Regulation of C2C12 myogenic terminal differentiation by MKK3/p38alpha pathway

The signal transduction pathwaysconnecting cell surface receptors to the activation of muscle-specificpromoters and leading to myogenesis are still largely unknown.Recently, a contribution of the p38 mitogen-activated protein kinase(MAPK) pathway to this process was evoked through the use ofpharmacological inhibitors. We used several mutants of the kinasescomposing this pathway to modulate the activity of the muscle-specificmyosin light chain and myogenin promoters in C2C12 cells by transienttransfections. In addition, we show for the first time, using a stableC2C12 cell line expressing a dominant-negative form of the p38activator MAPK kinase (MKK)3, that a functional p38 MAPK pathway isindeed required for terminal muscle cell differentiation. The mostobvious phenotype of this cell line, besides the inhibition of theactivation of p38, is its inability to undergo terminaldifferentiation. This phenotype is accompanied by a drastic inhibitionof cell cycle and myogenesis markers such as p21, p27, MyoD, andtroponin T, as well as a profound disorganization of the cytoskeleton.

     

p85alpha regulates osteoblast differentiation by cross-talking with the MAPK pathway

Class IA phosphoinositide 3-kinase (PI3K) is involved in regulating many cellular functions including cell growth, proliferation, cell survival, and differentiation. The p85 regulatory subunit is a critical component of the PI3K signaling pathway. Mesenchymal stem cells (MSC) are multipotent cells that can be differentiated into osteoblasts (OBs), adipocytes, and chondrocytes under defined culture conditions. To determine whether p85α subunit of PI3K affects biological functions of MSCs, bone marrow-derived wild type (WT) and p85α-deficient (p85α(-/-)) cells were employed in this study. Increased cell growth, higher proliferation rate and reduced number of senescent cells were observed in MSCs lacking p85α compare with WT MSCs as evaluated by CFU-F assay, thymidine incorporation assay, and β-galactosidase staining, respectively. These functional changes are associated with the increased cell cycle, increased expression of cyclin D, cyclin E, and reduced expression of p16 and p19 in p85α(-/-) MSCs. In addition, a time-dependent reduction in alkaline phosphatase (ALP) activity and osteocalcin mRNA expression was observed in p85α(-/-) MSCs compared with WT MSCs, suggesting impaired osteoblast differentiation due to p85α deficiency in MSCs. The impaired p85α(-/-) osteoblast differentiation was associated with increased activation of Akt and MAPK. Importantly, bone morphogenic protein 2 (BMP2) was able to intensify the differentiation of osteoblasts derived from WT MSCs, whereas this process was significantly impaired as a result of p85α deficiency. Addition of LY294002, a PI3K inhibitor, did not alter the differentiation of osteoblasts in either genotype. However, application of PD98059, a Mek/MAPK inhibitor, significantly enhanced osteoblast differentiation in WT and p85α(-/-) MSCs. These results suggest that p85α plays an essential role in osteoblast differentiation from MSCs by repressing the activation of MAPK pathway.     

Ebp1 regulates myogenic differentiation of myoblast cells via SMAD2/3 signaling pathway

Regulation of skeletal muscle development requires many of the regulatory networks that are fundamental to developmental myogenesis. ErbB3 binding protein‐1 (Ebp1) is involved in the control of myoblasts development in chicken. However, the expression and biological functions of Ebp1 in the progress of myogenesis are unclear. This study focused on determining the effect of Ebp1 on myogenic proliferation and differentiation using a primary myoblasts culture model. Ebp1 was found to upregulate in proliferating myoblasts and decrease at the early stage of myogenic differentiation. The level of endogenous Ebp1 increased from E9 to E20 chicken leg muscles. Knockdown of Ebp1 had no effect on myoblasts proliferation. However, myogenic differentiation into multinucleated myotubes was significantly reduced. The mRNA and protein expression of MRFs was decreased when Ebp1 was knocked down. Downregulation of Ebp1, accompanied by elevated levels of pSMAD2/3, suggests that Ebp1 is involved in regulating myogenic differentiation via SMAD2/3 inhibition. The phosphorylation of SMAD2/3 was activated and the expression of MYOD and MYOG was reduced in Ebp1 knockdown myoblasts, but addition of LY2109761 (an inhibitor specified to SMAD2/3) blocked these effects. Collectively, these results indicate that Ebp1 promotes myoblast differentiation by inhibition of SMAD2/3 signaling pathway during chicken myogenesis. These data provide new insights into the biological role of Ebp1 in embryonic chicken skeletal muscle development.     

