Cyclic stretch-induced cPLA2 mediates ERK 1/2 signaling in rabbit proximal tubule cells

BACKGROUND: Recent evidence from this laboratory have demonstrated a critical role of phospholipase A2 (PLA2) and arachidonic acid in angiotensin II type 2 (AT2) receptor-mediated kinase activation in renal epithelium independent of phosphoinositide- specific phospholipase C (PLC) and without the necessity of eicosanoid biosynthesis. In the present study, we investigated whether cyclic stress phosphorylates and activates the mitogen-activated protein kinase (MAPK) pathway and whether PLA2 activation mediates mechanotransduction in renal epithelial cells. The rational for studying kidney epithelial cells relates to their similarity to podocytes, which undergo mechanical stretch related to changes in intraglomerular pressure. METHODS: To produce strain or stretch, primary cultures of rabbit proximal tubular cell cells are grown in tissue culture wells having a collagen-coated Silastic deformable membrane bottoms and applying vacuum to the well to generate alternating cycles of stretch and relaxation (30 cycles/min). RESULTS: We found that cyclic stretching of rabbit proximal tubular cells caused a time- and intensity-dependent activation of extracellular signal-regulated kinases 1 and 2 (ERK 1/2) in proximal tubular cells as detected by its phosphorylation. In addition, mechanical stretch induced PLA2 activation and a subsequent rapid release of arachidonic acid. Inhibition of PLA2 by mepacrine and methyl arachidonyl fluorophosphonate ketone (AACOCF3) attenuated both arachidonic acid release and ERK 1/2 activation by cyclic stretch, supporting the importance of PLA2 as a mediator of mechanotransduction in renal proximal tubular cells. A requirement for extracellular Ca2+ and stretch-activated Ca2+ channels was also documented. Complete inhibition of ERK 1/2 by PD98059, a MAPK kinase (MEK) inhibitor, did not suppress stretch- induced PLA2 activation and arachidonic acid release, suggesting the later events were upstream of ERK 1/2. Cyclic stretch also caused rapid phosphorylation of the EGF receptor kinase and c-Src. Furthermore, arachidonic acid itself induced time- and dose-dependent phosphorylation of c-Src. In addition, the c-Src inhibitor PP2 and selective EGF receptor kinase inhibitor AG1478 attenuated both ERK 1/2 and EGF receptor phosphorylation by cyclic stretch. CONCLUSION: PLA2 dependence for ERK 1/2 activation in response to cyclic stretch in proximal tubular epithelial cells was established in this report. In addition, these findings indicate cyclic stretch increased the tyrosine phosphorylation of the EGF receptor and c-Src and that c-Src acts upstream of the EGF receptor to mediate its phosphorylation, whereby both are critical for stretch- induced ERK 1/2 activation in rabbit proximal tubular cells. These observations documents for the first time a mechanism of mechanical stretch-induced kinase activation mediated by stretch activated Ca2+ channels and PLA2-dependent release of arachidonic acid.     

Phosphate increases bone morphogenetic protein-2 expression through cAMP-dependent protein kinase and ERK1/2 pathways in human dental pulp cells

