成骨细胞特异性转录因子Osterix对骨形成作用的研 究进展

骨形成是一个涉及从间充质干细胞向成骨细胞分化的复杂发育过程,研究骨形成过程中的关键因子并阐明其作用的具体分子机制对骨代谢性疾病的治疗具有重要意义。Osterix(Osx)是迄今为止发现的唯一一个成骨细胞特异性转录因子,Osx只在骨组织细胞中表达,在干细胞向成骨细胞分化过程中起决定性作用,没有Oxs就没有骨形成和骨再生。Osx的发现为整个骨形成领域的研究开启了新的窗口。基于Osx在骨形成过程中的重要地位,研究者们迫切希望开发出作用于Osx的合成代谢分子,这对骨代谢性疾病的治疗将起到革命性的进步,因此对Osx的上下游因子及信号通路之间具体作用机制的进一步研究和阐明显得尤其重要。笔者对其研究进展做一综述。     

Regulation of osteoblast proliferation by leukemia inhibitory factor.

We recently showed that leukemia inhibitory factor (LIF) stimulates 45Ca release from neonatal mouse calvariae in vitro and that it increases DNA and protein synthesis in this model. To elucidate further the actions of LIF on bone we now report the effects of this cytokine on DNA synthesis and cell proliferation in isolated fetal rat osteoblasts and in the osteogenic sarcoma cell line, UMR-106. In both actively growing and growth-arrested rat osteoblasts, LIF stimulated [3H]thymidine incorporation in a dose-dependent manner. The increase in DNA synthesis was time dependent, was associated with an increase in the number of osteoblasts, and was not blocked by indomethacin. LIF-treated cells showed reduced [3H]thymidine incorporation in comparison with control, as they approached confluence, possibly because of the increased cell density in the LIF-treated cultures. In UMR-106 cells, treatment with LIF inhibited [3H]thymidine incorporation in both actively growing and growth-arrested cultures. The effect was dose dependent and sustained with time. There was a corresponding decrease in cell numbers. It is concluded that although LIF causes an early stimulation of proliferation in isolated osteoblasts, it has opposing effects on UMR-106 cells. It is not possible to determine which of these effects is more relevant to the actions of LIF in vivo. The demonstration of a LIF effect on both these cell types, however, provides further evidence that this cytokine acts directly on osteoblasts.     

Diclofenac inhibits proliferation and matrix formation of osteoblast cells

Cell culture studies have shown that NSAID may influence osteogenic activities of osteoblast cultures. However, these studies did not consider long-term effects on differentiating cells. The influence of Voltaren with the non-steroidal agent diclofenac on proliferation and gene expression of the osteoblast-like cell line SaOS-2 was investigated 2, 9, and 16 days after incubation. Two days after 24 h of incubation, 50 microg/ml diclofenac reduced the proliferation and collagen type I expression while 9 and 16 days later no effect was found on either of the parameters. In contrast, 50 microg/ml NSAID has no effect on alkaline phosphatase expression 2 days after incubation while 9 and 16 days later expression had been reduced. Lower concentrations (1.56 and 0.19 microg/ml) had no effects on the studied parameters. BrdU and MTT test showed that 50 microg/ml diclofenac reduced proliferative and metabolic activity. Lower concentrations (< or =25 microg/ml) had a lower or no influence. The findings indicate that the NSAID impairment depends on cellular differentiation stage and is not confined to the time during or immediately after NSAID incubation. According to these results in vitro testing of drugs should be performed over a longer time period to detect possible long-term impacts.     

成骨细胞特异性转录因子Osterix的研究进展

Osterix是目前所发现的调节成骨细胞分化的重要转录因子之一,在各种研究中其表达水平被作为成骨细胞分化程度的标志,其发现为人们进一步阐明骨代谢过程,特别是骨质疏松及其他骨病的机制提供了帮助,笔者将对近5年来关于此基因的研究作一综述.     

Age-related inhibitory activity of rat bone marrow supernatant on osteoblast proliferation.