Extracellular cysteine/cystine redox regulates the p44/p42 MAPK pathway by metalloproteinase-dependent epidermal growth factor receptor signaling

Previous research shows that stimulation of proliferation of colon carcinoma (Caco-2) cells by a more reduced extracellular cysteine/cystine (Cys/CySS) redox state occurs with no apparent effect on intracellular glutathione and that this stimulation is lost on addition of epidermal growth factor. The purpose of the present study was to determine whether a more reduced extracellular Cys/CySS redox state activates the mitogenic p44/p42 mitogen-activated protein kinase (MAPK) pathway and whether this is signaled through the epidermal growth factor receptor (EGFR). Caco-2 cells were exposed to a range of physiological extracellular redox conditions from -150 to 0 mV. In the absence of added growth factors, the most reduced (-150 mV) redox state induced an 80% increase in EGFR phosphorylation, and this was followed by a marked increase in phosphorylation of p44/p42 MAPK. Inhibitors of EGFR (AG1478) and p44/p42 MAPK (U0126) phosphorylation blocked redox-dependent p44/p42 phosphorylation, indicating that signaling occurred by EGFR. These effects were inhibited by pretreatment with a nonpermeant alkylating agent, showing that signaling involved thiols accessible to the extracellular space. The EGFR ligand TGF-alpha was increased in culture medium at more reduced redox states. Redox-dependent phosphorylation of EGFR was completely prevented by a metalloproteinase inhibitor (GM6001), and an antibody to TGF-alpha partially inhibited the phosphorylation of p44/p42 MAPK by redox. Thus the data show that a redox-dependent activation of metalloproteinase can stimulate the mitogenic p44/p42 MAPK pathway by a TGF-alpha-dependent mechanism. Because Cys availability and Cys/CySS redox are dependent on nutrition, disease, and environmental exposures, the results suggest that cell proliferation could be influenced physiologically by Cys-dependent redox effects on growth factor signaling pathways.     

Sema4C participates in myogenic differentiation in vivo and in vitro through the p38 MAPK pathway

Sema4C is a member of transmembrane semaphorin proteins which regulate axonal guidance in the developing nervous system. The expression of Sema4C was dramatically induced not only during differentiation of C2C12 mouse myoblasts, but also during injury-induced skeletal muscle regeneration. C2C12 cells stably or transiently expressing Sema4C both showed increased myogenic differentiation reflected by accelerated myotube formation and expression of muscle-specific proteins. Overexpression of Sema4C elicited p38 phosphorylation directly, and the effects of Sema4C during myogenic differentiation could be abolished by the p38alpha-specific inhibitor SB203580. Knockdown of Sema4C by siRNA transfection during C2C12 myoblasts differentiation could suppress the phosphorylation of p38 followed by dramatically diminished myotube formation. Sema4C could activate the myogenin promoter during myogenic differentiation. This activation could be abolished by p38 inhibitor SB203580. Taken together, these observations reveal novel functional potentialities of Sema4C which suggest that Sema4C promotes terminal myogenic differentiation in a p38 MAPK-dependent manner.     

