HtrA1 is upregulated during RANKL-induced osteoclastogenesis,and negatively regulates osteoblast differentiation and BMP2-induced Smad1/5/8, ERK and p38 phosphorylation

Bone remodeling is regulated by secreted factors in the bone microenvironment. However, data regarding osteoclast-derived factors that influence osteoblast differentiation are lacking. Here, we show that HtrA1 is produced as a secreted protein during osteoclastogenesis, and negatively regulates osteoblast differentiation. Exogenous addition of recombinant HtrA1 attenuates osteoblast differentiation and BMP2-induced Smad1/5/8, ERK1/2 and p38 phosphorylation in pre-osteoblasts. Our studies imply a unique mode of crosstalk in which HtrA1 is produced by both osteoclasts and osteoblasts and negatively regulates osteoblast differentiation, suggesting that HtrA1 may mediate the fine tuning of paracrine and autocrine regulations during bone remodeling processes.     

BMP10 positively regulates myogenic differentiation in C2C12 myoblasts via the Smad 1/5/8 signaling pathway

Molecular and Cellular Biochemistry - BMP10 plays an essential role in regulating cardiac growth, chamber maturation, and maintaining normal expressions of several key cardiogenic factors; however,...     

DUSP6 expression is associated with osteoporosis through the regulation of osteoclast differentiation via ERK2/Smad2 signaling

Osteoporosis-related fractures, such as femoral neck and vertebral fractures, are common in aged people, resulting in increased disability rate and health-care costs. Thus, it is of great importance to clarify the mechanism of osteoclast-related osteoporosis and find effective ways to avoid its complication. In this study, gene expression profile analysis and real-time polymerase chain reaction revealed that DUSP6 expression was suppressed in human and mice osteoporosis cases. In vitro experiments confirmed that DUSP6 overexpression prevented osteoclastogenesis, whereas inhibition of DUSP6 by small interference RNA or with a chemical inhibitor, (E/Z)-BCI, had the opposite effect. (E/Z)-BCl significantly accelerated the bone loss process in vivo by enhancing osteoclastogenesis. Bioinformatics analyses and in vitro experiments indicated that miR-181a was an upstream regulator of DUSP6. Moreover, miR-181a positively induced the differentiation and negatively regulated the apoptosis of osteoclasts via DUSP6. Furthermore, downstream signals by ERK2 and SMAD2 were also found to be involved in this process. Evaluation of ERK2-deficiency bone marrow-derived macrophages confirmed the role of ERK2 signaling in the DUSP6-mediated osteoclastogenesis. Additionally, immunoprecipitation assays confirmed that DUSP6 directly modified the phosphorylation status of SMAD2 and the subsequent nuclear transportation of NFATC1 to regulate osteoclast differentiation. Altogether, this study demonstrated for the first time the role of miRNA-181a/DUSP6 in the progression of osteoporosis via the ERK2 and SMAD2 signaling pathway. Hence, DUSP6 may represent a novel target for the treatment of osteoclast-related diseases in the future.Subject terms: Bone, Osteoporosis     

Ubiquitin C-terminal hydrolase-L3 regulates Smad1 ubiquitination and osteoblast differentiation

Ubiquitin C-terminal hydrolase-L3 (Uch-L3), a deubiquitinating enzyme, is upregulated in bone morphogenetic protein 2-induced osteoblast differentiation. The mechanism and role of Uch-L3 in the process of osteoblast differentiation is unknown. We found that Uch-L3 physically interacts with Smad1 and dramatically decreases the amount of poly-ubiquitinated Smad1. Osteoblast differentiation was enhanced in the C2C12 cells stably transfected with Uch-L3. Otherwise, the siRNA knock-down of Uch-L3 resulted in the decrease of osteoblast differentiation. These results suggest that Uch-L3 enhances osteoblast differentiation through the stabilization of Smad1 signaling. Thus, Uch-L3 acts to fine-tune the process of Smad1 activation.     

N-cadherin regulates cytoskeletally associated IQGAP1/ERK signaling and memory formation

Cadherin-mediated interactions are integral to synapse formation and potentiation. Here we show that N-cadherin is required for memory formation and regulation of a subset of underlying biochemical processes. N-cadherin antagonistic peptide containing the His-Ala-Val motif (HAV-N) transiently disrupted hippocampal N-cadherin dimerization and impaired the formation of long-term contextual fear memory while sparing short-term memory, retrieval, and extinction. HAV-N impaired the learning-induced phosphorylation of a distinctive, cytoskeletally associated fraction of hippocampal Erk-1/2 and altered the distribution of IQGAP1, a scaffold protein linking cadherin-mediated cell adhesion to the cytoskeleton. This effect was accompanied by reduction of N-cadherin/IQGAP1/Erk-2 interactions. Similarly, in primary neuronal cultures, HAV-N prevented NMDA-induced dendritic Erk-1/2 phosphorylation and caused relocation of IQGAP1 from dendritic spines into the shafts. The data suggest that the newly identified role of hippocampal N-cadherin in memory consolidation may be mediated, at least in part, by cytoskeletal IQGAP1/Erk signaling.     

