Notch signaling may negatively regulate neonatal rat cardiac fibroblast-myofibroblast transformation

Cardiac fibroblast-myofibroblast transformation (CMT) is a critical event in the initiation of myocardial fibrosis. Notch signaling has been shown to regulate myofibroblast transformation from other kinds of cells. However, whether Notch signaling is also involved in CMT remains unclear. In the present study, expressions of Notch receptors in cardiac fibroblasts (CFs) were examined, effects of Notch signaling inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) and transforming growth factor-beta1 (TGF-beta1) on CMT were determined by increasing alpha-smooth muscle actin (alpha-SMA) expression and collagen synthesis, and Notch signaling was examined by analyzing expressions of Notch receptors. The results showed that: (1) Notch receptor 1, 2, 3 and 4 were all expressed in CFs; (2) DAPT promoted CMT in a time-dependent manner; (3) During the period of CMT induced by TGF-beta1, expressions of Notch receptor 1, 3 and 4 in CFs were down-regulated, whereas there was no change for Notch receptor 2. Moreover, the downtrends of Notch 1, 3 and 4 were corresponding to the trend growth of alpha-SMA expression and collagen synthesis. These results suggested that inhibiting of Notch signaling might promote CMT. The down-regulations of Notch receptor 1, 3 and 4 induced by TGF-beta1 may facilitate CMT. In conclusion, inhibition of Notch signaling might be a novel mechanism of CMT in myocardial fibrosis.     

Notch-1 Signaling Regulates Microglia Activation via NF-κB Pathway after Hypoxic Exposure In Vivo and In Vitro

Neuroinflammation mediated by the activated microglia is suggested to play a pivotal role in the pathogenesis of hypoxic brain injury; however, the underlying mechanism of microglia activation remains unclear. Here, we show that the canonical Notch signaling orchestrates microglia activation after hypoxic exposure which is closely associated with multiple pathological situations of the brain. Notch-1 and Delta-1 expression in primary microglia and BV-2 microglial cells was significantly elevated after hypoxia. Hypoxia-induced activation of Notch signaling was further confirmed by the concomitant increase in the expression and translocation of intracellular Notch receptor domain (NICD), together with RBP-Jκ and target gene Hes-1 expression. Chemical inhibition of Notch signaling with N-[N-(3,5-difluorophenacetyl)-1-alany1- S-phenyglycine t-butyl ester (DAPT), a γ-secretase inhibitor, effectively reduced hypoxia-induced upregulated expression of most inflammatory mediators. Notch inhibition also reduced NF-κB/p65 expression and translocation. Remarkably, Notch inhibition suppressed expression of TLR4/MyD88/TRAF6 pathways. In vivo, Notch signaling expression and activation in microglia were observed in the cerebrum of postnatal rats after hypoxic injury. Most interestingly, hypoxia-induced upregulation of NF-κB immunoexpression in microglia was prevented when the rats were given DAPT pretreatment underscoring the interrelationship between Notch signaling and NF-κB pathways. Taken together, we conclude that Notch signaling is involved in regulating microglia activation after hypoxia partly through the cross talk between TLR4/MyD88/TRAF6/NF-κB pathways. Therefore, Notch signaling may serve as a prospective target for inhibition of microglia activation known to be implicated in brain damage in the developing brain.     

Timely Inhibition of Notch Signaling by DAPT Promotes Cardiac Differentiation of Murine Pluripotent Stem Cells

The Notch signaling pathway plays versatile roles during heart development. However, there is contradictory evidence that Notch pathway either facilitates or impairs cardiomyogenesis in vitro. In this study, we developed iPSCs by reprogramming of murine fibroblasts with GFP expression governed by Oct4 promoter, and identified an effective strategy to enhance cardiac differentiation through timely modulation of Notch signaling. The Notch inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester) alone drove the iPSCs to a neuronal fate. After mesoderm induction of embryoid bodies initiated by ascorbic acid (AA), the subsequent treatment of DAPT accelerated the generation of spontaneously beating cardiomyocytes. The timed synergy of AA and DAPT yielded an optimal efficiency of cardiac differentiation. Mechanistic studies showed that Notch pathway plays a biphasic role in cardiomyogenesis. It favors the early–stage cardiac differentiation, but exerts negative effects on the late-stage differentiation. Therefore, DAPT administration at the late stage enforced the inhibition of endogenous Notch activity, thereby enhancing cardiomyogenesis. In parallel, DAPT dramatically augmented the expression of Wnt3a, Wnt11, BMP2, and BMP4. In conclusion, our results highlight a practicable approach to generate cardiomyocytes from iPSCs based on the stage-specific biphasic roles of Notch signaling in cardiomyogenesis.     

