Cyclic strain activates redox-sensitive proline-rich tyrosine kinase 2 (PYK2) in endothelial cells

Proline-rich tyrosine kinase 2 (PYK2), structurally related to focal adhesion kinase, has been shown to play a role in signaling cascades. Endothelial cells (ECs) under hemodynamic forces increase reactive oxygen species (ROS) that modulate signaling pathways and gene expression. In the present study, we found that bovine ECs subjected to cyclic strain rapidly induced phosphorylation of PYK2 and Src kinase. This strain-induced PYK2 and Src phosphorylation was inhibited by pretreating ECs with an antioxidant N-acetylcysteine. Similarly, ECs exposed to H(2)O(2) increased both PYK2 and Src phosphorylation. An increased association of Src to PYK2 was observed in ECs after cyclic strain or H(2)O(2) exposure. ECs treated with an inhibitor to Src (PPI) greatly reduced Src and PYK2 phosphorylation, indicating that Src mediated PYK2 activation. Whereas the protein kinase C (PKC) inhibitor (calphostin C) pretreatment was shown to inhibit strain-induced NADPH oxidase activity, ECs treated with either calphostin C or the inhibitor to NADPH oxidase (DPI) reduced strain-induced ROS levels and then greatly inhibited the Src and PYK2 activation. In contrast to the activation of PYK2 and Src with calcium ionophore (ionomycin), ECs treated with a Ca(2+) chelator inhibited both phosphorylation, indicating that PYK2 and Src activation requires Ca(2+). ECs transfected with antisense to PKCalpha, but not antisense to PKCepsilon(,) reduced cyclic strain-induced PYK2 activation. These data suggest that cyclic strain-induced PYK2 activity is mediated via Ca(2+)-dependent PKCalpha that increases NADPH oxidase activity to produce ROS crucial for Src and PYK2 activation. ECs under cyclic strain thus activate redox-sensitive PYK2 via Src and PKC, and this PYK2 activation may play a key role in the signaling responses in ECs under hemodynamic influence.     

Inhibition of mechanical strain-induced fetal rat lung cell proliferation by gadolinium,a stretch-activated channel blocker

Normal growth of the fetal lung is dependent upon fetal breathing movements. We have previously demonsrated that mechanical strain, simulating fetal breathing movements, stimulated DNA synthesis and cell division by reaggregated alveolar-like structures of fetal rat lung cells. Herein, we report that both intracellular and extracellular calcium modulate strain-induced proliferative activity. Strain-induced cell proliferation was inhibited by BAPTA/AM, an intracellular calcium chelator. The intracellular calcium modulators, cyclopiazonic acid and 2,5-di-(tert-butyl)-1, 4-benzohydroquinone, increased DNA synthesis of unstrained cultures and partially reduced strain-induced cell growth activity. A similar effect was noted with the calcium ionophore A23187. Extracellular Ca²⁺ increased DNA synthesis in unstrained cultures in a concentration-dependent fashion. The stimulatory effect of strain on DNA synthesis was also dependent on the calcium concentration in the medium. Furthermore, strain-enhanced DNA synthesis was inhibited by the presence of a divalent ion chelator, EGTA, in the medium. Mechanical strain increased ⁴⁵Ca²⁺ influx within 1 min after the onset strain. This rapid entry of calcium was not affected by calcium channel blockers, such as verapamil or Ni²⁺. Calcium channel blockers verapamil, nifedipine, Ni²⁺, Co²⁺, or La³⁺ also did not inhibit strain-induced cell growth activity. In contrast, gadolinium, a stretch-activated channel blocker, inhibited strain-induced ⁴⁵Ca²⁺ influx and suppressed strain-enhanced DNA synthesis. We conclude that the entry of calcium into cells through stretch-activated ion channels plays a critical role in strain-induced fetal lung cell proliferation. © 1994 Wiley-Liss, Inc.     