Extracellular phosphate (Pi) is known to play a key role in promoting osteoblastic differentiation by altering gene expression and cellular function. Importantly, it may be possible to use this knowledge as a means to deliver Pi to local sites to regenerate mineralized tissues associated with the oral cavity. Therefore, we determined the ability of Pi to regulate differentiation of pulp cells toward an odontoblast phenotype and further determined if this was in part due to an increase in the expression of bone morphogenetic protein (BMP)-2, a crucial regulator of mineralization. Results showed that Pi increased BMP-2 expression at both mRNA and protein level and BMP-2 promoter activity. Signaling inhibitors revealed that increased BMP-2 expression was dependent on cAMP/protein kinase A but not the protein kinase C signaling pathway. Treatment with 8-Br-cAMP, a cell-permeable analog of cAMP, enhanced Pi-mediated BMP-2 expression, but treatment with 8-Br-cAMP alone did not increase BMP-2, suggesting that cAMP is indispensable but not sufficient for Pi-mediated BMP-2 expression. Pi activated ERK1/2, and treatment with PD98059, an ERK1/2 inhibitor, suppressed Pi-mediated BMP-2 increase, indicating a requirement for activation of ERK1/2. ERK1/2 pathway may operate independently of cAMP-dependent signaling because MDL12,330A, an adenylate cyclase inhibitor, did not inhibit phosphorylation of ERK1/2 in response to Pi. Pulp cells expressed the sodium-dependent Pi transporter (NaPi) III type, but not NaPi-I type or NaPi-II type. Pi-mediated BMP-2 increase was inhibited in the presence of phosphonoformic acid, an inhibitor not only of NaPi transport but also of crystal nucleation. Furthermore, a similar inhibition was observed in the presence of pyrophosphate, a mineralization inhibitor. These findings demonstrate, for the first time, that Pi regulates BMP-2 expression via cAMP/protein kinase A and ERK1/2 pathways in human dental pulp cells.     

Stretch‐induced inhibition of Wnt/β‐catenin signaling in mineralizing osteoblasts

Wnt signaling is important for bone formation and osteoblastic differentiation. Recent findings indicate a stimulating role of Wnt signaling in bone mechanotransduction. However, negative effects of Wnt signaling on osteoblast differentiation and mineralization have been described as well. We conducted in vitro stretch experiments using human pre‐osteoblasts to study short‐ and long‐term effects of mechanical loading on Wnt/β‐catenin signaling. As the extracellular regulated kinase (ERK) pathway is known to be involved in mechanotransduction in osteoblasts, we also evaluated its role in Wnt/β‐catenin signaling. Stretch experiments up to 21 days (using stretch episodes of 15 min, alternated with 90 min rest) resulted in higher mineralization compared to static control cultures. We found that 15 min of stretch initially increased nuclear β‐catenin, but ultimately resulted in significant decrease at 12 and 40 h after stretch. Downregulation of Wnt‐responsive element activity 16 h after stretch, using a luciferase construct, further supported these findings. The presence of the ERK inhibitor U0126 did not alter the stretch‐induced decrease of β‐catenin levels. Our data indicate a biphasic effect of mechanical loading on β‐catenin in mineralizing human differentiating osteoblasts, which is independent of the ERK pathway. The osteogenic potential of our loading regime was confirmed by an increase in osteogenic differentiation markers such as alkaline phosphatase activity and calcium deposition after 3 weeks of culture. We conjecture that the biphasic aspect of Wnt/β‐catenin signaling with a strong decrease up to 40 h after the stretch induction, is important for the anabolic effects of mechanical stretch on bone. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:390–396, 2010     

Albumin activates ERK via EGF receptor in human renal epithelial cells

Emerging clinical and experimental evidence strongly implicates proteinuria in the progression of kidney disease. One pathway involves the activation of NFkappaB by albumin, and it has been demonstrated that the activation of NFkappaB induced by albumin is dependent on mitogen-activated protein kinase ERK1/ERK2. To study the effect of albumin on gene expression, primary human renal tubular cells were exposed in vitro to albumin (1%) for 6 h, and gene expression profiling was performed with the human oligonucleotide microarray, U133A Affymetrix Gene Chip. In all, 223 genes were differentially regulated by albumin, including marked upregulation of the EGF receptor (EGFR) and IL-8. Accordingly, the authors sought to delineate the signaling pathway linking albumin to the EGFR and activation of ERK1/ERK2. It was found that albumin led to a dose- and time-dependent activation of ERK1/ERK2. Treatment with albumin led to EGFR phosphorylation, but the activation of ERK1/ERK2 was prevented by pretreatment of the cells with AG-1478, the EGFR kinase inhibitor, at a dose that inhibited EGF-induced ERK1/ERK2 activation. Exogenously administered reactive oxygen species (ROS) were found to activate ERK1/ERK2 via the EGFR and src tyrosine kinase activity and pretreatment of cells with the antioxidant N-acetylcysteine (NAC) and the NADPH oxidase inhibitor DPI abrogated albumin-induced activation of ERK1/ERK2. The src tyrosine kinase inhibitor, PP2, also inhibited the albumin-induced activation of ERK1/ERK2. Finally, pretreatment with AG-1478, the MEK inhibitor UO126, and NAC prevented the albumin-induced increase in IL-8 expression. The authors conclude that the EGF receptor plays a central role in the signaling pathway that links albumin to the activation of ERK1/ERK2 and increased expression of IL-8. Gene profiling studies suggest that there may be a positive feedback loop through the EGFR that amplifies the response of the proximal tubule cell to albumin. Taken together, these results suggest that the EGFR may be an important treatment target for kidney disease associated with proteinuria.     

