The cross-talk between transforming growth factor-beta1 and ultrasound stimulation during mechanotransduction of rat tenocytes

Ultrasound is an effective noninvasive treatment for various tendinopathies. However, how tenocytes convert ultrasound stimulation into cascades of cellular and molecular events is not well understood. The purpose of this study is to elucidate the signaling pathways of tenocytes during ultrasound stimulation. Primary cultures of tenocytes were harvested from Achilles tendons of Sprague-Dawley rats. The viability and proliferation of tenocytes, their genes expression, and the signaling pathways after ultrasound treatment with or without specific inhibitors were evaluated and analyzed. The results showed that ultrasound treatment (100 mW/cm(2) for 20 min) significantly enhanced matrix metalloproteinase 13 (MMP-13), c-Fos, and c-Jun gene expression, increased JNK and p38, but not extracellular signal-regulated kinase-1/2 (ERK1/2), phosphorylation at 5 min, and sustained up to 60 min. JNK inhibitor and p38 inhibitor, but not ERK1/2 inhibitor, attenuated ultrasound-dependent induction of MMP-13 expression, indicating that the JNK and p38 pathways are required for ultrasound-induced MMP-13 expression in tenocytes. We also found that SB431542 (transforming growth factor-beta (TGF-β) receptor kinases inhibitor) suppressed ultrasound-induced MMP?13 and c-Fos gene expression, and p38 phosphorylation. This study revealed that ultrasound treatment stimulates tenocytes proliferation and regulates their matrix metabolism through the cross-talk between TGF-β and ultrasound-induced mitogen-activated protein kinases (MAPKs) signaling pathways.     

Tension Force Downregulates Matrix Metalloproteinase Expression and Upregulates the Expression of Their Inhibitors through MAPK Signaling Pathways in MC3T3-E1 cells

Objective: Matrix metalloproteinases (MMPs), produced by osteoblasts, catalyze the turnover of extracellular matrix (ECM) molecules in osteoid, and the regulation of MMP activity depends on interactions between MMPs and tissue inhibitors of metalloproteinases (TIMPs). We focused on the degradation process of ECM in osteoid that was exposed to mechanical strain, and conducted an in vitro study using MC3T3-E1 osteoblastic cells to examine the effects of tension force (TF) on the expression of MMPs and TIMPs, and activation of mitogen-activated protein kinase (MAPK) pathways.Design: Cells were incubated on flexible-bottomed culture plates and stimulated with or without cyclic TF for 24 hours. The expression of MMPs and TIMPs was examined at mRNA and protein levels by real-time RT-PCR and Western blotting, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) were examined by Western blotting.Results: TF decreased the expression of MMP-1, -3, -13 and phosphorylated ERK1/2. In contrast, TF increased the expression of TIMP-2, -3 and phosphorylated SAPK/JNK. The expression of MMP-2, -14, TIMP-1, -4 and phosphorylated p38 MAPK was unaffected by TF. MMP-1, -3 and -13 expression decreased in cells treated with the ERK inhibitor PD98059 compared with untreated control cells. The JNK inhibitor SP600125 inhibited the TF-induced upregulation of TIMP-2 and -3.Conclusions: The results suggest that TF suppresses the degradation process that occurs during ECM turnover in osteoid via decreased production of MMP-1, -3 and -13, and increased production of TIMP-2 and -3 through the MAPK signaling pathways in osteoblasts.     

Differential activation and regulation of mitogen-activated protein kinases through the antigen receptor and CD40 in human B cells.

In human B cells, antigen receptor ligation and CD40 ligation are known to activate the extracellular-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) pathways, which in turn regulate many important B cell functions. We previously reported that antigen receptor ligation activated the ERK pathway whereas CD40 ligation activated the JNK/stress-activated protein kinase (SAPK) pathway. Here, we demonstrate that another SAPK, p38/Hog1, is activated by both antigen receptor ligation or CD40 ligation in a human B-lymphoblastoid cell line and tonsillar B cells. Wortmannin, an inhibitor of phosphatidylinositol 3-kinase, partially inhibited ERK2 and p38 activation triggered through the B cell receptor whereas activation of JNK1 and p38 through CD40 was not affected. PD98059, a specific inhibitor of mitogen-activated extracellular response kinase kinase (MEK), significantly inhibited ERK2 activation and partially inhibited p38 activation triggered by anti-IgM antibody treatment, but did not affect CD40-dependent signaling events. In addition, anti-IgM antibody-induced signaling pathways were shown to be PKC-dependent in contrast to the CD40-induced signaling pathways. Thus, the B cell receptor and CD40 recruit the ERK, JNK and p38 pathways by using different upstream effectors.     