Unc119 regulates myofibroblast differentiation through the activation of Fyn and the p38 MAPK pathway

Unc119 is an adaptor protein that is involved in the development of the vertebrate nervous system. We have shown that Unc119 stimulates the induction of alpha-smooth muscle actin (alpha-SMA) and myofibroblast differentiation by TGF-beta in human lung fibroblasts. Unc119 increases the kinase activity of Fyn and associates with it in coprecipitation and colocalization studies. Phosphorylation and activation of Fyn in response to TGF-beta and platelet-derived growth factor is delayed in Unc119-deficient cells. This delay translates into suppressed cell proliferation. In Src family kinase-deficient (SYF) cells, Unc119 knockdown does not affect cell proliferation. The result suggests that Unc119 interacts with Fyn in the early stages of signal generation and its presence is essential for conducive signal transduction. Unc119 overexpression does not stimulate alpha-SMA in SYF cells and this defect is restored upon reconstitution with Fyn indicating that Unc119 stimulation of alpha-SMA requires at least Fyn. Unc119 overexpression stimulated p38, but not JNK, phosphorylation. Blocking p38 MAPK resulted in reduced alpha-SMA expression by Unc119 suggesting that the p38 pathway regulates Unc119-induced myofibroblast differentiation. Unc119 stimulates the production of TGF-beta and IL-6, known inducers of myofibroblast differentiation. Thus, Unc119 regulates receptor-mediated signal transduction and myofibroblast differentiation by activating Fyn and the p38 MAPK pathway. Using primary lung fibroblasts from patients with fibrotic lung diseases and control subjects, we show that the expression of alpha-smooth muscle actin is highly correlated with that of Unc119. Taken together, our results suggest that Unc119 plays an important role in fibrotic processes through myofibroblast differentiation.     

Involvement of the p38 mitogen-activated protein kinase alpha, beta, and gamma isoforms in myogenic differentiation

We and others previously showed that p38 mitogen-activated protein kinase is indispensable for myogenic differentiation. However, it is less clear which of the four p38 isoforms in the mouse genome participates in this process. Using C2C12 myogenic cells as a model, we showed here that p38alpha, beta, and gamma are expressed with distinct expression patterns during differentiation. Knockdown of any of them by small interfering RNA inhibits myogenic differentiation, which suggests that the functions of the three p38 isoforms are not completely redundant. To further elucidate the unique role of each p38 isoform in myogenic differentiation, we individually knocked down one p38 isoform at a time in C2C12 cells, and we compared the whole-genome gene expression profiles by microarrays. We found that some genes are coregulated by all three p38 isoforms, whereas others are uniquely regulated by one particular p38 isoform. Furthermore, several novel p38 target genes (i.e., E2F2, cyclin D3, and WISP1) are found to be required for myogenin expression, which provides a molecular basis to explain why different p38 isoforms are required for myogenic differentiation.     

p38 MAPKα/β和ERK1/2在心肌缺氧预处理信号传递中的不同作用

建立培养乳鼠心肌细胞的缺氧／复氧(A／R)损伤模型和缺氧预处理(APC)模型,以细胞存活率、细胞内超氧化物趋化酶(SOD)活性、丙二醛(MDA)含量、培养上清液乳酸脱氢酶(LDH)活性作为反映心肌细胞损伤的指标。采用细胞外信号调节蛋白激酶(ERK1／2)抑制剂PD98059及丝裂素活化蛋白激酶p38α/β(p38α/β)阻滞剂SB203580干预模型,并以胶内原位磷酸化法测定ERK1／2和p38活性,借以探讨ERK1／2和p38α/β在缺氧预处理保护机制中的作用。结果表明：(1)在APC组,于预处理的缺氧时相给予PD98059,可以完全消除APC的延迟保护作用;在A／R组的缺氧时相加入PD98059对细胞损伤无影响;(2)在APC组的预处理缺氧时相给予p38α／β抑制剂SB203580并不能消除APC的保护作用,而在A／R组的持续缺氧时相给予SB203580则可显著减轻缺氧对细胞的损伤;(3)ERK1／2和p38总活性测定表明,缺氧可激活ERK1／2和p38,它们的活性在缺氧后4h时达到高峰,而经过APC处理后,两者活性高峰提前于缺氧后3h时出现,且峰值显著降低。上述结果提示,预处理过程中ERK1／2的激活可能是缺氧预处理延迟保护机制中细胞信号传递的重要环节,预处理阶段p38α/β的活化不参与APC诱导的延迟保护信号传递过程,p38的过度激活可能是缺氧／复氧损伤过程中的一个致损伤参与因素,而预处理抑制随后持续缺氧阶段p38的过度激活可能是其保护机制的一个环节。     