Renin-stimulated TGF-beta1 expression is regulated by a mitogen-activated protein kinase in mesangial cells

Recent evidence indicates that renin itself might be profibrotic, independent of angiotensin II; however, the signaling system by which renin exerts a direct effect is not known. We tested the hypothesis that renin receptor activation, in turn, activates the extracellular-signal regulated kinase 1 and 2 (ERK1/2) of the mitogen-activated protein kinase system in mesangial cells. Recombinant rat renin induced a rapid phosphorylation of ERK1/2 and subsequent cell proliferation in a dose- and time-dependent manner. ERK1/2 activation by renin addition was not altered by angiotensin-converting enzyme inhibition or angiotensin receptor blockade. An ERK kinase inhibitor significantly reduced the renin-induced ERK1/2 phosphorylation and the subsequent increase in transforming growth factor-beta1 (TGF-beta1) and plasminogen activator inhibitor-1 mRNA expression. A small-inhibiting RNA, siRNA, against the renin receptor completely blocked ERK1/2 activation by rat renin. We conclude that renin induces ERK1/2 activation though a receptor-mediated, angiotensin II-independent mechanism in mesangial cells. This renin-activated pathway triggers cell proliferation along with TGF-beta1 and plasminogen activator inhibitor-1 gene expression. This system may play an important role in the overall profibrotic actions of renin.     

Prostaglandin D2 induces apoptosis of human osteoclasts through ERK1/2 and Akt signaling pathways

In a recent study we have shown that prostaglandin D₂ (PGD₂) induces human osteoclast (OC) apoptosis through the activation of the chemoattractant receptor homologous molecule expressed on T-helper type 2 cell (CRTH2) receptor and the intrinsic apoptotic pathway. However, the molecular mechanisms underlying this response remain elusive. The objective of this study is to investigate the intracellular signaling pathways mediating PGD₂-induced OC apoptosis. OCs were generated by in vitro differentiation of human peripheral blood mononuclear cells (PBMCs), and then treated with or without the selective inhibitors of mitogen-activated protein kinase-extracellular signal-regulated kinase (ERK) kinase, (MEK)-1/2, phosphatidylinositol3-kinase (PI3K) and NF-κB/IκB kinase-2 (IKK2) prior to the treatments of PGD₂ as well as its agonists and antagonists. Fluorogenic substrate assay and immunoblotting were performed to determine the caspase-3 activity and key proteins involved in Akt, ERK1/2 and NF-κB signaling pathways. Treatments with both PGD₂ and a CRTH2 agonist decreased ERK1/2 (Thr202/Tyr204) and Akt (Ser473) phosphorylation, whereas both treatments increased β-arrestin-1 phosphorylation (Ser412) in the presence of naproxen, which was used to eliminate endogenous prostaglandin production. In the absence of naproxen, treatment with a CRTH2 antagonist increased both ERK1/2 and Akt phosphorylations, and reduced the phosphorylation of β-arrestin-1. Treatment of OCs with a selective MEK-1/2 inhibitor increased caspase-3 activity and OC apoptosis induced by both PGD₂ and a CRTH2 agonist. Moreover, a CRTH2 antagonist diminished the selective MEK-1/2 inhibitor-induced increase in caspase-3 activity in the presence of endogenous prostaglandins. In addition, treatment of OCs with a selective PI3K inhibitor decreased ERK1/2 (Thr202/Tyr204) phosphorylation caused by PGD₂, whereas increased ERK1/2 (Thr202/Tyr204) phosphorylation by a CRTH2 antagonist was attenuated with a PI3K inhibitor treatment. The DP receptor was not implicated in any of the parameters evaluated. Treatment of OCs with PGD₂ as well as its receptor agonists and antagonists did not alter the phosphorylation of RelA/p65 (Ser536). Moreover, the caspase-3 activity was not altered in OCs treated with a selective IKK2/NF-κB inhibitor. In conclusion, endogenous or exogenous PGD₂ induces CRTH2-dependent apoptosis in human differentiated OCs; β-arrestin-1, ERK1/2, and Akt, but not IKK2/NF-κB are probably implicated in the signaling pathways of this receptor in the model studied.     

