Biomedical subjects

B D Boyan

Publications and source records attributed to B D Boyan.

At least 37 records · Page 2Linked to original sources

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↗

PubMed2003

Osteoblasts generate an osteogenic microenvironment when grown on surfaces with rough microtopographies.

Osteoblasts respond to microarchitectural features of their substrate. On smooth surfaces (tissue culture plastic, tissue culture glass, and titanium), the cells attach and proliferate but they exhibit relatively low expression of differentiation markers in monolayer cultures, even when confluent. When grown on microrough Ti surfaces with an average roughness (Ra) of 4-7 mum, proliferation is reduced but differentiation is enhanced and in some cases, is synergistic with the effects of surface microtopography. In addition, cells on microrough Ti substrates form hydroxyapatite in a manner that is more typical of bone than do cells cultured on smooth surfaces. Osteoblasts also respond to growth factors and cytokines in a surface-dependent manner. On rougher surfaces, the effects of regulatory factors like 1alpha,25(OH)2D3 or 17beta-estradiol are enhanced. The response to the surface is mediated by integrins, which signal to the cell through many of the same mechanisms used by growth factors and hormones. Studies using PEG-modified surfaces indicate that increased differentiation may be related to altered attachment to the surface. When osteoblasts are grown on surfaces with chemistries or microarchitectures that reduce cell attachment and proliferation, and enhance differentiation, the cells tend to increase production of factors like TGF-beta1 that promote osteogenesis while decreasing osteoclastic activity. Thus, on microrough Ti surface, osteoblasts create a microenvironment conducive to new bone formation.

Calcium↗

PubMed2003

1alpha,25(OH)2D3 causes a rapid increase in phosphatidylinositol-specific PLC-beta activity via phospholipase A2-dependent production of lysophospholipid.

1alpha,25(OH)(2)D(3) activates protein kinase C (PKC) in rat growth plate chondrocytes via mechanisms involving phosphatidylinositol-specific phospholipase C (PI-PLC) and phospholipase A(2) (PLA(2)). The purpose of this study was to determine if 1alpha,25(OH)(2)D(3) activates PI-PLC directly or through a PLA(2)-dependent mechanism. We determined which PLC isoforms are present in the growth plate chondrocytes, and determined which isoform(s) of PLC is(are) regulated by 1alpha,25(OH)(2)D(3). Inhibitors and activators of PLA(2) were used to assess the inter-relationship between these two phospholipid-signaling pathways. PI-PLC activity in lysates of prehypertrophic and upper hypertrophic zone (growth zone) cells that were incubated with 1alpha,25(OH)(2)D(3), was increased within 30s with peak activity at 1-3 min. PI-PLC activity in resting zone cells was unaffected by 1alpha,25(OH)(2)D(3). 1beta,25(OH)(2)D(3), 24R,25(OH)(2)D(3), actinomycin D and cycloheximide had no effect on PLC in lysates of growth zone cells. Thus, 1alpha,25(OH)(2)D(3) regulation of PI-PLC enzyme activity is stereospecific, cell maturation-dependent, and nongenomic. PLA(2)-activation (mastoparan or melittin) increased PI-PLC activity to the same extent as 1alpha,25(OH)(2)D(3); PLA(2)-inhibition (quinacrine, oleyloxyethylphosphorylcholine (OEPC), or AACOCF(3)) reduced the effect of 1alpha,25(OH)(2)D(3). Neither arachidonic acid (AA) nor its metabolites affected PI-PLC. In contrast, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) activated PI-PLC (LPE>LPC). 1alpha,25(OH)(2)D(3) stimulated PI-PLC and PKC activities via Gq; GDPbetaS inhibited activity, but pertussis toxin did not. RT-PCR showed that the cells express PLC-beta1a, PLC-beta1b, PLC-beta3 and PLC-gamma1 mRNA. Antibodies to PLC-beta1 and PLC-beta3 blocked the 1alpha,25(OH)(2)D(3) effect; antibodies to PLC-delta and PLC-gamma did not. Thus, 1alpha,25(OH)(2)D(3) regulates PLC-beta through PLA(2)-dependent production of lysophospholipid.

Animals↗

PubMed2003

Pulsed electromagnetic fields affect phenotype and connexin 43 protein expression in MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells.

