Force-independent expression of c-fos mRNA by endothelin-1 in rat intact small mesenteric arteries

AIM: Wall stress-independent signalling pathways were studied for endothelin-1 (ET-1)-induced c-fos expression in rat intact mesenteric small arteries. METHODS: Arteries were kept unmounted in Krebs buffer, equilibrated for 1 h and stimulated with vasoactive substances for 15-60 min. The c-fos mRNA expression was determined by real-time polymerase chain reaction. RESULTS: Stimulation with fetal bovine serum (FBS), phorbol 12-myristate 13-acetate (PMA) and ET-1 caused about a doubling of c-fos mRNA. The ET-1-induced c-fos expression was steady (15-60 min) and was inhibited by the inhibitor of the ET(A) receptor, BQ-123. Platelet-derived growth factor-B, angiotensin II and U46619 did not cause increased c-fos mRNA levels. The broad specificity inhibitor staurosporine inhibited the response to ET-1, but inhibitors of Rho-A kinase and phosphatidylinositol 3-kinase had no effect. However, inhibitors to tyrosine kinases, the MAP kinases [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun amino-terminal kinase, p38], and to conventional protein kinase C showed no inhibition. Consistent with these findings, ET-1 did not cause activation of ERK1/2, a finding also seen in vessels held under pressure. In contrast, ET-1-induced c-fos expression was inhibited by the calcium chelator BAPTA, suggesting a role for intracellular calcium. This possibility was supported by the finding that raising the extracellular K(+) concentration caused increased expression of c-fos in a concentration-dependent manner. CONCLUSION: The results suggest that in the absence of wall stress, ET-1 is able to induce increased expression of c-fos independent of traditional growth pathways, such as MAP kinase. The mechanism appears to be calcium-dependent.     

Biochemical characterization of mitogen-activated protein (MAP) kinase activity in Toxoplasma gondii

Mitogen-activated protein (MAP) kinase or extracellular signal-regulated kinase (ERK) are activated by many extracellular stimuli. In this study, we investigated whether MAP kinase and tyrosine kinases were involved in transducing signals in Toxoplasma gondii. Using anti-phosphotyrosine and anti-active ERK antibodies, we identified several phosphorylated proteins in Toxoplasma. In particular, phosphorylation of a 47 kDa and a 43 kDa protein increased strongly after calcium influx. MAP kinase activity, caused by calcium influx, was determined using either a specific synthetic peptide, or an in gel kinase assay. Conversely, calcium chelators (BAPTA and EGTA) and a calcium channel blocker (nifedipine) inhibited this activation. Also, a specific inhibitor of MAP kinase kinase (PD 098059) blocked MAP kinase activity. Three specific anti-MAP kinase antibodies recognized the 47 kDa and 43 kDa proteins, which were putatively identified as ERK1- and ERK2-homologs, respectively. These findings provide early evidence of signal transduction involving members of the MAP kinase family in T. gondii. Received: 28 September 1999 / Accepted: 12 October 1999     

Fractalkine functions as a chemoattractant for osteoarthritis synovial fibroblasts and stimulates phosphorylation of mitogen‐activated protein kinases and Akt

Fractalkine (FKN/CX3CL1) has been detected in synovial fluids from osteoarthritis (OA) patients. Additionally, low‐level expression of the FKN receptor, CX3CR1, has been demonstrated in OA synovial lining. This study aimed to determine a biological function for this ligand/receptor pair in OA and to assess a potential signalling mechanism for FKN in this predominant synovial lining cell type, using chemotaxis assays, Western blotting and F‐actin staining. Chemotaxis assays demonstrate that the chemokine domain of FKN effectively induces migration of OA fibroblasts. Consistent with this finding, visualization of F‐actin demonstrates that 1 or 10 nM FKN induces noticeable reorganization of cytoskeletal structure in OA fibroblasts after 30 min stimulation with a maximal enhancement at approximately 2 h. In addition, Western blotting analysis demonstrates that FKN stimulates phosphorylation of the mitogen‐activated protein (MAP) kinases p38, c‐Jun N‐terminal kinase (JNK) and extracellular‐regulated kinase (ERK) 1/2 as well as the serine‐threonine kinase Akt at Ser 473 and Thr 308. All these phosphorylation events occur in a time‐dependent manner, with little or no activation within 1 min, and maximal activation occurring typically between 5 and 30 min. Moreover, inhibition of ERK 1/2 significantly reduces FKN‐induced OA fibroblast migration. These results suggest that FKN is a novel chemoattractant for OA fibroblasts, consistent with FKN‐induced alterations in cytoskeletal structure. In addition, FKN induces OA fibroblast signalling via the MAP kinases p38, JNK and ERK 1/2, as well as Akt.     

