Neuropeptide W regulates proliferation and differentiation of ATDC5: Possible involvement of GPR7 activation,PKA and PKC‐dependent signalling cascades

Various neuropeptides related to the energy equilibrium affect bone growth in humans and animals. Neuropeptides W (NPW) are identical in the internal ligands of the two G‐protein receptors (GPRs) included in subtypes 7 and 8. Neuropeptides W inhibits proliferation in the cultivated rat calvarial osteoblast‐like (ROB) cells. This study examines the expression of NPW and GPR7 in murine chondrocyte and their function. An immunohistochemical analysis showed that NPW and GPR7 were expressed in the proliferative chondrocytes of the growth plates in the hind limbs of mice. The NPW mRNA quickly elevated in the early differentiation (7‐14 days) of ATDC5 cells, while NPW and GPR7 mRNA were reduced during the late stage (14‐21 days) of differentiation. Neuropeptide W‐23 (NPW‐23) promoted the proliferation of ATDC5 cells, which was attenuated by inhibiting the GPR7, protein kinase A (PKA), protein kinase C (PKC) and ERK1/2 pathways. Neuropeptide W‐23 enhanced the early cell differentiation, as evaluated by collagen type II and the aggrecan gene expression, which was unaffected by inhibiting the ERK1/2 pathway, but significantly decreased by inhibiting the PKA, PKC and p38 MAPK pathways. In contrast, NPW‐23 was not involved in the terminal differentiation of the chondrocytes, as evaluated by the mineralization of the chondrocytes and the activity of the alkaline phosphatase. Neuropeptides W stimulated the PKA, PKC, p38 MAPK and ERK1/2 activities in a dose‐ and time‐dependent manner in the ATDC5 cells. These results show that NPW promotes the proliferation and early differentiation of murine chondrocyte via GPR7 activation, as well as PKA and PKC‐dependent signalling cascades, which may be involved in endochondral bone formation.     

Gas1 cooperates with Cdo and promotes myogenic differentiation via activation of p38MAPK

Skeletal myogenesis is a multistep process that involves cell cycle exit, expression of muscle-specific genes and formation of multinucleated myotubes. Growth arrest specific gene 1 (Gas1) is a GPI-linked membrane protein and originally identified as a growth arrest-linked gene in fibroblasts. Promyogenic cell surface protein, Cdo functions as a component of multiprotein complexes that include other cell adhesion molecules, like Cadherins to mediate cell contact signaling. Here we report that Gas1 and Cdo are coexpressed in muscle cells and form a complex in differentiating myoblasts. Interestingly, Cdo^−/− myoblasts display defects in Gas1 induction during differentiation. Overexpression or depletion of Gas1 enhances or decreases myogenic differentiation, respectively. During myoblast differentiation, Gas1 depletion causes defects in downregulation of Cdk2 and Cyclin D1 and up-regulation of miR-322, a negative regulator of Cdk2 activities. Furthermore overexpression or knockdown of Gas1 either enhances or decreases activation of p38MAPK that functions downstream of Cdo. Additionally, Gas1 overexpression in Cdo-depleted C2C12 cells restores p38MAPK activities and differentiation abilities. These data suggest that Gas1 promotes myogenic differentiation through regulation of cell cycle arrest and is critical to activate p38MAPK, most likely via association with Cdo/Cadherin multiprotein complexes.     

IL-6通过MAPK/ERK信号通路调节BMSCs的软骨定向分化

白细胞介素-6（IL-6）是参与骨髓间充质干细胞（BMSCs）软骨定向分化的重要调节因子. MAPK/ERK信号通路可介导骨关节炎软骨损伤. 然而,IL-6调节BMSCs定向分化为软骨细胞的分子机制尚不清楚. IL-6通过激活MAPK/ERK信号途径,抑制BMSCs的成软骨分化. 本文发现,BMSCs在体外向软骨细胞分化时, Il-6基因表达水平显著下调,同时分泌到培养基中的IL-6蛋白水平亦明显降低. 重组IL-6可抑制BMSCs向软骨细胞分化,软骨分化标志蛋白Runx2和Sox9的诱导表达亦相应下调. IL-6可诱导MAPK/ERK信号通路活化,加入ERK特异性阻断剂后,Runx2和Sox9的诱导表达恢复正常.结果提示,IL-6通过激活MAPK/ERK信号通路抑制BMSCs的软骨细胞分化.炎症因子IL-6对软骨细胞的再生具有不利的影响,该研究为软骨组织工程研究和骨关节炎等软骨疾病的治疗提供有价值的参考.     

