BMP9 and BMP10: Two close vascular quiescence partners that stand out

Bone morphogenetic proteins (BMPs) are dimeric transforming growth factor ß (TGFß) family cytokines that were first described in bone and cartilage formation but have since been shown to be involved in many pleiotropic functions. In human, there are 15 BMP ligands, which initiate their cellular signaling by forming a complex with two copies of type I receptors and two copies of type II receptors, both of which are transmembrane receptors with an intracellular serine/threonine kinase domain. Within this receptor family, ALK1 (activin receptor-like kinase 1), which is a type I receptor mainly expressed on endothelial cells, and BMPRII (BMP Receptor type II), a type II receptor also highly expressed on endothelial cells, have been directly linked to two rare vascular diseases: hereditary hemorrhagic telangiectasia (HHT), and pulmonary arterial hypertension (PAH), respectively. BMP9 (gene name GDF2) and BMP10, two close members of the BMP family, are the only known ligands for the ALK1 receptor. This specificity gives them a unique role in physiological and pathological angiogenesis and tissue homeostasis. The aim of this current review is to present an overview of what is known about BMP9 and BMP10 on vascular regulation with a particular emphasis on recent results and the many questions that remain unanswered regarding the roles and specificities between BMP9 and BMP10.     

LPS激活TLR4抑制BMP9诱导iMEFs成骨分化

目的研究脂多糖(LPS)激活的Toll样受体4(TLR4)信号对骨形态发生蛋白9(BMP9)诱导永生化小鼠胚胎成纤维细胞(i MEFs)成骨分化的影响。方法细胞免疫荧光检测TLR4/NF-κB信号通路的激活;LPS,BAY11-7082和BMP9处理iMEFs,ALP染色和活性检测i MEFs早期成骨分化能力;茜素红S染色检测晚期成骨分化能力;半定量PCR和Western blot检测晚期成骨基因OCN和OPN表达;Western blot检测Smad1/5/8磷酸化水平;半定量PCR和Western blot检测成骨关键转录因子Runx2和Dlx5的表达。结果 LPS成功激活TLR4/NF-κB信号通路;LPS抑制BMP9诱导的ALP染色和活性(P0.01)、钙盐沉积、OCN的mRNA和蛋白质表达(P0.05)、OPN的mRNA(P0.01)和蛋白质(P0.05)表达、Smad1/5/8信号通路激活(P0.01)、Runx2的mRNA和蛋白质表达(P0.05)、Dlx5的mRNA(P0.01)和蛋白质(P0.05)表达,BAY11-7082可以部分逆转LPS的抑制作用(P0.05)。结论 LPS激活TLR4可以通过NF-κB信号通路抑制BMP9诱导的iMEFs成骨分化。     

骨形态发生蛋白9抑制人乳腺癌MDA-MB-231细胞体外侵袭和迁移

目的 探讨骨形态发生蛋白9(BMP9)对乳腺癌MDA-MB-231细胞侵袭、迁移能力的影响.方法 RT-PCR法检测MDA-MB-231细胞系中BMP9的表达;扩增高滴度的BMP9表达腺病毒,感染MDA-MB-231细胞,制备高表达BMP9的重组MDA-MB-231/BMP9细胞,以此作为实验组;同时以含GFP的窄载腺病毒感染该细胞为MDA-MB-231/GFP,联合空白MDA-MB-231共同作为对照组;通过平板集落形成实验、细胞划痕实验、Transwell侵袭实验检测重组MDA-MB-231/BMP9细胞侵袭及迁移能力.结果 与对照组细胞相比,实验组细胞中BMP9 mRNA表达水平明显增高;实验组细胞集落形成率由对照的90.6%±2.5%与85.6%±3.5%显著下降至57.3%±4.5%(P＜0.05);实验组划痕愈合率由对照的95.4%±3.1%与92.5%±2.4%下降至74.0%±3.6%(P＜0.05);实验组穿膜细胞数由对照的(255.3±16.1)个与(267.3±14.9)个下降至(150.3±14.6)个(P＜0.05).结论 BMP9可以在体外抑制乳腺癌MDA-MB-231细胞的侵袭与迁移.     