Macrophage migration inhibitory factor induces MMP-9 expression in macrophages via the MEK-ERK MAP kinase pathway.

We have shown previously that macrophage migration inhibitory factor (MIF) may play a role in the destabilization of atherosclerotic plaques by activating matrix metalloproteinase protein-9 (MMP-9). The aim of this study is to investigate the signaling mechanism by which MIF induces MMP-9 expression and activation in a murine macrophage line (RAW264.7). MIF was able to activate extracellular signal-regulated kinase 1/2 (ERK1/2), to a less extent JNK, but not p38 mitogen-activated protein (MAP), MAP kinase to induce MMP9 mRNA and protein expression in RAW264.7 murine macrophages. This was confirmed by the findings that addition of an ERK MAP kinase inhibitor (PD98059) but not a p38 inhibitor (SB203589) abolished MIF-induced MMP-9 expression and activation, whereas addition of a JNK inhibitor (SP600125) produced a partially inhibitory effect. The functional role of mitogen-activated protein kinase kinase (MEK)-ERK MAP kinase in MIF-induced MMP-9 expression was further confirmed by overexpressing dominant negative MEK (DN-MEK) and DN-ERK MAP kinases. Interestingly, constitutive expression of a wild-type (WT)-MEK alone was also capable of inducing a low, but significant MMP-9 mRNA and protein expression but did not cause a further increase in MMP-9 in response to MIF. MIF activates the MEK-ERK MAP kinase pathway to induce MMP-9 expression by murine macrophages. Activation of this pathway is necessary for MMP-9 expression and activation in response to MIF stimulation.     

p38 MAPK- Pim-3通路可能介导预处理对抗心肌细胞缺氧/复氧损伤

目的： 研究MAPK通路在原癌基因Pim-3抗心肌急性缺氧复氧损伤中的作用。方法：采用原代培养新生大鼠的心肌细胞,随机分为4组：正常对照组（control）、缺氧复氧组(A/R)、缺氧预适应组(APC+A/R)、阻断剂组。在缺氧预处理前分别用终浓度为10 μmol/L SB203850(p38 MAPK阻断剂)、U0126（ERK1/2阻断剂）、SP600125（SAPK/JNK阻断剂）与细胞孵育30 min。实验结束后测定MAPKs通路中ERK1/2、JNK、p38 MAPK 磷酸化蛋白表达水平及Pim-3蛋白的表达水平,同时检测培养液中乳酸脱氢酶(LDH) 活性、四唑盐（MTT）比色试验测定细胞存活率、TUNEL法检测细胞凋亡。结果： SB203850、U0126、SP600125能分别取消由APC或A/R所诱导ERK1/2、JNK、p38 MAPK的磷酸化水平的升高;由APC所诱导的Pim-3表达的升高在p38 MAPK通路被阻断后明显下调(P<0.01),并且心肌细胞LDH值升高,细胞存活率则下降,心肌细胞的凋亡指数升高。结论： p38 MAPK的激活可上调原癌基因Pim-3的表达,从而可能对心肌细胞起到保护作用。     

Low-intensity pulsed ultrasound stimulates matrix metabolism of human annulus fibrosus cells mediated by transforming growth factor β1 and extracellular signal-regulated kinase pathway

Purpose: There are limited strategies to restore the damaged annulus fibrosus (AF) of the intervertebral disc. Low-intensity pulsed ultrasound (LIPUS) has positive effects on the proliferation of several types of cells and the repair of damage tissue in vivo. However, scientific evidence of therapeutic effects of LIPUS on AF cells remains limited. The purpose of this study is to evaluate the feasibility of applying LIPUS to the repair of the AF.