Effects of compressive force on the differentiation of pluripotent mesenchymal cells

The purpose of this study was to determine the effect of mechanical stress on the differentiation of the pluripotent mesenchymal cell line C2C12. C2C12 cells were cultured continuously under compressive force (0.25-2.0 g/cm(2)). After mechanical stress loading, the levels of expression of mRNAs and proteins for phenotype-specific markers of osteoblasts (Runx2, Msx2, Dlx5, Osterix, AJ18), chondroblasts (Sox5, Sox9), myoblasts (MyoD), and adipocytes (PPAR gamma) were measured by real-time polymerase chain reaction analysis and Western blot analysis, respectively. The expression of activated p38 mitogen-activated protein kinase (p38 MAPK) was measured by Western blotting and/or ELISA. Loading 0.5 g/cm(2) of compressive force significantly increased the expression levels of Runx2, Msx2, Dlx5, Osterix, Sox5, and Sox9. In contrast, the expression levels of AJ18, MyoD, and PPAR gamma were decreased by exposure to 0.5 g/cm(2) of compressive force. Loading 0.5 g/cm(2) of compressive force also induced the phosphorylation of p38 MAPK. SB203580, which is a specific inhibitor of p38 MAPK, inhibited the compressive force-induced phosphorylation of p38 MAPK and partially blocked compressive force-induced Runx2 mRNA expression. These results demonstrate that compressive force stimulation directs the differentiation pathway of C2C12 cells into the osteoblast and chondroblast lineage via activated phosphorylation of p38 MAPK.     

BDNF alters ERK/p38 MAPK activity ratios to promote differentiation in growth plate chondrocytes

The ERK and p38 MAPK pathways are well-known transducers of signals that regulate proliferation and differentiation, but precisely how these pathways control growth plate chondrocyte development is unclear. For example, the ERK pathway has been reported to be required by some investigators but inhibitory to chondrocyte development by others. Moreover, how these two pathways interact to regulate chondrocyte development is even less clear. Using primary bovine growth plate chondrocytes and murine ATDC5 cells, we demonstrate that the ERK and p38 pathways have opposing effects on proliferation but are both absolutely required for differentiation. Two factors that promote chondrocyte differentiation, brain-derived neurotrophic factor (BDNF) and C-type natriuretic peptide, increase p38 activity while decreasing, but not completely inhibiting, ERK activity. The attenuation of ERK activity by BDNF occurs via p38-dependent raf-1 inhibition. The inhibition of raf-1 by p38 is direct, because purified p38 protein inhibits the kinase activity of purified active raf-1 as well as raf-1 immunoprecipitated from chondrocyte lysates. Moreover, IGF-I, which stimulates proliferation, suppresses p38 activation. This work describes a model wherein unopposed IGF-I promotes high ERK/p38 activity ratios favoring proliferation, whereas BDNF signals a transition to differentiation by decreasing the ERK/p38 activity ratio without completely inhibiting ERK, which involves the direct inhibition of raf-1 by p38.     

BMP-2-enhanced chondrogenesis involves p38 MAPK-mediated down-regulation of Wnt-7a pathway

The bone morphogenetic protein (BMP) family has been implicated in control of cartilage development. Here, we demonstrate that BMP-2 promotes chondrogenesis by activating p38 mitogen-activated protein kinase (MAPK), which in turn downregulates Wnt-7a/b-catenin signaling responsible for proteasomal degradation of Sox9. Exposure of mesenchymal cells to BMP-2 resulted in upregulation of Sox9 protein and a concomitant decrease in the level of b-catenin protein and Wnt-7a signaling. In agreement with this, the interaction of Sox9 with b-catenin was inhibited in the presence of BMP-2. Inhibition of the p38 MAPK pathway using a dominant negative mutant led to sustained Wnt-7a signaling and decreased Sox9 expression, with consequent inhibition of precartilage condensation and chondrogenic differentiation. Moreover, overexpression of b-catenin caused degradation of Sox9 via the ubiquitin/26S proteasome pathway. Our results collectively indicate that the increase in Sox9 protein resulting from downregulation of b-catenin/Wnt-7a signaling is mediated by p38 MAPK during BMP-2 induced chondrogenesis in chick wing bud mesenchymal cells.     

