BMP2 Transfer to Neighboring Cells and Activation of Signaling

Morphogen gradients and concentration are critical features during early embryonic development and cellular differentiation. Previously we reported the preparation of biologically active, fluorescently labeled BMP2 and quantitatively analyzed their binding to the cell surface and followed BMP2 endocytosis over time on the level of single endosomes. Here we show that this internalized BMP2 can be transferred to neighboring cells and, moreover, also activates downstream BMP signaling in adjacent cells, indicated by Smad1/5/8 phosphorylation and activation of the downstream target gene id1. Using a 3D matrix to modulate cell–cell contacts in culture we could show that direct cell–cell contact significantly increased BMP2 transfer. Using inhibitors of vesicular transport, transfer was strongly inhibited. Interestingly, cotreatment with the physiological BMP inhibitor Noggin increased BMP2 uptake and transfer, albeit activation of Smad signaling in neighboring cells was completely suppressed. Our findings present a novel and interesting mechanism by which morphogens such as BMP2 can be transferred between cells and how this is modulated by BMP antagonists such as Noggin, and how this influences activation of Smad signaling by BMP2 in neighboring cells.      

Overexpression of LIM Kinase 1 Renders Resistance to Apoptosis in PC12 Cells by Inhibition of Caspase Activation

LIM kinases (LIMKs) regulate actin polymerization by phosphorylating cofilin and are predominantly expressed in neural tissue. In this study, the effect of LIMK1 overexpression in PC12 cell apoptosis was investigated. PC12 cells overexpressing the wild-type LIMK1 were more resistant to serum-withdrawal-induced cell death and the level of caspase 3 activation in these cells was lower than in the control PC12 cells or than in the PC12 cells expressing a mutant LIMK1 lacking the kinase domain. The inhibition of JNK activation was observed in the PC12 cells overexpressing the wild-type LIMK1 after serum withdrawal. These results suggest that the LIMK1 might allow resistance to apoptosis in PC12 cells by inhibiting JNK activation.     

BMP antagonism by Spemann's organizer regulates rostral-caudal fate of mesoderm

Recent revisions to the Xenopus fate map challenge the interpretation of previous maps and current models of amphibian axial patterning (Lane, M.C., Smith, W.C., 1999. The origins of primitive blood in Xenopus: implications for axial patterning. Development 126 (3), 423-434.; Lane, M.C., Sheets, M.D., 2000. Designation of the anterior/posterior axis in pregastrula Xenopus laevis. Dev. Biol. 225, 37-58). We determined the rostralmost contributions to both dorsal and ventral mesoderm concomitantly from marginal zone progenitors in stage 6 embryos. Data reveal an unequivocal rostral-to-caudal progression of both dorsal and ventral mesoderm across the pre-gastrula axis historically called the dorsal-ventral axis, and a dorsal-to-ventral progression from animal-to-vegetal in the marginal zone. These findings support the proposed revisions to the fate and axis orientation maps. Most importantly, these results raise questions about the role of the organizer grafts and organizer-derived BMP antagonists in the "induction" of secondary axes. We re-examine both phenomena, and find that organizer grafts and BMP antagonists evoke caudal-to-rostral mesodermal fate transformations, and not ventral-to-dorsal transformations as currently believed. We demonstrate that BMP antagonism evokes a second axis because it stimulates precocious mediolateral intercalation of caudal, dorsal mesoderm. The implications of these findings for models of organizer function in vertebrate axial patterning are discussed.     

Up‐regulation of LIMK1 expression in prostate cancer is correlated with poor pathological features,lymph node metastases and biochemical recurrence

