L.-Q. Yuan

Insulin-Like Effects of Visfatin on Human Osteoblasts

Visfatin (also known as pre-B cell colony-enhancing factor or PBEF) is a novel adipocytokine that is highly expressed in visceral fat and upregulated in obesity and type 2 diabetes mellitus. Visfatin binds to and activates the insulin receptor (IR), thereby exerting insulin-mimetic effects in various cell lines. IR has been detected in osteoblasts, which is consistent with the role of insulin as an important osteotropic hormone. This study investigated the actions of visfatin on human primary osteoblasts. The expression and tyrosine phosphorylation of IR, IR substrate-1 (IRS-1), and IRS-2 were determined by immunoprecipitation and immunoblotting. Cell proliferation was determined by measuring [3H]thymidine incorporation and cell number. Glucose uptake was determined by measuring 2-[3H]deoxyglucose incorporation. Real-time quantitative reverse-transcription polymerase chain reaction (PCR) was used for determining alkaline phosphatase (ALP), osteocalcin, and type I collagen mRNA expression. Enzyme-linked immunosorbent assay and radioimmunoassay were used for measuring ALP activity, osteocalcin secretion, and type I collagen production. We found that visfatin induced tyrosine phosphorylation of IR, IRS-1, and IRS-2. Moreover, the effects of visfatin – glucose uptake, proliferation, and type I collagen enhancement of cultured human osteoblast-like cells – bore a close resemblance to those of insulin and were inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester, a specific inhibitor of IR tyrosine kinase activity. We also unexpectedly found that visfatin downregulated osteocalcin secretion from human osteoblast-like cells. These data indicate that the regulation of glucose uptake, proliferation, and type I collagen production by visfatin in human osteoblasts involves IR phosphorylation, the same signal-transduction pathway used by insulin.

Taurine promotes connective tissue growth factor (CTGF) expression in osteoblasts through the ERK signal pathway

Summary.Taurine is found in bone tissue, but its function in skeletal tissue is not fully understood. The present study was undertaken to investigate regulation of gene expression of connective tissue growth factor (CTGF), and the roles of mitogen-activated protein kinases (MAPKs) in murine osteoblast MC3T3-E1 cells treated with taurine. Western blot analysis showed taurine stimulated CTGF protein secretion in a dose- and time-dependent manner. Taurine induced activation of extracellular signal-regulated kinase (ERK), but not p38 and c-jun N-terminal Kinase (JNK), in osteoblasts. Furthermore, pretreatment of osteoblasts with the ERK inhibitor PD98059 abolished the taurine-induced CTGF production. These data indicate that taurine induces CTGF secretion in MC3T3-E1 cells mediated by the ERK pathway, and suggest that osteoblasts are direct targets of taurine.

l-Carnitine protects against apoptosis of murine MC3T3-E1 osteoblastic cells

Summary.l-Carnitine (LC), an amino acid with a major role in cellular energy metabolism, has positive effects on bone metabolism. However, the effect of LC on apoptosis of osteoblast in vitro has not been reported. The aim of this study was to investigate the action of LC on apoptosis of mouse osteoblastic cell line MC3T3-E1. Cell apoptosis was measured by sandwich-enzyme-immunoassay. Release of cytochrome c from mitochondria into cytosol and Bcl-2, Bax protein levels were determined by Western blot analysis. The enzyme substrate was used to assess the activation of caspase-3 and caspase-9. LC inhibited MC3T3-E1 cell apoptosis induced by serum deprivation. Our study also shows that LC decreased cytochrome c release and caspase-3 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Furthermore, LC protected against MC3T3-E1 cell apoptosis induced by the glucocorticoid (GC) dexamethasone (Dex).