Osteocytes, the predominant cells in bone, are postulated to be responsible for sensing mechanical and electrical stimuli, transducing signals via gap junctions. Osteocytes respond to induced shear by increasing connexin 43 (Cx43) levels, suggesting that they might be sensitive to physical stimuli like low-frequency electromagnetic fields (EMF). Immature osteoblasts exhibit decreased intercellular communication in response to EMF but no change in Cx43. Here, we examined long term effects of pulsed EMF (PEMF) on MLO-Y4 osteocyte-like cells and ROS 17/2.8 osteoblast-like cells. In MLO-Y4 cell cultures, PEMF for 8 h/day for one, two or four days increased alkaline phosphatase activity but had no effect on cell number or osteocalcin. Transforming growth factor beta-1 (TGF-beta 1) and prostaglandin E(2) were increased, and NO(2-) was altered. PEMFs effect on TGF-beta1 was via a prostaglandin-dependent mechanism involving Cox-1 but not Cox-2. In ROS 17/2.8 cells, PEMF for 24, 48 or 72 h did not affect cell number, osteocalcin mRNA or osteocalcin protein. PEMF reduced Cx43 protein in both cells. Longer exposures decreased Cx43 mRNA. This indicates that cells in the osteoblast lineage, including well-differentiated osteoblast-like ROS 17/2.8 cells and terminally differentiated osteocyte-like MLO-Y4 cells, respond to PEMF with changes in local factor production and reduced Cx43, suggesting decreased gap junctional signaling.

Alkaline Phosphatase↗

PubMed2003

Pretreatment of bone with osteoclasts affects phenotypic expression of osteoblast-like cells.

Implant surface morphology regulates osteoblast phenotypic expression. Osteoblast sensitivity to non-biologic surfaces suggests that native bone surface features may also affect osteoblast response. To test this, MG63 osteoblast-like cells were grown for 7 days on bovine cortical bone wafers pretreated with rat bone marrow osteoclasts for 0, 10 or 20 days. Response to osteoclast-treated surfaces was compared to the response of MG63 cells to titanium surfaces with smooth and rough microtopographies. Cell number, differentiation (alkaline phosphatase activity and osteocalcin levels), and local factors (PGE(2) and TGF-beta1) were measured in confluent cultures. Compared to culture on plastic, cell number was reduced on all three types of bone wafers; this effect was dose-dependent with increasing resorption of the surface. Alkaline phosphatase specific activity was increased (P<or=0.05) on all three surfaces compared with plastic, but this increase was not dependent on resorption time, indicating this parameter was sensitive to the surface (bovine bone vs. plastic) but not to osteoclast-resorption. There was a direct correlation between the area of the bone surface resorbed and the amount of osteocalcin, TGF-beta1 and PGE(2) (R(2)=0.8025, 0.8689, 0.8896, respectively). With 20 days of osteoclast pretreatment, there was a 20-fold increase in osteocalcin over plastic and a 7-fold increase over cultures on untreated bone wafers. Similar increases were found for TGF-beta1 and PGE(2). Thus, surface changes resulting from osteoclast pretreatment have a strong effect on osteoblast phenotypic expression, and suggest that microtopography may play a role.

Alkaline Phosphatase↗

PubMed2003

Estrogen-dependent rapid activation of protein kinase C in estrogen receptor-positive MCF-7 breast cancer cells and estrogen receptor-negative HCC38 cells is membrane-mediated and inhibited by tamoxifen.

We examined protein kinase C (PKC) in the regulation of breast cancer cells by estrogen. Estrogen receptor (ER)- positive (+) MCF-7 and ER-negative (-) HCC38 cells were treated with 17 beta-estradiol (E(2)) or E(2)-BSA, which cannot enter the cell. E(2) and E(2)-BSA rapidly increased PKC-alpha in both cells via phosphatidylinositol-dependent phospholipase C and G protein, but not phospholipase A(2) or arachidonic acid. In MCF-7 cells, E(2) and E(2)-BSA had comparable effects, maximal at 90 min. In HCC38 cells, PKC was maximal at 9 min, with E(2)-BSA more than E(2). Tamoxifen blocked estrogen-dependent PKC in MCF-7 cells and reduced it in HCC38 cells. ER-antagonist ICI 182780, ER-agonist diethylstilbestrol, and antibodies to ER alpha and ER beta had no effect. E(2) stimulated [(3)H]thymidine incorporation in MCF-7 only; E(2)-BSA had no effect. Tamoxifen did not alter E(2)-dependent increases in MCF-7 cells, whereas ICI 182780 reduced DNA synthesis in control and E(2)-treated cultures. PKC activity was positively correlated with tumor severity in 133 breast cancer specimens and was greater in ER(-) tumors. Tamoxifen treatment reduced recurrence, and recurrent tumors had higher PKC activity. This indicates that E(2) rapidly increases PKC activity via membrane pathways not involving ER alpha or ER beta and suggests that tamoxifen works by reducing PKC activity through non-ER alpha/ER beta-dependent mechanisms.