This study aimed to explore the association between LIM domain kinase 1 (LIMK1) expression in prostate cancer (PCa) tissues with advanced pathological features, lymph node metastases and biochemical recurrence. A total of 279 PCa specimens from patients who underwent radical prostatectomy and 50 benign prostatic hyperplasia (BPH) specimens were collected to construct tissue microarray, which were subjected to immunohistochemical staining for LIMK1 expression subsequently. Logistic and Cox regression analysis were used to evaluate the relationship between LIMK1 expression and clinicopathological features of patients with PCa. Immunohistochemical staining assay demonstrated that LIMK1 expression was significantly higher in PCa than BPH specimens (77.1% vs 26.0%; P < .001). LIMK1 expression was significantly higher in positive lymph node specimens than corresponding PCa specimens (P = .002; P < .001). Up‐regulation of LIMK1 was associated with prostate volume, prostate‐specific antigen, prostate‐specific antigen density, Gleason score, T stage, lymph node metastases, extracapsular extension and seminal vesicle invasion, and positive surgical margin. Multivariate logistic regression analysis demonstrated that LIMK1 was an independent risk factor for PCa lymph node metastasis (P < .05). Multivariate Cox regression analysis revealed that the up‐regulation of LIMK1 was an independent risk factor for biochemical recurrence. Kaplan‐Meier analysis indicated that up‐regulation LIMK1 was associated with shortened biochemical‐free survival (BFS) after radical prostatectomy (P < .001). In conclusion, LIMK1 was significantly up‐regulated in PCa and positive lymph node specimens and correlated with lymph node metastasis and shortened BFS of PCa. The underlying molecular mechanism of LIMK1 in PCa should be further evaluated.     

Regulation of human erythropoiesis by activin A,BMP2, and BMP4, members of the TGFbeta family

Activin A, BMP2, and BMP4, members of the TGFbeta family, have been implicated in the regulation of hematopoiesis. Here we explore and compare, for the first time in human primary cells, the role of activin A, BMP2, and BMP4 during erythropoiesis. Using in vitro erythroid differentiation of CD34(+) primary cells, we obtained the main stages of early erythropoiesis, characterized at the molecular, biochemical, and functional levels. Our results indicate that BMP2 acts on early erythroid cells and activin A on a more differentiated population. We report an insight into the mechanism of commitment of erythropoiesis by activin A and BMP2 involving two key events, increase in EPO-R and decrease in GATA2 expression. Simultaneous addition of activin A with BMP molecules suggests that BMP2 and BMP4 differently affect activin A induction of erythropoiesis. Follistatin and FLRG proteins downmodulate the effects of activin A and BMP2 on erythroid maturation.     

Syndecan-1 regulates BMP signaling and dorso-ventral patterning of the ectoderm during early Xenopus development

Extracellular regulation of growth factor signaling is a key event for embryonic patterning. Heparan sulfate proteoglycans (HSPG) are among the molecules that regulate this signaling during embryonic development. Here we study the function of syndecan1 (Syn1), a cell-surface HSPG expressed in the non-neural ectoderm during early development of Xenopus embryos. Overexpression of Xenopus Syn1 (xSyn1) mRNA is sufficient to reduce BMP signaling, induce chordin expression and rescue dorso-ventral patterning in ventralized embryos. Experiments using chordin morpholinos established that xSyn1 mRNA can inhibit BMP signaling in the absence of chordin. Knockdown of xSyn1 resulted in a reduction of BMP signaling and expansion of the neural plate with the concomitant reduction of the non-neural ectoderm. Overexpression of xSyn1 mRNA in xSyn1 morphant embryos resulted in a biphasic effect, with BMP being inhibited at high concentrations and activated at low concentrations of xSyn1. Interestingly, the function of xSyn1 on dorso-ventral patterning and BMP signaling is specific for this HSPG. In summary, we report that xSyn1 regulates dorso-ventral patterning of the ectoderm through modulation of BMP signaling.     

BMP9经JNK激酶途径调控间充质干细胞C3H10T1/2成骨分化

为了证实JNK激酶在骨形态发生蛋白9(bone morphogenetic proteins 9,BMP9) 诱导间充质干细胞C3H10T1/2成骨分化中的作用,利用重组腺病毒将BMP9导入间充质干细胞C3H10T1/2. 通过碱性磷酸酶(ALP)活性测定、钙盐沉积实验、荧光素酶报告基因检测、Western印迹和组织化学染色等方法,检测BMP9是否可经JNK激酶途径调控间充质干细胞C3H10T1/2向成骨分化.动物实验验证在RNA沉默JNK蛋白激酶后,对BMP9诱导间充质干细胞C3H10T1/2向成骨分化的影响．结果发现,BMP9可以增强JNK 激酶的磷酸化;利用JNK抑制剂SP600125抑制JNK激酶活性后,BMP9诱导的间充质干细胞C3H10T1/2的早期成骨指标ALP活性和晚期指标钙盐沉积均受到抑制,而且经典SMAD信号的活化也相应受到抑制;RNA干扰沉默JNK基因表达后,同样也可抑制BMP9 诱导的C3H10T1/2细胞的ALP活性和裸鼠皮下异位成骨．因此表明,BMP9可活化JNK激酶途径从而诱导间充质干细胞C3H10T1/2向成骨分化．     