The role of mitogen-activated protein kinases in eotaxin-induced cytokine production from bronchial epithelial cells

Eotaxin is a critical chemokine eliciting migration of eosinophils and basophils in the pathogenesis of bronchial asthma. Recent studies have shown that the specific receptor for eotaxin, CCR3, is expressed in bronchial epithelial cells. Although mitogen-activated protein (MAP) kinases are involved in diverse cell functions of bronchial epithelial cells, their role in eotaxin signaling is unknown. In this study, we studied the activation and functional relevance of MAP kinases in bronchial epithelial cells stimulated with eotaxin. Eotaxin (1-100 nM) induced tyrosine/threonine phosphorylation and activation of extracellular regulated kinase (ERK) 1/2 and p38 in NCI-H(292) cells and normal human bronchial epithelial cells. The phosphorylation of these MAP kinases was detectable after 30 s, and peaked at 5 min. Eotaxin stimulated production of interleukin-8 and granulocyte macrophage colony-stimulating factor. Pretreatment of Compound X (a specific CCR3 antagonist), pertussis toxin, genistein, and wortmannin reduced the MAP kinase phosphorylation and cytokine production. The eotaxin-induced cytokine production was inhibited by specific inhibitors for MAP/ERK kinase (PD98059) and p38 MAP kinase (SB202190). These results suggest that both ERK1/2 and p38 MAP kinase activated by eotaxin have a critical role in the pathogenesis of asthma.     

Resveratrol-induced activation of the mitogen-activated protein kinases, ERK1 and ERK2, in human neuroblastoma SH-SY5Y cells.

Phosphorylation of the mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1 (ERK1) and extracellular signal-regulated kinase 2 (ERK2), induced by resveratrol, a natural antioxidant present in grapes and wine, has been studied in vitro on undifferentiated and differentiated (induction by retinoic acid) SH-SY5Y human neuroblastoma cells. In undifferentiated cells resveratrol 1 microM induced phosphorylation of ERK1 and ERK2, which was already evident at 2 min, peaked at 10 min and persisted at 30 min. A wide range (from 1 pM to 10 microM) of resveratrol concentrations were able to induce phosphorylation of ERK1 and ERK2, while higher concentrations (50-100 microM) inhibited MAP kinases phosphorylation. In retinoic acid (RA) differentiated cells resveratrol (1 microM) induced an evident increase in ERK1 and ERK2 phosphorylation. This study demonstrates that resveratrol, even at very low concentrations, may have a biological effect on neuron-like cells.     

Platelet-activating factor activates mitogen-activated protein kinases through the activation of phosphatidylinositol 3-kinase and tyrosine kinase in human eosinophils