Materials and methods: We used an in vitro model of human AF cells subjected to LIPUS stimulation to examine its effects on cell proliferation and matrix metabolism. Cell viability, synthesis of collagen and glycosaminoglycan (GAG), expression of matrix metalloproteinases (MMPs) and transforming growth factor β1 and pathways involving mitogen-activated protein kinases (MAPKs) were investigated.

Results: LIPUS significantly enhanced proliferation of AF cells after 5 days of treatment. LIPUS with an intensity of 0.5?W/cm² increased the collagen and GAG synthesis and decreased the expressions of MMP-1 and -3 of human AF cells. Real-time polymerase chain reactions and western blotting analysis revealed that LIPUS could increase transforming growth factor β1 (TGF-β1) and activate extracellular signal-regulated kinase (ERK) pathway. In addition, TGF-β receptor kinase inhibitor could suppress the ultrasound-induced alterations in cell viability and matrix metabolism.

Conclusions: The findings suggested that LIPUS could be useful as a physical stimulation of cell metabolism for the repair of the AF.     

Protective effect of sauchinone against regional myocardial ischemia/reperfusion injury: inhibition of p38 MAPK and JNK death signaling pathways

Sauchinone has been known to have anti-inflammatory and antioxidant effects. We determined whether sauchinone is beneficial in regional myocardial ischemia/reperfusion (I/R) injury. Rats were subjected to 20 min occlusion of the left anterior descending coronary artery, followed by 2 hr reperfusion. Sauchinone (10 mg/kg) was administered intraperitoneally 30 min before the onset of ischemia. The infarct size was measured 2 hr after resuming the perfusion. The expression of cell death kinases (p38 and JNK) and reperfusion injury salvage kinases (phosphatidylinositol-3-OH kinases-Akt, extra-cellular signal-regulated kinases [ERK1/2])/glycogen synthase kinase (GSK)-3β was determined 5 min after resuming the perfusion. Sauchinone significantly reduced the infarct size (29.0% ± 5.3% in the sauchinone group vs 44.4% ± 6.1% in the control, P < 0.05). Accordingly, the phosphorylation of JNK and p38 was significantly attenuated, while that of ERK1/2, Akt and GSK-3β was not affected. It is suggested that sauchinone protects against regional myocardial I/R injury through inhibition of phosphorylation of p38 and JNK death signaling pathways.     

Coronarin D suppresses proliferation,invasion and migration of glioma cells via activating JNK signaling pathway

Coronarin D (CD) is one of the primary components of the Hedychium coronarium rhizomes and possesses strong anticancer effects via preventing cell growth in many cancer cells. The study was aimed to explore the molecular mechanisms underlying effects of CD on proliferation, invasion and migration of gliomas cells. Gliomas cell lines U251 was employed for detecting cells viability and proliferation by Cell Counting Kit-8 assay and colony formation assay. In addition, scratch wound healing and transwell assays were performed for the analysis of U251 cells invasion and migration respectively. Furthermore, the expression of p-Akt/Akt, p-p38/p38, p-ERK/ERK, p-JNK/JNK, p-STAT3/STAT3, cyclinE, cyclinD1, CTGF, MMP-2 and MMP-9 were measured by Western blotting. CD could suppress proliferation, invasion and migration of glioma cells and induced reduction of cyclinE, cyclinD1, CTGF, MMP-2 and MMP-9 expression via activating JNK signaling pathway. CD treatment suppressed expression of p-AKT, p38, and ERK and elevated expression of p-JNK in concentration- and time-dependent manners. Moreover, CD significantly induced reduction of phosphorylated STAT3 expression. Exposure of cells to the JNK-specific inhibitor SP600125 reduced the cytotoxicity effects of CD, combination of CD and SP600125 corrected overexpression of phosphorylated JNK and reduction of phosphorylated STAT3. Pretreatment of SP600125 also improves gliomas cells viability and invasion. The results revealed that CD may remarkably suppress gliomas cell growth through JNK and STAT3 signaling. In present study, these findings revealed that CD induces suppression of cell viability in gliomas cells and possesses therapeutic effect on gliomas.     