腺病毒介导的BMP9过表达抑制人骨肉瘤细胞系143B的增殖与迁移

目的 探讨骨形态发生蛋白9(BMP9)过表达对人骨肉瘤细胞系143B的生物学行为的影响.方法 用RTPCR和Western blot检测骨肉瘤细胞系中BMP9的内源性表达,用表达BMP9的腺病毒重组体(adBMP9)感染内源性表达BMP9相对较低的骨肉瘤细胞系,Transwell法检测细胞侵袭,划痕愈合实验检测细胞迁移,MTT法和结晶紫染色法检测细胞的增殖,Hoechst 33258染色法检测凋亡,平板集落形成实验检测集落形成能力.结果 adBMP9可以明显减弱骨肉瘤细胞系143B的增殖、迁移、侵袭及集落形成能力(P＜0.05),并促进细胞凋亡.结论 adBMP9能抑制人骨肉瘤细胞系143B的增殖和迁移,促进细胞凋亡.     

Identification of a mutation causing deficient BMP1/mTLD proteolytic activity in autosomal recessive osteogenesis imperfecta

Herein, we have studied a consanguineous Egyptian family with two children diagnosed with severe autosomal recessive osteogenesis imperfecta (AR-OI) and a large umbilical hernia. Homozygosity mapping in this family showed lack of linkage to any of the previously known AR-OI genes, but revealed a 10.27 MB homozygous region on chromosome 8p in the two affected sibs, which comprised the procollagen I C-terminal propeptide (PICP) endopeptidase gene BMP1. Mutation analysis identified both patients with a Phe249Leu homozygous missense change within the BMP1 protease domain involving a residue, which is conserved in all members of the astacin group of metalloproteases. Type I procollagen analysis in supernatants from cultured fibroblasts demonstrated abnormal PICP processing in patient-derived cells consistent with the mutation causing decreased BMP1 function. This was further confirmed by overexpressing wild type and mutant BMP1 longer isoform (mammalian Tolloid protein [mTLD]) in NIH3T3 fibroblasts and human primary fibroblasts. While overproduction of normal mTLD resulted in a large proportion of proα1(I) in the culture media being C-terminally processed, proα1(I) cleavage was not enhanced by an excess of the mutant protein, proving that the Phe249Leu mutation leads to a BMP1/mTLD protein with deficient PICP proteolytic activity. We conclude that BMP1 is an additional gene mutated in AR-OI.     

Systemic inhibition of BMP1-3 decreases progression of CCl4-induced liver fibrosis in rats

Liver fibrosis is a progressive pathological process resulting in an accumulation of excess extracellular matrix proteins. We discovered that bone morphogenetic protein 1-3 (BMP1-3), an isoform of the metalloproteinase Bmp1 gene, circulates in the plasma of healthy volunteers and its neutralization decreases the progression of chronic kidney disease in 5/6 nephrectomized rats. Here, we investigated the potential role of BMP1-3 in a chronic liver disease. Rats with carbon tetrachloride (CCl₄)-induced liver fibrosis were treated with monoclonal anti-BMP1-3 antibodies. Treatment with anti-BMP1-3 antibodies dose-dependently lowered the amount of collagen type I, downregulated the expression of Tgfb1, Itgb6, Col1a1, and Acta2 and upregulated the expression of Ctgf, Itgb1, and Dcn. Mehanistically, BMP1-3 inhibition decreased the plasma levels of transforming growth factor beta 1(TGFβ1) by prevention of its activation and lowered the prodecorin production further suppressing the TGFβ1 profibrotic effect. Our results suggest that BMP1-3 inhibitors have significant potential for decreasing the progression of fibrosis in liver cirrhosis.     

BMP4-siRNA重组腺病毒的构建及鉴定

目的构建靶向抑制骨形态发生蛋白BMP4基因RNA的siRNA腺病毒,为进一步研究其在乳腺癌骨转移中的作用打下基础。方法以PCR扩增获得BMP4全长片段,插入筛选质粒pSOS载体,同时设计6段靶向BMP4基因RNA的干扰片段,分别经酶切、连接及鉴定后获得pSOS-sirBMP4。脂质体转染293细胞,根据荧光表达量筛选获得干扰效率较高的4个干扰片段,插入穿梭质粒pSES-HUS,经重组、HEK-293细胞包装获得BMP4-siRNA腺病毒。感染乳腺癌细胞系MDA-MB-231,RT-PCR、Western blot检测其干扰效率。MTT法检测其对MDA-MB-231细胞增殖的影响。结果筛选获得4个干扰效率较高的片段,插入穿梭质粒,经重组、包装获得腺病毒pAd-sirBMP4。感染乳腺癌细胞系MDA-MB-231后,明显抑制BMP4的表达并促进MDA-MB-231细胞增殖。结论成功构建具有较高干扰效率的腺病毒pAd-sirBMP4,为进一步研究BMP4在乳腺癌中的作用打下基础。     