Expression of the Proenkephalin A Gene and [Met5]-Enkephalin Secretion Induced by Arachidonic Acid in Bovine Adrenal Medullary Chromaffin Cells: Involvement of Second Messengers

Abstract: We have previously reported that arachidonic acid (AA) increases the long-term secretion of [Met⁵]-enkephalin (ME) and the expression of proenkephalin A (proENK) mRNA in bovine adrenal medullary chromaffin (BAMC) cells. To characterize the underlying signal transductional mechanisms for the AA-induced responses, the interactions of AA with several second messenger systems were studied. Long-term (24-h) treatment with AA (100 µ M ) increased both the secretion of ME and the expression of proENK mRNA. Pretreatment of BAMC cells with nimodipine (1 µ M ), but not with ω-conotoxin GVIA (1 µ M ), inhibited the secretion of ME and the expression of proENK mRNA induced by AA. Calmidazolium (1 µ M ), a calmodulin antagonist, also significantly inhibited AA-induced responses. However, a protein kinase C (PKC) inhibitor, sphingosine (36 µ M ), was ineffective in blocking AA-induced responses. In addition, the down-regulation of PKC by phorbol 12-myristate 13-acetate (0.1 µ M ) for 48 h did not inhibit the AA-induced responses. Forskolin (5 µ M ), an adenyl cyclase activator, alone increased the secretion of ME as well as proENK mRNA levels and, when coincubated with AA, showed an additive effect on the secretion of ME and the levels of proENK mRNA. The results suggest that the Ca²⁺/calmodulin pathway, but not the protein kinase A or PKC pathway, is partially involved in mediating the AA-induced increases of the long-term secretion of ME and the levels of proENK mRNA.     

Endothelin-1 Increases Arachidonic Acid Release in C6 Glioma Cells Through a Potassium-Modulated Influx of Calcium

Abstract: Endothelin-1 (Et-1) but not a range of other receptor agonists stimulated the release of arachidonic acid (AA) in C6 glioma. Et-1 activation was concentration dependent and was inhibited by chelation of extracellular calcium. The calcium ionophores A23187 and ionomycin could also stimulate release of AA. Et-1 caused an early increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) followed by a sustained but lower plateau level. The sensitivity of the response to quinacrine, its dependence on Ca²⁺, and the demonstration of an increase in phospholipase A₂ (PLA₂) activity that was insensitive to dithiothreitol suggested that the release of AA was due to activation of cytosolic PLA₂ in the cells. Staurosporine, a protein kinase C (PKC) inhibitor, had no effect on Et-1-induced AA release but abolished that by phorbol 12-myristate 13-acetate, demonstrating that the Et-1 response was PKC independent. Raised levels of extracellular KCI inhibited both AA release and the increase in [Ca²⁺]_i triggered by Et-1, whereas valinomycin, which causes K⁺ efflux, not only caused a rapid rise in [Ca²⁺]_i but also caused AA mobilisation. The results therefore suggest that Et-1 activation of PLA₂ in this cell type requires calcium influx dependent on K⁺ efflux.     

17beta-estradiol inhibits cyclic strain-induced endothelin-1 gene expression within vascular endothelial cells

It has been well documented previously that 17beta-estradiol (E2) exerts a protective effect on cardiovascular tissue. The possible role of E2 in the regulation of endothelin (ET)-1 production has been previously reported, although the complex mechanisms by which E2 inhibits ET-1 expression are not completely understood. The aims of this study were to examine whether E2 was able to alter strain-induced ET-1 gene expression and also to identify the putative underlying signaling pathways that exist within endothelial cells. For cultured endothelial cells, E2 (1-100 nM), but not 17alpha-estradiol, inhibited the level of strain-induced ET-1 gene expression and also peptide secretion. This inhibitory effect elicited by E2 was able to be prevented by the coincubation of endothelial cells with the estrogen receptor antagonist ICI-182,780 (1 microM). E2 also inhibited strain-enhanced NADPH oxidase activity and intracellular reactive oxygen species (ROS) generation as measured by the redox-sensitive fluorescent dye 2',7'-dichlorofluorescin diacetate and the level of extracellular signal-regulated kinase (ERK) phosphorylation. Furthermore, the presence of E2 and antioxidants such as N-acetylcysteine and diphenylene iodonium were able to elicit a decrease in the level of strain-induced ET-1 secretion, ET-1 promoter activity, ET-1 mRNA, ERK phosphorylation, and activator protein-1 binding activity. In summary, we demonstrated, for the first time, that E2 inhibits strain-induced ET-1 gene expression, partially by interfering with the ERK pathway via the attenuation of strain-induced ROS generation. Thus this study delivers important new insight regarding the molecular pathways that may contribute to the proposed beneficial effects of estrogen on the cardiovascular system.     