Kinases and kinase signaling pathways: potential therapeutic targets in Parkinson's disease

Complex molecular mechanisms underlying the pathogenesis of Parkinson's disease (PD) are gradually being elucidated. Accumulating genetic evidence implicates dysfunction of kinase activities and phosphorylation pathways in the pathogenesis of PD. Causative and risk gene products associated with PD include protein kinases (such as PINK1, LRRK2 and GAK) and proteins related phosphorylation signaling pathways (such as SNCA, DJ-1). PINK1, LRRK2 and several PD gene products have been associated with mitogen-activated protein (MAP) and protein kinase B (AKT) kinase signaling pathways. C-Jun N-terminal kinase (JNK), extracellular signal-regulated kinases (ERK) and p38, signaling pathways downstream of MAP, are particularly important in PD. JNK and p38 play an integral role in neuronal death. Targeting JNK or p38 signaling may offer an effective therapy for PD. Inhibitors of the ERK signaling pathway, which plays an important role in the development of l-DOPA-induced dyskinesia (LID), have been shown to attenuate this condition in animal models. In this review, we summarize experimental evidence gathered over the last decade on the role of PINK1, LRRK2 and GAK and their related phosphorylation signaling pathways (JNK, ERK, p38 and PI3K/AKT) in PD. It is speculated that improvement or modulation of these signaling pathways will reveal potential therapeutic targets for attenuation of the cardinal symptoms and motor complications in patients with PD in the future.     

柚皮苷在促大鼠骨髓间充质干细胞骨向分化过程中对MAPK信号通路的影响

目的: 研究中药单体柚皮苷(NG)对体外培养的大鼠骨髓间充质干细胞(MSCs)向成骨细胞分化过程中MAPK信号通路的影响。方法: 观察在正常、加入p38、ERK和JNK通路抑制剂SB203580、PD98059、SP600125及3种抑制剂全部加入的情况下,各组碱性磷酸酶(ALP)、骨钙素(BGP)、I型胶原(Col I)等骨向分化指标的差异。用Western blotting技术检测各组p38、ERK1/2和JNK蛋白的磷酸化水平,用荧光定量PCR技术检测细胞因子转化生长因子β₁(TGF-β₁)、骨形成蛋白2(BMP-2)和核心结合因子α1(Cbfα1) mRNA的表达。结果: (1)10^-7mol/L为本实验中NG的最佳促骨向分化浓度。(2) NG最佳浓度组的ALP和BGP含量比其它各组都高(P<0.05),Col I含量无明显差异(P>0.05);与NG组相比,加入不同抑制剂组的ALP、BGP和ColⅠ表达量出现不同程度的降低。(3)与空白组相比,NG组JNK蛋白的磷酸化水平升高(P<0.05),p38蛋白的磷酸化水平降低(P<0.01),ERK1/2蛋白的磷酸化水平无明显差异(P>0.05)。与NG组相比,加入不同抑制剂组的p38、ERK1/2和JNK蛋白的磷酸化水平有升高也有降低。(4) NG组上调BMP-2的表达(P<0.05),下调Cbfα1的表达(P<0.05),而对TGF-β₁的表达无明显影响(P>0.05)。与NG组相比,加入不同抑制剂组的TGF-β₁、BMP-2和Cbfα1 mRNA表达量出现不同程度的降低。结论: NG主要通过激活MAPK信号通路中ERK通路、JNK通路以及上调BMP-2的表达,促进MSCs的骨向分化。NG上调BMP-2的表达受MAPK通路中p38通路的影响较大。     

Cinobufagin exerts anti-proliferative and pro-apoptotic effects through the modulation ROS-mediated MAPKs signaling pathway

