Interaction between caspase-3 and caspase-5 in the stretch-induced programmed cell death in the human periodontal ligament cells

In our previous studies, programmed cell death (PCD) was induced in human periodontal ligament (PDL) cells, through activation of caspase-3 and upregulation of CASP5 gene (encoding caspase-5 protein), in response to mechanical stretch loading. The aim of this study is to explore the relationship between the inflammatory caspase, caspase-5, and the apoptotic executioner protein, caspase-3, in human PDL cells. Here, we found that cyclic stretching upregulated the activity and the protein expression level of caspase-3 and -5 and the addition of the caspase-3 inhibitor or caspase-5 inhibitor significantly inhibited the stretch-induced PCD. Meanwhile, the inhibition of caspase-5 inhibited the activation of caspase-3 and vice versa. The result of coimmunoprecipitation also demonstrated that the expression of caspase-3 was immunoprecipitated with caspase-5. Thus, our study revealed that the in vitro application of cyclic stretching induced PCD by activation of caspase-3 and -5 in human PDL cells, and these two caspases could interact with each other after mechanical stretch loading. The study may facilitate further studies on the mechanism of stretch-induced PCD and help us understand the force-related periodontal homeostasis and remodeling better.     

Caffeine decreases vitamin D receptor protein expression and 1,25(OH)2D3 stimulated alkaline phosphatase activity in human osteoblast cells

Of the various risk factors contributing to osteoporosis, dietary/lifestyle factors are important. In a clinical study we reported that women with caffeine intakes >300 mg/day had higher bone loss and women with vitamin D receptor (VDR) variant, tt were at a greater risk for this deleterious effect of caffeine. However, the mechanism of how caffeine effects bone metabolism is not clear. 1,25-Dihydroxy vitamin D₃ (1,25(OH)₂D₃) plays a critical role in regulating bone metabolism. The receptor for 1,25(OH)₂D₃, VDR has been demonstrated in osteoblast cells and it belongs to the superfamily of nuclear hormone receptors. To understand the molecular mechanism of the role of caffeine in relation to bone, we tested the effect of caffeine on VDR expression and 1,25(OH)₂D₃ mediated actions in bone. We therefore examined the effect of different doses of caffeine (0.2, 0.5, 1.0 and 10 mM) on 1,25(OH)₂D₃ induced VDR protein expression in human osteoblast cells. We also tested the effect of different doses of caffeine on 1,25(OH)₂D₃ induced alkaline phosphatase (ALP) activity, a widely used marker of osteoblastic activity. Caffeine dose dependently decreased the 1,25(OH)₂D₃ induced VDR expression and at concentrations of 1 and 10 mM, VDR expression was decreased by about 50–70%, respectively. In addition, the 1,25(OH)₂D₃ induced alkaline phosphatase activity was also reduced at similar doses thus affecting the osteoblastic function. The basal ALP activity was not affected with increasing doses of caffeine. Overall, our results suggest that caffeine affects 1,25(OH)₂D₃ stimulated VDR protein expression and 1,25(OH)₂D₃ mediated actions in human osteoblast cells.     

Inhibition of prostate cancer growth by vitamin D: Regulation of target gene expression

Prostate cancer (PCa) cells express vitamin D receptors (VDR) and 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits the growth of epithelial cells derived from normal, benign prostate hyperplasia, and PCa as well as established PCa cell lines. The growth inhibitory effects of 1,25(OH)(2)D(3) in cell cultures are modulated tissue by the presence and activities of the enzymes 25-hydroxyvitamin D(3) 24-hydroxylase which initiates the inactivation of 1,25(OH)(2)D(3) and 25-hydroxyvitamin D(3) 1alpha-hydroxylase which catalyses its synthesis. In LNCaP human PCa cells 1,25(OH)(2)D(3) exerts antiproliferative activity predominantly by cell cycle arrest through the induction of IGF binding protein-3 (IGFBP-3) expression which in turn increases the levels of the cell cycle inhibitor p21 leading to growth arrest. cDNA microarray analyses of primary prostatic epithelial and PCa cells reveal that 1,25(OH)(2)D(3) regulates many target genes expanding the possible mechanisms of its anticancer activity and raising new potential therapeutic targets. Some of these target genes are involved in growth regulation, protection from oxidative stress, and cell-cell and cell-matrix interactions. A small clinical trial has shown that 1,25(OH)(2)D(3) can slow the rate of prostate specific antigen (PSA) rise in PCa patients demonstrating proof of concept that 1,25(OH)(2)D(3) exhibits therapeutic activity in men with PCa. Further investigation of the role of calcitriol and its analogs for the therapy or chemoprevention of PCa is currently being pursued.     