Inhibition of gamma-secretases alters both proliferation and differentiation of mesenchymal stem cells

INTRODUCTION: Human mesenchymal stem cell (hMSC) proliferation and development is regulated by many signalling pathways. gamma-Secretases play an important role in Notch signalling as well as other processes that are involved in developmental decisions, but their role in hMSC proliferation and cell fate decisions has not been explored. OBJECTIVE: To investigate the role of gamma-secretases in hMSC proliferation and differentiation. MATERIALS AND METHODS: Using the gamma-secretase inhibitor N-[N-(3,5-Difluorophenacetyl-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), we investigated their role in hMSC growth and differentiation to chondrogenic, osteogenic and adipogenic fates. RESULTS: We found that inhibiting gamma-secretases reduced the rate of hMSC proliferation, and altered hMSC differentiation in vitro. Addition of DAPT had an inhibitory effect on chondrogenesis resulting in impaired cartilage matrix production and altered chondrocyte morphology. DAPT treated chrodrocytic pellets had reduced levels of Hes1 and Hey1 suggesting that these effects are mediated via Notch signalling. Addition of the DAPT inhibitor to osteogenic cultures did not alter the appearance of bone markers, however, adipogenesis occurred in these cultures in a DAPT concentration-dependent manner. DAPT did not enhance adipogenesis in the presence of a potent adipogenic cocktail, but had an adipogenic effect when combined with dexamethasone only. CONCLUSION: We conclude that gamma-secretases play an important role in both hMSC proliferation and differentiation.     

Notch inhibition promotes fetal liver stem/progenitor cells differentiation into hepatocytes via the inhibition of HNF-1β

In a previous study, the Notch pathway inhibited with N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (also called DAPT) was shown to promote the differentiation of fetal liver stem/progenitor cells (FLSPCs) into hepatocytes and to impair cholangiocyte differentiation. The precise mechanism for this, however, was not elucidated. Two mechanisms are possible: Notch inhibition might directly up-regulate hepatocyte differentiation via HGF (hepatocyte growth factor) and HNF (hepatocyte nuclear factor)-4α or might impair cholangiocyte differentiation thereby indirectly rendering hepatocyte differentiation as the dominant state. In this study, HGF and HNF expression was detected after the Notch pathway was inhibited. Although our initial investigation indicated that the inhibition of Notch induced hepatocyte differentiation with an efficiency similar to the induction via HGF, the results of this study demonstrate that Notch inhibition does not induce significant up-regulation of HGF or HNF-4α in FLSPCs. This suggests that Notch inhibition induces hepatocyte differentiation without the influence of HGF or HNF-4α. Moreover, significant down-regulation of HNF-1β was observed, presumably dependent on an impairment of cholangiocyte differentiation. To confirm this presumption, HNF-1β was blocked in FLSPCs and was followed by hepatocyte differentiation. The expression of markers of mature cholangiocyte was impaired and hepatocyte markers were elevated significantly. The data thus demonstrate that the inhibition of cholangiocyte differentiation spontaneously induces hepatocyte differentiation and further suggest that hepatocyte differentiation from FLSPCs occurs at the expense of the impairment of cholangiocyte differentiation, probably being enhanced partially via HNF-1β down-regulation or Notch inhibition.     

Stretch-induced retinal vascular endothelial growth factor expression is mediated by phosphatidylinositol 3-kinase and protein kinase C (PKC)-zeta but not by stretch-induced ERK1/2, Akt, Ras, or classical/novel PKC pathways.