Adult↗

PubMed2003

Use of orthodontic treatment as an aid to third molar extraction: a method for prevention of mandibular nerve injury and improved periodontal status.

BACKGROUND: Impaction of mandibular third molars predisposes to pathological conditions including periodontal disease. Extraction of these teeth also may lead to damage to the nerve and to periodontal involvement of the second molars. This report describes a series of cases in which the third molars were orthodontically induced to erupt to prevent the sequelae associated with extraction. METHODS: Impacted mandibular third molars in 18 patients were surgically exposed following placement of an orthodontic appliance. Depending on the individual case, 1 of 3 approaches was used: attachment of a bracket, placement of a post in the root canal, or placement of an orthodontic wire through a bucco-lingual canal. After suturing the mucoperiosteal flap, the orthodontic appliance was activated. After the tooth erupted, it was removed and periodontal parameters were measured on the second molar. RESULTS: No damage to the inferior alveolar nerve was found. Probing depths on the second molar were reduced from 7.9 +/- 1.6 mm on the buccal and 7.4 +/- 1.0 mm on the lingual to 1.8 +/- 0.7 mm and 1.9 +/- 0. 7 mm, respectively. There was an average gain of 5.0 mm in attachment. Keratinized tissue increased from 2.9 +/- 0.7 to 3.8 +/- 0.6 mm. CONCLUSIONS: The interdisiplinary use of periodontics and orthodontics results in non-surgical removal of impacted mandibular third molars without damage to the inferior alveolar nerve and iatrogenic periodontal sequelae to the second molars.

Adolescent↗

PubMed2002

Osteoblast-mediated mineral deposition in culture is dependent on surface microtopography.

Osteoblast phenotypic expression in monolayer culture depends on surface microtopography. Here we tested the hypothesis that mineralized bone nodule formation in response to osteotropic agents such as bone morphogenetic protein-2 (BMP-2) and dexamethasone is also influenced by surface microtopography. Fetal rat calvarial (FRC) cells were cultured on Ti implant materials (PT [pretreated], Ra = 0.6 microm; SLA [course grit blasted and acid etched], Ra = 4.0 microm; TPS [Ti plasma sprayed], Ra = 5.2 microm) in the presence of either BMP-2 (20 ng/ml) or 10(-8) M dexamethasone (Dex). At 14 days post-confluence, a homogenous layer of cells covered the surfaces, and stacks of cells that appeared to be nodules emerging from the culture surface were present in some areas on all three Ti surfaces. Cell proliferation decreased while alkaline phosphatase specific activity (ALPase) and nodule number generally increased with increasing surface roughness in both control and treated cultures. There was no difference in cell number between the control and Dex-treated cultures for a particular surface, but BMP-2 significantly reduced cell number compared with control or Dex-treated cultures. Treatment with Dex or BMP-2 further increased ALPase on all surfaces except for PT cultures with Dex. Dex had no effect on nodule area in cultures grown on PT or SLA disks, yet increased nodule number by more than 100% in cultures on PT disks. Though the effect of BMP-2 on nodule number was the same as Dex, BMP-2 increased nodule area on all surfaces except TPS, where area was decreased. Ca and P content of the cell layers in control cultures did not vary with surface roughness. However, cultures treated with Dex had increased Ca content on all surfaces, but the greatest increase was seen on SLA and TPS. BMP-2 increased Ca content in cultures on all surfaces, with the greatest increase on the PT surface. BMP-2 treatment increased P content on all surfaces, whereas Dex only increased P on rough surfaces. Of all cultures examined, the Ca/P weight ratio was 2:1 only on rough surfaces with BMP-2, indicating the presence of bone-like apatite. This was further validated by Fourier transform infrared (FTIR) imaging showing a close association between mineral and matrix on TPS and SLA surfaces with BMP-2-treated cells, and individual spectra indicated the presence of an apatitic mineral phase comparable to bone. In contrast, mineral on the smooth surface of BMP-2-treated cultures and on all surfaces where cultures were treated with Dex was not associated with the matrix and the spectra, not typical of bone apatite, implying dystrophic mineralization. This demonstrates that interactions between growth factor or hormone and surface microtopography can modulate bone cell differentiation and mineralization.