Membrane actions of vitamin D metabolites 1alpha,25(OH)2D3 and 24R,25(OH)2D3 are retained in growth plate cartilage cells from vitamin D receptor knockout mice

1alpha,25(OH)(2)D(3) regulates rat growth plate chondrocytes via nuclear vitamin D receptor (1,25-nVDR) and membrane VDR (1,25-mVDR) mechanisms. To assess the relationship between the receptors, we examined the membrane response to 1alpha,25(OH)(2)D(3) in costochondral cartilage cells from wild type VDR(+/+) and VDR(-/-) mice, the latter lacking the 1,25-nVDR and exhibiting type II rickets and alopecia. Methods were developed for isolation and culture of cells from the resting zone (RC) and growth zone (GC, prehypertrophic and upper hypertrophic zones) of the costochondral cartilages from wild type and homozygous knockout mice. 1alpha,25(OH)(2)D(3) had no effect on [(3)H]-thymidine incorporation in VDR(-/-) GC cells, but it increased [(3)H]-thymidine incorporation in VDR(+/+) cells. Proteoglycan production was increased in cultures of both VDR(-/-) and VDR(+/+) cells, based on [(35)S]-sulfate incorporation. These effects were partially blocked by chelerythrine, which is a specific inhibitor of protein kinase C (PKC), indicating that PKC-signaling was involved. 1alpha,25(OH)(2)D(3) caused a 10-fold increase in PKC specific activity in VDR(-/-), and VDR(+/+) GC cells as early as 1 min, supporting this hypothesis. In contrast, 1alpha,25(OH)(2)D(3) had no effect on PKC activity in RC cells isolated from VDR(-/-) or VDR(+/+) mice and neither 1beta,25(OH)(2)D(3) nor 24R,25(OH)(2)D(3) affected PKC in GC cells from these mice. Phospholipase C (PLC) activity was also increased within 1 min in GC chondrocyte cultures treated with 1alpha,25(OH)(2)D(3). As noted previously for rat growth plate chondrocytes, 1alpha,25(OH)(2)D(3) mediated its increases in PKC and PLC activities in the VDR(-/-) GC cells through activation of phospholipase A(2) (PLA(2)). These responses to 1alpha,25(OH)(2)D(3) were blocked by antibodies to 1,25-MARRS, which is a [(3)H]-1,25(OH)(2)D(3) binding protein identified in chick enterocytes. 24R,25(OH)(2)D(3) regulated PKC in VDR(-/-) and VDR(+/+) RC cells. Wild type RC cells responded to 24R,25(OH)(2)D(3) with an increase in PKC, whereas treatment of RC cells from mice lacking a functional 1,25-nVDR caused a time-dependent decrease in PKC between 6 and 9 min. 24R,25(OH)(2)D(3) dependent PKC was mediated by phospholipase D, but not by PLC, as noted previously for rat RC cells treated with 24R,25(OH)(2)D(3). These results provide definitive evidence that there are two distinct receptors to 1alpha,25(OH)(2)D(3). 1alpha,25(OH)(2)D(3)-dependent regulation of DNA synthesis in GC cells requires the 1,25-nVDR, although other physiological responses to the vitamin D metabolite, such as proteoglycan sulfation, involve regulation via the 1,25-mVDR.     