Because histomorphometric indices of bone formation (osteoblastic index, tetracyclin-labeled perimeter) are deeply depressed in aged rats, while in vitro proliferation of trabecular bone cells was found increased, we hypothesized that a signal to proliferate, correctly induced by increased strains on scarce bone, could be opposed in vivo by an inhibitor present in the bone marrow extracellular medium. Thus, we tested the effect of bone marrow extracellular fluid (BM supernatant) of rat femoral diaphysis on cultures of primary osteoblasts and osteoblastic cell lines and found that it inhibited bone cell proliferation. In a group of 69 female rats aged 4, 12, and 15/21 months, there was a stepwise increase in the inhibitory activity of the BM supernatant. The double reciprocal plots relating inhibition power of the medium to BM supernatant dilution suggest that we deal with a simple system and that the kinetics of the phenomenon are the same in older and younger animals. Moreover, proliferation inhibition by BM supernatant and trabecular bone surface measured by histomorphometry in the distal femoral metaphysis were inversely correlated. Because the extracellular fluid of bone marrow is also the medium surrounding the osteoblasts and their precursor cells, our results suggest that the bone marrow negatively regulates osteogenic cells and that this inhibition could contribute to the inability of older animals to supply osteoblasts to bone in proportion to the demand. Preliminary biochemical characterization of the inhibitor suggests it to be a protein of 30-40 kDa with an isoelectric point (pI) of about 6.5.     

The Sox2 high mobility group transcription factor inhibits mature osteoblast function in transgenic mice

We have previously shown that in osteoblasts Sox2 expression can be induced by Fgfs, and can inhibit Wnt signaling and differentiation. Furthermore, in mice in which Sox2 is conditionally deleted in the osteoblastic lineage, bones are osteopenic, and Sox2 inactivation in cultured osteoblasts leads to a loss of proliferative ability with a senescent phenotype. To help understand the role of Sox2 in osteoblast development we have specifically expressed Sox2 in bone from a Col1α1 promoter, which extended Sox2 expression into more mature osteoblasts. In long bones, trabecular cartilage remodeling was delayed and the transition from endochondral to cortical bone was disrupted, resulting in porous and undermineralized cortical bone. Collagen deposition was disorganized, and patterns of osteoclast activity were altered. Calvarial bones were thinner and parietal bones failed to develop the diploic space. Microarray analysis showed significant up- or downregulation of a variety of genes coding for non-collagenous extracellular matrix proteins, with a number of genes typical of mature osteoblasts being downregulated. Our results position Sox2 as a negative regulator of osteoblast maturation in vivo.     

健骨方对破骨细胞形成和成骨细胞增殖分化的影响

目的　探讨健骨方水提物对破骨细胞分化及成骨细胞增殖分化的影响。方法　制备健骨方水提物,通过MTT法测定药物对骨髓单核巨噬细胞（BMMs）细胞的毒性,采用核因子κB受体活化因子配体（RANKL）诱导BMMs分化形成破骨细胞,加入不同浓度药物进行干预,采用抗酒石酸酸性磷酸酶( TRACP) 染色法测定破骨细胞分化抑制作用,采用Western Blot 法测定RANKL诱导的NF-κB破骨细胞分化信号通路,运用RT-qPCR法测定信号通路下游破骨细胞分化关键基因NFATc1、C-FOS等的mRNA表达水平。以MC3T3-E1细胞作为前体成骨细胞,加入不同浓度药物进行干预,通过CCK8法测定细胞增殖能力、PNPP法检测碱性磷酸酶（ALP）活性、茜素红S染色法测定细胞矿化能力。结果　MTT法结果显示,健骨方细胞有毒性浓度大于500 μg/mL（P＜0.05）,破骨细胞分化抑制IC50为1.25 μg/mL。机制研究显示健骨方显著下调了RANKL-NF-κB信号通路中的p-P65、P53的蛋白表达（P＜0.05）,显著抑制了通路下游C-FOS、NFATc1等的mRNA表达水平（P＜0.01,P＜0.05）。此外,成骨细胞活性检测显示,健骨方能明显促进MC3T3-E1细胞增殖、提高ALP活性及增加成骨细胞钙化的能力。结论 健骨方具有抑制破骨细胞分化和促进成骨前体细胞增殖、分化、矿化的药效作用。其作用与抑制破骨细胞分化RANKL-NF-κB信号通路及其下游C-FOS、NFATc1等基因,上调成骨细胞分化促进因子CAL1A2、SPARC和FOSL1基因的表达有关。     