We determined whether platelet-activating factor (PAF) activates mitogen-activated protein (MAP) kinases in human eosinophils, and if so, which signaling pathways are utilized for the MAP kinase activation. PAF activated 42-and 44-kDa MAP kinases (ERK1/ERK2) in eosinophils, which became maximal at 1 min after stimulation. The PAF receptor antagonist E6123 and pertussis toxin inhibited the PAF-induced MAP kinase activation in eosinophils. The phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin, tyrosine kinase inhibitors herbimycin A and genistein, and an intracellular Ca2+ chelator BAPTA/AM inhibited PAF-induced MAP kinase activation in eosinophils, whereas protein kinase C inhibitors staurosporine and calphostin C had no effect. Furthermore, wortmannin as well as herbimycin A and genistein, but not BAPTA/AM, prevented PAF-induced tyrosine phosphorylation of Shc adapter protein in eosinophils. Finally, the specific MEK inhibitor PD98059 inhibited PAF-induced chemotaxis in eosinophils. Taken together, these results indicate that PAF activates MAP kinases in eosinophils through the activation of PI 3-kinase and a tyrosine kinase and the increase in intracellular Ca2+ and that PAF-induced MAP kinase activation mediates chemotaxis in eosinophils.     

猫视皮层ERK mRNA表达的研究

丝裂原激活的蛋白激酶又称细胞外信号调节激酶,具有神经元信号传导功能,参与多种发育过程。它由细胞外特殊信号激活,将酪氨酸磷酸化过程转化为丝氨酸／苏氨酸磷酸化过程,后者进一步调节下游的蛋白激酶。因此,细胞外信号调节激酶在磷酸化的连锁反应过程中起着枢纽作用。本文应用地高辛标记寡核苷酸探针的原位杂交技术,研究了细胞外信号调节激酶的２ 种亚型 ＥＲＫ１ 和ＥＲＫ２ ｍ ＲＮＡ 在生后４ 周龄的幼猫视皮层各层的分布。结果表明,ＥＲＫ１ ｍ ＲＮＡ 在视皮层的表达层次明确,以第Ⅴ层和第Ⅲ层的表达为最强,Ⅱ层呈中等表达,Ⅳ层较弱,Ⅵ层最弱,即在视皮层的表达强度为Ⅴ、Ⅲ＞ Ⅱ＞ Ⅳ＞ Ⅵ。与ＥＲＫ１相比,ＥＲＫ２ 的表达普遍较强,但层次欠清晰,其表达强度为Ⅴ、Ⅱ＞ Ⅲ、Ⅳ＞ Ⅵ。ＥＲＫ 的２ 种亚型在视皮层的Ⅰ层即分子层均不表达。ＥＲＫ１ 和ＥＲＫ２ ｍ ＲＮＡ 在视皮层的这种特征性分布提示,在幼猫的视皮层发育过程中,ＥＲＫ 可能具有独特的信号传递作用。     

Differential activation of mitogen‐activated protein kinase signalling pathways by isometric contractions in isolated slow‐ and fast‐twitch rat skeletal muscle

Activation of mitogen‐activated protein (MAP) kinases has been implicated in the signal transduction pathways linking exercise to adaptive changes of muscle protein expression. In the present study, we investigated whether contractions of isolated muscles induced phosphorylation of extracellular signal‐regulated kinase 1 and 2 (ERK1/2) and p38 MAPK in a fibre‐type dependent manner. Slow‐twitch (soleus) and fast‐twitch (epitrochlearis, extensor digitorum longus) rat skeletal muscles were exposed to intermittent tetanic stimulation. Compared with the contralateral non‐stimulated muscle, contractions increased ERK1/2 phosphorylation to the same extent in fast‐ and slow‐twitch muscles. Significant increase in phosphorylation of p38 MAPK was observed in the fast‐twitch muscles only. The total amount of ERK1/2 and p38 MAPK proteins was higher in the slow‐twitch soleus muscle. In conclusion, MAP kinase signalling pathways are differentially activated and expressed in slow‐ and fast‐twitch muscles. In addition, this activation is owing to muscle contraction per se and do not demand additional external influence.     

