SATB2促进BMP9诱导C2C12细胞的成骨分化

目的:研究特异AT序列结合蛋白2(Special AT-rich sequence-binding protein 2,SATB2)在骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9)诱导的间充质干细胞(mesenchymal stem cells,MSCs)C2C12成骨分化中的作用。方法:首先用Ad-BMP9感染C2C12细胞,RT-PCR检测SATB2在基因水平上的表达变化,Western blot检测SATB2在蛋白水平上的变化;构建过表达SATB2重组腺病毒Ad-SATB2,感染C2C12细胞后,Western blot验证Ad-SATB2表达情况;用AdSATB2处理BMP9诱导的C2C12细胞,碱性磷酸酶(ALP)活性和染色检测成骨早期ALP的变化,茜素红S染色检测成骨晚期指标钙盐沉积的变化。结果:Ad-BMP9处理C2C12细胞后,比对照组SATB2在基因水平和蛋白水平上表达量上调;Ad-SATB2可以在细胞中成功表达SATB2蛋白;用Ad-SATB2处理BMP9诱导的C2C12细胞后,ALP以及钙盐的沉积与对照比较均上调。结论:SATB2可以促进BMP9诱导的间充质干细胞C2C12的成骨分化。     

mTORC1 Signaling Promotes Osteoblast Differentiation from Preosteoblasts

Preosteoblasts are precursor cells that are committed to the osteoblast lineage. Differentiation of these cells to mature osteoblasts is regulated by the extracellular factors and environmental cues. Recent studies have implicated mTOR signaling in the regulation of osteoblast differentiation. However, mTOR exists in two distinct protein complexes (mTORC1 and mTORC2), and the specific role of mTORC1 in regulating the progression of preosteoblasts to mature osteoblastis still unclear. In this study, we first deleted Raptor, a unique and essential component of mTORC1, in primary calvarial cells. Deletion of Raptor resulted in loss of mTORC1 but an increase in mTORC2 signaling without overtly affecting autophagy. Under the osteogenic culture condition, Raptor-deficient cells exhibited a decrease in matrix synthesis and mineralization. qPCR analyses revealed that deletion of Raptor reduced the expression of late-stage markers for osteoblast differentiation (Bglap, Ibsp, and Col1a), while slightly increasing early osteoblast markers (Runx2, Sp7, and Alpl). Consistent with the findings in vitro, genetic ablation of Raptor in osterix-expressing cells led to osteopenia in mice. Together, our findings have identified a specific role for mTORC1 in the transition from preosteoblasts to mature osteoblasts.     

Promotion of Bone Morphogenetic Protein Signaling by Tetraspanins and Glycosphingolipids

Bone morphogenetic proteins (BMPs) belong to the transforming growth factor β (TGFβ) superfamily of secreted molecules. BMPs play essential roles in multiple developmental and homeostatic processes in metazoans. Malfunction of the BMP pathway can cause a variety of diseases in humans, including cancer, skeletal disorders and cardiovascular diseases. Identification of factors that ensure proper spatiotemporal control of BMP signaling is critical for understanding how this pathway is regulated. We have used a unique and sensitive genetic screen to identify the plasma membrane-localized tetraspanin TSP-21 as a key new factor in the C. elegans BMP-like “Sma/Mab” signaling pathway that controls body size and postembryonic M lineage development. We showed that TSP-21 acts in the signal-receiving cells and genetically functions at the ligand-receptor level. We further showed that TSP-21 can associate with itself and with two additional tetraspanins, TSP-12 and TSP-14, which also promote Sma/Mab signaling. TSP-12 and TSP-14 can also associate with SMA-6, the type I receptor of the Sma/Mab pathway. Finally, we found that glycosphingolipids, major components of the tetraspanin-enriched microdomains, are required for Sma/Mab signaling. Our findings suggest that the tetraspanin-enriched membrane microdomains are important for proper BMP signaling. As tetraspanins have emerged as diagnostic and prognostic markers for tumor progression, and TSP-21, TSP-12 and TSP-14 are all conserved in humans, we speculate that abnormal BMP signaling due to altered expression or function of certain tetraspanins may be a contributing factor to cancer development.     