Serelaxin inhibits differentiation and fibrotic behaviors of cardiac fibroblasts by suppressing ALK-5/Smad2/3 signaling pathway

Serelaxin, a recombinant form of human relaxin-2, is currently regarded as a novel drug for treatment of acute heart failure. However, whether therapeutic effects of serelaxin are achieved by inhibiting cardiac fibrosis remains unclear. In this study, we investigate effects of serelaxin on inhibiting cardiac fibrosis. Cardiac fibroblasts (CFs) were isolated from the hearts of adult rats. Effects of serelaxin on differentiation of CFs towards myofibroblasts (MFs) and their fibrotic behaviors after induction with TGF-β1 were examined. Synthesis and degradation of collagens, secretion of IL-10, and expression of ALK-5 and p-Smad2/3 of TGF-β1-induced cells were assessed after treatment with serelaxin. Serelaxin inhibited differentiation of TGF-β1-induced CFs towards MFs, and reduced proliferation and migration of the induced cells. Moreover, serelaxin down-regulated expression of collagen I/III and TIMP-2, and up-regulated expression of MMP-2 and MMP-9 in the cells. After treatment with serelaxin, activity of MMP-2 and MMP-9 and secretion of IL-10 increased, expression of ALK-5 and the level of Smad2/3 phosphorylation was reduced significantly. These results suggest that serelaxin can inhibit differentiation of TGF-β1-induced CFs towards MFs, reduce production of collagens by suppressing ALK-5/Smad2/3 signaling pathway, and enhance extracellular matrix degradation by increasing MMP-2/TIMP-2 ratio and IL-10 secretion. Serelaxin may be a potential therapeutic drug for inhibiting cardiac fibrosis.     

Lanthanum enhances in vitro osteoblast differentiation via pertussis toxin-sensitive gi protein and ERK signaling pathway

Converging lines of evidence suggest that lanthanum tends to deposit in bone. The influence of lanthanum ion (La3+) on osteoblast differentiation and the related mechanism are essential to understanding its effect on bone metabolism. In this study, La3+ treatment enhanced in vitro osteoblast differentiation as evidenced by promoting alkaline phosphatase (ALP) activity, osteocalcin (OC) secretion, and matrix mineralization. The expressions of osteoblast-specific genes of Cbfa-1, osteopontin (OPN), and bone sialoprotein (BSP) were all increased in the presence of La3+, but no change was observed in that of type I collagen (COL-I). Further studies demonstrated that La3+ treatment enhanced phosphorylation of extracellular signal-regulated kinase (ERK). Inhibition of ERK activation by U0126 suppressed the effects of La3+ on osteoblast activity. Moreover, pretreatment of the cells with pertussis toxin (PTx), a Gi protein inhibitor, suppressed the La3+-enhanced ERK phosphorylation and osteoblast differentiation. These findings suggest that La3+ exposure enhances in vitro osteoblast differentiation and the effect depends on ERK phosphorylation via PTx-sensitive Gi protein signaling.     

Dok5 regulates proliferation and differentiation of osteoblast via canonical Wnt/β-catenin signaling

Objective:In bone tissue engineering, the use of osteoblastic seed cells has been widely adopted to mediate the osteogenic differentiation so as to prompt bone regeneration and repair. It is hypothesized that Dok5 can regulate the proliferation and differentiation of osteoblasts. In this study, the role of Dok5 in osteoblast proliferation and differentiation was investigated.Methods:A lentiviral vector to silence Dok5 was transferred to C3H10, 293T and C2C12 cells. CCK-8 assay was used to detect the cell proliferation. Cells were stained by ALP and AR-S staining. Western blot and RT-PCR were used to detect the expression levels of related factors.Results:Dok5 expression level was gradually up-regulated during the osteoblast differentiation. Dok5 silencing down-regulated the expression levels of osteogenic biosignatures OPN, OCN, and Runx2 and suppressed the osteogenesis. Additionally, the osteoblast proliferation and canonical Wnt/β-catenin signaling were suppressed upon Dok5 knockdown, β-catenin expression level was significantly down-regulated in the knockdown group, while the expression levels of GSK3-β and Axin, negative regulators in the Wnt signaling pathway, were up-regulated. Furthermore, overexpression of Dok5 promoted the proliferation and osteogenesis and activated the canonical Wnt/β-catenin signaling pathway.Conclusion:Dok5 may regulate the osteogenic proliferation and differentiation via the canonical Wnt/β-catenin signaling pathway.     