Relationship of circulating MMP–2, MMP–1, and TIMP–1 levels with bone biochemical markers and bone mineral density in postmenopausal Chinese women

IntroductionOsteoblast-derived matrix metalloproteinase (MMP)–2, MMP–1 and tissue inhibitor of metalloproteinase (TIMP)–1 have been shown to play a role in bone metabolism by degrading the bone matrix.MethodsThe present study was performed to investigate the relationships between serum MMP–2, MMP–1, or TIMP–1 levels and bone mineral density (BMD), as well as bone biochemical markers, in 297 Chinese postmenopausal women aged 42–80 years.ResultsWe found a significant negative weak correlation between MMP–2 and BMD at various skeletal regions. After adjustment for age and BMI, the correlation with BMD at the femoral neck and total hip disappeared. Multiple linear stepwise regression analysis showed that MMP–2 was not a determinant factor for BMD. The significant positive correlations between MMP–2 and bone cross-linked N–telopeptides of type I collagen (NTX), alkaline phosphatase (BAP), and osteocalcin (OC) and were found, and remained significant after adjustment for age and BMI. Moreover, serum MMP–2 concentrations were significantly higher in postmenopausal women with osteoporosis than in age-matched normal controls. There were no significant correlations between MMP–1, TIMP–1 and BMD. There were no significant relationships between MMP–1 and BAP, OC, and NTX. The associations between TIMP–1 and BAP and OC were not specific and constant.ConclusionsIn conclusion, our results suggest that circulating MMP–2 and markers of bone turnover are correlated, and serum MMP–2 levels may rise with increase in bone turnover.

Taurine transporter is expressed in osteoblasts

Summary.Taurine influences bone metabolism and is taken up by cells via a specific transport system, the taurine transporter (TAUT). We report a link between taurine and bone homeostasis by demonstrating transcription and translation of TAUT in bone-forming cells. TAUT was expressed in human primary osteoblasts, the human osteosarcoma osteoblast-like cell line MG63, and the mouse osteoblastic cell line MC3T3-E1. Immunostaining with polyclonal antibodies also demonstrated the presence of TAUT in both human and murine osteoblasts. TAUT mRNA expression and [3H]taurine uptake increased during differentiation of MG63 cells in culture. Supplementation of culture medium with taurine enhanced alkaline phosphatase activity and osteocalcin secretion. The regulation and detailed function of taurine and TAUT in bone remain unclear, but our findings suggest a functional role for them in bone homeostasis.

Ascorbic acid inhibits osteoclastogenesis of RAW264.7 cells induced by receptor activated nuclear factor kappaB ligand (RANKL) in vitro

Ascorbic acid (AA) plays a key role in the regulation of differentiation and activation of osteoclast (OCL). It was reported that AA might induce the formation of OCL in co-cultures of mouse bone marrow cells and ST2 cells, but it is not clear whether AA has a direct impact on the OCL precursors. The purpose of this study was to examine the effect of AA on the differentiation of OCL precursor RAW264.7 cells, cultured with receptor-activated nuclear factor kappaB ligand (RANKL). The results showed that AA remarkably inhibited the cell proliferation at a higher concentration and RANKL alone is sufficient for osteoclastogenesis. The expression of carbonic anhydrase (CAII) mRNA and protein, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs), and the percentage area of resorption lacunae induced by RANKL were decreased when AA was added to the cultures. The results demonstrate that AA inhibits RANKL-induced differentiation of OCL precursor cells into mature OCL and reduces the formation of bone resorption pits in vitro.

Tissue inhibitor of matrix metalloproteinase-1 suppresses apoptosis of mouse bone marrow stromal cell line MBA-1

We investigated the action of tissue inhibitor of metalloproteinase-1 (TIMP-1) on apoptosis and differentiation of mouse bone marrow stromal cell line MBA-1. TIMP-1 did not affect alkaline phosphatase (ALP) activity, suggesting that it is not involved in osteoblastic differentiation in MBA-1 cells. However, TIMP-1 inhibited MBA-1 apoptosis induced by serum deprivation in a dose-dependent manner. Our study also showed increased Bcl-2 protein expression and decreased Bax protein expression with TIMP-1 treatment. TIMP-1 decreased cytochrome c release and caspase-3 activation in MBA-1 cells. TIMP-1 activated phosphatidylinositol 3-kinase (PI3-kinase) and c-Jun N-terminal kinase (JNK), and the PI3-kinase inhibitor LY294002 or the JNK inhibitor SP600125 abolished its antiapoptotic activity. To investigate whether antiapoptotic action of TIMP-1 was mediated through its inhibition on MMP activities, we constructed mutant TIMP-1 by side-directed mutagenesis, which abolished the inhibitory activity of MMPs by deletion of Cys1 to Ala4. Wild-type TIMP-1 and mutant TIMP-1 expression plasmids were transfected in MBA-1 cells, and results showed that mutant TIMP-1 still protected the induced MBA-1 cell against apoptosis. These data suggest that TIMP-1 antiapoptotic actions are mediated via the PI3-kinase and JNK signaling pathways and independent of TIMP-1 inhibition of MMP activities.

