation at twenty-eight days in a dose-dependent manner. This inhibitory effect was resolved by fifty-six days, except in tissues in which demineralized bone matrix and 10 microg of PDGF had been implanted. In sites treated with 10 microg of PDGF, the area of new bone was decreased and the area of bone marrow was reduced at twenty-eight and fifty-six days. PDGF also appeared to retard resorption of demineralized bone matrix in a dose-dependent manner. Platelet-rich plasma reduced osteoinduction by human demineralized bone matrix that had high osteoinductive activity and had no effect on osteoinduction by demineralized bone matrix with low activity. CONCLUSIONS: PDGF inhibits, in a dose-dependent manner, intramuscular osteoinduction and chondrogenesis by demineralized bone matrix in immunocompromised mice. Platelet-rich plasma also reduces the osteoinductivity of active demineralized bone matrix.

Analysis of Variance↗
PubMed2005

Surface microtopography regulates osteointegration: the role of implant surface microtopography in osteointegration.

Increased surface roughness of dental implants enhances the process of osseointegration. It increases bone conduction and increases BIC in all types of bone, resulting in elevated removal torque values. Surface roughness elevated the CSR of implants implanted in adverse conditions as augmented ridges and sinuses and areas of poor bone, such as the posterior maxilla, and in some cases abolished the deleterious effect of smoking. A growing number of clinical studies suggest that early and immediate loading of rough-surfaced implants may lead to predictable osseointegration. However, it is important to note that these studies provide short-term results based on radiographic observation and clinical mobility only. Before we adopt new surgical and prosthetic guidelines, longer and broader studies are needed. Most recent research has examined the effect of surface roughness on bone healing around implants in vivo and the influence of surface roughness on osteoblasts in vitro. In a study just published, it was found that changing the surface chemistry by submerging an implant in an isotonic sodium chloride solution following acid etching to avoid contamination with molecules from the atmosphere significantly increased osteoblast differentiation in vitro and BIC in vivo. This finding may lead us to a new era in dental implants.

Animals↗
PubMed2004

Rapid vitamin D-dependent PKC signaling shares features with estrogen-dependent PKC signaling in cartilage and bone.

Our work is based on the hypothesis that steroid hormones regulate cells through traditional cytoplasmic and nuclear receptor-mediated mechanisms, as well as by rapid effects that are mediated by membrane-associated pathways. We have used the rat costochondral growth plate chondrocyte culture model to study the signaling mechanisms used by steroid hormones to elicit rapid responses and to modulate gene expression in target cells. Our studies show that the secosteroids 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] and 24,25-dihydroxyvitamin D3 [24R,25(OH)2D3], and the steroid hormone 17beta-estradiol, cause rapid increases in protein kinase C alpha (PKCalpha) activity, and many of the physiological responses of the cells to these regulators are PKC-dependent. Target cell specificity and the mechanisms by which PKCalpha is activated vary with each hormone. PKC activation initiates a signaling cascade that results in activation of the ERK1/2 family of mitogen activated protein kinases (MAPK), providing an alternate method for the steroids to modulate gene expression other than by traditional steroid hormone receptor-mediated pathways. In addition to their effects on growth plate chondrocytes, steroid hormones secreted by the cells also control events in the extracellular matrix through direct non-genomic regulation of matrix vesicles.

Animals↗
PubMed2004

Stimulation of growth factor synthesis by electric and electromagnetic fields.

Biophysical input, including electric and electromagnetic fields, regulate the expression of genes in connective tissue cells for structural extracellular matrix (ECM) proteins resulting in an increase in cartilage and bone production. In in vivo models and clinical situations, this can be manifested as enhanced repair and a gain in mechanical properties of the repairing tissues. The mechanisms by which cell functions are regulated by biophysical input is the subject of this review. Biophysical interactions of electric and electromagnetic fields at the cell membrane are not well understood and require considerable additional study. We review information on transmembrane signaling, channel activation and receptor stimulation or blockade. Understanding physical interactions and transmembrane signaling will most likely be necessary to establish dosing paradigms and improve therapeutic efficacy. Considerable information has been generated on an intermediary mechanism of activity - growth factor stimulation. Electric and electromagnetic fields increase gene expression for, and synthesis of, growth factors and this may function to amplify field effects through autocrine and paracrine signaling. Electric and electromagnetic fields can produce a sustained upregulation of growth factors, which enhance, but do not disorganize endochondral bone formation. Progress in the areas of signal transduction and growth factor synthesis is very rapid and future directions are suggested.

Animals↗
PubMed2003

Steroid hormone action in musculoskeletal cells involves membrane receptor and nuclear receptor mechanisms.