ERK5信号通路介导流体剪切力对MC3T3-E1成骨细胞MMPs、TIMPs表达的影响

目的观察流体剪切力(fluid shear stress,FSS)作用下,MC3T3-E1成骨细胞中细胞基质金属蛋白酶(matrix metalloproteinases,MMPs)和基质金属蛋白酶抑制剂(tissue inhibitors of metalloproteinases,TIMPs)的表达情况,并探讨ERK5信号通路在其中的作用。方法对MC3T3-E1成骨细胞进行不同的处理,分为正常组、XMD8-92组、FSS组和FSS+XMD8-92组,对FSS组施加12 dyn/cm~2流体剪切力,采用蛋白免疫印迹法分别检测P-ERK5、ERK5、MMPs和TIMPs蛋白水平的变化。结果生理强度(12 dyn/cm~2)的流体剪切力作用于MC3T3-E1成骨细胞45 min后能显著上调MMPs的表达,下调TIMPs的表达,但此效应可被ERK5高选择性抑制剂XMD8-92阻断。结论 ERK5信号通路调控流体剪切力对成骨细胞MMPs、TIMPs蛋白的表达。     

Prostaglandin-dependent activation of ERK mediates cell proliferation induced by transforming growth factor beta in mouse osteoblastic cells

Transforming growth factor beta (TGF(beta)) is a major coupling factor for bone turnover and is known to stimulate osteoblastic proliferation. Recent information indicates that, in addition to the Smad pathway, TGF(beta) also activates MAP kinases in osteoblastic cells. The role of these signaling cascades in cell proliferation induced by TGF(beta) as well as the cellular and molecular mechanisms of their activation by TGF(beta) has been investigated in this study. In MC3T3-E1 cells, TGF(beta) enhanced cell proliferation by about 2-fold and induced activation of the three MAP kinases, extracellular regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Surprisingly, however, whereas activation of Smad2 was rapid and maximal after 15-min incubation, activation of MAP kinases was delayed with p38 stimulation detected after 1-h exposure and activation of ERK and JNK after 3 h, suggesting indirect activation of MAP kinases by TGF(beta). Among factors known to be released in response to TGF(beta) in osteoblastic cells and influence their growth, prostaglandins (PGs) were good candidates that were further investigated for mediating TGF(beta)-induced activation of MAP kinases and cell proliferation. Indomethacin, a selective inhibitor of PG synthesis, completely blunted cell proliferation induced by TGF(beta) and markedly reduced activation of MAP kinases without influencing Smad2 phosphorylation. EP4A, a specific PGE2 receptor antagonist, also blunted TGF(beta)-induced osteoblastic proliferation. In addition to these effects, PGE2 rapidly activated MAP kinases in MC3T3-E1 cells and increased cell proliferation by about 2-fold. The role of each MAP kinases in mediating TGF(beta)- and PGE2-induced cell proliferation was investigated using selective inhibitors. U0126, a specific inhibitor of the ERK pathway, completely blocked both TGF(beta)- and PGE2-induced cell proliferation whereas SB203580 and SP600125, which are selective inhibitors of, respectively, p38 and JNK pathways, had no effect. Finally, the effect of PGE2 on activation of ERK was mimicked by phorbol esters and not by forskolin, and was associated with activation of protein kinase C. This latter effect and the stimulation of ERK induced by PGE2 were completely blocked by a specific inhibitor of PKC. In conclusion, data presented in this study strongly suggest that the local release of PGE2 is involved in cell proliferation induced by TGF(beta) in osteoblastic cells. This effect is mediated by the ERK pathway activated by a PKC-dependent mechanism.     