Cellular and molecular responses in progressive pseudorheumatoid dysplasia articular cartilage associated with compound heterozygous WISP3 gene mutation.

Progressive pseudorheumatoid dysplasia (PPD) is characterized by continuous degeneration and loss of articular cartilage, which has been attributed to mutations in the gene encoding WISP3. We collected a PPD family and analyzed their WISP3 genes mutation. Articular chondrocytes (ACs) were purified from the femurs of a PPD patient after hip replacement surgery. Cell growth, proliferation, and viability were examined. Gene expression profiling and analyses of matrix metalloproteinases (MMP)-1, -3, and -13 proteins were carried out using cDNA differential microarrays, real-time reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and Western blot analysis. We found that two probands carried a deletion (840delT) mutation in maternal allele, which leads to truncated WISP3 protein missing 43 residues in C terminus; and a 1000T>C substitution in paternal allele, which was also passed on to four other members in the PPD kindred. PPD ACs were heterogeneous in size with an enhanced rate of cell proliferation and viability compared with the normal ACs. MMP-1, -3, and -13 mRNA expressions were dereased in PPD ACs. MMP-1, -3, and -13 protein levels, however, were increased in cell lysates from PPD ACs, but markedly decreased in the supernatants from cultured ACs. WISP3 mRNA expression in PPD ACs was also decreased. Our results show, for the first time, a compound heterozygous mutation of WISP3 and a series of cellular and molecular changes disturbing the endochondral ossification in this PPD patient.

Age-related bone mineral density, osteoporosis rate and risk of vertebral fracture in mainland Chinese women with type 2 diabetes mellitus

Few data are available regarding bone mineral density (BMD) and the risk of vertebral fracture among mainland Chinese women with type 2 diabetes mellitus (T2DM). A decrease in the bone projective area (BPA) can be an indirect marker reflecting compressed vertebral fracture. We investigated age-related BMD, BPA, and the prevalence of osteoporosis in women with T2DM in mainland China. BMD and BPA of the posteroanterior lumbar spine (L1–L4) and hip were measured by dual-energy X-ray absorptiometry in 1253 women with T2DM and 1194 control subjects without diabetes aged 40–80 yr. BMD of the lumbar spine and hip decreased with age. BMD of the lumbar spine was higher in T2DM than controls (p<0.05–0.001), as was BPA at some vertebral bodies (p<0.05–0.001), whereas no significant intergroup differences in BPA were observed at the hip. The prevalence of osteoporosis in the women with T2DM increased with age: 0–2.58% at age 40–49 yr, 6.94–28.4% at age 50–59 yr, 32.7–76.7% at age 70–80 yr, with the range reflecting differences between skeletal sites. In subjects over 60 yr, the rates of osteoporosis at posteroanterior spine were significantly lower in T2DM patients than in controls (p<0.05–0.001). In conclusion, women with T2DM had higher BMD and lower risk of osteoporosis. Higher BPA of the vertebrae indicated that women with T2DM in mainland China would have a lower risk of vertebral fracture than non-diabetic women.

Taurine transporter is expressed in vascular smooth muscle cells

Summary.The regulation of vascular smooth muscle cells (VSMCs) function by taurine has been a subject of increasing interest and investigation, and taurine is taken up into cells through a specific transporter system, the taurine transporter (TAUT). In the present study, we examined the expression of TAUT in VSMCs and the kinetic parameters of the uptake process of TAUT in VSMCs. RT-PCR and western blot demonstrated that the mRNA and protein of TAUT was expressed in VSMCs in vitro. Immunohistochemistry using antibody for TAUT revealed the expression of this protein in rat thoracic aorta. The maximal [3H]taurine uptake rate in VSMCs was 37.75 ± 3.13 pmol/min per mg of protein, with a Km value of 5.42 ± 0.81 µM. Thus, VSMCs are able to express a functional taurine transporter. The regulation and detailed function of taurine and TAUT in VSMCs remain unclear, but our findings suggest a functional role for them in VSMCs metabolism.