Steroid hormones regulate target cells through traditional nuclear mechanisms as well as by membrane mechanisms. 1alpha,25(OH)2D3 and 24R,25(OH)2D3 bind membrane receptors (mVDR) and mediate their effects on the physiological responses of musculoskeletal cells via protein kinase C (PKC). In cultures of costochondral growth plate chondrocytes, 1alpha,25(OH)2D3 binds the 1,25-mVDR in growth zone cells, activating phospholipase C (PLC), leading to diacylglycerol (DAG) production and PKC translocation to the plasma membrane. It also activates PLA2, increasing arachidonic acid release and prostaglandin synthesis. 24R,25(OH)2D3 binds its membrane receptor in resting zone chondrocytes, activating phospholipase D (PLD), and increasing DAG and PKC activity, but translocation does not occur. PLA2 activity is decreased, reducing arachidonic acid and prostaglandin production. 17Beta-estradiol (E2) activates PKC in both cartilage cells, but DAG is not involved. 1alpha,25(OH)2D3 and 24R,25(OH)2D3 also increase PKC in osteoblasts in a cell-specific manner. Antibodies to the 1,25-mVDR block PKC activation. Membrane-mediated events influence gene expression via signaling cascades, including the ERK1/2 MAP kinases. The ability of steroid hormones to initiate events nongenomically is important for regulation of matrix vesicle (MV) function in the extracellular matrix. MVs have mVDRs, but ligand binding inhibits PKC-zeta (PKCzeta) via a mechanism that differs from PKCalpha activation in the plasma membranes. Treatment of MVs from growth zone chondrocyte cultures with 1alpha,25(OH)2D3 releases stromelysin-1 (MMP-3) and increases TGF-beta activation. MMP-3 is also involved in proteoglycan degradation, facilitating calcification. 24R,25(OH)2D3 inhibits PKCzeta in MV from resting zone cell cultures and inhibits MMP-3 release. Chondrocytes and osteoblasts produce 1,25(OH)2D3, 24,25(OH)2D3, and E2; thus, locally produced steroids may function as autocrine regulators of matrix events, including matrix vesicle enzyme activity and matrix protein remodelling during longitudinal growth, calcification, and growth factor activation.

24,25-Dihydroxyvitamin D 3↗
PubMed2002

Transforming growth factor-beta1 regulation of growth zone chondrocytes is mediated by multiple interacting pathways.

Transforming growth factor beta 1 (TGF-beta1) affects growth plate chondrocytes through Smad-mediated mechanisms and has been shown to increase protein kinase C (PKC). This study determined if PKC mediates the physiological response of rat costochondral growth zone (GC) chondrocytes to TGF-beta1; if the physiological response occurs via type II or type III TGF-beta receptors, and, if so, which receptor mediates the increase in PKC; and the signal transduction pathways involved. Treatment of confluent GC cells with TGF-beta1 stimulated [(3)H]thymidine and [(35)S]sulfate incorporation as well as alkaline phosphatase (ALPase) and PKC specific activities. Inhibition of PKC with chelerythrine, staurosporine, or H-7 caused a dose-dependent decrease in these parameters, indicating that PKC signaling was involved. TGF-beta1-dependent PKC and the physiological response of GC cells to TGF-beta1 was reversed by anti-type II TGF-beta receptor antibody and soluble type II TGF-beta receptor, showing that TGF-beta1 mediates these effects through the type II receptor. The increase in [3H]thymidine incorporation and ALPase specific activity were also regulated by protein kinase A (PKA) signaling, since the effects of TGF-beta1 were partially blocked by the PKA inhibitor H-8. The mechanism of TGF-beta1 activation of PKC is through phospholipase A(2) (PLA(2)) and not through phospholipase C (PLC). Arachidonic acid increased PKC in control cultures and was additive with TGF-beta1. Prostanoids are required, as indomethacin blocked the effect of TGF-beta1, and Cox-1, but not Cox-2, is involved. TGF-beta1 stimulates prostaglandin E(2) (PGE(2)) production and exogenous PGE(2) stimulates PKC, but not as much as TGF-beta1, suggesting that PGE(2) is not sufficient for all of the prostaglandin effect. In contrast, TGF-beta1 was not regulated by diacylglycerol; neither dioctanoylglycerol (DOG) nor inhibition of diacylglycerol kinase with R59022 had an effect. G-proteins mediate TGF-beta1 signaling at different levels in the cascade. TGF-beta1-dependent increases in PGE(2) levels and PKC were augmented by the G protein activator GTP gamma S, whereas inhibition of G-protein activity via GDP beta S, pertussis toxin, or cholera toxin blocked stimulation of PKC by TGF-beta1, indicating that both G(i) and G(s) are involved. Inhibition of PKA with H-8 partially blocked TGF-beta1-dependent PKC, suggesting that PKA inhibition on the physiological response was via PKA regulation of PKC signaling. This indicates that multiple interacting signaling pathways are involved: TGF-beta1 stimulates PLA(2) and prostaglandin release via the action of Cox-1 on arachidonic acid. PGE(2) activates the EP2 receptor, leading to G-protein-dependent activation of PKA. PKA signaling results in increased PKC activity and PKC signaling regulates proliferation, differentiation, and matrix synthesis.

Alkaline Phosphatase↗