Parathyroid hormone mediates bone growth through the regulation of osteoblast proliferation and differentiation

PTH (1-34) is the only FDA-approved anabolic agent for osteoporosis treatment in the U.S., but its mechanisms are not completely understood. This study investigated PTH effects on osteogenic cells at various stages of differentiation and proliferation using an engineered bone growth model in vivo. Ossicles were generated from bone marrow stromal cells (BMSCs) implanted in immunocompromised mice. Three weeks of PTH (40 microg/kg/day) or vehicle treatment initiated 1 day, 1, 2, or 3 weeks after BMSC implantation resulted in an anabolic response in PTH-treated implants (via histomorphometry and muCT) in all treatment groups. A novel in vivo tracking strategy with luciferase tagged BMSCs and weekly bioluminescent imaging of ossicles revealed increased donor cell proliferation in PTH-treated ossicles. The greatest increase occurred during the first week, and the activity remained elevated in PTH-treated implants over time. Zoledronic acid (ZA) was combined with PTH to delineate interactive mechanisms of these bone active agents. Combining ZA with PTH treatment reduced the PTH-mediated increase in luciferase BMSC activity, serum osteocalcin, and serum tartrate resistant acid phosphotase-5b (TRAP-5b) but ZA did not reduce the PTH-induced increase in total bone. Since zoledronic acid reduced PTH-induced proliferation without reducing bone volume, these data suggest that combining PTH and bisphosphonate therapy warrants further investigation in the treatment of bone disorders.     

Role of platelet-derived growth factor in osteoblast function and bone synthesis

In vitro studies have shown that platelet-derived growth factor binds to specific receptors on osteoblasts and induces proliferation. Some reports also indicate that platelet-derived growth factor may inhibit differentiation of bone cells. Such findings are difficult to interpret, however, because many of these studies used impure platelet-derived growth factor extracted from platelets (rather than pure, recombinant platelet-derived growth factor), used mixed cell populations, and added serum to the culture media (likely introducing additional growth factors). In vivo studies also have yielded conflicting results regarding the effects of platelet-derived growth factor on bone formation. Platelet-derived growth factor may act in concert with insulin-like growth factor I to induce bone formation in a canine periodontal system. Other investigators, however, have shown that platelet-derived growth factor inhibits the effects of osteoinductive proteins such as osteogenin. No evidence of bone formation has been detected in patients receiving platelet-derived growth factor treatment of soft tissue wounds overlying bony prominences in clinical trials. Thus, despite the observations that osteoblasts can specifically bind and proliferate in response to platelet-derived growth factor in tissue culture, platelet-derived growth factor alone has not yet been proved to be osteoinductive in vivo. These observations suggest that in the presence of the many cytokines and multiple cell types found in wounds and fractures, the direct effects of platelet-derived growth factor on osteoblasts are eclipsed.     

Effects of geranylgeranoic acid in bone: induction of osteoblast differentiation and inhibition of osteoclast formation.

Retinoids are known to be of special importance for normal bone growth and development. Recently, we reported that retinoids not only induced osteoblast differentiation, but also inhibited osteoclast formation in vitro. In this study, we examined the osteogenic effects of geranylgeranoic acid (GGA), a chemically synthesized acyclic retinoid, in bone in vitro and in vivo. GGA not only suppressed proliferation of osteoblastic MC3T3-E1 cells, but also up-regulated differentiation markers of osteoblasts such as alkaline phosphatase (ALP) activity and expression of osteopontin (OP) messenger RNA (mRNA). In contrast, GGA inhibited osteoclast formation induced by 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] in cocultures of mouse bone marrow cells and primary osteoblasts. Treatment of stromal ST2 cells with GGA restored the 1alpha,25(OH)2D3- or prostaglandin E2 (PGE2)-induced suppression of osteoprotegerin (OPG) mRNA expression. GGA inhibited osteoclast formation induced by macrophage colony-stimulating factor (M-CSF) and soluble receptor activator of nuclear factor kappaB ligand (sRANKL) in the culture of bone marrow macrophages. Thus, it is likely that GGA inhibits osteoclast formation by affecting both osteoblasts and osteoclast progenitors in the coculture system. Furthermore, in vivo, GGA increased bone mineral density (BMD) of total as well as distal femur in a P6 strain of senescence-accelerated mice (SAMP6). These results indicate that GGA increases bone mass by maintaining a positive balance of bone turnover by inducing osteoblast differentiation and suppressing osteoclast formation.     