ROR2 promotes the epithelial-mesenchymal transition by regulating MAPK/p38 signaling pathway in breast cancer

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) is a tyrosine-protein kinase receptor highly implicated in the growth plate and cartilage development, which may be involved in epithelial-mesenchymal transition (EMT) in breast cancer (BC) cells. Although ROR2 is known to promote the migration of BC cells, the detailed mechanism of this event is still not clear. Here, we found that ROR2 expression was significantly increased in BC lymphatic metastatic tissue as well as BC samples compared to normal adjacent breast tissues. A higher expression of ROR2 in MDA-MB-231 and a lower expression of ROR2 in MCF-7 cells were observed. MDA-MB-231-siROR2 cells with ROR2 knockdown inhibited MDA-MB-231 cell invasion, migration, and clonal formation, while MCF-7-OvROR2 cells with overexpression showed the opposite results. The underlying mechanisms involved in ROR2-induced EMT in MDA-MB-231 and MCF-7 cells were further investigated. ROR2 may activate EMT progression in BC cells by altering MAPK kinase 3/6 (MKK3/6) expression. The expressions of transforming growth factor-β, matrix metalloproteinase-2 (MMP-2), and MMP-9, which were related to tumor cell invasion activities, were notably increased in MCF-7-OvROR2 cells. The EMT markers, including snail, N-cadherin, tissue inhibitor of metalloproteinases-1, and vimentin, were significantly upregulated in MCF-7-OvROR2 cells. On the contrary, E-cadherin was obviously reduced expressed in MCF-7-OvROR2 cells. ROR2 may regulate the malignant phenotype of BC cells possibly via activation of mitogen-activated protein kinase (MAPK)/p38 signaling pathway. Collectively, ROR2 promotes BC carcinogenesis by mediating the MAPK/p38 pathway, which is independent of Wnt5α.     

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.     

Tetraspanin CD9 regulates osteoclastogenesis via regulation of p44/42 MAPK activity

Tetraspanin CD9 has been shown to regulate cell-cell fusion in sperm-egg fusion and myotube formation. However, the role of CD9 in osteoclast, another multinucleated cell type, is not still clear. Therefore, we investigated the role of CD9 in osteoclast differentiation. CD9 was expressed in osteoclast lineage cells and its expression level increased during the progression of RANKL-induced osteoclastogenesis. KMC8, a neutralizing antibody specific to CD9, significantly suppressed RANKL-induced multinucleated osteoclast formation and the mRNA expression of osteoclast differentiation marker genes. To define CD9-regulated osteoclastogenic signaling pathway, MAPK pathways were examined. KMC8 induced long-term phosphorylation of p44/42 MAPK, but not of p38 MAPK. Constitutive activation of p44/42 MAPK by overexpressing constitutive-active mutant of MEK1 almost completely blocked osteoclast differentiation. Taken together, these results suggest that CD9 expressed on osteoclast lineage cells might positively regulate osteoclastogenesis via the regulation of p44/42 MAPK activity.     