Death effector domain DEDa, a self-cleaved product of caspase-8/Mch5, translocates to the nucleus by binding to ERK1/2 and upregulates procaspase-8 expression via a p53-dependent mechanism

Activation of the apical caspase-8 is crucial to the extrinsic apoptotic pathway. Although the death effector domain (DED) of caspase-8 has been reported to be involved in death-inducing signaling complex formation, the detailed mechanism of how DED functions in regulating apoptosis remains largely unknown. Here, we demonstrate that the prodomain of the caspase-8/Mch5 can be further cleaved between two tandemly repeated DEDs (DEDa-DEDb) at the amino-acid residue Asp129 by caspase-8 itself. The DEDa fragment generated from the endogenous caspase-8 was detected in isolated nucleoli upon treatment with TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). Cleaved DEDa appears to translocate into the nucleus by association with extracellular signal-regulated protein kinases-1/2 (ERK1/2). Elimination of ERK1/2 expression by RNA interference resulted in a significant attenuation of nuclear entry of DEDa and reduced caspase-8-dependent apoptosis. In the nucleus, DEDa interacts with TOPORS, a p53 and topoisomerase I binding protein, and possibly displaces p53 from TOPORS, allowing p53 to stimulate caspase-8 gene expression. In summary, we postulate a positive feedback loop involving DEDa, which enables the continual replenishment of procaspase-8 during apoptosis.     

Mxi2 sustains ERK1/2 phosphorylation in the nucleus by preventing ERK1/2 binding to phosphatases

ERK1/2 (extracellular-signal-regulated kinase 1/2) MAPKs (mitogen-activated protein kinases) are tightly regulated by the cellular microenvironment in which they operate. Mxi2 is a p38α splice isoform capable of binding to ERK1/2 and ensuring their translocation to the nucleus. Therein Mxi2 sustains ERK1/2 phosphorylation levels and, as a consequence, ERK1/2 nuclear signals are enhanced. However, the molecular mechanisms underlying this process are still unclear. In the present study, we show that Mxi2 prevents nuclear but not cytoplasmic phosphatases from binding to and dephosphorylating ERK1/2, disclosing an unprecedented mechanism for the spatial regulation of ERK1/2 activation. We also demonstrate that the kinetics of ERK1/2 extranuclear signals can be significantly altered by artificially tethering Mxi2 to the cytoplasm. In this case, Mxi2 abolishes ERK1/2 inactivation by cytoplasmic phosphatases and potentiates ERK1/2 functions at this compartment. These results highlight Mxi2 as a key spatial regulator of ERK1/2 functions, playing a pivotal role in the balance between ERK1/2 nuclear and cytoplasmic signals.     

Smurf1 facilitates myogenic differentiation and antagonizes the bone morphogenetic protein-2-induced osteoblast conversion by targeting Smad5 for degradation

Controlled proteolysis mediated by Smad ubiquitination regulatory factors (Smurfs) plays a crucial role in modulating cellular responses to signaling of the transforming growth factor-beta (TGF-beta) superfamily. However, it is not clear what influences the selectivity of Smurfs in the individual signaling pathway, nor is it clear the biological function of Smurfs in vivo. Using a mouse C2C12 myoblast cell differentiation system, which is subject to control by both TGF-beta and bone morphogenetic protein (BMP), here we examine the role of Smurf1 in myogenic differentiation. We show that increased expression of Smurf1 promotes myogenic differentiation of C2C12 cells and blocks the BMP-induced osteogenic conversion but has no effect on the TGF-beta-induced differentiation arrest. Consistent with an inhibitory role in the BMP signaling pathway, the elevated Smurf1 markedly reduces the level of endogenous Smad5, whereas it leaves unaltered that of Smad2, Smad3, and Smad7, which are components of the TGF-beta pathway. Adding back Smad5 from a different source to the Smurf1-overexpressing cells restores the BMP-mediated osteoblast conversion. Finally, by depletion of the endogenous Smurf1 through small interfering RNA-mediated RNA interference, we demonstrate that Smurf1 is required for the myogenic differentiation of C2C12 cells and plays an important regulatory role in the BMP-2-mediated osteoblast conversion.     