Activation of JNKs is essential for BMP9-induced osteogenic differentiation of mesenchymal stem cells

Although BMP9 is highly capable of promoting osteogenic differentiation of mesenchymal stem cell (MSCs), the molecular mechanism involved remains to be fully elucidated. Here, we explore the possible involvement and detail role of JNKs (c-Jun N-terminal kinases) in BMP9-induced osteogenic differentiation of MSCs. It was found that BMP9 stimulated the activation of JNKs in MSCs. BMP9-induced osteogenic differentiation of MSCs was dramatically inhibited by JNKs inhibitor SP600125. Moreover, BMP9-activated Smads signaling was decreased by SP600125 treatment in MSCs. The effects of inhibitor are reproduced with adenoviruses expressing siRNA targeted JNKs. Taken together, our results revealed that JNKs was activated in BMP9-induced osteogenic differentiation of MSCs. What is most noteworthy, however, is that inhibition of JNKs activity resulted in reduction of BMP9-induced osteogenic differentiation of MSCs, implying that activation of JNKs is essential for BMP9 osteoinductive activity. [BMB Reports 2013; 46(8):422-427]     

Cross‐talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross‐talks with BMP9 and regulates BMP9‐induced osteogenic differentiation. We find that EGF potentiates BMP9‐induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG‐1478 and AG‐494 in a dose‐ and time‐dependent manner. Furthermore, EGF significantly augments BMP9‐induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9‐induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up‐regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross‐talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine.     

Formation of stable homomeric and transient heteromeric bone morphogenetic protein (BMP) receptor complexes regulates Smad protein signaling

The type I and type II bone morphogenetic protein receptors (BMPRI and BMPRII) are present at the plasma membrane as monomers and homomeric and heteromeric complexes, which are modulated by ligand binding. The complexes of their extracellular domains with ligand were shown to form heterotetramers. However, the dynamics of the oligomeric interactions among the full-length receptors in live cell membranes were not explored, and the roles of BMP receptor homodimerization were unknown. Here, we investigated these issues by combining patching/immobilization of an epitope-tagged BMP receptor at the cell surface with measurements of the lateral diffusion of a co-expressed, differently tagged BMP receptor by fluorescence recovery after photobleaching (FRAP). These studies led to several novel conclusions. (a) All homomeric complexes (without or with BMP-2) were stable on the patch/FRAP time scale (minutes), whereas the heterocomplexes were transient, a difference that may affect signaling. (b) Patch/FRAP between HA- and myc-tagged BMPRII combined with competition by untagged BMPRIb showed that the heterocomplexes form at the expense of homodimers. (c) Stabilization of BMPRII·BMPRIb heterocomplexes (but not homomeric complexes) by IgG binding to same-tag receptors elevated phospho-Smad formation both without and with BMP-2. These findings suggest two mechanisms that may suppress the tendency of preformed BMP receptor hetero-oligomers to signal without ligand: (a) competition between homo- and heterocomplex formation, which reduces the steady-state level of the latter, and (b) the transient nature of the heterocomplexes, which limits the time during which BMPRI can be phosphorylated by BMPRII in the heterocomplex.     

Activation of the orphan receptor tyrosine kinase ALK by zinc

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase essentially and transiently expressed during development of the central and peripheral nervous system. The nature of the cognate ligand of this receptor in Vertebrates is still a matter of debate. During synaptic transmission the release of ionic zinc found in vesicles of certain glutamatergic and gabaergic terminals may act as a neuromodulator by binding to pre- or post-synaptic receptors. Recently, zinc has been shown to activate the receptor tyrosine kinase, TrkB, independently of neurotrophins. This activation occurs via increasing the Src family kinase activity. In the present study, we investigated whether the ALK activity could be modulated by extracellular zinc. We first showed that zinc alone rapidly activates ALK. This activation is dependent of ALK tyrosine kinase activity and dimerization of the receptor but is independent of Src family kinase activity. In contrast, addition of sodium pyrithione, a zinc ionophore, led to a further activation of ALK. This stronger activation is dependent of Src family kinase but independent of ALK activity and dimerization. In conclusion, zinc could constitute an endogenous ligand of ALK in vertebrates.     