Stretch-induced expression of vascular endothelial growth factor (VEGF) is thought to be important in mediating the exacerbation of diabetic retinopathy by systemic hypertension. However, the mechanisms underlying stretch-induced VEGF expression are not fully understood. We present novel findings demonstrating that stretch-induced VEGF expression in retinal capillary pericytes is mediated by phosphatidylinositol (PI) 3-kinase and protein kinase C (PKC)-zeta but is not mediated by ERK1/2, classical/novel isoforms of PKC, Akt, or Ras despite their activation by stretch. Cardiac profile cyclic stretch at 60 cpm increased VEGF mRNA expression in a time- and magnitude-dependent manner without altering mRNA stability. Stretch increased ERK1/2 phosphorylation, PI 3-kinase activity, Akt phosphorylation, and PKC-zeta activity. Signaling pathways were explored using inhibitors of PKC, MEK1/2, and PI 3-kinase; adenovirus-mediated overexpression of ERK, PKC-alpha, PKC-delta, PKC-zeta, and Akt; and dominant negative (DN) mutants of ERK, PKC-zeta, Ras, PI 3-kinase and Akt. Although stretch activated ERK1/2 through a Ras- and PKC classical/novel isoform-dependent pathway, these pathways were not responsible for stretch-induced VEGF expression. Overexpression of DN ERK and Ras had no effect on VEGF expression in these cells. In contrast, DN PI 3-kinase as well as pharmacologic inhibitors of PI 3-kinase blocked stretch-induced VEGF expression. Although stretch-induced PI 3-kinase activation increased both Akt phosphorylation and activity of PKC-zeta, VEGF expression was dependent on PKC-zeta but not Akt. In addition, PKC-zeta did not mediate stretch-induced ERK1/2 activation. These results suggest that stretch-induced expression of VEGF involves a novel mechanism dependent upon PI 3-kinase-mediated activation of PKC-zeta that is independent of stretch-induced activation of ERK1/2, classical/novel PKC isoforms, Ras, or Akt. This mechanism may play a role in the well documented association of concomitant hypertension with clinical exacerbation of neovascularization and vascular permeability.     

Inhibition of Notch Signaling by a γ-Secretase Inhibitor Attenuates Hepatic Fibrosis in Rats

Notch signaling is essential to the regulation of cell differentiation, and aberrant activation of this pathway is implicated in human fibrotic diseases, such as pulmonary, renal, and peritoneal fibrosis. However, the role of Notch signaling in hepatic fibrosis has not been fully investigated. In the present study, we show Notch signaling to be highly activated in a rat model of liver fibrosis induced by carbon tetrachloride (CCl₄), as indicated by increased expression of Jagged1, Notch3, and Hes1. Blocking Notch signaling activation by a γ-secretase inhibitor, DAPT, significantly attenuated liver fibrosis and decreased the expression of snail, vimentin, and TGF-β1 in association with the enhanced expression of E-cadherin. The study in vitro revealed that DAPT treatment could suppress the EMT process of rat hepatic stellate cell line (HSC-T6). Interestingly, DAPT treatment was found not to affect hepatocyte proliferation in vivo. In contrast, DAPT can inhibit hepatocyte apoptosis to some degree. Our study provides the first evidence that Notch signaling is implicated in hepatic fibrogenesis and DAPT treatment has a protective effect on hepatocytes and ameliorates liver fibrosis. These findings suggest that the inhibition of Notch signaling might present a novel therapeutic approach for hepatic fibrosis.     

Effects of cyclic stretch on the molecular regulation of myocardin in rat aortic vascular smooth muscle cells

Background

The expression of myocardin, a cardiac-restricted gene, increases during environmental stress. How mechanical stretch affects the regulation of myocardin in vascular smooth muscle cells (VSMCs) is not fully understood. We identify the mechanisms and pathways through which mechanical stretch induces myocardin expression in VSMCs.