LRP4 induces extracellular matrix productions and facilitates chondrocyte differentiation

Endochondral ossification is an essential step for skeletal development, which requires chondrocyte differentiation in growth cartilage. The low-density lipoprotein receptor-related protein 4 (LRP4), a member of LDLR family, is an inhibitor for Wnt signaling, but its roles in chondrocyte differentiation remain to be investigated. Here we found by laser capture microdissection that LRP4 expression was induced during chondrocyte differentiation in growth plate. In order to address the roles, we overexpressed recombinant human LRP4 or knocked down endogenous LRP4 by lentivirus in mouse ATDC5 chondrocyte cells. We found that LRP4 induced gene expressions of extracellular matrix proteins of type II collagen (Col2a1), aggrecan (Acan), and type X collagen (Col10a1), as well as production of total proteoglycans in ATDC5 cells, whereas LRP4 knockdown had opposite effects. Interestingly, LRP4-knockdown reduced mRNA expression of Sox9, a master regulator for chondrogenesis, as well as Dkk1, an extracellular Wnt inhibitor. Analysis of Wnt signaling revealed that LRP4 blocked the Wnt/β-catenin signaling activity in ATDC5 cells. Finally, the reduction of these extracellular matrix productions by LRP4-knockdown was rescued by a β-catenin/TCF inhibitor, suggesting that LRP4 is an important regulator for extracellular matrix productions and chondrocyte differentiation by suppressing Wnt/β-catenin signaling.     

p21Cip1 and p27Kip1 induce distinct cell cycle effects and differentiation programs in myeloid leukemia cells

The cyclin-dependent kinase (Cdk) inhibitors p21(Cip1) and p27(Kip1) have been proposed to exert redundant functions in cell cycle progression and differentiation programs, although nonoverlapping functions have also been described. To gain further insights into the relevant mechanisms and to detect possible functional differences between both proteins, we conditionally expressed p21(Cip1) and p27(Kip1) in K562, a multipotent human leukemia cell line. Temporal ectopic expression of either p21(Cip1) or p27(Kip1) arrested proliferation, inhibited Cdk2 and Cdk4 activities, and suppressed retinoblastoma phosphorylation. However, whereas p21(Cip1) arrested cells in both G(1) and G(2) cell cycle phases, p27(Kip1) blocked the G(1)/S-phase transition. Furthermore, although both p21(Cip1) and p27(Kip1) associated with Cdk6, only p27(Kip1) significantly inhibited its activity. Most importantly, each protein promoted differentiation along a distinct pathway; p21(Cip1) triggered megakaryocytic maturation, whereas p27(Kip1) resulted in the expression of erythroid markers. Consistently, p21(Cip1) and p27(Kip1) were rapid and transiently up-regulated when K562 cells are differentiated into megakaryocytic and erythroid lineages, respectively. These findings demonstrate distinct functions of p21(Cip1) and p27(Kip1) in cell cycle regulation and differentiation and indicate that these two highly related proteins possess unique biological activities and are not functionally interchangeable.     

Pannexin 3 Regulates Intracellular ATP/cAMP Levels and Promotes Chondrocyte Differentiation

Pannexin 3 (Panx3) is a new member of the gap junction pannexin family, but its expression profiles and physiological function are not yet clear. We demonstrate in this study that Panx3 is expressed in cartilage and regulates chondrocyte proliferation and differentiation. Panx3 mRNA was expressed in the prehypertrophic zone in the developing growth plate and was induced during the differentiation of chondrogenic ATDC5 and N1511 cells. Panx3-transfected ATDC5 and N1511 cells promoted chondrogenic differentiation, but the suppression of endogenous Panx3 inhibited differentiation of ATDC5 cells and primary chondrocytes. Panx3-transfected ATDC5 cells reduced parathyroid hormone-induced cell proliferation and promoted the release of ATP into the extracellular space, possibly by action of Panx3 as a hemichannel. Panx3 expression in ATDC5 cells reduced intracellular cAMP levels and the activation of cAMP-response element-binding, a protein kinase A downstream effector. These Panx3 activities were blocked by anti-Panx3 antibody. Our results suggest that Panx3 functions to switch the chondrocyte cell fate from proliferation to differentiation by regulating the intracellular ATP/cAMP levels.     