Regulation of postnatal trabecular bone formation by the osteoblast endothelin A receptor

Endothelin‐1 (ET‐1) is a potent vasoconstrictor that also stimulates cells in the osteoblast lineage by binding to the endothelin A receptor (ETAR). ET‐1 ligand is widely secreted, particularly by the vasculature. However, the contributions of ETAR signaling to adult bone homeostasis have not been defined. ETAR was inactivated in osteoblasts by crossing ETAR‐floxed and osteocalcin‐Cre mice. Histomorphometric analyses were performed on 4‐, 8‐, and 12‐week‐old osteoblast‐targeted ETAR knockout (KO) and wild‐type (WT) male and female mice. Tibial trabecular bone volume was significantly lower from 12 weeks in KO versus WT mice in both males and females. Bone‐formation rate, osteoblast density, and in vitro osteoblast differentiation were reduced by targeted inactivation of ETAR. A separate longitudinal analysis was performed between 8 and 64 weeks to examine the effect of aging and castration on bone metabolism in ETAR KO mice. Hypogonadism did not change the rate of bone accrual in WT or KO females. However, eugonadal KO males had a significantly larger increase in tibial and femoral bone acquisition than WT mice. Male mice castrated at 8 weeks of age showed the reverse: KO mice had reduced rates of tibial and femoral BMD acquisition compared with WT mice. In vitro, ET‐1 increased osteoblast proliferation, survival, and differentiation. Dihydrotestosterone also increased osteoblast differentiation using a mechanism distinct from the actions of ET‐1. These results demonstrate that endothelin signaling in osteoblasts is an important regulator of postnatal trabecular bone remodeling and a modulator of androgen effects on bone. © 2011 American Society for Bone and Mineral Research     

Decreased in vitro osteoblast proliferation and low turnover bone disease in nonuremic proteinuric patients

Patients with proteinuria, even those with normal glomerular filtration rate, often present abnormal bone histology. We evaluated bone histology and the in vitro proliferation of osteoblasts in samples obtained from 17 proteinuric patients with primary glomerulopathies. Histomorphometric analysis of bone biopsies was performed, and bone fragments were obtained for osteoblast culture, in which we evaluated cell proliferation. In comparison to controls, patients presented lower trabecular bone volume (20.9+/-14.5% vs 26.8+/-5.9%; P=0.0008); lower trabecular number (1.7+/-0.2/mm vs 2.0+/-0.3/mm; P=0.004); and greater trabecular separation (475.5+/-96.4 microm vs 368.3+/-86.2 microm, P=0.0002). We also found alterations in bone formation and resorption: lower osteoid volume (0.9+/-0.7% vs 2.0+/-1.4%; P=0.0022); lower osteoid thickness (6.4+/-2.8 microm vs 11.5+/-3.2 microm; P<0.0001); less mineralizing surface (4.6+/-3.1% vs 13.5+/-6.0%; P<0.0001); lower bone formation rate (0.03+/-0.04 microm(3)/microm(2)/day vs 0.09+/-0.05 microm(3)/microm(2)/day; P<0.0001); and greater osteoclast surface (0.35+/-0.6 vs 0.05+/-0.1%, P=0.0016). Mean in vitro osteoblast proliferation was lower in patients than in controls (910.2+/-437.1 vs 2261.0+/-1121.0 d.p.m./well, P=0.0016). Serum concentrations of 25-hydroxyvitamin-D(3) correlated negatively with proteinuria and positively with in vitro osteoblast proliferation. Our results demonstrate that nonuremic proteinuric glomerulonephritic patients present bone structure disorder, low bone formation and high bone resorption, as well as low osteoblast proliferation.