Analogs of 1alpha,25-dihydroxyvitamin D3 as pluripotent immunomodulators

The active form of vitamin D(3), 1,25(OH)(2)D(3), is known, besides its classical effects on calcium and bone, for its pronounced immunomodulatory effects that are exerted both on the antigen-presenting cell level as well as directly on the T lymphocyte level. In animal models, these immune effects of 1,25(OH)(2)D(3) are reflected by a strong potency to prevent onset and even recurrence of autoimmune diseases. A major limitation in using 1,25(OH)(2)D(3) in clinical immune therapy are the adverse side effects on calcium and on bone. TX527 (19-nor-14,20-bisepi-23-yne-1,25(OH)(2)D(3)) is a structural 1,25(OH)(2)D(3) analog showing reduced calcemic activity associated with enhanced in vitro and in vivo immunomodulating capacity compared to the mother-molecule. Indeed, in vitro TX527 is more potent that 1,25(OH)(2)D(3) in redirecting differentiation and maturation of dendritic cells and in inhibiting phytohemagglutinin-stimulated T lymphocyte proliferation. In vivo, this enhanced potency of TX527 is confirmed by a stronger potential to prevent type 1 diabetes in nonobese diabetic (NOD) mice and to prolong the survival of syngeneic islets grafts, both alone and in combination with cyclosporine A, in overtly diabetic NOD mice. Moreover, these in vivo effects of TX527 are obtained without the adverse side effects observed for 1,25(OH)(2)D(3) itself. We believe therefore that TX527 is a potentially interesting candidate to be considered for clinical intervention trails in autoimmune diseases.     

Stimulatory effect of 1,25-dihydroxyvitamin D3 on the glucose-6-phosphate dehydrogenase activity in the MCF-7 human breast cancer cell line

Human breast cancer cell lines have been shown to possess high affinity receptors for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) and their growth is inhibited by this steroid. The present study examines the effect of 1,25(OH)2D3 on the activity of glucose-6-phosphate dehydrogenase (G6PD) in cells of a human breast cancer cell line MCF-7. G6PD, an enzyme which controls the hexose monophosphate shunt, is elevated and sensitive to 17 beta-estradiol in breast tumors. G6PD activity was stimulated by 1,25(OH)2D3 in a dose-dependent manner at very low concentrations of steroid (10(-10)-10(-12) M). 1,25(OH)2D3 increased maximum velocity without modifying the affinity constant of the enzyme for glucose-6-phosphate.     

Modulation of 1alpha,25-dihydroxyvitamin D3-membrane associated, rapid response steroid binding protein expression in mouse odontoblasts by 1alpha,25-(OH)2D3

The rapid, nongenomic effects of 1alpha,25-dihydroxyvitamin D3 (1alpha,25-(OH)2D3 have been related to a 1,25D3-membrane associated, rapid response steroid binding protein or 1,25D3-[MARRS]bp, with a molecular weight of 65 kDa, in several tissues and species. Currently, no information is available concerning the nongenomic responses to 1alpha,25-(OH)2D3 in dental tissues. In order to investigate the expression of 1,25D3-[MARRS]bp in dental cells, in the presence or absence of 1alpha,25-(OH)2D3, we have used rabbit polyclonal antibodies directed against the N-terminus of the 1,25D3-[MARRS]bp (Ab099) that recognizes the 1alpha,25-(OH)2D3 binding protein in chick intestinal basolateral membranes and a mouse odontoblast-like cell line (MO6-G3). Western blotting and flow cytometric analyses with Ab099 specifically detected 1,25D3-[MARRS]bp in MO6-G3 cells. Moreover, 1,25D3-[MARRS]bp was up-regulated, in vivo, in differentiated dental cells. Electron microscopic analysis confirmed the plasma membrane localization of this binding protein and also showed its intracellular presence. Incubation of MO6-G3 cells with different doses of 1alpha,25-(OH)2D3 for 36 h resulted in an inhibition of 1,25D3-[MARRS]bp expression with a maximal effect at 50 nM steroid. In addition, the culture media of MO6-G3 cells contains immunoreactive 1,25D3-[MARRS]bp. Immunogold positive membrane vesicle-like structures are present in the extracellular matrix of MO6-G3 cells. Altogether, these results indicate that the 1,25D3-[MARRS]bp expression in MO6-G3 cells is modulated by 1alpha,25-(OH)2D3. In conclusion, this 1alpha,25-(OH)2D3 binding protein could play an important role in the rapid, nongenomic responses to 1alpha,25-(OH)2D3 in dental cells.     