Alkaline Phosphatase↗

PubMed2002

Shear force modulates osteoblast response to surface roughness.

Previous studies have shown that osteoblasts are sensitive to surface roughness. When cultured on Ti, MG63 osteoblast-like cells exhibit decreased proliferation and increased differentiation with increasing surface roughness. In vivo, osteoblasts also are subjected to shear force during osseointegration. To examine how shear force modulates osteoblast response to surface roughness, MG63 cells were cultured on glass disks or Ti disks with three different R(a) values and topographies (PT: R(a) = 0.60 microm; SLA: R(a) = 3.97 microm; TPS: R(a) = 5.21 microm) in a continuous flow device, resulting in shear forces of 0, 1, 5, 14, and 30 dynes/cm(2). Confluent cultures were exposed to fluid flow for 1 h. After an additional 23 h, cell number, alkaline-phosphatase-specific activity, and levels of osteocalcin, TGF-beta1, and PGE2 in the conditioned media were determined. Cell numbers on smooth surfaces (glass and PT) were unaffected by shear force. In contrast, shear force caused a dose-dependent reversal of the decrease in cell numbers seen on rough SLA and TPS surfaces. Alkaline-phosphatase-specific activity was unaffected on glass or PT, but shear force caused a biphasic reduction in the roughness-dependent increase on SLA and TPS that was maximal at 14 dynes/cm(2). There was a similar effect seen with TGF-beta1 levels. Osteocalcin was unaffected on smooth surfaces; shear force caused a dose-dependent reduction in the roughness-stimulated increase seen on SLA and TPS. PGE2 production was increased by shear force on all surfaces. There was a twofold increase in PGE2 levels in the media of MG63 cells cultured on glass and PT in response to 14 dynes/cm(2), but on SLA and TPS, 14 dynes/cm(2) shear force caused a 9-10-fold increase. These results show that osteoblastic response to shear force is modulated by surface topography. The shear-force-mediated decrease in osteoblast differentiation seen in cultures on rough surfaces may be due to increased production of PGE2.

Alkaline Phosphatase↗

PubMed2002

Response of normal female human osteoblasts (NHOst) to 17beta-estradiol is modulated by implant surface morphology.

Titanium (Ti) surfaces with rough microtopographies enhance osteogenic differentiation, local factor production, and response to osteogenic agents in vitro and increase pullout strength of dental implants in vivo. Estrogens regulate bone formation, resorption, and remodeling in females and may be important in implant success. Here, we tested the hypothesis that estrogen modulates osteoblast response to implant surface morphology. Primary female human osteoblasts were cultured to confluence on three Ti surfaces (pretreatment, PT - R(a) 0.60 microm; sandblasted and acid-etched, SLA - R(a) 3.97 microm; and Ti plasma-sprayed, TPS - R(a) 5.21 microm) and treated for 24 h with 10(-7) or 10(-8) M 17beta-estradiol (E(2)). Cell number decreased with increasing surface roughness, but was not sensitive to E(2). Alkaline phosphatase specific activity of isolated cells and cell layer lysates was lower on rough surfaces. E(2) increased both parameters on smooth surfaces, whereas on rough surfaces, the stimulatory effect of E(2) on alkaline phosphatase was evident only when measuring cell layer lysates. Osteocalcin levels were higher in the conditioned media of cells grown on rough surfaces; E(2) had no effect in cultures on the plastic surfaces, but increased osteocalcin production on all Ti surfaces. TGF-beta1 and PGE(2) production was increased on rough surfaces, and E(2) augmented this effect in a synergistic manner; on smooth surfaces, there was no change in production with E(2). The response of osteoblasts to surface topography was modulated by E(2). On smooth surfaces, E(2) affected only alkaline phosphatase, but on rough surfaces, E(2) increased levels of osteocalcin, TGF-beta1, and PGE(2). These results show that normal adult human female osteoblasts are sensitive to surface microtopography and that E(2) can alter this response.