FAM3D inhibits glucagon secretion via MKP1-dependent suppression of ERK1/2 signaling

Dysregulated glucagon secretion is a hallmark of type 2 diabetes (T2D). To date, few effective therapeutic agents target on deranged glucagon secretion. Family with sequence similarity 3 member D (FAM3D) is a novel gut-derived cytokine-like protein, and its secretion timing is contrary to that of glucagon. However, the roles of FAM3D in metabolic disorder and its biological functions are largely unknown. In the present study, we investigated whether FAM3D modulates glucagon production in mouse pancreatic alpha TC1 clone 6 (αTC1-6) cells. Glucagon secretion, prohormone convertase 2 (PC2) activity, and mitogen-activated protein kinase (MAPK) pathway were assessed. Exogenous FAM3D inhibited glucagon secretion, PC2 activity, as well as extracellular-regulated protein kinase 1/2 (ERK1/2) signaling and induced MAPK phosphatase 1 (MKP1) expression. Moreover, knockdown of MKP1 and inhibition of ERK1/2 abolished and potentiated the inhibitory effect of FAM3D on glucagon secretion, respectively. Taken together, FAM3D inhibits glucagon secretion via MKP1-dependent suppression of ERK1/2 signaling. These results provide rationale for developing the therapeutic potential of FAM3D for dysregulated glucagon secretion and T2D.     

miR-145-5p attenuates hypertrophic scar via reducing Smad2/Smad3 expression

The study was designed to explore the underlying mechanism of micro ribonucleic acids (miR)-145-5p in the process of hypertrophic scar (HS). The difference in the relative content of miR-145-5p between HS and adjacent normal skin collected from 5 patients was detected via RT-PCR. Expressions of Smad2 and Smad3 with or without TGF-β1 was detected by western blotting. Fibroblasts apoptosis rate was examined by Annexin V/Propidium Iodide double staining. HS fibroblasts (HSFs) were isolated from HS tissues, cultured and then divided into control group, miR-145-5p inhibitor group (transfected with miR-145-5p inhibitor) and miR-145-5p mimic group (transfected with miR-145-5p plasmid) based on different treatment methods. Next, CCK-8 was employed to examine the function of miR-145-5p in HSF proliferation. Luciferase assay was conducted to confirm whether Smad2/3 were direct targets of miR-145-5p, and RT-PCR was done to measure the expression of miR-145-5p, Smad2/Smad3 and fibrosis-related genes of fibroblasts in three groups. Wound injury mice model was established to determine the function of miR-145-5p in regulating scar formation. miR-145-5p was found lowly expressed in HS tissues. Compared with Control group, miR-145-5p mimic decreased the levels of Smad2/3, arrested the activation and proliferation of HSFs and induced HSFs apoptosis. Overexpressing miR-145-5p achieved the contrary results. Smad2/3 was confirmed as the target of miR-145-5p. Moreover, miR-145-5p mimic decreased the recruitment of fibroblasts in vivo and decreased the expression of fibrosis-related genes after wound injury. In conclusion, miR-145-5p arrests the development of fibrogenesis and decreases HS formation by reducing the expression of Smad2/3. miR-145-5p may be an optional novel molecular target for treating HS.     

CCN3/NOV inhibits BMP-2-induced osteoblast differentiation by interacting with BMP and Notch signaling pathways

We elucidate the role of CCN3/NOV, a member of the CCN family proteins, in osteoblast differentiation using MC3T3-E1 osteoblastic cells. Transduction with CCN3 adenovirus (AdCCN3) alone induced no apparent changes in the expression of osteoblast-related markers, whereas cotransduction with BMP-2 adenovirus (AdBMP-2) and AdCCN3 significantly inhibited the AdBMP-2-induced mRNA expression of Runx2, osterix, ALP, and osteocalcin. Immunoprecipitation-western analysis revealed that CCN3 associated with BMP-2. Compared to transduction with AdBMP-2 alone, cotransduction with AdBMP-2 and AdCCN3 attenuated the expression of phosphorylated Smad1/5/8 and the mRNA for Id1, Id2, and Id3. Transduction with AdCCN3 stimulated the expression of cleaved Notch1, the mRNA expression of Hes1 and Hey1/Hesr1, and the promoter activities of Hes1 and Hey1. The inhibitory effects of CCN3 on the expression of BMP-2-induced osteoblast-related markers were nullified in Hey1-deficient osteoblastic cells. These results indicate that CCN3 exerts inhibitory effects on BMP-2-induced osteoblast differentiation by its involvement of the BMP and Notch signaling pathways.