Lactacystin, a proteasome inhibitor, enhances BMP-induced osteoblastic differentiation by increasing active Smads

Proteasome inhibitors enhance bone formation and osteoblastic differentiation in vivo and in vitro. In the present study, we examined whether the molecular mechanisms of lactacystin, one of many proteasome inhibitors, stimulated the osteoblastic differentiation of C2C12 cells that is induced by bone morphogenetic proteins (BMPs). Pretreatment with lactacystin enhanced the alkaline phosphatase (ALP) activity induced by BMP2, BMP4 or BMP7, but lactacystin did not induce ALP in the absence of BMPs. In addition, lactacystin-stimulated BMP2 induced mRNA expression of ALP, type I collagen, osteonectin, osteocalcin, Id1, Osterix, and Runx2. Lactacystin maintained BMP2-induced phosphorylation of Smad1/5/8 and increased the length of time that these Smads were bound to target DNA. Moreover, lactacystin prevented BMP receptor-induced Smad degradation. This enhancement of BMP2-induced ALP activity and Smad phosphorylation by lactacystin was also observed in primary osteoblasts. These findings suggest that pretreatment with lactacystin accelerates BMP-induced osteoblastic differentiation by increasing the levels of phosphorylated Smads, which are maintained because BMP receptor-induced degradation is inhibited. We propose that optimized stimulation by proteasome inhibitors in a clinical setting may facilitate autogenous or BMP-induced bone formation in areas of defective bone.     

Psoralen stimulates osteoblast differentiation through activation of BMP signaling

Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture. In order to improve the treatment of osteoporosis, identification of anabolic and orally available agents with minimal side effects is highly desirable. Psoralen is a coumarin-like derivative extracted from Chinese herbs, which have been used to treat bone diseases for thousands of years. However, the role of Psoralen in osteoblast function and the underlying molecular mechanisms remain poorly understood. In this study, we found that Psoralen promoted osteoblast differentiation in primary mouse calvarial osteoblasts in a dose-dependent manner, demonstrated by up-regulation of expressions of osteoblast-specific marker genes including type I collagen, osteocalcin and bone sialoprotein and enhancement of alkaline phosphatase activity. We further demonstrated that Psoralen up-regulated the expression of Bmp2 and Bmp4 genes, increased the protein level of phospho-Smad1/5/8, and activated BMP reporter (12xSBE-OC-Luc) activity in a dose-dependent manner, as well as enhanced the expression of Osx, the direct target gene of BMP signaling. Deletion of the Bmp2 and Bmp4 genes abolished the stimulatory effect of Psoralen on the expression of osteoblast marker genes, such as Col1, Alp, Oc and Bsp. Our results suggest that Psoralen acts through the activation of BMP signaling to promote osteoblast differentiation and demonstrate that Psoralen could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.     

Bone morphogenetic protein -4 and -5 in pancreatic cancer--novel bidirectional players

Bone morphogenetic proteins (BMPs) are multifunctional signaling molecules that have gained increasing interest in cancer research. To obtain a systematic view on BMP signaling in pancreatic cancer we first determined the mRNA expression levels of seven BMP ligands (BMP2–BMP8) and six BMP specific receptors in pancreatic cancer cell lines and normal pancreatic tissue. BMP receptor expression was seen in all cancer and normal samples. Low expression levels of BMP5 and BMP8 were detected in cancer cells compared to the normal samples, whereas BMP4 expression was elevated in 25% of the cases. The impact of BMP4 and BMP5 signaling on cell phenotype was then evaluated in five pancreatic cancer cell lines. Both ligands suppressed the growth of three cell lines (up to 79% decrease in BMP4-treated PANC-1 cells), mainly due to cell cycle changes. BMP4 and BMP5 concurrently increased cell migration and invasion (maximally a 10.8-fold increase in invaded BMP4-treated PANC-1 cells). The phenotypic changes were typically associated with the activation of the canonical SMAD pathway, although such activation was not observed in the PANC-1 cells. Taken together, BMP4 and BMP5 simultaneously inhibit the growth and promote migration and invasion of the same pancreatic cells and thus exhibit a biphasic role with both detrimental and beneficial functions in pancreatic cancer progression.     