Crosstalk between neuropeptides SP and CGRP in regulation of BMP2-induced bone differentiation

ABSTRACT

Aim of the study: The peripheral nervous system is involved in regulation of bone metabolism via sensory and sympathetic innervation. Substance P (SP) and calcitonin gene-related peptide (CGRP) are two sensory neuropeptides that have been associated with regulation of osteogenic differentiation. However, the interaction between SP and CGRP both with each other and the bone morphogenetic protein 2 (BMP2) in regulation of osteogenic differentiation has not been studied. Therefore, the aim of this study was to investigate the interaction between SP and CGRP on BMP2-induced bone differentiation using model progenitor cells. Materials and methods: C2C12 myoblasts and MC3T3 pre-osteoblasts were treated with SP and CGRP, both individually and in combination, in the presence of BMP2. The effects of the neuropeptides on BMP2-induced osteogenic differentiation were assessed by measuring alkaline phosphatase (ALP) activity, mineralization, and expression of osteogenic markers. Results: Both SP and CGRP enhanced BMP2 signaling, Runx2 mRNA expression, as well as mineralization in vitro. Co-stimulation with SP and CGRP resulted in down-regulation of BMP2-induced bone differentiation, suggesting potential crosstalk between the two neuropeptides in regulation of BMP2 signaling. Conclusions: Based on the results shown here, CGRP can mitigate augmenting effects of SP on BMP2 signaling and the three pathways potentially converge on Runx2 to regulate BMP2-induced bone differentiation.     

Antagonists of retinoic acid and BMP4 affect fetal mouse osteogenesis and odontoblast differentiation

Retinoic acid and bone morphogenetic protein (BMP4) are endogenous factors indispensable for the physiological development of vertebrates. The proximate aim of the present study was to investigate whether the natural compound citral (a retinoic acid synthesis inhibitor) and a monoclonal, anti-BMP4 antibody, administered to pregnant mice affect in the fetuses cranial osteogenesis and odontoblast differentiation. The present investigation was motivated by the fact that, retinoic acid inhibitors and BMP4 neutralizers may frequently contact human tissues (both intentional and unintentional, and/or unconsciously) inducing unanticipated effects. Our ultimate goal is the prevention of side effects and, future clinical implementation of the results. To this end, pregnant, white mice (balb-c Mus musculus) were intra-abdominally injected with either citral or anti-BMP4 antibody at the 9th gestational day. Newborns were processed within 5 h, postnatal. Results were evaluated (a) macroscopically, (b) stereoscopically, following histochemical double staining of cartilage and osseous tissues and, (c) microscopically after (c₁) histological staining of paraffin sections, and, (c₂) immunohistochemical detection of apoptosis. Data indicate that in vivo administration of citral (biomimicking hypovitaminosis A) caused restriction/retardation of cranial chondrogenesis and osteogenesis. Apoptosis was not detected in teeth tissues. In vivo administration of anti-BMP4 antibody resulted in a transitory interference with the normal course of odontoblast differentiation and the production of pre-dentin, whereas, delay in the ossification also included the alveoli. Animals inspected in adulthood displayed a fairly normal phenotype. It is concluded that those two substances, under their concentrations experienced, are quite safe for the public.     