Angiotensin II regulation of TGF-beta in murine mesangial cells involves both PI3 kinase and MAP kinase

INTRODUCTION: Chronic activation of the angiotensin II (AngII) type 1 receptor (AT-1) is a central event in the development of chronic kidney disease (CKD), in part through enhanced expression of TGF-beta, and AT-1 receptor blockade inhibits the progression to CKD in a variety of disease states. The AT-1 receptor is a heptahelical Gaq/11-coupled receptor that initiates phospholipase C activity and release of intracellular calcium; recent data suggest that the AT-1 receptor can also activate the epidermal growth factor receptor (EGFR), although the roles of specific EGF-mediated signaling cascades in AT-1 effects on mesangial cell biology are uncertain. We hypothesized that 2 EGFR-activated pathways, PI3 kinase and MAP kinase, are stimulated by the AT-1 receptor and, in part, regulate the effects of AngII on TGF-beta1 levels in mesangial cells. METHODS: We examined the effects of AT-1 receptor activation on EGFR, PI3 kinase, and MAP kinase activation in murine mesangial cells. Upon achieving 60-80% confluence, the medium was changed to low-serum for 48 hr and cells were exposed to either the AT-1 receptor blocker, losartan, the EGFR blocker, AG1478, or control medium, and then stimulated with AngII. Similar experiments were performed using LY294002 and U0126, specific inhibitors of PI3 kinase and MEK, respectively. Total cellular protein lysates and RNA were isolated. Activation of the receptors and pathways was evaluated by immunoblotting and levels of TGF-beta mRNA were measured using real-time quantitative RT-PCR. RESULTS: AngII induced autophosphorylation of EGFR (pY1068) and activated Akt and ERK, downstream targets of PI3 kinase and MAP kinase, respectively. AngII-mediated EGFR autophosphorylation was inhibited by losartan and AG1478. AG1478 also inhibited both basal and AngII-mediated activation of Akt and ERK. Finally, AngII-mediated increase in TGF-beta mRNA was inhibited by losartan, AG1478, LY249002, and U0126. CONCLUSIONS: Stimulation of the AT-1 receptor in murine mesangial cells results in activation of the EGF receptor with subsequent signaling through PI3 kinase and MAP kinase, thereby regulating TGF-beta mRNA levels. These data suggest that AT-1 receptor signaling pathways through EGFR may serve as a therapeutic target to inhibit the development of CKD.     

Human skeletal muscle cell differentiation is associated with changes in myogenic markers and enhanced insulin‐mediated MAPK and PKB phosphorylation

Aim: We hypothesized that myogenic differentiation of HSMC would yield a more insulin responsive phenotype. Methods: We assessed expression of several proteins involved in insulin action or myogenesis during differentiation of primary human skeletal muscle cultures (HSMC). Results: Differentiation increased creatine kinase activity and expression of desmin and myocyte enhancer factor (MEF)2C. No change in expression was observed for big mitogen‐activated protein kinase (BMK1/ERK5), MEF2A, insulin receptor (IR), hexokinase II, and IR substrates 1 and 2, while expression of glycogen synthase, extracellular signal‐regulated kinase 1 and 2 (ERK1/2 MAP kinase) and the insulin responsive aminopeptidase increased after differentiation. In contrast to protein kinase B (PKB)a, expression of (PKB)b increased, with differentiation. Both basal and insulin‐stimulated PI 3‐kinase activity increased with differentiation. Insulin‐mediated phosphorylation of PKB and ERK1/2 MAP kinase increased after differentiation. Conclusion: Components of the insulin‐signalling machinery are expressed in myoblast and myotube HSMC; however, insulin responsiveness to PKB and ERK MAP kinase phosphorylation increases with differentiation.     

Beta 1/beta 3 integrin ligation is uncoupled from ERK1/ERK2 activation in cytotoxic T lymphocytes

