1α,25-dihydroxyvitamin D3 upregulates tumour necrosis factor superfamily member 11 (TNFSF11) that codes for the receptor activator of nuclear factor κB ligand (RANKL), and downregulates osteoprotegerin (OPG) expression. We have analyzed the individual effects of polymorphisms in the vitamin D receptor gene (VDR), OPG and TNFSF11, and searched for interactions between them. Six hundred and forty one subjects were evaluated: 239 osteoporotic and 228 non-osteoporotic post-menopausal, 57 pre-menopausal women and 117 elderly men. The subjects were genotyped for BsmI, FokI and Cdx2 in VDR, K3N in OPG and −290C>T, −643C>T and −693G>C in TNFSF11 gene. Bone mineral density (BMD) and biochemical markers were measured. In the osteoporotic women, femoral neck BMD (BMD-fn) showed associations with BsmI(VDR) and Cdx2(VDR) (P=0.015 and 0.047 respectively), and lumbar spine BMD (BMD-ls) with K3N(OPG) and −290C>T(TNFSF11) (P=0.021 and 0.017). No association with BMD was found in the non-osteoporotic women. In the pre-menopausal women, the Cdx2(VDR) polymorphism was associated with BMD-fn and total hip BMD (P=0.011 and 0.011). In elderly men, FokI(VDR) was associated with BMD-fn and BMD-ls (P=0.040 and 0.036). Interestingly, the −290C>T(TNFSF11)-K3N(OPG) combination was associated with BMD-th (P=0.041) in the osteoporotic women. In the non-osteoporotic women, the combination K3N(OPG)-Cdx2(VDR) was associated with BMD-ls, BMD-th and BMD-fn (P=0.032, 0.049 and 0.022), and the combination −290C>T(TNFSF11)-K3N(OPG) with BMD-fn (P=0.029). For the first time, the presence of gene–gene interactions between VDR, OPG and TNFSF11 genes was studied. Our results strongly suggest further confirmation of their combined influence on larger cohorts.
Simona Mencej-Bedrač, Janez Preželj, Tomaž Kocjan, Karmen Teskač, Barbara Ostanek, Mojca Šmelcer and Janja Marc
KM Wiren, AR Toombs and XW Zhang
Non-aromatizable androgens have significant beneficial effects on skeletal homeostasis independently of conversion to estradiol, but the effects of androgens on bone cell metabolism and cell proliferation are still poorly understood. Using an osteoblastic model with enhanced androgen responsiveness, MC3T3-E1 cells stably transfected with androgen receptor (AR) under the control of the type I collagen promoter (colAR-MC3T3), the effects of androgens on mitogenic signaling were characterized. Cultures were treated with the non-aromatizable androgen 5alpha-dihydrotestosterone (DHT) and the effects on osteoblast viability were determined as measured by an MTT assay. A complex response was observed in that continuous short-term DHT treatment enhanced osteoblast viability, but with longer-term DHT treatment inhibition was observed. The inhibition by DHT was prevented by the specific AR antagonist hydroxyflutamide, and was also observed in primary cultures of normal rat calvarial osteoblasts. In order to identify potential mediators of this effect, mitogenic pathway-specific cDNA microarrays were interrogated. Reduced hybridization of several genes important in MAP kinase-mediated signaling was observed, with the most dramatic effect on Elk-1 expression. Analysis of phosphorylation cascades demonstrated that DHT treatment inhibited phosphoERK1/2 levels, MAP kinase activation of Elk-1, Elk-1 protein and phosphoElk-1 levels, and downstream AP-1/luciferase reporter activity. Together, these data provide the first evidence that androgen inhibition of the MAP kinase signaling pathway is a potential mediator of osteoblast growth, and are consistent with the hypothesis that the MAP cascade may be a specific downstream target of DHT.