Pancreatic cancer cell proliferation is phosphatidylinositol 3-kinase dependent

BACKGROUND: Genetic mutations found in pancreatic cancer (K-ras, p16, p53) lead to inappropriate cellular proliferation. Mitogens stimulate proliferation via the phosphatidylinositol 3-kinase (PI3K)- and/or the p44/42-mitogen-activated protein kinase [p44/42-MAPK or extracellular signal-regulated kinase (ERK)] signaling pathways. We examined whether inhibition of either PI3K or ERK could limit proliferation in human pancreatic cancer. METHODS: Proliferation was stimulated in quiescent human pancreatic cancer cell lines (BxPC3 and Panc-1) by 10% fetal calf serum (FCS). In certain samples, PD98059 (an ERK inhibitor) or LY294002 (a PI3K inhibitor) was also added. AKT phosphorylation (indicating PI3K activity) and ERK phosphorylation (ERK activation) were determined by Western blot. Cell viability was determined by MTT assay. Cell cycle progression and apoptosis were determined by flow cytometry. A two-tailed t test was used for statistical analysis of the data (significance P < 0.05). RESULTS: LY294002 inhibited the PI3K pathway without affecting ERK activation in response to serum. PD98059 inhibited the ERK pathway specifically. In both BxPC-3 and Panc-1 cell lines, LY294002 inhibited serum-induced proliferation. This was associated with G(1) cell cycle arrest and with an increase in the rate of apoptosis. PD98059 inhibited proliferation only in BxPC3 cells, and to a lesser degree than did LY294002. CONCLUSIONS: PI3K signaling appears to be necessary for G(1)-to-S phase progression and proliferation in pancreatic cancer cells. ERK plays a lesser role in mitogen-induced proliferation. Pharmacological inhibition of PI3K may decrease proliferation, increase apoptosis, and potentially confer therapeutic benefit in pancreatic cancer.     

Autocrine activation of the IGF-I signaling pathway in mesangial cells isolated from diabetic NOD mice.

Mesangial cells isolated from NOD mice after the onset of diabetes have undergone a stable phenotypic change. This phenotype is characterized by increased expression of IGF-I and downregulation of collagen degradation, which is associated with decreased MMP-2 activity. Here, we investigated the IGF-I signaling pathway in mesangial cells isolated from NOD mice before (nondiabetic NOD mice [ND-NOD]) and after (diabetic NOD mice [D-NOD]) the onset of diabetes. We found that the IGF-I signaling pathway in D-NOD cells was activated by autocrine IGF-I. They had phosphorylation of the IGF-I receptor beta-subunit, phosphorylation of insulin receptor substrate (IRS)-1, and association of the p85 subunit (phosphatidylinositol 3-kinase [PI3K]) with the IGF-I receptor and IRS-1 in D-NOD cells in the basal state. This was also associated with increased phosphorylation of ERK2 in D-NOD mesangial cells. Inhibiting autocrine IGF-I from binding to its receptor using an IGF-I-neutralizing antibody or inhibiting IGF-I signaling pathways using a specific PI3K inhibitor or a specific mitogen-activated protein kinase/extracellular response kinase kinase inhibitor decreased phosphorylated ERKs in D-NOD cells. Importantly, this was associated with increased MMP-2 activity. The addition of exogenous IGF-I to ND-NOD activated signal transduction. Therefore, we conclude that the IGF-I signaling pathway is intact in both D-NOD and ND-NOD cells. However, the phenotypic change in D-NOD cells is associated with constitutive activation of the IGF-I signaling pathways, which may participate in the development and progression of diabetic glomerulosclerosis.     