Vibration loading promotes osteogenic differentiation of bone marrow-derived mesenchymal stem cells via p38 MAPK signaling pathway

Low magnitude high frequency vibration (LMHFV) exhibits effectively anabolic effects on the bone tissue, and can promote osteogenic differentiation of mesenchymal stem cells (MSCs) in vitro. The role of p38 MAPK signaling in LMHFV-induced osteogenesis remains unclear. In this current study, LMHFV loading was applied to BMSCs in vitro, and cell proliferation, alkaline phosphatase (ALP), matrix mineralization, as well as osteogenic genes expression were assayed. The mechanism of mechanical signal transduction was analysed using PCR array, qRT-PCR and Western blot. LMHFV increased cell proliferation in the growth medium, while inhibited proliferation in the osteogenic medium. ALP activity, matrix mineralization and osteogenic genes expression of Runx2, Col-I, ALP, OPN and OC were increased by LMHFV. p38 and MKK6 genes expression, and p38 phosphorylation were promoted in LMHFV-induced osteogenesis. Inhibition of p38 MAPK with SB203580 and targeted p38 siRNA blunted the increased ALP activity and osteogenic genes expression by LMHFV. These findings suggest that LMHFV promotes osteogenic differentiation of BMSCs, and p38 MAPK signaling shows an important function in LMHFV-induced osteogenesis.     

p38 MAPK regulates group IIa phospholipase A2 expression in interleukin-1beta -stimulated rat neonatal cardiomyocytes

Group IIa phospholipase A(2) (GIIa PLA(2)) is released by some cells in response to interleukin-1beta. The purpose of this study was to determine whether interleukin-1beta would stimulate the synthesis and release of GIIa PLA(2) from cardiomyocytes, and to define the role of p38 MAPK and cytosolic PLA(2) in the regulation of this process. Whereas GIIa PLA(2) mRNA was not identified in untreated cells, exposure to interleukin-1beta resulted in the sustained expression of GIIa PLA(2) mRNA. Interleukin-1beta also stimulated a progressive increase in cellular and extracellular GIIa PLA(2) protein levels and increased extracellular PLA(2) activity 70-fold. In addition, interleukin-1beta stimulated the p38 MAPK-dependent activation of the downstream MAPK-activated protein kinase, MAPKAP-K2. Treatment with the p38 MAPK inhibitor, SB202190, decreased interleukin-1beta stimulated MAPKAP-K2 activity, GIIa PLA(2) mRNA expression, GIIa PLA(2) protein synthesis, and the release of extracellular PLA(2) activity. Infection with an adenovirus encoding a constitutively active form of MKK6, MKK6(Glu), which selectively phosphorylates p38 MAPK, induced cellular GIIa PLA(2) protein synthesis and the release of GIIa PLA(2) and increased extracellular PLA(2) activity 3-fold. In contrast, infection with an adenovirus encoding a phosphorylation-resistant MKK6, MKK6(A), did not result in GIIa PLA(2) protein synthesis or release by unstimulated cardiomyocytes. In addition, infection with an adenovirus encoding MKK6(A) abrogated GIIa PLA(2) protein synthesis and release by interleukin-1beta-stimulated cells. These results provide direct evidence that p38 MAPK activation was necessary for interleukin-1beta-induced synthesis and release of GIIa PLA(2) by cardiomyocytes.     

Activation by 2-arachidonoylglycerol of platelet p38MAPK/cPLA2 pathway

The endogenous cannabinoid 2-arachidonoylglycerol (2-AG) is described as a platelet agonist able to induce aggregation and to increase intracellular calcium. In the present report we have confirmed these data and demonstrated that the inhibitor of p38MAPK SB203580 and the inhibitor of cPLA(2) metabolism ETYA affect both these parameters. Thus, we aimed to define the role of p38MAPK/cytosolic phospholipase A(2) (cPLA(2)) pathway in 2-AG-induced human platelet activation. p38MAPK activation was assayed by phosphorylation. cPLA(2) activation was assayed by phosphorylation and as arachidonic acid release and thromboxane B(2) formation. It was shown that 2-AG in a dose- and time-dependent manner activates p38MAPK peaking at 10 μM after 1 min of incubation. The 2-AG effect on p38MAPK was not impaired by apyrase, indomethacin or RGDS peptide but it was significantly reduced by SR141716, specific inhibitor of type-1 cannabinoid receptor and unaffected by the specific inhibitor of type-2 cannabinoid receptor SR144528. Moreover, the incubation of platelets with 2-AG led to the phosphorylation of cPLA(2) and its activation. Platelet pretreatment with SB203580, inhibitor of p38MAPK, abolished both cPLA(2) phosphorylation and activation. In addition SR141716 strongly impaired cPLA(2) phosphorylation, arachidonic acid release and thromboxane B(2) formation, whereas SR144528 did not change these parameters. Finally platelet stimulation with 2-AG led to an increase in free oxygen radical species. In conclusion, data provide insight into the mechanisms involved in platelet activation by 2-AG, indicating that p38MAPK/cPLA(2) pathway could play a relevant role in this complicated process.     