Results

Rat VSMCs grown on a flexible membrane base were stretched to 20% of maximum elongation, at 60 cycles per min. An in vivo model of aorta-caval shunt in adult rats was also used to investigate myocardin expression. Cyclic stretch significantly increased myocardin and angiotensin II (AngII) expression after 18 and 6 h of stretch. Addition of extracellular signal-regulated kinases (ERK) pathway inhibitor (PD98059), ERK small interfering RNA (siRNA), and AngII receptor blocker (ARB; losartan) before stretch inhibited the expression of myocardin protein. Gel shift assay showed that myocardin-DNA binding activity increased after stretch. PD98059, ERK siRNA and ARB abolished the binding activity induced by stretch. Stretch increased while myocardin-mutant plasmid, PD98059, and ARB abolished the promoter activity. Protein synthesis by measuring [³H]proline incorporation into the cells increased after cyclic stretch, which represented hypertrophic change of VSMCs. An in vivo model of aorta-caval shunt also demonstrated increased myocardin protein expression in the aorta. Confocal microscopy showed increased VSMC size 24 h after cyclic stretch and VSMC hypertrophy after creation of aorta-caval shunt for 3 days.

Conclusions

Cyclic stretch enhanced myocardin expression mediated by AngII through the ERK pathway in cultured rat VSMCs. These findings suggest that myocardin plays a role in stretch-induced VSMC hypertrophy.     

C-terminal fragment of presenilin is the molecular target of a dipeptidic gamma-secretase-specific inhibitor DAPT (N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester)

Gamma-secretase is a multimeric membrane protein complex composed of presenilin (PS), nicastrin, Aph-1 and, Pen-2 that is responsible for the intramembrane proteolysis of various type I transmembrane proteins, including amyloid beta-precursor protein and Notch. The direct labeling of PS polypeptides by transition-state analogue gamma-secretase inhibitors suggested that PS represents the catalytic center of gamma-secretase. Here we show that one of the major gamma-secretase inhibitors of dipeptidic type, N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT), targets the C-terminal fragment of PS, especially the transmembrane domain 7 or more C-terminal region, by designing and synthesizing DAP-BpB (N-[N-(3,5-difluorophenacetyl)-l-alanyl]-(S)-phenylglycine-4-(4-(8-biotinamido)octylamino)benzoyl)benzyl)methylamide), a photoactivable DAPT derivative. We also found that DAP-BpB selectively binds to the high molecular weight gamma-secretase complex in an activity-dependent manner. Photolabeling of PS by DAP-BpB is completely blocked by DAPT or its structural relatives (e.g. Compound E) as well as by arylsulfonamides. In contrast, transition-state analogue inhibitor L-685,458 or alpha-helical peptidic inhibitor attenuated the photolabeling of PS1 only at higher concentrations. These data illustrate the DAPT binding site as a novel functional domain within the PS C-terminal fragment that is distinct from the catalytic site or the substrate binding site.     

The Notch pathway attenuates interleukin 1β (IL1β)-mediated induction of adenylyl cyclase 8 (AC8) expression during vascular smooth muscle cell (VSMC) trans-differentiation

Vascular smooth muscle cell (VSMC) trans-differentiation, or their switch from a contractile/quiescent to a secretory/inflammatory/migratory state, is known to play an important role in pathological vascular remodeling including atherosclerosis and postangioplasty restenosis. Several reports have established the Notch pathway as tightly regulating VSMC response to various stress factors through growth, migration, apoptosis, and de-differentiation. More recently, we showed that alterations of the Notch pathway also govern VSMC acquisition of the inflammatory state, one of the major events accelerating atherosclerosis. We also evidenced that the inflammatory context of atherosclerosis triggers a de novo expression of adenylyl cyclase isoform 8 (AC8), associated with the properties developed by trans-differentiated VSMCs. As an initial approach to understanding the regulation of AC8 expression, we examined the role of the Notch pathway. Here we show that inhibiting the Notch pathway enhances the effect of IL1β on AC8 expression, amplifies its deleterious effects on the VSMC trans-differentiated phenotype, and decreases Notch target genes Hrt1 and Hrt3. Conversely, Notch activation resulted in blocking AC8 expression and up-regulated Hrt1 and Hrt3 expression. Furthermore, overexpressing Hrt1 and Hrt3 significantly decreased IL1β-induced AC8 expression. In agreement with these in vitro findings, the in vivo rat carotid balloon-injury model of restenosis evidenced that AC8 de novo expression coincided with down-regulation of the Notch3 pathway. These results, demonstrating that the Notch pathway attenuates IL1β-mediated AC8 up-regulation in trans-differentiated VSMCs, suggest that AC8 expression, besides being induced by the proinflammatory cytokine IL1β, is also dependent on down-regulation of the Notch pathway occurring in an inflammatory context.     