beta 3 integrins mediate fibronectin binding and enhanced activation of cytotoxic T lymphocytes (CTL). The intracellular signals initiated by beta 3 integrins in lymphocytes are not well characterized, but in many cell types, beta 1 integrin ligation activates mitogen-activated protein (MAP) kinases. In the present study, we find that fibronectin can synergize with very low levels of CD3 stimulation to activate the extracellular signal-regulated kinase (ERK)1 and ERK2 MAP kinases but that fibronectin alone induces no detectable MAP kinase activation in CTL. Surprisingly, antibodies to beta1 or beta 3 integrins were also unable to stimulate MAP kinase activation, suggesting that although beta 1 integrins are capable of stimulating MAP kinase activation in other cells, they cannot do so in CTL. In CTL, phosphorylation of proline-rich tyrosine kinase 2 downstream of integrin stimulation did not result in recruitment of the adaptor protein Grb2. Additionally, we examined the role of MAP kinases in regulating integrin-mediated adhesion. Anti-CD3-triggered adhesion to fibronectin was largely insensitive to the MAP kinase kinase inhibitor PD98059. Triggered cell-spreading on fibronectin was inhibited by PD98059 but not by U0126. In summary, ligation of beta 3 integrin by antibodies or fibronectin or of beta1 integrin by monoclonal antibodies fails to activate ERK MAP kinases, but integrin ligation synergizes with T cell receptor stimulation upstream of MAP kinases.     

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.     

Mechanisms of H4/ICOS costimulation: effects on proximal TCR signals and MAP kinase pathways

H4/ICOS is a costimulatory molecule related to CD28. Its effects on early TCR signals have been analyzed in mouse CD4(+) Th2 cells, expressing H4/ICOS at higher levels than Th1 clones. Anti-H4/ICOS antibodies strongly enhanced CD3-mediated tyrosine phosphorylation of ZAP-70, zeta, or Vav, as well as extracellular signal-regulated kinase (ERK), Jun N-terminal kinase (JNK) and p38 MAP kinase activation in these cells. The association of phosphoinositide 3-kinase (PI-3K) to H4/ICOS was enhanced by H4/ICOS cross-linking, and PI-3K inhibitors inhibited ERK and JNK activation and IL-4/IL-10 secretion, but not p38 MAP kinase or ZAP-70 activation. H4/ICOS-mediated activation of JNK, but not ERK or p38, is partially dependent on the expression of CD4 by the cells, whereas H4/ICOS costimulation is partially independent on CD28 expression. Cytochalasin D, an inhibitor of actin polymerization, inhibited ZAP-70, MAP kinase activation, or IL-4/IL-10 secretion. Neither cyclosporin A nor inhibitors of PKC produced detectable inhibition of ZAP-70 phosphorylation or MAP kinase activation in these Th2 cells. Cyclosporin A strongly inhibited IL-4, but not IL-10 secretion. ERK or JNKinhibitors partially inhibited IL-4 and IL-10 secretion, while PKC or p38 inhibitors had no significant effects on IL-4 or IL-10 secretion. Taken together, our data show clear similarities of costimulation mechanisms between H4/ICOS and CD28 during the early steps of TCR activation.     

Mitogen-activated protein kinases and apoptosis in PIN

 Mitogen-activated protein (MAP) kinases are key elements of the signalling systems needed to transduce different extracellular messages into cellular responses. At least three parallel MAP kinase pathways have been identified: one, stimulated by serum and growth factors to activate extracellular signal-regulated protein kinases (ERKs) by dual tyrosine and threonine phosphorylation, triggers cell proliferation or differentiation; the other two, induced by a variety of cellular stresses to activate c-jun N-terminal kinases (JNKs) and reactivating kinase (p38/RK), result in growth arrest and induction of apoptosis. Mitogen-activated protein kinase phosphatases (MKPs) inactivate MAP kinases through dephosphorylation and, thus, can modulate the MAP kinase pathways. Expression of JNK-1, ERK-1, p38/RK and MKP-1 proteins was investigated by immunohistochemistry and expression of MKP-1 mRNA by in situ hybridisation in 50 cases of high-grade prostatic intraepithelial neoplasia (PIN), thought to represent the precursor of prostate cancer. The frequency of apoptotic cells was also determined in these cases. Overexpression of the three MAP kinases and MKP-1 mRNA was found in all cases of high-grade PIN compared with normal prostate. Immunoreactivity for MKP-1 protein was found to be as intense as in normal glands in 30% and weaker in 56% of the PIN cases. Fourteen per cent of PIN cases did not stain with MKP-1 antibody. The proportion of apoptosis was significantly higher (P < 0.008) in PIN lesions that did not express MKP-1 protein than in those that did. These results are consistent with our previous demonstration of preferential inhibition of the apoptosis-related kinases by MKP-1 and further support the contention that MKP-1, even in PIN, may shift the balance existing between cell proliferation and death. When expressed, it may inhibiting those pathways that lead to apoptosis. Received: 27 August 1997 / Accepted: 29 December 1997     

Modulation of bronchial epithelial cells by IL-17.