Differential effects of growth factors and cytokines on the synthesis of SPARC, DNA, fibronectin and alkaline phosphatase activity in human periodontal ligament cells

Growth factors and cytokines play an important role in tissue development and repair. However, it remains unknown how they act on proliferation and differentiation of periodontal ligament cells. In this study, we investigated the effects of several growth factors and cytokines on the synthesis of DNA, alkaline phosphatase (ALPase), fibronectin, and secreted protein acidic and rich in cysteine (SPARC) in human periodontal ligament (HPL) cells. Transforming growth factor-beta (TGF-beta) increased the synthesis of DNA, fibronectin and SPARC, whereas it decreased ALPase activity. Basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) and tumor necrosis factor-alpha (TNF-alpha) decreased SPARC and ALPase levels, whereas these peptides increased DNA synthesis and did not affect fibronectin synthesis. Epidermal growth factor (EGF) up-regulated the synthesis of DNA and fibronectin and inhibited SPARC and ALPase levels. Interleukin-1beta (IL-1beta) decreased the synthesis of DNA, ALPase, fibronectin and SPARC. These findings demonstrate that TGF-beta, bFGF, EGF, PDGF, TNF-alpha and IL-1beta have characteristically different patterns of action on DNA, SPARC, fibronectin and ALPase synthesis by HPL cells. The differences in regulation of function of periodontal ligament cells by these peptides may be involved in the regeneration and repair of periodontal tissue.     

Expression of UNCL during development of periodontal tissue and response of periodontal ligament fibroblasts to mechanical stress in vivo and in vitro

Mutations in two genes, uncoordinated (unc) and uncoordinated-like (uncl), lead to a failure of mechanotransduction in Drosophila. UNCL, the human homolog of unc and uncl, is preferentially expressed in periodontal ligament (PDL) fibroblasts compared with gingival fibroblasts. However, the precise role of UNCL in the PDL remains unclear. The aim of the present study has been to examine whether mechanical stimuli modulate the expression of UNCL in the human PDL in vivo and in vitro and to examine the roles of UNCL in the development, regeneration, and repair of the PDL. We have investigated the expression pattern of UNCL during the development of periodontal tissue and the response of PDL fibroblasts to mechanical stress in vivo and in vitro. The expression of UNCL mRNA and protein increases with PDL fibroblast differentiation from the confluent to multilayer stage but slightly decreases on mineralized nodule formation. UNCL has also been localized in ameloblasts and adjacent cells, differentiating cementoblasts, and osteoblasts of the developing tooth. Strong distinct UNCL expression has further been observed in the differentiating cementoblasts of the tooth periodontium at the site of tension after orthodontic tooth movement. Application of cyclic mechanical stress on PDL fibroblasts increases the expression of UNCL mRNA. These results indicate that UNCL plays important roles in the development, differentiation, and maintenance of periodontal tissues and also suggest a potential role of UNCL in the mechanotransduction of PDL fibroblasts.This work was supported by a grant from the Korea Health 21R&D Project, Ministry of Health & Welfare, Republic of Korea (03-PJ1-PG1-CH08-0001).     