Suppression of c-Src activity stimulates muscle differentiation via p38 MAPK activation

Role of c-Src in muscle differentiation has been controversial. Here, we investigated if c-Src positively or negatively regulates muscle differentiation, using H9c2 and C2C12 cell lines. Inhibition of c-Src by treatment with PP1 and SU6656, pharmacologic inhibitors of Src family kinases, or by expression of a dominant negative c-Src, all induced muscle differentiation in proliferation medium (PM). In differentiating cells in differentiation medium (DM), c-Src activity gradually decreased and reached basal level 3 days after induction of differentiation. Inhibition of c-Src suppressed Raf/MEK/ERK pathway but activated p38 MAPK. Inhibition of p38 MAPK did not affect c-Src activity in PM. However, it reactivated Raf/MEK/ERK pathway in c-Src-inhibited cells regardless of PM or DM. Concomitant inhibition of c-Src and p38 MAPK activities blocked muscle differentiation in both media. In conclusion, suppression of c-Src activity stimulates muscle differentiation by activating p38 MAPK uni-directionally.     

Non-Classical P38 Map Kinase Functions: Cell Cycle Checkpoints and Survival

The p38 MAPK kinase pathway is activated in response to a wide range of cellular stress stimuli and cytokines. Our understanding of the important functions of p38 MAPK in the process of differentiation and cell death has grown considerably in the recent years and is now relatively established. Here we discuss the role of p38 MAPK in the mediation of cell cycle checkpoints and cell survival, processes that have received less attention. We describe how p38 MAPK regulates both the G2/M as well as a G1/S cell cycle checkpoint in response to cellular stress such as DNA damage. While p38 MAPK has classically been associated with the induction of apoptosis, we discuss that p38 MAPK can also mediate cell survival in specific situations, such as in response to DNA damage. It is important to recognize these less appreciated functions of p38 MAPK when considering the potential use of pharmacological inhibitors of p38 MAPK in therapeutic treatments for disease.     

Modulation of vascular smooth muscle cell growth by magnesium-role of mitogen-activated protein kinases

We tested the hypothesis that Mg(2+) influences growth of vascular smooth muscle cells (VSMCs) by modulating cell cycle activation through mitogen-activated protein (MAP) kinase-dependent pathways. Rat VSMCs were grown in culture medium containing normal Mg(2+) (1.02 mmol/L, control) and increasing concentrations of Mg(2+) (2-4 mmol/L) for 1-8 days. Effects of varying extracellular Mg(2+) concentration ([Mg(2+)](e)) on intracellular free Mg(2+) concentration ([Mg(2+)](i)) were assessed using mag-fura. Growth actions of Mg(2+) were evaluated by measuring cell cycle activation, DNA synthesis, and protein synthesis. Expression of cell cycle promoters, cyclin D1, cyclin E, Cdk2, and Cdk4 was assessed by immunoblotting. Phosphorylation of cell cycle inhibitors p21(cip1) and p27(kip1) and MAP kinases, ERK1/2, p38MAP kinase, and JNK was evaluated using phospho-specific antibodies. [Mg(2+)](i) increased in a dose-dependent manner in response to increasing [Mg(2+)](e). These effects were evident within 2 days and maximal responses were obtained after 6 days. High [Mg(2+)](e) induced cell cycle activation with a lower proportion of cells in G(1) phase (75 +/- 1.0%) and a higher fraction of cells in S phase (12 +/- 0.7%) versus control (G(1), 88.5 +/- 1.4%; S, 6.8 +/- 1.2%; P < 0.05). This was associated with increased protein content of cyclin D1 and Cdk4 and decreased activation of p21(cip1) and p27(kip1). In cells exposed to 2 mmol/L Mg(2+), DNA and protein synthesis was increased approximately threefold. Phosphorylation of MEK1/2 and ERK1/2 was enhanced two to threefold in cells grown in 2 mmol/L Mg(2+). These effects were rapid, occurring within 2 days. Phosphorylation of MEK3/6, p38 MAP kinase, and JNK was unaltered by increasing [Mg2](e). PD98059 (10(-5) mol/L), specific MEK1/2 inhibitor, but not SB202190 (10(-5) mol/L) (specific p38 MAP kinase inhibitor), attenuated Mg(2+)-induced growth actions. These data demonstrate the novel findings that cell cycle activation and growth regulation by Mg(2+) occurs via ERK1/2-dependent, p38 MAP kinase-independent pathways.