Alkaline Phosphatase↗

PubMed2002

Tamoxifen elicits its anti-estrogen effects in growth plate chondrocytes by inhibiting protein kinase C.

17 beta-Estradiol (E(2)) regulates growth plate cartilage cells via classical nuclear receptor mechanisms, as well as by direct effects on the chondrocyte membrane. These direct effects are stereospecific, causing a rapid increase in protein kinase C (PKC) specific activity, are only found in cells from female rats and are mimicked by E(2)-bovine serum albumin (BSA), which cannot penetrate the cell membrane. E(2) and E(2)-BSA stimulate alkaline phosphatase specific activity and proteoglycan sulfation in female rat costochondral cartilage cell cultures, but traditional nuclear receptors do not appear to be involved. This study examined the effect of the anti-estrogen tamoxifen on these markers of chondrocyte differentiation; the gender-specificity of tamoxifen's effect on PKC, if tamoxifen has an effect on vitamin D metabolite-stimulated PKC, which is mediated via specific membrane receptors (1,25-mVDR; 24,25-mVDR) and whether the effect of tamoxifen is mediated by nuclear estrogen receptors. Tamoxifen dose-dependently inhibited the effect of E(2)-BSA on PKC, alkaline phosphatase and proteoglycan sulfation in confluent cultures of female resting zone (RC) cells and growth zone (GC) (prehypertrophic/upper hypertrophic zones) cells, suggesting that its action is at the membrane and not cell maturation-dependent. Neither the estrogen receptor (ER) antagonist ICI 182780 nor the ER agonist diethylstilbesterol affected E(2) or E(2)-BSA-stimulated PKC in female chondrocytes. Tamoxifen also inhibited the increase in PKC activity due to 1 alpha,25-(OH)(2)D(3) or 24R,25-(OH)(2)D(3) in growth plate cells derived from either female or male rats. Inhibition of PKC by tamoxifen may be a general property of membrane receptors involved in rapid responses to hormones.

Alkaline Phosphatase↗

PubMed2002

Rat costochondral chondrocytes produce 17beta-estradiol and regulate its production by 1alpha,25(OH)(2)D(3).

Prior studies have shown that 17beta-estradiol (17beta-E(2)) regulates growth plate chondrocyte maturation and differentiation. This study examines the hypothesis that 17beta-E(2) is a local regulator of rat costochondral growth plate chondrocytes by determining whether these cells express aromatase mRNA and enzyme activity, produce 17beta-E(2), and regulate 17beta-E(2) production by vitamin D(3) metabolites in a gender-specific and cell-maturation-dependent manner. Aromatase gene expression was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and northern analysis of total RNA from male and female chondrocytes. Aromatase specific activity was measured in cell layer lysates of confluent male and female rat costochondral resting zone (RC) and growth zone (GC) cartilage cells that had been treated for 24 h with 1alpha, 25(OH)(2)D(3), 24R,25(OH)(2)D(3), or transforming growth factor (TGF)-beta1. 17beta-E(2) released into the culture media of treated cells was measured by radioimmunoassay (RIA). Female RC cells expressed the highest levels of aromatase mRNA compared with male RC cells and both male and female GC cells. Aromatase activity was present in male and female cells and was 1.6 times greater in female RC cells than female GC cells; male RC and GC cells displayed comparable levels. All cultures produced 17beta-E(2), with a 2.5-fold greater production by female RC cells than female GC cells or either cell type from male rats. Treatment of cultures with 1alpha,25(OH)(2)D(3) caused a dose-dependent increase in 17beta-E(2) production by female RC (1.5-fold greater than control cells) and female GC (threefold greater than control cells) cells. In contrast, 1alpha,25(OH)(2)D(3) had no effect on male GC cells and increased production in male RC cells by only 10% at the highest concentration of 1alpha,25(OH)(2)D(3) used. Neither 24R, 25(OH)(2)D(3) nor TGF-beta1 had an effect on 17beta -E(2) production. These results support our hypothesis and indicate that 17beta-E(2) is most likely a local regulator of rat costochondral growth plate chondrocytes.

24,25-Dihydroxyvitamin D 3↗

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↗

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↗