Jacob Lund, Mads T Søndergaard, Cheryl A Conover and Michael T Overgaard
IGF1 and IGF2 are potent stimulators of diverse cellular activities such as differentiation and mitosis. Six IGF-binding proteins (IGFBP1–IGFBP6) are primary regulators of IGF half-life and receptor availability. Generally, the binding of IGFBPs inhibits IGF receptor activation. However, it has been shown that IGFBP2 in complex with IGF2 (IGF2/IGFBP2) stimulates osteoblast function in vitro and increases skeletal mass in vivo. IGF2 binding to IGFBP2 greatly increases the affinity for 2- or 3-carbon O-sulfated glycosaminoglycans (GAGs), e.g. heparin and heparan sulfate, which is hypothesized to preferentially and specifically target the IGF2/IGFBP2 complex to the bone matrix. In order to obtain a more detailed understanding of the interactions between the IGF2/IGFBP2 complex and GAGs, we investigated heparin-binding properties of IGFBP2 and the IGF2/IGFBP2 complex in a quantitative manner. For this study, we mutated key positively charged residues within the two heparin-binding domains (HBDs) in IGFBP2 and in one potential HBD in IGF2. Using heparin affinity chromatography, we demonstrate that the two IGFBP2 HBDs contribute differentially to GAG binding in free IGFBP2 and the IGF2/IGFBP2 protein complex. Moreover, we identify a significant contribution from the HBD in IGF2 to the increased IGF2/IGFBP2 heparin affinity. Using molecular modeling, we present a novel model for the IGF2/IGFBP2 interaction with heparin where all three proposed HBDs constitute a positively charged and surface-exposed area that would serve to promote the increased heparin affinity of the complex compared with free intact IGFBP2.
Michael Chau, Patricia Forcinito, Anenisia C Andrade, Anita Hegde, Sohyun Ahn, Julian C Lui, Jeffrey Baron and Ola Nilsson
In embryonic growth cartilage, Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP) participate in a negative feedback loop that regulates chondrocyte differentiation. Postnatally, this region undergoes major structural and functional changes. To explore the organization of the Ihh–PTHrP system in postnatal growth plate, we microdissected growth plates of 7-day-old rats into their constituent zones and assessed expression of genes participating in the Ihh–PTHrP feedback loop. Ihh, Patched 1, Smoothened, Gli1, Gli2, Gli3, and Pthr1 were expressed in regions analogous to the expression domains in embryonic growth cartilage. However, PTHrP was expressed in resting zone cartilage, a site that differs from the embryonic source, the periarticular cells. We then used mice in which lacZ has replaced coding sequences of Gli1 and thus serves as a marker for active hedgehog signaling. At 1, 4, 8, and 12 weeks of age, lacZ expression was detected in a pattern analogous to that of embryonic cartilage. The findings support the hypothesis that the embryonic Ihh–PTHrP feedback loop is maintained in the postnatal growth plate except that the source of PTHrP has shifted to a more proximal location in the resting zone.
Yingyu Feng, Lei Su, Xing Zhong, Wei Guohong, Haipeng Xiao, Yanbing Li and Lingling Xiu
Glucagon-like peptide-1 (GLP1) and its receptor agonist have been previously reported to play a positive role in bone metabolism in aged ovariectomized rats and insulin-resistant models. However, whether GLP1 has a direct effect on the proliferation and differentiation of osteoblasts or any cellular mechanism for this potential role is unknown. We examined the effects of the GLP1 receptor agonist exendin-4 on the proliferation, differentiation, and mineralization of mouse osteoblastic MC3T3-E1 cells. GLP1 receptor was detected in MC3T3-E1 cells by polymerase chain reaction (PCR) and Western blot assay. Cell proliferation was assessed using MTT assay, revealing that exendin-4 increased cell proliferation at effective concentrations between 10−10 and 10−5 M. Quantitative PCR analysis showed that exendin-4 increased the mRNA expression of the differentiation markers alkaline phosphatase (ALP), collagen-1 (COL1), osteocalcin (OC), and runt-related transcription factor 2 (RUNX2) under osteogenic conditions. Alizarin red staining confirmed that 10−7 M exendin-4 increased osteoblast mineralization by 18.7%. Exendin-4 upregulated the phosphorylation of ERK1/2, p38, and JNK, with the peak effect at 1.5 h in the Western blot analysis. The use of selective MAPK inhibitors, namely PD98059, SB203580, and SP600125, blocked the effects of exendin-4 on kinase activation (ERK1/2, p38, and JNK), as well as cell proliferation and differentiation in MC3T3-E1 cells. These findings demonstrate that exendin-4 promotes both the proliferation and differentiation of preosteoblasts MC3T3-E1 via activation of the MAPK pathway.