Direct BMP2/4 signaling through BMP receptor IA regulates fetal thymocyte progenitor homeostasis and differentiation to CD4+CD8+ double-positive cell

BMP2/4 signaling is required for embryogenesis and involved in thymus morphogenesis and T-lineage differentiation. In vitro experiments have shown that treatment of thymus explants with exogenous BMP4 negatively regulated differentiation of early thymocyte progenitors and the transition from CD4−CD8− (DN) to CD4+CD8+ (DP). Here we show that in vivo BMP2/4 signaling is required for fetal thymocyte progenitor homeostasis and expansion, but negatively regulates differentiation from DN to DP cell. Unexpectedly, conditional deletion of BMPRIA from fetal thymocytes (using the Cre-loxP system and directing excision to hematopoietic lineage cells with the Vav promoter) demonstrated that physiological levels of BMP2/4 signaling directly to thymocytes through BMPRIA are required for normal differentiation and expansion of early fetal DN thymocytes. In contrast, the arrest in early thymocyte progenitor differentiation caused by exogenous BMP4 treatment of thymus explants is induced in part by direct signaling to thymocytes through BMPRIA, and in part by indirect signaling through non-hematopoietic cells. Analysis of the transition from fetal DN to DP cell, both by ex vivo analysis of conditional BMPRIA-deficient thymocytes and by treatment of thymus explants with the BMP4-inhibitor Noggin demonstrated that BMP2/4 signaling is a negative regulator at this stage. We showed that at this stage of fetal T-cell development BMP2/4 signals directly to thymocytes through BMPRIA.     

利用同源蛋白的信号肽和前肽序列在CHO细胞中表达重组人BMP6

BMP6属于TGF-β超家族,具有较强的骨诱导作用。主要利用BMP6自身的信号肽、前肽与成熟肽基因序列构建了表达质粒pcDNA-BMP6;同时利用BMP2的信号肽、前肽与BMP6的成熟肽基因序列构建了表达质粒pcDNA-BMP2/6。将两种质粒分别瞬时转染Cos7细胞,发现质粒pcDNA-BMP2/6表达rhBMP6的效率高于质粒pcDNA-BMP6。然后将质粒pcDNA-BMP2/6与含二氢叶酸还原酶基因(dhfr)的表达质粒共转染dhfr缺陷型的中华仓鼠卵巢(CHO)细胞,经G418筛选、氨甲蝶呤(MTX)介导目的基因扩增、亚克隆后得到表达rhBMP6成熟肽的单克隆细胞株。将表达产物rhBMP6初步纯化后,能诱导前成肌细胞系C2C12向成骨细胞方向转化,显示其具有骨诱导作用。     

Regulation of actin function by protein kinase A‐mediated phosphorylation of Limk1

Proper regulation of the cAMP-dependent protein kinase (protein kinase A, PKA) is necessary for cellular homeostasis, and dysregulation of this kinase is crucial in human disease. Mouse embryonic fibroblasts (MEFs) lacking the PKA regulatory subunit Prkar1a show altered cell morphology and enhanced migration. At the molecular level, these cells showed increased phosphorylation of cofilin, a crucial modulator of actin dynamics, and these changes could be mimicked by stimulating the activity of PKA. Previous studies of cofilin have shown that it is phosphorylated primarily by the LIM domain kinases Limk1 and Limk2, which are under the control of the Rho GTPases and their downstream effectors. In Prkar1a^−/− MEFs, neither Rho nor Rac was activated; rather, we showed that PKA could directly phosphorylate Limk1 and thus enhance the phosphorylation of cofilin. These data indicate that PKA is crucial in cell morphology and migration through its ability to modulate directly the activity of LIM kinase.