1,25-Dihydroxyvitamin D3 inhibits tumor necrosis factor-alpha-induced adhesion molecule expression in endothelial cells

This study tested the hypothesis that 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] plays a role in human umbilical vein endothelial cells (HUVEC) cultures. HUVEC were incubated with 10 or 100 nM 1,25(OH)(2)D(3) for 24 h, in the absence or presence of 40 ng/ml tumor necrosis factor-alpha (TNF-alpha) or 2 ng/ml interleukin-1alpha (IL-1alpha). 1,25(OH)(2)D(3) did not affect HUVEC viability and proliferation, while TNF-alpha, alone or in combination with the hormone, significantly inhibited HUVEC viability. [(3)H]thymidine incorporation in HUVEC treated with TNF-alpha or IL-1alpha significantly decreased, in the absence or in the presence of the hormone, while the levels of vitamin D receptor markedly increased in the presence of 1,25(OH)(2)D(3) alone or associated with TNF-alpha or IL-1alpha, in comparison to the control. The noteworthy increase in protein levels of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) induced by TNF-alpha was significantly decreased after incubation of the cells with 1,25(OH)(2)D(3), this effect not being seen on E-selectin expression. Neither apoptosis nor nuclear translocation of NF-kappaB, induced in HUVEC by TNF-alpha was influenced by 1,25(OH)(2)D(3) treatment.     

Dexamethasone-induced up-regulation of two-pore domain K+ channel genes, TASK-1 and TWIK-2, in cultured human periodontal ligament fibroblasts

Two-pore domain K(+) channels are widely expressed in many types of cells, and have various important functions, especially maintaining the resting membrane potential. In the previous report, we have confirmed the presence of several kinds of two-pore domain K(+) channels in the periodontal ligament (PDL) fibroblasts. It is well known that dexamethasone (Dex) regulates the functions of various kinds of ion channels. In this work, we investigate if Dex affects the gene expressions of the two-pore domain K(+) channels in the PDL fibroblasts. We also examined the effects of other steroid hormones on the K(+) channels gene expression. The mRNA levels of two-pore domain K(+) channels in human PDL fibroblasts were examined in the presence or absence of Dex by RT-PCR. The effects of other steroid hormones (aldosterone, estrogen, 1α,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], and retinoic acid) were also examined. Dex significantly induced the expression of TASK-1 and TWIK-2 in mRNA levels in both a dose- and a time-dependent manner. The stimulatory effects of Dex were completely abolished by a glucocorticoid receptor antagonist. 1,25-(OH)(2)D(3) also increased the TASK-1 mRNA levels but had no effect on TWIK-2 expression. Dex, one of the potent glucocorticoid, probably have a protective role against external stimuli by maintaining the membrane potential of PDL fibroblasts through the up-regulation of TASK-1 and TWIK-2 K(+) channels.     

Membrane receptor-initiated signaling in 1,25(OH)2D3-stimulated calcium uptake in intestinal epithelial cells

Demonstrating 1,25(OH)2D3-stimulated calcium uptake in isolated chick intestinal epithelial cells has been complicated by simultaneous enhancement of both uptake and efflux. We now report that in intestinal cells of adult birds, or those of young birds cultured for 72 h, 1,25(OH)2D3-stimulates 45Ca uptake to greater than 140% of corresponding controls within 3 min of addition. Such cells have lost hormone-stimulated protein kinase C (PKC) activity, believed to mediate calcium efflux. To further test this hypothesis, freshly isolated cells were preincubated with calphostin C, and calcium uptake monitored in the presence or absence of steroid. Only cells treated with the PKC inhibitor demonstrated a significant increase in 45Ca uptake in response to 1,25(OH)2D3, relative to corresponding controls. In addition, phorbol ester was shown to stimulate efflux, while forskolin stimulated uptake. To further investigate the mechanisms involved in calcium uptake, we assessed the role of TRPV6 and its activation by beta-glucuronidase. beta-Glucuronidase secretion from isolated intestinal epithelial cells was significantly increased by treatment with 1,25(OH)2D3, PTH, or forskolin, but not by phorbol ester. Treatment of cells with beta-glucuronidase, in turn, stimulated 45Ca uptake. Finally, transfection of cells with siRNA to either beta-glucuronidase or TRPV6 abolished 1,25(OH)2D3-enhanced calcium uptake relative to controls transfected with scrambled siRNA. Confocal microscopy further indicated rapid redistribution of enzyme and calcium channel after steroid. 1,25(OH)2D3 and PTH increase calcium uptake by stimulating the PKA pathway to release beta-glucuronidase, which in turn activates TRPV6. 1,25(OH)2D3-enhanced calcium efflux is mediated by the PKC pathway.     