Metalloproteinases and transforming growth factor-alpha mediate substance P-induced mitogen-activated protein kinase activation and proliferation in human colonocytes

Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of proinflammatory cytokines from colonic epithelial cells. SP also stimulates cell proliferation, a critical event in tissue healing during chronic colitis, via transactivation of the epidermal growth factor (EGF) receptor (EGFR) and activation of mitogen-activated protein kinase (MAPK). Here we examined the mechanism by which SP induces EGFR and MAPK activation. We used non-transformed human NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) as well as untransfected U373 MG cells expressing high levels of endogenous NK-1R. Exposure of both cell lines to SP (10(-7) m) stimulated EGFR activation (1 min) followed by extracellular signal-regulated protein kinase (ERK1/2) activation (2-5 min). SP-induced ERK1/2 activation was blocked by pretreatment with the metalloproteinase inhibitor Batimastat/GM6001, the EGFR phosphorylation inhibitor AG1478, and the tumor necrosis factor-alpha-converting enzyme (TACE) inhibitor TAPI-1. Pretreatment with antibodies against potential EGFR ligands suggested that transforming growth factor-alpha (TGFalpha), but not the other EGFR ligands EGF, heparin-binding EGF, or amphiregulin, mediates SP-induced EGFR transactivation. SP stimulated TGFalpha release into the extracellular space that was measurable within 2 min, and this release was inhibited by metalloproteinase inhibitors and the TACE inhibitor TAPI-1. SP also induced MAPK-mediated cell proliferation that was inhibited by TACE, matrix metalloproteinase (MMP), EGFR, and MEK1 inhibitors. Thus, in human colonocytes, NK-1R-induced EGFR and MAPK activation and cell proliferation involve matrix metalloproteinases (most likely TACE) and the release of TGFalpha. These signaling mechanisms may be involved in the protective effects of NK-1R in chronic colitis.     

Metalloprotease disintegrin-mediated ectodomain shedding of EGFR ligands promotes intestinal epithelial restitution

EGF receptor (EGFR) promotes intestinal epithelial restitution, an important early process in the reepithelialization of ulcers. During epithelial restitution, the mechanism of EGFR activation is not known. We evaluated the role of TNF-converting enzyme (TACE), a metalloprotease disintegrin that proteolytically processes plasma membrane-anchored EGFR ligand precursors into their mature active forms, in wound-induced EGFR activation and epithelial restitution. With the use of scrape-wounded rat intestinal epithelial-1 (RIE-1) cell monolayers to model epithelial ulceration and restitution, we observed the rapid wound-dependent release of EGFR ligands into culture medium. RIE-1 cells express TACE, and treatment with phorbol ester, an established TACE stimulus, triggered the extracellular release of an EGFR ligand, transforming growth factor-alpha. Blockade of TACE using TNF processing inhibitor (TAPI-1), a specific hydroxamate inhibitor of metalloprotease disintegrins, prevented release of EGFR ligands from wounded RIE-1 cell monolayers. The restitution of wounded RIE-1 cell monolayers was also dose-dependently inhibited by TAPI-1, establishing the role of metalloprotease disintegrins in this process. These results have established a mechanism of EGFR activation in wounded intestinal epithelium and show an important functional role for metalloprotease disintegrin-mediated ectodomain shedding during intestinal epithelial restitution. Therefore, activation of the TACE-EGFR system might promote the healing of intestinal tract ulcers in patients.     