Loss-of-function of ACVR1 in osteoblasts increases bone mass and activates canonical Wnt signaling through suppression of Wnt inhibitors SOST and DKK1

BMPs (Bone morphogenetic proteins) such as BMP2 and BMP7 have been used about one decade as bone anabolic agents in orthopaedics. The BMP receptor ACVR1, which is a key receptor of BMP7, is expressed in bone. The pathological role of ACVR1 in humans has been reported: a point mutation in ACVR1 can cause fibrodysplasia ossificans progressiva (FOP) in which ectopic ossification occurs in skeletal muscles and deep connective tissues. The physiological function of ACVR1 in bone, however, is totally unknown. The purpose of this study is to investigate the endogenous role of ACVR1 in osteoblasts, one of the most dominant cell-types in bone. We generated Acvr1-null mice in an osteoblast-specific manner using an inducible Cre-loxP system. Surprisingly, we found that bone mass was increased in the Acvr1-null mice. Interestingly, canonical Wnt signaling was increased and expression levels of Wnt inhibitors Sost and Dkk1 were both suppressed in the null bones during the developmental stages. In addition, we confirmed that expression levels of both Sost and Dkk1 were upregulated by BMP7 dose-dependently in vitro. These results suggest that the Acvr1-deficiency can increase bone mass by activating Wnt signaling in which both Sost and Dkk1 expression levels are diminished. This study leads to a new concept of the BMP7-ACVR1-SOST/DKK1 axis in osteoblasts, in which BMP7 signaling through ACVR1 can reduce Wnt signaling via SOST/DKK1 and then inhibits osteogenesis. Although this concept is beyond the current known function of BMP7, it can explain the varied outcomes of BMP7 treatment. We believe BMP signaling can exhibit multifaceted effects by context and cell type.     

Sprouty2 is involved in the control of osteoblast proliferation and differentiation through the FGF and BMP signaling pathways

Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) play essential roles in bone formation and osteoblast activity through the extracellular signal‐regulated kinase 1/2 (ERK1/2) and Smad pathways. Sprouty family members are intracellular inhibitors of the FGF signaling pathway, and four orthologs of Sprouty have been identified in mammals. In vivo analyses have revealed that Sprouty2 is associated with bone formation. However, the mechanism by which the Sprouty family controls bone formation has not been clarified. In this study, we investigated the involvement of Sprouty2 in osteoblast proliferation and differentiation. We examined Sprouty2 expression in MC3T3‐E1 cells, and found that high levels of Sprouty2 expression were induced by basic FGF stimulation. Overexpression of Sprouty2 in MC3T3‐E1 cells resulted in suppressed proliferation compared with control cells. Sprouty2 negatively regulated the phosphorylation of ERK1/2 after basic FGF stimulation, and of Smad1/5/8 after BMP stimulation. Furthermore, Sprouty2 suppressed the expression of osterix, alkaline phosphatase, and osteocalcin mRNA, which are markers of osteoblast differentiation. Additionally, Sprouty2 inhibited osteoblast matrix mineralization. These results suggest that Sprouty2 is involved in the control of osteoblast proliferation and differentiation by downregulating the FGF‐ERK1/2 and BMP‐Smad pathways, and suppresses the induction of markers of osteoblast differentiation.     

BMP-Non-Responsive Sca1+CD73+CD44+ Mouse Bone Marrow Derived Osteoprogenitor Cells Respond to Combination of VEGF and BMP-6 to Display Enhanced Osteoblastic Differentiation and Ectopic Bone Formation