The p53 pathway is synergized by p38 MAPK signaling to mediate 11,11'-dideoxyverticillin-induced G2/M arrest

The phytochemical 11,11'-dideoxyverticillin, derived from the fungus Shiraia bambusicola, has been shown to possess potent anticancer activity in vitro and in vivo. Here, we investigated the effect of 11,11'-dideoxyverticillin on cell cycle progression, and explored the potential mechanisms for this effect. A concentration- and time-dependent cell cycle blockade at G2/M phase was observed in human colon cancer cells (HCT-116) following 11,11'-dideoxyverticillin treatment and was associated with marked increases in levels of p53, phospho-p53(ser20) and phospho-Chk2(Thr 68). When wild type p53 expression was specifically inhibited by RNA interference, HCT-116 cells treated with 11,11'-dideoxyverticillin failed to arrest in G2/M and did not show increased phospho-Chk2(Thr 68). On the other hand, 11,11'-dideoxyverticillin treatment also elicited p38 MAP kinase activity and expression of phospho-p38 MAPK. Treatment with a specific p38 MAPK inhibitor (SB203580) successfully inhibited p38 MAPK and delayed the onset of G2/M arrest induced by 0.5 microM 11,11'-dideoxyverticillin after approximately 6 h, but did not abolish the induction of G2/M arrest. Additionally, SB203580 did not alter the levels of p53, phospho-p53 (ser20), or phospho-Chk2 (Thr68) proteins in 11,11'-dideoxyverticillin-treated cells. Together, these findings indicate that p53-mediated phosphorylation of Chk2 maybe plays a vital role in 11,11'-dideoxyverticillin-induced G2/M arrest, and that p38 MAPK might accelerate this progression. Our work suggests a new possibility of interactions among p53, Chk2 and p38 MAPK signaling in G2/M arrest.     

A Novel MAPK phosphatase MKP-7 acts preferentially on JNK/SAPK and p38 alpha and beta MAPKs

Mitogen-activated protein kinases (MAPKs) are inactivated via dephosphorylation of either the threonine or tyrosine residue or both in the P-loop catalyzed by protein phosphatases which include serine/threonine phosphatases, tyrosine phosphatases, and dual specificity phosphatases. Nine members of the dual specificity phosphatases specific for MAPKs, termed MKPs, have been reported. Each member has its own substrate specificity, tissue distribution, and subcellular localization. In this study, we have cloned and characterized a novel MKP, designated MKP-7. MKP-7 is most similar to hVH5, a member of previously known MKPs, in the primary structure. MKP-7 is predominantly localized in the cytoplasm when expressed in cultured cells, whereas hVH5 is both in the nucleus and the cytoplasm. MKP-7 binds to and inactivates p38 MAPK and JNK/SAPK, but not ERK. Furthermore, we have found that MKPs have the substrate specificity toward the isoforms of the p38 family (alpha, beta, gamma, and delta). MKP-7 binds to and inactivates p38 alpha and -beta, but not gamma or delta. MKP-5 and CL100/MKP-1 also bind to p38 alpha and -beta, but not gamma or delta. Finally, we propose a tentative classification of MKPs based on the sequence characteristics of their MAPK-docking site.