J Tucci, V Hammond, P V Senior, A Gibson and F Beck
During pregnancy, a placental calcium pump maintains the fetus in a hypercalcaemic state relative to the mother, a condition which has been thought to facilitate normal development of the fetal skeleton. Based on experiments performed in the sheep, parathyroid hormone-related protein (PTHrP) has been implicated as the hormone responsible for maintaining the placental calcium pump. In the present study on mice in which the PTHrP gene has been ablated by homologous recombination, we have measured both fetal and maternal circulating total and ionised calcium levels, as well as fetal total body calcium, in order to determine whether absence of PTHrP during fetal development has an effect on fetal calcium levels. Our results show that, in fetuses lacking PTHrP, circulating ionised calcium levels are significantly lower than those of heterozygote and wild-type littermates, but circulating total calcium levels show no difference. Total body calcium levels of null mutants are significantly higher than those of normal littermates.
The role of PTHrP in maintaining the integrity of the transplacental calcium pump in the rodent thus remains unclear. It may be that the lower levels of fetal blood ionised calcium in mutant animals are due to disruption of the placental pump, but, if this is the case, compensatory mechanisms have operated to allow the excessive calcium deposition seen in the skeletons of these animals. Alternatively, the increased avidity of the bones for calcium may in itself have produced a lower equilibrium level of available ionised calcium.
Hanwei Cui, Qian Yi, Jianguo Feng, Li Yang and Liling Tang
IGF1Ec in humans or IGF1Eb in rodents (known as mechano growth factor (MGF)) has a unique E domain, and the C-terminal end of the E domain (MGF E peptide) plays important roles in proliferation, migration and differentiation of many cell types. Bone marrow mesenchymal stem cells (BMSCs) have multiple differentiation potentials and are considered as perfect seed cells for tissue repair. But the role of MGF E peptide on BMSCs is seldom investigated and the mechanism is still unclear. In this study, we investigated the effects of MGF E peptide on rat BMSCs (rBMSCs). Our results revealed that treatment with MGF E peptide had no effect on BMSC proliferation. However, both wound-healing and transwell assays indicated that MGF E peptide could significantly enhance rBMSCs migration ability. Further analysis indicated that MGF E peptide also reduced the expression levels of osteogenic genes, but increased the expression levels of adipogenic genes. Analysis of molecular mechanism showed that phosphorylation-Erk1/2 was activated by MGF E peptide and blockage of either Erk1/2 or IGF1 receptor could repress the migration effect of MGF E peptide. In conclusion, MGF E peptide is able to inhibit osteogenic differentiation but promote adipogenic differentiation. In addition, the migration effect of MGF E peptide on rBMSCs depends on IGF1 receptor via Erk1/2 signal pathway.
M Nakamura, S Morimoto, Q Yang, T Hisamatsu, N Hanai, Y Nakamura, I Mori and K Kakudo
Receptor activity modifying proteins (RAMPs) act as receptor modulators that determine the ligand specificity of receptors for the calcitonin (CT) family. The purpose of this study was to analyze the expression of RAMPs in osteoclast-like cells using the laser capture microdissection (LCM) technique. Mouse bone marrow and spleen cells were co-cultured on a film designed for LCM. After 10 days, 250 osteoclast-like cells were captured using the LCM system. Total RNA from these cells was used to synthesize cDNA and RT-PCR analysis was performed. Osteoclast-like cells expressed CT receptor (CTR), CT receptor-like receptor (CRLR) and RAMP2, but did not express RAMP1 or RAMP3. These results indicated (1) that a pure population of osteoclast-like cells can be prepared by LCM and gene expression of this population can be analyzed by RT-PCR and (2) that RT-PCR shows that osteoclast-like cells express RAMP2, CTR and CRLR, suggesting the potential for adrenomedullin binding to osteoclast-like cells. This is the first report that osteoclast-like cells express RAMP2.