Clinical trials on fracture repair have challenged the effectiveness of bone morphogenetic proteins (BMPs) but suggest that delivery of mesenchymal stem cells (MSCs) might be beneficial. It has also been reported that BMPs could not increase mineralization in several MSCs populations, which adds ambiguity to the use of BMPs. However, an exogenous supply of MSCs combined with vascular endothelial growth factor (VEGF) and BMPs is reported to synergistically enhance fracture repair in animal models. To elucidate the mechanism of this synergy, we investigated the osteoblastic differentiation of cloned mouse bone marrow derived MSCs (D1 cells) in vitro in response to human recombinant proteins of VEGF, BMPs (-2, -4, -6, -9) and the combination of VEGF with BMP-6 (most potent BMP). We further investigated ectopic bone formation induced by MSCs pre-conditioned with VEGF, BMP-6 or both. No significant increase in mineralization, phosphorylation of Smads 1/5/8 and expression of the ALP, COL1A1 and osterix genes was observed upon addition of VEGF or BMPs alone to the cells in culture. The lack of CD105, Alk1 and Alk6 expression in D1 cells correlated with poor response to BMPs indicating that a greater care in the selection of MSCs is necessary. Interestingly, the combination of VEGF and BMP-6 significantly increased the expression of ALP, COL1A1 and osterix genes and D1 cells pre-conditioned with VEGF and BMP-6 induced greater bone formation in vivo than the non-conditioned control cells or the cells pre-conditioned with either VEGF or BMP-6 alone. This enhanced bone formation by MSCs correlated with higher CADM1 expression and OPG/RANKL ratio in the implants. Thus, combined action of VEGF and BMP on MSCs enhances osteoblastic differentiation of MSCs and increases their bone forming ability, which cannot be achieved through use of BMPs alone. This strategy can be effectively used for bone repair.     

Construction of recombinant adenovirus vector containing hBMP2 and hVEGF165 genes and its expression in rabbit Bone marrow mesenchymal stem cells

To construct an adenovirus vector co-expressing human bone morphogenetic protein (hBMP2) and human vascular endothelial growth factor (hVEGF165) as well as green fluorescence protein (GFP) as a marker, with which the intracellular expression of the inserted genes could be identified in Bone marrow mesenchymal stem cells (BM-MSCs). BMP2 and VEGF165 genes were PCR amplified from a cDNA library and inserted to the polyclonal site of adenovirus shuttle plasmid pAd-MCMV-GFP. The virus solution (Ad-BMP2-VEGF165) was generated by co-transfecting HEK293 cells with the constructed recombinant shuttle plasmid pAd-MCMV-BMP2-VEGF165 and adenovirus helper plasmid pBHGloxΔ (delta) E1, 3Cre. The virus solution was further purified and virus titer was determined accordingly. The expression of the target genes was subsequently detected and quantified in rabbit BM-MSCs by using real time PCR, ELISA and Western blotting. The recombinant adenovirus vector containing BMP2 and VEGF165 (Ad-BMP2-VEGF165) was successfully constructed, which was confirmed by Sanger sequencing, colony PCR, as well as visually detection of GFP, and the titer of the adenovirus was 1 × 10¹⁰ PFU/mL, and the proteins level of BMP2 and VEGF165 secreted in the supernatant are significantly higher than the control. Recombinant adenovirus vector containing hBMP2 and hVEGF165 genes was successfully constructed. The transfection rate of BM-MSCs by the adenovirus was high (95% at 100 MOI) and the BMP2 and VEGF165 genes was highly expressed in the cells. The present study provides a method to efficiently express the target genes in BM-MSCs and an vector for further research of bone defect repair using dual genes of BMP2 and VEGF165.     

Characterization of neuritin as a novel angiogenic factor

Neuritin (NRN1), a neurotrophic factor, plays an important role in neurite growth and neuronal survival. In this study, we identify a new function of neuritin as a novel angiogenic factor in vitro and in vivo. Recombinant neuritin protein had no effect on the proliferation and adhesion of human umbilical vein endothelial cells (HUVEC), but it dose-dependently increased endothelial cell migration. Furthermore, overexpression of neuritin significantly promoted tumor angiogenesis, and surprisingly, it inhibited tumor growth in a xenograft tumor model. Thus, our results indicate that neuritin may act as an important angiogenic factor and serve as a potential target for cancer therapy.     