In vitro stretch injury induces time- and severity-dependent alterations of STEP phosphorylation and proteolysis in neurons

Striatal-enriched tyrosine phosphatase (STEP) has been identified as a component of physiological and pathophysiological signaling pathways mediated by N-methyl-d-aspartate (NMDA) receptor/calcineurin/calpain activation. Activation of these pathways produces a subsequent change in STEP isoform expression or activation via dephosphorylation. In this study, we evaluated changes in STEP phosphorylation and proteolysis in dissociated cortical neurons after sublethal and lethal mechanical injury using an in vitro stretch injury device. Sublethal stretch injury produces minimal changes in STEP phosphorylation at early time points, and increased STEP phosphorylation at 24 h that is blocked by the NMDA-receptor antagonist APV, the calcineurin-inhibitor FK506, and the sodium channel blocker tetrodotoxin. Lethal stretch injury produces rapid STEP dephosphorylation via NR2B-containing NMDA receptors, but not calcineurin, and a subsequent biphasic phosphorylation pattern. STEP(61) expression progressively increases after sublethal stretch with no change in calpain-mediated STEP(33) formation, while lethal stretch injury results in STEP(33) formation via a NR2B-containing NMDA receptor pathway within 1 h of injury. Blocking calpain activation in the initial 30 min after stretch injury increases the ratio of active STEP in cells and blocks STEP(33) formation, suggesting that STEP is an early substrate of calpain after mechanical injury. There is a strong correlation between the amount of STEP(33) formed and the degree of cell death observed after lethal stretch injury. In summary, these data demonstrate that previously characterized pathways of STEP regulation via the NMDA receptor are generally conserved in mechanical injury, and suggest that calpain-mediated cleavage of STEP(33) should be further examined as an early marker of neuronal fate after stretch injury.     

Extracellular signal-regulated kinases and calcium channels are involved in the proliferative effect of bisphosphonates on osteoblastic cells in vitro.

Bisphosphonates (BPs) are analogues of pyrophosphate, which are widely used for the treatment of different pathologies associated with imbalances in bone turnover. Recent evidence suggested that cells of the osteoblastic lineage might be targets of the action of BPs. The objective of this work was to determine whether BPs induce proliferation of osteoblasts and whether this action involves activation of the extracellular signal-regulated kinases (ERKs). We have shown that three different BPs (olpadronate, pamidronate, and etidronate) induce proliferation in calvaria-derived osteoblasts and ROS 17/2.8 as measured by cell count and by [3H]thymidine uptake. Osteoblast proliferation induced by all BPs diminished to control levels in the presence of U0126, a specific inhibitor of the upstream kinase MEK 1 responsible for ERK phosphorylation. Consistent with this, BPs induced ERK activation as assessed by in-gel kinase assays. Phosphorylation of ERK1/2 was induced by the BPs olpadronate and pamidronate within 30 s, followed by rapid dephosphorylation, whereas etidronate induced phosphorylation of ERKs only after 90 s of incubation and returned to basal levels within 15-30 minutes. In addition, both BP-induced cell proliferation and ERK phosphorylation were reduced to basal levels in the presence of nifedipine, an L-type voltage-sensitive calcium channel (VSCC) inhibitor. These results show that BP-induced proliferation of osteoblastic cells is mediated by activation of ERKs and suggest that this effect requires influx of Ca2+ from the extracellular space through calcium channels.     

Connexin 43 and ERK regulate tension‐induced signal transduction in human periodontal ligament fibroblasts

Periodontal ligament (PDL) fibroblasts play an important role in preserving periodontal homeostasis and transmitting mechanical signals to alveolar bone. Connexin 43 (Cx43), a gap junction protein, is essential for bone homeostasis and regulates bone remodeling. However, the function of Cx43 in human PDL fibroblast‐regulated bone remodeling has not yet been elucidated. In this study, human PDL fibroblasts were exposed to cyclic mechanical tension with a maximum 5% elongation for different durations. We then examined the expression of signaling molecules related to osteogenesis and osteoclastogenesis at both the mRNA and protein levels as well as the activity of extracellular signal‐regulated kinase (ERK) in human PDL fibroblasts after loading. We found that mechanical tension increased Cx43, which further upregulated osteogenic (e.g., RUNX2, Osterix, and OPG) and down‐regulated osteoclastogenic (e.g., RANKL) signaling molecules. Suppressing Cx43 gene (Gja1) by siRNA inhibited the increase in osteogenesis‐related molecules but enhanced RANKL expression. Similar to Cx43, activated ERK1/2 was also enhanced by mechanical tension and suppressed by Cx43 siRNA. Inhibition of ERK1/2 signaling using PD98059 reduced the tension‐regulated increase in osteogenesis‐related molecules but enhanced that of osteoclastogenesis‐related ones. These findings suggest that cyclic tension may involve into the osteogenic or osteoclastogenetic differentiation potential of human PDL fibroblasts via the Cx43‐ERK1/2 signaling pathway. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:1008–1014, 2015.     