Gamma-secretase inhibitor suppressed Notch1 intracellular domain combination with p65 and resulted in the inhibition of the NF-κB signaling pathway induced by IL-1β and TNF-α in nucleus pulposus cells

In this experiment, the cross-talk betweenNotch and the NF-κB signaling pathway was examined to reveal the mechanism of slowing down the type II collagen (ColII) and aggrecan degeneration affected by inflammatory cytokines. The expression levels of ColII and aggrecan in the intervertebral disc were observed through immunohistochemistry and hematoxylin-eosin staining+alcian blue staining, respectively. The expression levels of ColII, aggrecan, Runx2, and NF-κB in the nuclei of human nucleus pulposus cells (hNPCs) in each group, as well as the phosphorylation and acetylation levels of p65, were examined through Western blot analysis. The 293T cells were transfected with a plasmid containing the overexpressed relative domain of Notch1 intracellular domain (NICD1), and immunoprecipitation (IP) was performed to observe the combination of NICD1 and p65. HNPCs were transfected with a lentiviral-contained overexpression lacking the ANK region of NICD1, and IP was performed to observe the combination of NICD1 and p65. The expression of ColII and aggrecan in the intervertebral disc culture increased when γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-1-alanyl]-Sphenylglycine t-butyl ester (DAPT) was added to the disc culture medium. Western blot revealed that DAPT inhibited p65 phosphorylation and acetylation, and the p65 and p50 levels in the nucleus decreased. NICD1 was found to be combined with p65 in contrast to the reverse consequences after ANK domain deletion in hNPCs. In nucleus pulposus cells, the combination of p65 and the ANK domain of NICD1 is a critical procedure for the degeneration related to the NF-κB signaling pathway activation induced by IL-1β and TNF-α.     

Effects of N-[N-(3, 5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) on cell proliferation and apoptosis in Ishikawa endometrial cancer cells

Endometrial cancer is one of the most common gynecological malignancies in Japan, where the disease shows an increasing morbidity. However, surgical therapy remains the treatment of choice for endometrial cancers that tend to be insensitive to radiation therapy and chemotherapy. Therefore, novel therapeutic strategies are required. The Notch signaling pathway regulates embryogenesis and cellular development, but deregulated Notch signaling may contribute to tumorigenesis in several cancers. Moreover, γ-secretase inhibitors have been shown to be potent inhibitors of the Notch signaling pathway; they suppress cellular proliferation and induce apoptosis in several cancer cells. In the present study, we investigated the effect of N-[N-(3, 5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT, γ-secretase inhibitor) on the cell proliferation and apoptosis in Ishikawa endometrial cancer cells. Real-time PCR detected mRNA derived from NOTCH1 and HES1, which are target genes of the Notch signaling pathway, in Ishikawa endometrial cancer cells. After blocking Notch signaling, cellular proliferation decreased, accompanied by increased expression of p21 mRNA and decreased expression of the cyclin A protein. Furthermore, blockade of Notch signaling induced apoptosis. These results suggest that the Notch signaling pathway may be involved in cell proliferation through cell cycle regulation and apoptosis in Ishikawa endometrial cancer cells. Inhibition of the Notch signaling pathway by γ-secretase inhibitors is expected to be a potential target of novel therapeutic strategies for endometrial cancer.     

Uniaxial cyclic stretch induces focal adhesion kinase (FAK) tyrosine phosphorylation followed by mitogen-activated protein kinase (MAPK) activation

We investigated the role of tyrosine phosphorylation of FAK in the stretch-induced MAPKs (extracellular signal-regulated kinase (ERK), p38MAPK) activation in mutant FAK-transfected fibroblasts. In response to uniaxial cyclic stretch (1 Hz, 120% in length), the levels of tyrosine phosphorylation of the Tyr-397 and Tyr-925 of FAK in control cells increased and peaked at 5 min (2.75 +/- 0.51, n = 3), and 20 min (2.98 +/- 0.58, n = 3), respectively, and the activities of MAPKs increased and peaked at approximately 10 min. On the other hand, in the mutant FAK-transfected cells, the stretch-induced MAPKs activation was significantly inhibited. The stretch-induced activation of MAPKs was also significantly abolished by either treatment with Gd(3+) or extracellular Ca(2+) removal which may inhibit intracellular Ca(2+) increase caused by the activation of cation selective (Ca(2+)-permeable) stretch activated (SACatC) channels. These results suggest that the stretch-induced tyrosine-phosphorylation of FAK via SACatC activation is critical for the stretch-induced MAPKs activation.