TurboFect介导骨形态发生蛋白2 转染CHO 细胞的研究

目的:构建真核表达载体p IRES-EGFP-BMP-2,通过Turbo Fect转染得到表达BMP-2蛋白的CHO细胞系。方法:利用逆转录PCR方法扩增获得人的BMP-2基因c DNA,克隆入p MD18-T载体,经PCR、酶切和基因测序分析等方法鉴定重组质粒;将BMP-2连入p IRES-EGFP真核表达载体中,经限制性酶切和PCR扩增鉴定重组质粒。以壳聚糖和Turbo Fect分别作为基因载体转染CHO细胞,荧光显微镜检测分析转染结果;G418筛选富集转染阳性细胞。结果:成功的克隆得到了BMP-2基因,酶切鉴定成功构建了p IRES-EGFP-BMP-2质粒。与壳聚糖组相比,Turbo Fect用量为1:1时,细胞阳性率为(31.92±1.31)%,高于壳聚糖的细胞阳性率(6.33±1.53)%。目的基因与Turbo Fect比例为1:2时转染效率为(42.90±1.10)%高于1:1的(28.59±2.38)%和1:3的(37.52±2.14)%。细胞密度调节到5×103 cells/cm2阳性细胞率可达到(44.43±3.23)%。荧光检测可见荧光阳性细胞得到稳定传代。Western Blot检测可见BMP-2蛋白表达。结论:Turbo Fect成功的介导了p IRES-EGFP-BMP-2载体转染CHO细胞,建立了稳定表达BMP-2和EGFP的CHO细胞株。     

Distinct Modes of Inhibition by Sclerostin on Bone Morphogenetic Protein and Wnt Signaling Pathways

Sclerostin is expressed by osteocytes and has catabolic effects on bone. It has been shown to antagonize bone morphogenetic protein (BMP) and/or Wnt activity, although at present the underlying mechanisms are unclear. Consistent with previous findings, Sclerostin opposed direct Wnt3a-induced but not direct BMP7-induced responses when both ligand and antagonist were provided exogenously to cells. However, we found that when both proteins are expressed in the same cell, sclerostin can antagonize BMP signaling directly by inhibiting BMP7 secretion. Sclerostin interacts with both the BMP7 mature domain and pro-domain, leading to intracellular retention and proteasomal degradation of BMP7. Analysis of sclerostin knock-out mice revealed an inhibitory action of sclerostin on Wnt signaling in both osteoblasts and osteocytes in cortical and cancellous bones. BMP7 signaling was predominantly inhibited by sclerostin in osteocytes of the calcaneus and the cortical bone of the tibia. Our results suggest that sclerostin exerts its potent bone catabolic effects by antagonizing Wnt signaling in a paracrine and autocrine manner and antagonizing BMP signaling selectively in the osteocytes that synthesize simultaneously both sclerostin and BMP7 proteins.     

Low Density Lipoprotein Receptor-related Protein 1 Promotes Anti-apoptotic Signaling in Neurons by Activating Akt Survival Pathway

The low density lipoprotein receptor-related protein 1 (LRP1) is a multi-ligand receptor abundantly expressed in neurons. Previous work has shown that brain LRP1 levels are decreased during aging and in Alzheimer disease. Although mounting evidence has demonstrated a role for LRP1 in the metabolism of apolipoprotein E/lipoprotein and amyloid-β peptide, whether LRP1 also plays a direct role in neuronal survival is not clear. Here, we show that LRP1 expression is critical for the survival of primary neurons under stress conditions including trophic withdrawal, the presence of apoptosis inducers, or amyloid-β-induced neurotoxicity. Using lentiviral short hairpin RNA to knock down endogenous LRP1 expression, we showed that a depletion of LRP1 leads to an activation of caspase-3 and increased neuronal apoptosis, an effect that was rescued by a caspase-3 inhibitor. A correlation between decreased Akt phosphorylation and the activation of caspase-3 was demonstrated in LRP1 knocked down neurons. Notably, LRP1 knockdown decreased insulin receptor levels in primary neurons, suggesting that decreased neuronal survival might be a consequence of an impaired insulin receptor signaling pathway. Correspondingly, both insulin receptor and phospho-Akt levels were decreased in LRP1 forebrain knock-out mice. These results demonstrate that LRP1 mediates anti-apoptotic function in neurons by regulating insulin receptor and the Akt survival pathway and suggest that restoring LRP1 expression in Alzheimer disease brain might be beneficial to inhibiting neurodegeneration.     