Modulation of vascular smooth muscle cell alignment by cyclic strain is dependent on reactive oxygen species and P38 mitogen-activated protein kinase

PURPOSE: The aim of this study was to investigate the molecular targets of reactive oxygen species (ROS) and to determine whether cyclic strain induces smooth muscle cell (SMC) alignment via the ROS system. We assessed stretch-induced nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activation and the redox sensitivity of cyclic strain-stimulated activation of the mitogen-activated protein kinase (MAPK) family. METHODS: SMCs were seeded on flexible collagen I-coated plates and exposed to cyclic strain. NAD(P)H oxidase activation was measured with lucigenin-enhanced chemiluminescent detection of superoxide. Activation of MAPK was detected by determining phosphorylation of extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK1/2), and p38 MAPK with immunoblotting. In other experiments, SMCs were exposed to diphenylene iodonium (DPI), an NAD(P)H inhibitor, 30 minutes before stretch. MAPK activation and cell orientation were then assessed. RESULTS: Cyclic strain elicits a rapid increase in intracellular NADH/NADPH oxidase in SMCs. There was also a rapid and robust phosphorylation of ERK1/2, JNK1/2, and p38 MAPK. Cyclic strain-induced intracellular NAD(P)H generation was almost completely blocked with DPI. DPI also inhibited the strain-induced phosphorylation of ERK1/2, JNK1/2, and p38 MAPK. Both the p38 MAPK specific inhibitor, SB 202190, and DPI blocked cyclic strain-induced cell alignment, but PD98059, an ERK1/2-specific inhibitor, and SP600125, an anthrazolone inhibitor of JNK, did not. CONCLUSION: Our results provide evidence that p38 MAPK is a critical component of the oxidant stress ROS-sensitive signaling pathway and plays a crucial role in vascular alignment induced by cyclic stain.     

Cytoskeletal signaling by way of alpha-actinin-1 mediates ERK1/2 activation by repetitive deformation in human Caco2 intestinal epithelial cells

BACKGROUND: Repetitive deformation stimulates proliferation in human Caco2 intestinal epithelial cells by way of an ERK1/2-dependent pathway. We examined the effects of cytoskeletal perturbation on deformation-induced signaling in Caco2 cells. METHODS: The Caco2 cell cytoskeleton was disrupted with either cytochalasin D, phalloidin, colchicine, or paclitaxel. Levels of alpha-actinin-1 and -4 and paxillin were reduced by specific small interfering RNA. Cells on collagen I-precoated membranes were subjected to 10% repetitive deformation at 10 cycles/min. After 1 hour, cells were lysed for Western blot analysis. RESULTS: Strain-activated ERK1/2, focal adhesion kinase, and Src phosphorylation in dimethyl sulfoxide- and/or nontargeting small interfering RNA-treated control cell populations. Cytochalasin D and paclitaxel, but not phalloidin and colchicine, blocked ERK1/2 phosphorylation. A decrease in alpha-actinin-1, but not in alpha-actinin-4 or paxillin, inhibited ERK1/2 and focal adhesion kinase phosphorylation, whereas Src activation appears to be independent of these effects. CONCLUSIONS: The intestinal epithelial cell cytoskeleton may transduce mechanical signals by way of alpha-actinin-1 into the focal adhesion complex, culminating in ERK1/2 activation and proliferation.