BACKGROUND: The induction of epithelial cytokines/chemokines is crucial in the migration of leukocytes, and its regulatory mechanisms remain incompletely defined. OBJECTIVE: To determine the role of IL-17, a CD4(+) T cell-derived cytokine, in modulation of primary bronchial epithelial cells, the expression of IL-6, IL-8, and intercellular adhesion molecule 1 (ICAM-1) and the potential involvement of mitogen-activated protein (MAP) kinases in IL-17-mediated signaling were examined. METHODS: The levels of gene expression and protein production for IL-6 and IL-8 in IL-17-treated cells, in the presence or absence of MAP kinase inhibitors, were analyzed by RT-PCR and ELISA, respectively, and activation of MAP kinases was determined by Western blot analyses. RESULTS: We showed first that IL-17 induced time-dependent expression of IL-6 and IL-8 but not of the chemokines eotaxin and RANTES. In addition, IL-17 induced activation of extracellular signal-regulated kinase 1/2 but not of p38 or JNK kinases. A selective MAP kinase kinase inhibitor, PD98059, inhibited IL-17-induced IL-6 and IL-8. A combination of IL-17 and each of the cytokines IL-4, IL-13, and IFN-gamma further enhanced IL-8 expression. IL-17 alone did not induce ICAM-1 expression and showed no effect on IL-4- or IL-13-induced ICAM-1 expression. In contrast, a combination of IL-17 and IFN-gamma augmented IL-6 and ICAM-1 expression. CONCLUSION: These findings suggest that IL-17, alone or in combination with other cytokines, modulates airway inflammation via-in part-the expression of epithelial IL-6, IL-8, and ICAM-1.     

Angiotensin II up-regulates angiotensin I-converting enzyme (ACE), but down-regulates ACE2 via the AT1-ERK/p38 MAP kinase pathway

The recent discovery of the angiotensin II (Ang II)-breakdown enzyme, angiotensin I converting enzyme (ACE) 2, suggests the importance of Ang II degradation in hypertension. The present study explored the signaling mechanism by which ACE2 is regulated under hypertensive conditions. Real-time PCR and immunohistochemistry showed that ACE2 mRNA and protein expression levels were high, whereas ACE expression levels were moderate in both normal kidney and heart. In contrast, patients with hypertension showed marked ACE up-regulation and ACE2 down-regulation in both hypertensive cardiopathy and, particularly, hypertensive nephropathy. The inhibition of ACE2 expression was shown to be associated with ACE up-regulation and activation of extracellular regulated (ERK)1/2 and p38 mitogen-activated protein (MAP) kinases. In vitro, Ang II was able to up-regulate ACE and down-regulate ACE2 in human kidney tubular cells, which were blocked by an angiotensin II (AT)1 receptor antagonist (losartan), but not by an AT2 receptor blocker (PD123319). Furthermore, blockade of ERK1/2 or p38 MAP kinases by either specific inhibitors or a dominant-negative adenovirus was able to abolish Ang II-induced ACE2 down-regulation in human kidney tubular cells. In conclusion, Ang II is able to up-regulate ACE and down-regulate ACE2 expression levels under hypertensive conditions both in vivo and in vitro. The AT1 receptor-mediated ERK/p38 MAP kinase signaling pathway may be a key mechanism by which Ang II down-regulates ACE2 expression, implicating an ACE/ACE2 imbalance in hypertensive cardiovascular and renal damage.     

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.