Biochemical significance of enhanced activity of fluorinated 1,25-dihydroxyvitamin D3 in human cultured cell lines

Several human cancer cells possess receptors for 1,25-dihydroxyvitamin D3[1,25-(OH)2D3]. In these cells 1,25-(OH)2D3 has a biphasic concentration-dependent regulatory effect on cell replication and specifically induces its own metabolism. We have studied the effects on these parameters of the native hormone together with those of two analogues fluorinated at the 24-carbon and of 1,24R,25-trihydroxyvitamin D3[1,24R,25-(OH)3D3]. The difluorinated analogue 24,24-difluoro-1,25-(OH)2D3[24,24-F2-1,25-(OH)2D3] is an approximately fivefold more potent inhibitor of cellular replication than the native hormone, while 1,24R,25-(OH)3D3 is about fivefold less potent. This enhanced potency of the fluorinated analogue parallels its enhanced potency in in vivo studies of its effects on calcium and mineral metabolism. However, although the analogue retains replication stimulatory activity, it is clearly no more potent than the native hormone in this activity: 1,24R,25-(OH)3D3 has no significant stimulatory activity. Exposure of the cells to 1,25-(OH)2D3 at 0.05 nM for 6 h increases the subsequent conversion of labelled hormone to aqueous phase soluble compounds by 6.7-fold. None of the other compounds had a similar effect at this concentration. At 10 nM all 1-hydroxylated compounds increased aqueous phase radioactivity about equally (13 to 17-fold); this effect is still specific since 25-OH D3 had no such effect even at 10 nM. Studies on the effects of the fluorinated analogues upon receptor binding of hormone in cell cytosols and uptake of hormone by intact cells clearly demonstrate that the enhanced activity of these analogues is not due to higher receptor affinity or more rapid access to intracellular receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vivo relevance for photoprotection by the vitamin D rapid response pathway

Vitamin D is produced by exposure of 7-dehydrocholesterol in the skin to UV irradiation (UVR) and further converted in the skin to the biologically active metabolite, 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) and other compounds. UVR also results in DNA damage producing cyclobutane pyrimidine dimers (CPD). We previously reported that 1,25(OH)₂D₃ at picomolar concentrations, protects human skin cells from UVR-induced apoptosis, and decreases CPD in surviving cells. 1,25(OH)₂D₃ has been shown to generate biological responses via two pathways—the classical steroid receptor/genomic pathway or a rapid, non-genomic pathway mediated by a putative membrane receptor. Whether the rapid response pathway is physiologically relevant is unclear. A cis-locked, rapid-acting agonist 1,25(OH)₂lumisterol₃ (JN), entirely mimicked the actions of 1,25(OH)₂D₃ to reduce fibroblast and keratinocyte loss and CPD damage after UVR. The effects of 1,25(OH)₂D₃ were abolished by a rapid-acting antagonist, but not by a genomic antagonist. Skh:hr1 mice exposed to three times the minimal erythemal dose of solar-simulated UVR and treated topically with 1,25(OH)₂D₃ or JN immediately after UVR showed reduction in UVR-induced UVR-induced sunburn cells (p < 0.01 and <0.05, respectively), CPD (p < 0.01 for both) and immunosuppression (p < 0.001 for both) compared with vehicle-treated mice. These results show for the first time an in vivo biological response mediated by a rapid-acting analog of the vitamin D system. The data support the hypothesis that 1,25(OH)₂D₃ exerts its photoprotective effects via the rapid pathway and raise the possibility that other D compounds produced in skin may contribute to the photoprotective effects.     