Abstract

Cinobufagin (CBG) is a cardiotoxic bufanolide steroid secreted by the skin and parotid venom glands of the Asiatic toad Bufo bufo gargarizans (called Chan-Su). Although CBG is known to exhibit anti-cancer activities, very little is known about its potential mechanism(s) of action. In this study, we investigated whether CBG mediates its effect through the modulation of the mitogen-activated protein kinases (MAPKs) signaling pathway in human multiple myeloma (MM) U266 cells. We found that CBG caused the significant activation of ERK, JNK and p38 MAPK in U266 cells. CBG showed much higher cytotoxicity against U266 cells as compared to peripheral blood mononuclear cells (PBMC). Induction of CBG increased reactive oxygen species (ROS) generation from mitochondria, which is associated with the induction of apoptosis as characterized by increased sub-G1 DNA contents of cell cycle, positive Annexin V binding, activation of caspase-3 and cleavage of PARP. Inhibition of ROS generation by N-acetyl-l-cysteine (NAC) significantly prevented CBG-induced ERK, JNK and p38 MAPK activation and apoptosis. CBG also down-regulated the expression of various downstream gene products that mediate cell proliferation, survival, angiogenesis and metastasis. Interestingly, ERK, JNK and p38MAPK pharmacological inhibitors blocked CBG-induced MAPKs activation and ERK inhibitor (PD98059) also prevented the CBG-induced caspase-3 activation and PARP cleavage in U266 cells. Taken together, these findings suggest that CBG can act as a potent anticancer agent against MM and possibly exerts its effects through the ROS-mediated activation of ERK, JNK and p38 MAPK leading to the activation of caspase-3 in U266 cells.     

Mitogen-activated protein kinase and activator protein-1 dependent signals are essential for Bacteroides fragilis enterotoxin-induced enteritis

The approximately 20-kDa heat-labile toxin produced by enterotoxigenic Bacteroides fragilis is known to be associated with the development of enteritis. However, the molecular mechanism involved is not yet fully understood. In this study, we assessed whether B. fragilis enterotoxin (BFT)-induced enteritis is related to mitogen-activated protein kinase (MAPK) signaling pathways. In human colon epithelial cells, BFT activated three major MAPK cascades. The activation of p38 was sustained for a relatively long period, while the stimulation of extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) was transient. BFT stimulation also activated AP-1 signals composed of c-Jun/c-Fos heterodimers. The p38 inhibitor SB203580 and the ERK inhibitor U0126 reduced not only AP-1 activity, but also decreased IL-8 and MCP-1 expression. In addition, the overexpression of superrepressors for c-Jun and Ras induced by BFT stimulation decreased the levels of IL-8 and MCP-1 production. Furthermore, SB203580 prevented BFT-induced colitis in the mouse ileum, as evidenced by significant decreases in villous destruction, neutrophil infiltration, and mucosal congestion. These results suggest that a pathway, including Ras, MAPK, and subsequent AP-1 activation, is required for IL-8 and MCP-1 expression in intestinal epithelial cells exposed to BFT, and can be involved in the development of enteritis.     

Matrix metalloproteinases (MMP), EMMPRIN (extracellular matrix metalloproteinase inducer) and mitogen-activated protein kinases (MAPK): Co-expression in metastatic serous ovarian carcinoma

Activation or suppression of intracellular signaling via the mitogen-activated protein kinase (MAPK) family has been linked to expression of matrix metalloproteinases (MMP) in experimental models, but this association has not been demonstrated in clinical material. The objective of this study was to investigate the possible association between expression and activity of MMP, expression of the MMP inducer EMMPRIN, and the expression (level) and phosphorylation status (activity) of the extracellular-regulated kinase (ERK), c-Jun amino-terminal kinase (JNK) and high osmolarity glycerol response kinase (p38) in effusions from patients diagnosed with serous ovarian carcinoma. MAPK level and activity were studied in 55 effusions using immunoblotting. MMP-1, MMP-2, MMP-9 and EMMPRIN expression was studied using immunocytochemistry (ICC) and mRNA in situ hybridization (ISH). The gelatinolytic activity of MMP-2 and MMP-9 was measured by zymography. ERK and phospho-ERK (p-ERK) were detected in 54/55 (98%) and 50/55 (91%) specimens, respectively. JNK and p-JNK were detected in 53/55 (96%) and 38/55 (69%) specimens, respectively. p38 was expressed in 54/55 (98%) specimens, and its phosphorylated form was found in 51/55 (92%). MMP-2 mRNA expression (P=0.048), protein expression (P=0.046) and gelatinolytic activity (P=0.039) correlated with ERK phosphorylative activity. MMP-2 activity also correlated with p38 activity (P=0.017). MMP-9 protein expression correlated with phosphorylation of p38 (P=0.046), but enzyme activity showed inverse relationship with both p-ERK (P=0.05) and p-p38 (P=0.033) expression. EMMPRIN expression correlated with MMP-1 (P<0.001), MMP-2 (P=0.042) and MMP-9 (P=0.029) expression, as well as with ERK activity (P=0.001). Our results present the first evidence of a possible link between MAPK signaling and MMP expression and activity in vivo. These data may expand our understanding regarding the mechanisms by which MMP synthesis is regulated in effusions and possibly affect treatment strategies for this form of malignancy. This revised version was published online in July 2006 with corrections to the Cover Date.     