Cumulin,an Oocyte-secreted Heterodimer of the Transforming Growth Factor-β Family,Is a Potent Activator of Granulosa Cells and Improves Oocyte Quality

Growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-specific growth factors with central roles in mammalian reproduction, regulating species-specific fecundity, ovarian follicular somatic cell differentiation, and oocyte quality. In the human, GDF9 is produced in a latent form, the mechanism of activation being an open question. Here, we produced a range of recombinant GDF9 and BMP15 variants, examined their in silico and physical interactions and their effects on ovarian granulosa cells (GC) and oocytes. We found that the potent synergistic actions of GDF9 and BMP15 on GC can be attributed to the formation of a heterodimer, which we have termed cumulin. Structural modeling of cumulin revealed a dimerization interface identical to homodimeric GDF9 and BMP15, indicating likely formation of a stable complex. This was confirmed by generation of recombinant heterodimeric complexes of pro/mature domains (pro-cumulin) and covalent mature domains (cumulin). Both pro-cumulin and cumulin exhibited highly potent bioactivity on GC, activating both SMAD2/3 and SMAD1/5/8 signaling pathways and promoting proliferation and expression of a set of genes associated with oocyte-regulated GC differentiation. Cumulin was more potent than pro-cumulin, pro-GDF9, pro-BMP15, or the two combined on GC. However, on cumulus-oocyte complexes, pro-cumulin was more effective than all other growth factors at notably improving oocyte quality as assessed by subsequent day 7 embryo development. Our results support a model of activation for human GDF9 dependent on cumulin formation through heterodimerization with BMP15. Oocyte-secreted cumulin is likely to be a central regulator of fertility in mono-ovular mammals.     

Activation of the JNK pathway by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are increasingly recognized as a novel and unique tool in various life science fields, including electroporation and cancer therapy, although their mode of action in cells remains largely unclear. Here, we show that nsPEFs induce strong and transient activation of a signaling pathway involving c-Jun N-terminal kinase (JNK). Application of nsPEFs to HeLa S3 cells rapidly induced phosphorylation of JNK1 and MKK4, which is located immediately upstream of JNK in this signaling pathway. nsPEF application also elicited increased phosphorylation of c-Jun protein and dramatically elevated c-jun and c-fos mRNA levels. nsPEF-inducible events downstream of JNK were markedly suppressed by the JNK inhibitor SP600125, which confirmed JNK-dependency of these events in this pathway. Our results provide novel mechanistic insights into the mode of nsPEF action in human cells.     

合浦珠母贝外套膜细胞engrailed、BMP2和Smad3的mRNA表达水平的相关性

软体动物engrailed蛋白和骨形成相关蛋白对胚胎贝壳区域边界形成可能具有重要作用,engrailed还被推测为调节基质蛋白在外套膜组织区域化表达的重要调控因子．因此,弄清调控engrailed在软体动物中特征表达的分子机制有着重要的研究意义．但是,由于贝类基因组测序尚不完整,目前也没有建立获得贝类细胞系,以致于许多预测可能参与调控的基因需要通过克隆来鉴定,而且经典的研究细胞信号通路的方法也很难得到应用．目前,在中国南海广泛养殖的合浦珠母贝中,已获知其BMP2和Smad3的cDNA全长,以该贝的基因组为模板,PCR扩增获得了一段engrailed编码区片段．经软件分析,该片段含有EH4结构域,且与其他物种engrailed蛋白具有很高的同源性．研究的贝中,特别是外套膜组织中,engrailed、BMP2和Smad3三者表达之间的相关性,将有助于我们理解贝壳形成的分子机制．贝壳缺刻后半定量PCR试验结果表明,三者均参与贝壳修复,且在贝壳缺刻后的修复过程中,engrailed和Smad3的mRNA表达变化规律非常相似,提示它们之间可能存在相互影响的联系．用地塞米松(DXM)和过氧化氢(H₂O₂)分别处理原代培养的贝外套膜组织迁出细胞,实时相对定量PCR检测engrailed、BMP2和Smad3的mRNA表达水平,统计分析结果表明,三者具有显著的相关性．上述所有结果为进一步研究贝类生物矿化的发育和信号转导机制提供了新的思路和基础．     

The role of BMP‐7 in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells in vitro