BMP4/Smad Signaling Pathway Induces the Differentiation of Mouse Spermatogonial Stem Cells via Upregulation of Sohlh2

Spermatogonial stem cells (SSCs) capable of self‐renewal and differentiation are the foundation for spermatogenesis. Although several factors that govern these processes have been investigated, the underlying molecular mechanisms have not been fully elucidated. Here, we investigated the role of BMP4 in mouse SSC differentiation, and found that SSCs cultured in the presence of BMP4 underwent differentiation, characterized by downregulation of SSC self‐renewal markers, Plzf, and upregulation of SSC differentiation marker, c‐kit. Smad1/5/8 proteins were phosphorylated during BMP4‐induced differentiation. The effects of BMP4 on SSCs were blocked by BMP4 inhibitor (Dorsomorphin). The activation of BMP4/Smad signaling pathway in SSCs increased the expression of Sohlh2, which is involved in the early differentiation of spermatogonia. Knockdown sohlh2 expression by RNA interference abolished the effect of BMP4 on SSC differentiation and the upregulation of c‐kit expression. Overall, our results suggest that BMP4 plays an important role during the early differentiation of SSCs via upregulation of sohlh2. Anat Rec, 297:749–757, 2014. © 2014 Wiley Periodicals, Inc.     

胃癌中ANP表达与BMP信号通路相互作用对细胞侵袭、转移的影响

目的探讨心房钠尿肽(atrial natriuretic peptide,ANP)在胃癌组织和细胞中的表达及ANP对胃癌细胞侵袭转移的作用。方法采用免疫组化EnVision法检测60例胃癌组织及正常胃黏膜组织中ANP的表达;培养胃癌细胞MGC-803,并将其分为两组:加入ANP组(ANP阳性组)、未加入ANP组(ANP阴性组)。采用Transwell实验检测两组胃癌细胞MGC-803的侵袭性,利用CCK8法检测两组胃癌细胞MGC-803的增殖能力。采用Western blot法检测两组胃癌细胞MGC-803中BMP信号通路相关蛋白表达量的变化及Ki-67、MMP-2和MMP-9蛋白表达量的变化。结果免疫组化检测ANP定位于胃黏膜细胞的胞质中,且在胃癌组织中的阳性率明显低于正常胃组织,差异有统计学意义(P<0.05);CCK8法、Transwell实验结果显示,ANP阳性组胃癌细胞的增殖速度及侵袭性比ANP阴性组低,差异有统计学意义(P均<0.05)。Western blot法检测ANP阳性组中BMP信号通路相关蛋白Smad1/5表达量比ANP阴性组低,差异有统计学意义(P<0.05);BMP6/7和p-Smad1/5的表达升高,差异有统计学意义(P均<0.05)。Western blot法检测ANP阳性组中Ki-67、MMP-2和MMP-9蛋白比ANP阴性组低,差异有统计学意义(P均<0.05)。结论胃癌组织中ANP的表达低于正常胃组织,ANP可能通过激活BMP信号通路抑制胃癌细胞MGC-803的增殖、侵袭和转移,为胃癌的发病机制及靶向治疗提供新思路。     

DLX1联合BMP9促进人骨肉瘤细胞向成骨分化

目的探讨DLX1联合BMP9可否影响人骨肉瘤细胞MG63的成骨分化。方法用构建有DLX1和BMP9基因的重组腺病毒Ad DLX1和Ad BMP9,单独或联合感染MG63细胞,分为DLX1组(Ad DLX1+AdRFP感染组)、DLX1+BMP9组(Ad DLX1+Ad BMP9感染组)、BMP9组(Ad BMP9+AdRFP感染组)、RFP组(AdRFP感染组)。用RT-PCR和Western blot验证DLX1和BMP9的表达情况,Transwell实验检测细胞迁移、侵袭能力;碱性磷酸酶(ALP)染色和读数分析细胞早期成骨能力,茜素红染色检测细胞晚期成骨能力,Western blot检测骨桥蛋白(OPN)蛋白表达水平。结果 Ad DLX1和Ad BMP9感染MG63细胞后,DLX1和BMP9的表达水平上调(P0.05);与RFP组相比,DLX1组、DLX1+BMP9组和BMP9组细胞迁移能力和侵袭能力下降(P0.05),DLX1+BMP9组细胞迁移能力和侵袭能力下降更为明显(P0.05);ALP活性、钙盐沉积和OPN蛋白表达在DLX1+BMP9组和BMP9组增加(P0.05),且DLX1+BMP9组较BMP9组显著增强(P0.05)。结论 DLX1与BMP9联合作用可抑制骨肉瘤细胞迁移和侵袭,同时可诱导骨肉瘤细胞向成骨分化。     