Involvement of ILK/ERK1/2 and ILK/p38 pathways in mediating the enhanced osteoblast differentiation by micro/nanotopography

The hierarchical micro/nanotextured topography (MNT) on titanium (Ti) implant surface significantly enhances osteoblast differentiation. We have demonstrated that integrin-linked kinase (ILK) is a key underlying signal molecule and β-catenin is one of its downstream mediators in MNT-regulated osteoblast behavior. Here we propose that mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 1/2 (ERK1/2), p38 and c-Jun NH₂-terminal kinase (JNK), are other mediators downstream of ILK, and this study aims to confirm this. Firstly, the levels of ILK and MAPK activity in MG63 cells on MNT are examined by Western blot analysis. The ILK, ERK1/2 and p38 signals are significantly up-regulated by MNT, whereas the JNK activity is undetectable by Western blot. The MG63 cell morphology, proliferation and differentiation are studied in the absence and presence of the MAPK subgroup inhibitors to confirm their roles in cell functions on the Ti surface. The MAPK subgroup inhibitors obviously change the cell shape and depress cell proliferation. Blocking the ERK1/2 or p38 signaling, but not the JNK signaling, significantly down-regulates the cell osteogenesis-related gene expression, ALP production, collagen secretion and matrix mineralization. Afterwards, the ILK expression is down-regulated using ILK-specific siRNA (ILKsi) and then the MAPK activity is determined. ILKsi significantly attenuates the phosphorylated ERK1/2 and p38 levels on MNT, explicitly demonstrating that the ERK1/2 and p38 signalings are downstream effectors of ILK. In conclusion, these data demonstrate that both ILK/ERK1/2 and ILK/p38 pathways are involved in the mechanisms mediating the enhanced osteoblast differentiation by biomaterial surface topography, hopefully directing the biomaterial modification and biofunctionalization.     

Implication of mitogen-activated protein kinases in T84 cell responses to enteropathogenic Escherichia coli infection

Enteropathogenic Escherichia coli (EPEC) infection of T84 cells induces a decrease in transepithelial resistance, the formation of attaching and effacing (A/E) lesions, and cytokine production. The purpose of this study was to investigate the ability of EPEC to activate mitogen-activated protein (MAP) kinases in T84 cells and to correlate these signaling pathways with EPEC-induced cell responses. T84 cells were infected with either the wild-type (WT) EPEC strain E2348/69 or two mutants, intimin deletion strain CVD206 (deltaeaeA) and type III secretion apparatus mutant strain CVD452 (deltaescN::aphA). Infection of T84 cells with WT but not mutant EPEC strains induced tyrosine phosphorylation of several proteins in T84 cells, including the p46 and p52 Shc isoforms. Kinetics studies revealed that ERK1/2, p38, and c-Jun N-terminal kinase (JNK) MAP kinases were activated in cells infected with strain E2348/69 but not with the mutant strains. Inhibition of MAP kinases with PD98059 or SB203580 did not affect the EPEC-induced decrease in transepithelial resistance or actin accumulation beneath the WT bacteria, but these two inhibitors significantly decreased interleukin-8 (IL-8) synthesis. We demonstrate that EPEC induces activation of ERK1/2, p38, and JNK cascades, which all depend on bacterial adhesion and expression of the bacterial type III secretion system. ERK1/2 and p38 MAP kinases were equally implicated in IL-8 expression but did not participate in A/E lesion formation or transepithelial resistance modification, indicating that the signaling pathways involved in these events are distinct.