This study addresses the role of bone morphogenetic protein‐7 (BMP‐7) in chondrogenic and osteogenic differentiation of human bone marrow multipotent mesenchymal stromal cells (BM MSCs) in vitro. BM MSCs were expanded and differentiated in the presence or absence of BMP‐7 in monolayer and three‐dimensional cultures. After 3 days of stimulation, BMP‐7 significantly inhibited MSC growth in expansion cultures. When supplemented in commonly used induction media for 7–21 days, BMP‐7 facilitated both chondrogenic and osteogenic differentiation of MSCs. This was evident by specific gene and protein expression analyses using real‐time PCR, Western blot, histological, and immunohistochemical staining. BMP‐7 supplementation appeared to enhance upregulation of lineage‐specific markers, such as type II and type IX collagens (COL2A1, COL9A1) in chondrogenic and secreted phosphoprotein 1 (SPP1), osteocalcin (BGLAP), and osterix (SP7) in osteogenic differentiation. BMP‐7 in the presence of TGF‐β3 induced superior chondrocytic proteoglycan accumulation, type II collagen, and SOX9 protein expression in alginate and pellet cultures compared to either factor alone. BMP‐7 increased alkaline phosphatase activity and dose‐dependently accelerated calcium mineralization of osteogenic differentiated MSCs. The potential of BMP‐7 to promote adipogenesis of MSCs was restricted under osteogenic conditions, despite upregulation of adipocyte gene expression. These data suggest that BMP‐7 is not a singular lineage determinant, rather it promotes both chondrogenic and osteogenic differentiation of MSCs by co‐ordinating with initial lineage‐specific signals to accelerate cell fate determination. BMP‐7 may be a useful enhancer of in vitro differentiation of BM MSCs for cell‐based tissue repair. J. Cell. Biochem. 109: 406–416, 2010. © 2009 Wiley‐Liss, Inc.     

Extracellular acidification induces connective tissue growth factor production through proton-sensing receptor OGR1 in human airway smooth muscle cells

Asthma is characterized by airway inflammation, hyper-responsiveness and remodeling. Extracellular acidification is known to be associated with severe asthma; however, the role of extracellular acidification in airway remodeling remains elusive. In the present study, the effects of acidification on the expression of connective tissue growth factor (CTGF), a critical factor involved in the formation of extracellular matrix proteins and hence airway remodeling, were examined in human airway smooth muscle cells (ASMCs). Acidic pH alone induced a substantial production of CTGF, and enhanced transforming growth factor (TGF)-β-induced CTGF mRNA and protein expression. The extracellular acidic pH-induced effects were inhibited by knockdown of a proton-sensing ovarian cancer G-protein-coupled receptor (OGR1) with its specific small interfering RNA and by addition of the G_q/11 protein-specific inhibitor, YM-254890, or the inositol-1,4,5-trisphosphate (IP₃) receptor antagonist, 2-APB. In conclusion, extracellular acidification induces CTGF production through the OGR1/G_q/11 protein and inositol-1,4,5-trisphosphate-induced Ca²⁺ mobilization in human ASMCs.     

Growth rate suppression of cultured mammalian cells enhances protein productivity

Suppression of proliferation of cells which contain stable or stabilized mRNA coded for a protein to be produced, a partial mimic of cell differentiation, was examined for enhancing protein production by cultured mammalian cells. Hybridoma 2E3 cells which were adapted to be interleukin-6 sensitively growth-suppressed accumulated the mRNA of IgG₁ which is reported stable, and IgG₁ production rate increased as a result when their growth was suppressed with interleukin-6. A myeloma cell line was similarly adapted; the obtained myeloma cells can be used as host cells for enhancing production of exogenous proteins by suppressing growth with interleukin-6. Temperature-sensitively growth-suppressible mutants of mouse mammary carcinoma FM3A were transfected with cDNA of IgM ₁ chain and cultured at nonpermissive temperature to enhance production of ₁. Addition of various growth-suppressive reagents to culture medium was studied for finding methods suitable for suppressing growth while maintaining high cell viability. Caffeine yielded the best results among these reagents. Deprivation of various growth-supporting components in culture medium was also tested; simultaneous deprivation of insulin and transferrin viably suppressed growth of hybridoma 2E3 cells, resulting in enhanced antibody productivity.Abbreviations IL6 recombinant human interleukin-6 - TGF- recombinant human TGF-₁ - X63.653-P3X63 Ag8.653 myeloma     