小鼠4-1 BB分子的配体识别主要区域定位及结构分析

目的 明确4-1 BB分子的配体结合4-1 BB分子胞外区的关键区域和基本结构.方法 分区表达鼠4-1 BB胞外区结构域及其天然配体,利用ELISA和Western blot等方法 对4-1 BB分子的配体识别结构域进行定位.结果 4-1 BB分子半胱氨酸寓集结构域(cysteine-rich domain,CRD)Ⅱ是配体结合的主要结构域,不结合CRD Ⅰ和CRDⅢ.免疫刺激性抗4-1 BB单克隆抗体2A,主要结合4-1 BB分子CRD与跨膜区之间的连接区(STP区),同时对包括CRDⅡ的各个CRDs有弱识别,可封阻4-1 BB与其配体结合.结论 4-1 BB分子配体的主要识别区位于4-1 BB分子胞外CRD Ⅱ,具有空间结构特点,此为进一步分析结合区及其关键氨基酸提供了资料.     

Identification and In Vivo Functional Characterization of Novel Compound Heterozygous BMP1 Variants in Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) comprises a heterogeneous group of disorders that are characterized by susceptibility to bone fractures, and range in severity from a subtle increase in fracture frequency to death in the perinatal period. Most patients have defects in type I collagen biosynthesis with autosomal‐dominant inheritance, but many autosomal‐recessive genes have been reported. We applied whole‐exome sequencing to identify mutations in a Korean OI patient who had an umbilical hernia, frequent fractures, a markedly short stature, delayed motor development, scoliosis, and dislocation of the radial head, with a bowed radius and ulna. We identified two novel variants in the BMP1 gene: c.808A>G and c.1297G>T. The former variant caused a missense change p.(Met270Val) and the latter variant caused the skipping of exon 10. The hypofunctional nature of the two variants was demonstrated in a zebrafish assay.     

Specific Preferences in Lineage Choice and Phenotypic Plasticity of Glioma Stem Cells Under BMP4 and Noggin Influence

Although BMP4‐induced differentiation of glioma stem cells (GSCs) is well recognized, details of the cellular responses triggered by this morphogen are still poorly defined. In this study, we established several GSC‐enriched cell lines (GSC‐ECLs) from high‐grade gliomas. The expansion of these cells as adherent monolayers, and not as floating neurospheres, enabled a thorough study of the phenotypic changes that occurred during their differentiation. Herein, we evaluated GSC‐ECLs’ behavior toward differentiating conditions by depriving them of growth factors and/or by adding BMP4 at different concentrations. After analyzing cellular morphology, proliferation and lineage marker expression, we determined that GSC‐ECLs have distinct preferences in lineage choice, where some of them showed an astrocyte fate commitment and others a neuronal one. We found that this election seems to be dictated by the expression pattern of BMP signaling components present in each GSC‐ECL. Additionally, treatment of GSC‐ECLs with the BMP antagonist, Noggin, also led to evident phenotypic changes. Interestingly, under certain conditions, some GSC‐ECLs adopted an unexpected smooth muscle‐like phenotype. As a whole, our findings illustrate the wide differentiation potential of GSCs, highlighting their molecular complexity and paving a way to facilitate personalized differentiating therapies.     

BMP4沉默对骨肉瘤细胞系MG-63上皮间质转换及侵袭转移能力的影响

目的探讨沉默骨形成蛋白-4(BMP4)对骨肉瘤细胞系MG-63上皮间质转换(EMT)和侵袭迁移能力的影响。方法脂质体法转染MG-63细胞BMP4 shRNA表达质粒,将MG-63细胞分为MG-63对照组(正常培养对照)、空白对照组(转染空白质粒)和BMP4沉默组(转染BMP4 shRNA质粒)。反转录聚合酶链反应(RT-PCR)法和Western blot检测细胞E-cadherin、vimentin、twist和snail的mRNA及蛋白水平。Transwell侵袭实验和伤口愈合实验检测细胞侵袭和迁移能力。结果沉默BMP4后MG-63细胞的间质细胞标志物vimentin、twist1和snail水平降低,上皮细胞标志物E-cadherin表达增高(P0.05)。BMP4沉默组细胞穿膜细胞数和迁移距离显著少于MG-63对照组和空白对照组(P0.05)。结论沉默BMP4基因可以使间质型MG-63骨肉瘤细胞向上皮细胞表型转变,从而有效地降低肿瘤细胞的侵袭迁移能力。