Lei Yang, Hongzheng Meng and Maowei Yang
Patients with type II diabetes are susceptible to fracture; however, these patients typically have normal bone mineral density. Thus, such fractures cannot be entirely explained by advanced glycation end products (AGEs)-induced osteoblast apoptosis. Autophagy is a molecular process allowing cells to degrade unnecessary or dysfunctional cellular organelles, and closely interacts with apoptosis. The aim of this study was to determine whether autophagy participated in the pathology of AGEs-treated osteoblasts, and the possible mechanism of such an involvement. Osteoblastic MC3T3-E1 cells were used. Autophagy was evaluated by detecting the level of LC3 via western blotting and immunofluorescence. p62/SQSTM1 expression was also assessed by western blotting. The autophagy inducer rapamycin (RA) and the autophagy inhibitor 3-methyladenine were used to determine whether autophagy has effect on AGEs-induced apoptosis. N-Acetylcysteine (NAC), reactive oxygen species (ROS) inhibitor, was used to determine whether ROS and mitochondrial damage were involved in autophagy regulation. The results showed that the autophagy level was increased in MC3T3-E1 cells treated with AGEs, as represented by an increase in both the total LC3 level and the LC3II/LC3I ratio, as well as a decrease in p62/SQSTMI expression. Further inducing autophagy by RA attenuated AGEs-induced apoptosis. The antioxidant NAC suppresses AGEs-induced autophagy in osteoblastic MC3T3-E1 cells. These results demonstrate that autophagy participates in the pathology of AGEs-treated osteoblasts, and may play a protective role in AGEs-induced apoptosis in osteoblastic MC3T3-E1 cells. ROS and mitochondrial damage are essential in upregulating AGEs-induced autophagy.
Simona Mencej, Omar M E Albagha, Janez Preželj, Tomaž Kocjan and Janja Marc
Tumour necrosis factor superfamily member 11 (TNFSF11) gene, that codes for receptor activator of nuclear factor-κB ligand, is one of the candidate genes for the genetic susceptibility to osteoporosis. As variations in the TNFSF11 gene promoter could alter its expression, the aim of the study was to evaluate the functional influence of three polymorphisms in the promoter and to investigate their association with bone mineral density (BMD) and biochemical markers in postmenopausal women. A total of 404 postmenopausal women were genotyped for the presence of TNFSF11 gene promoter polymorphisms −290C>T, −643C>T and −693G>C. Two common haplotypes, CCG and TTC, which occur in 44.3 and 49.3% of subjects respectively, were subjected to functional analysis. Amplified fragments were cloned into pGL3-basic reporter plasmid, which was co-transfected with pRL-TK plasmid into HEK293 cells. Dual luciferase reporter assay was performed. BMD and biochemical markers were measured. Reporter gene analysis showed significantly higher luciferase activity in CCG than in TTC haplotype (P=0.018). Both showed association with lumbar spine BMD (BMD-ls; P=0.005 and 0.007 for TTC and CCG respectively), whereas in femoral neck there was no association with BMD. In postmenopausal osteoporosis, association with BMD-ls was established in −290C>T, −643C>T and −693G>C (P values: 0.001, 0.041 and 0.013 respectively). Association with femoral neck BMD was shown in −693G>C (P=0.049). No association was found with biochemical markers in any of the groups. Our results suggest that in postmenopausal osteoporosis, TNFSF11 gene promoter polymorphisms −290C>T, −643C>T and −693G>C play a functional role in the genetic regulation of BMD.