Inhibition of autophagy stimulate molecular iodine-induced apoptosis in hormone independent breast tumors

Estrogen receptor negative (ER^−ve) and p53 mutant breast tumors are highly aggressive and have fewer treatment options. Previously, we showed that molecular Iodine (I₂) induces apoptosis in hormone responsive MCF-7 breast cancer cells, and non-apoptotic cell death in ER^−ve–p53 mutant MDA-MB231 cells (Shrivastava, 2006). Here we show that I₂ (3 μM) treatment enhanced the features of autophagy in MDA-MB231 cells. Since autophagy is a cell survival response to most anti-cancer therapies, we used both in vitro and in vivo systems to determine whether ER^−ve mammary tumors could be sensitized to I₂-induced apoptosis by inhibiting autophagy. Autophagy inhibition with chloroquine (CQ) and inhibitors for PI3K (3MA, LY294002) and H+/ATPase (baflomycin) resulted in enhanced cell death in I₂ treated MDA-MB231 cells. Further, CQ (20 μM) in combination with I₂, showed apoptotic features such as increased sub-G1 fraction (∼5-fold), expression of cleaved caspase-9 and -3 compared to I₂ treatment alone. Flowcytometry of I₂ and CQ co-treated cells revealed increase in mitochondrial membrane permeability (p < 0.01) and translocation of cathepsin D activity to cytosol relative to I₂ treatment. For in vivo studies ICRC mice were transplanted subcutaneously with MMTV-induced mammary tumors. A significant reduction in tumor volumes, as measured by MRI, was found in I₂ and CQ co-treated mice relative to I₂ or vehicle treated mice. These data indicate that inhibition of autophagy renders ER^−ve breast tumor cells more sensitive to I₂ induced apoptosis. Thus, I₂ together with autophagy inhibitor could have a potential tumorostatic role in ER^−ve aggressive breast tumors that may be evaluated in future studies.     

Mechanotransduction activates α5β1 integrin and PI3K/Akt signaling pathways in mandibular osteoblasts

It is unclear how bone cells at different sites detect mechanical loading and how site-specific mechanotransduction affects bone homeostasis. To differentiate the anabolic mechanical responses of mandibular cells from those of calvarial and long bone cells, we isolated osteoblasts from C57B6J mouse bones, cultured them for 1 week, and subjected them to therapeutic low intensity pulsed ultrasound (LIPUS). While the expression of the marker proteins of osteoblasts and osteocytes such as alkaline phosphatase and FGF23, as well as Wnt1 and β-catenin, was equally upregulated, the expression of mandibular osteoblast messages related to bone remodeling and apoptosis differed from that of messages of other osteoblasts, in that the messages encoding the pro-remodeling protein RANKL and the anti-apoptotic protein Bcl-2 were markedly upregulated from the very low baseline levels. Blockage of the PI3K and α₅β₁ integrin pathways showed that the mandibular osteoblast required mechanotransduction downstream of α₅β₁ integrin to upregulate expression of the proteins β-catenin, p-Akt, Bcl-2, and RANKL. Mandibular osteoblasts thus must be mechanically loaded to preserve their capability to promote remodeling and to insure osteoblast survival, both of which maintain intact mandibular bone tissue. In contrast, calvarial Bcl-2 is fully expressed, together with ILK and phosphorylated mTOR, in the absence of LIPUS. The antibody blocking α₅β₁ integrin suppressed both the baseline expression of all calvarial proteins examined and the LIPUS-induced expression of all mandibular proteins examined. These findings indicate that the cellular environment, in addition to the tridermic origin, determines site-specific bone homeostasis through the remodeling and survival of osteoblastic cells. Differentiated cells of the osteoblastic lineage at different sites transmit signals through transmembrane integrins such as α₅β₁ integrin in mandibular osteoblasts, whose signaling may play a major role in controlling bone homeostasis.