Bone Morphogenetic Protein-2 Promotes Survival and Differentiation of Striatal GABAergic Neurons in the Absence of Glial Cell Proliferation

We examined the potential neurotrophic effects of bone morphogenetic protein (BMP)-2 on the survival and differentiation of neurons cultured from the rat developing striatum at embryonic day 16, a period during which the mRNAs for BMP-2 and its receptor subunits (types IA, IB, and II) were detected. BMP-2 exerted potent activity to promote the survival of striatal neurons and increased the number of surviving microtubule-associated protein-2-positive cells by 2.4-fold as compared with the control cultures after 4 days in vitro. Although basic fibroblast growth factor (bFGF) also showed relatively high activity to promote the survival of striatal neurons, transforming growth factor-beta1, -beta2, and -beta3, glial cell line-derived neurotrophic factor, or brain-derived neurotrophic factor promoted their survival weakly. Striatal neurons cultured in the presence of BMP-2 or bFGF possessed extensive neurite outgrowths, the majority of which were GABA-immunoreactive. Inhibition of glial cell proliferation by 5-fluorodeoxyuridine did not affect the capacity of BMP-2 to promote the survival of striatal GABAergic neurons. In contrast, the ability of bFGF to promote the survival of striatal neurons was inhibited significantly by the treatment of cells with 5-fluorodeoxyuridine. All these results suggest that BMP-2 exerts potent neurotrophic effects on the striatal GABAergic neurons in a glial cell-independent manner.     

Notch信号参与BMP4诱导的间充质干细胞成骨分化及其机制的初步探讨

目的:探讨Notch信号对骨形态发生蛋白4(bone morphogenetic protein 4,BMP4)诱导间充质干细胞成骨分化的影响以及作用机制。方法:(1)DAPT或Ad-dominant-negative mutants of Notch1(Addn Notch1)和BMP4-CM处理小鼠胚胎成纤维细胞,检测早期成骨指标碱性磷酸酶(alkaline phosphatase,ALP);(2)茜素红S染色实验检测晚期成骨钙盐沉积情况;(3)半定量反转录聚合酶链反应(RT-PCR)检测成骨分化相关基因ALP,Runx2,Col1a1的表达;(4)免疫细胞化学检测p-Smad1/5/8的表达;(5)结晶紫染色和流式细胞术检测细胞的增殖及周期改变。结果:(1)DAPT抑制BMP4诱导的早期成骨分化,且呈浓度依赖性;(2)Delta-like 1(DLL1)促进BMP4诱导的成骨分化,DAPT和dn Notch1抑制BMP4诱导的成骨分化;(3)DLL1促进BMP4诱导的成骨相关基因ALP,Runx2,Col1a1的表达,DAPT抑制这些基因的表达;(4)DLL1促进BMP4诱导的细胞核内p-Smad1/5/8的表达,而DAPT抑制其表达;(5)DLL1促进BMP4诱导的细胞增殖,而DAPT抑制BMP4诱导的细胞增殖。结论:Notch信号通过BMP/Smads信号通路促进BMP4诱导的MSCs成骨分化,在此过程中也有促细胞增殖的作用。     

骨形态发生蛋白2通过Smad途径上调Osterix的表达

Osterix（Osx）是一种重要的调控成骨细胞分化的具有锌指结构的转录因子．骨形态发生蛋白2（bone morphogenetic protein 2, BMP2）能够上调Osx的表达,但其分子机制并不清楚．采用实时定量RT-PCR方法检测到BMP2诱导成骨相关细胞C3H10T1/2, MC3T3-E1, C2C12中Osx的转录水平显著上调,并且与成骨分化指标Col1a1, osteocalcin具有相似的表达动力学特征．而且,在C3H10T1/2细胞中过表达负显性（dominant negative, DN）Osx基因,能够有效抑制BMP2诱导的成骨分化．过表达BMP/Smad信号通路抑制蛋白Smad6,能够抑制Osx转录水平的上调．但是通过荧光素酶报告载体对Osx的启动子-1254～+85区域进行分析后未发现接受BMP通路调控的启动子区域．上述结果表明,BMP2能够通过Smad途径上调Osx的表达,并对成骨分化的过程具有十分重要的作用．