Chemical synthesis of 20S-hydroxyvitamin D3, which shows antiproliferative activity

20S-hydroxyvitamin D3 (20S-(OH)D3), an in vitro product of vitamin D3 metabolism by the cytochrome P450scc, was recently isolated, identified and shown to possess antiproliferative activity without inducing hypercalcemia. The enzymatic production of 20S-(OH)D3 is tedious, expensive, and cannot meet the requirements for extensive chemical and biological studies. Here we report for the first time the chemical synthesis of 20S-(OH)D3 which exhibited biological properties characteristic of the P450scc-generated compound. Specifically, it was hydroxylated to 20,23-dihydroxyvitamin D3 and 17,20-dihydroxyvitamin D3 by P450scc and was converted to 1α,20-dihydroxyvitamin D3 by CYP27B1. It inhibited proliferation of human epidermal keratinocytes with lower potency than 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) in normal epidermal human keratinocytes, but with equal potency in immortalized HaCaT keratinocytes. It also stimulated VDR gene expression with similar potency to 1,25(OH)2D3, and stimulated involucrin (a marker of differentiation) and CYP24 gene expression, showing a lower potency for the latter gene than 1,25(OH)2D3. Testing performed with hamster melanoma cells demonstrated a dose-dependent inhibition of cell proliferation and colony forming capabilities similar or more pronounced than those of 1,25(OH)2D3. Thus, we have developed a chemical method for the synthesis of 20S-(OH)D3, which will allow the preparation of a series of 20S-(OH)D3 analogs to study structure-activity relationships to further optimize this class of compound for therapeutic use.     

Human periodontal ligament cells derived from deciduous teeth induce osteoclastogenesis in vitro

The receptor activator of NF-kappa B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG), are the important proteins involved in osteoclastogenesis. In this study, we investigated the expressions of RANKL and OPG in cultured human periodontal ligament cells derived from deciduous teeth (DPDL cells) and their roles in osteoclastogenesis. Northern blotting revealed that the OPG mRNA was down-regulated by application of 10(-8) M 1 alpha, 25(OH)2 vitamin D3 [1,25-(OH)2D3] and 10(-7) M dexamethasone (Dex). In contrast, RANKL mRNA was up-regulated by the same treatment. Western blotting demonstrated a decrease in OPG following application of 1, 25-(OH)2D3 and Dex. Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNCs) were induced when DPDL cells were co-cultured with mouse bone marrow cells in the presence of 1,25-(OH)2D3 and Dex. TRAP-positive MNCs increased significantly when the DPDL cells were co-cultured with bone marrow cells in the presence of anti-human OPG antibody together with 1, 25-(OH)2D3 and Dex. These results indicate that PDL cells derived from deciduous teeth synthesize both RANKL and OPG and could regulate the differentiation of osteoclasts.     

病理性周期性张应力诱导人牙周膜细胞凋亡的机制研究

目的:阐明病理性周期性张应力诱导人牙周膜细胞凋亡的分子机制。方法:人牙周膜细胞取自健康前磨牙,经过3?5代传代,细胞受到20%牵张力,时间为6 h或24 h,通过用膜联蛋白异硫氰酸荧光素(V-FITC)和碘化丙啶(PI)结合流式细胞仪检测细胞凋亡,用Western Blot法研究caspase-3,cleaved caspase-3,116 kDa PARP-1和85 k Da PARP-1蛋白的表达变化。结果:人PDL细胞受到病理性周期性张应力时存在凋亡,并以一种时间依赖的方式增加。受到病理性周期性张应力后裂解的caspase-3和PARP蛋白随着时间增加,然而抑制caspase-3的活性却可以抑制细胞的凋亡,但并不能抑制由其他通路导致的凋亡。结论:病理性周期性张应力通过caspase-3/PARP途径诱导人牙周膜细胞的凋亡。