Cyclic strain stimulates monocyte chemotactic protein-1 mRNA expression in smooth muscle cells

Hemodynamic forces are important determinants for the formation of atherosclerotic plaques. The recruitment of circulating monocytes into the arterial wall is an important step during atherogenesis. Monocyte chemotactic protein-1 (MCP-1) has been shown to be a key factor for monocyte transmigration. This study examined the effects of cyclic strain on MCP-1 mRNA expression levels of cultured rat aortic smooth muscle cells. The MCP-1 mRNA levels of aortic smooth muscle cells first increased as the duration of cyclic strain increased, reaching the maximum at 6-12 h, maintained at high levels throughout the 48-h strain period. To explore signaling pathways mediating cyclic strain-stimulated MCP-1 mRNA expression, we examined the involvement of tyrosine kinase and protein kinase C (PKC). Tyrosine kinase inhibitors, genistein and tyrphostin 51, at 50 microM blocked cyclic strain-stimulated MCP-1 mRNA expression. Preincubation with a PKC activator, phorbol 12-myristate 13-acetate (PMA), 2 microM, for 24 h to downregulate PKC did not decrease cyclic strain-induced MCP-1 mRNA expression. A 6-h incubation with 0. 1 microM PMA to activate PKC, which stimulated MCP-1 expression when applied alone, abolished the stimulatory effects of cyclic strain. A specific PKC inhibitor, calphostin C (0.1 microM), diminished cyclic strain-stimulated MCP-1 mRNA expression. Angiotensin II at 10 or 1,000 nM induced a moderate upregulation of MCP-1 mRNA, and no synergistic effects were observed between angiotensin II and cyclic strain. These results indicate that cyclic strain stimulates MCP-1 mRNA expression in smooth muscle cells through signaling pathway(s) mediated by tyrosine kinase activation.     

Unloading of intercellular tension induces the directional translocation of PKCα

The migration of endothelial cells (ECs) is closely associated with a Ca²⁺-dependent protein, protein kinase Cα (PKCα). The disruption of intercellular adhesion by single-cell wounding has been shown to induce the directional translocation of PKCα. We hypothesized that this translocation of PKCα is induced by mechanical stress, such as unloading of intercellular tension, or by intercellular communication, such as gap junction-mediated and paracrine signaling. In the current study, we found that the disruption of intercellular adhesion induced the directional translocation of PKCα even when gap junction-mediated and paracrine signaling were inhibited. Conversely, it did not occur when the mechanosensitive channel was inhibited. In addition, the strain field of substrate attributable to the disruption of intercellular adhesion tended to be larger at the areas corresponding with PKCα translocation. Recently, we found that a direct mechanical stimulus induced the accumulation of PKCα at the stimulus area, involving Ca ²⁺ influx from extracellular space. These results indicated that the unloading of intercellular tension induced directional translocation of PKCα, which required Ca ²⁺ influx from extracellular space. The results of this study indicate the involvement of PKCα in the Ca ²⁺ signaling pathway in response to mechanical stress in ECs.     

Heparan sulfate proteoglycan,integrin, and syndecan-4 are mechanosensors mediating cyclic strain-modulated endothelial gene expression in mouse embryonic stem cell-derived endothelial cells

It is widely believed that the differentiation of embryonic stem cells (ESCs) into viable endothelial cells (ECs) for use in vascular tissue engineering can be enhanced by mechanical forces. In our previous work, we reported that shear stress enhanced important EC functional genes on a CD31⁺/CD45⁻ cell population derived from mouse ESC committed to the EC lineage. In the present study, in contrast to the effects of shear stress on this cell population, we observed that cyclic strain significantly reduced the expression of EC-specific marker genes (vWF, VE-cadherin, and PECAM-1), tight junction protein genes (ZO-1, OCLD, and CLD5), and vasoactive genes (eNOS and ET1), while it did not alter the expression of COX2. Taken together, these studies indicate that only shear stress, not cyclic strain, is a useful mechanical stimulus for enhancing the properties of CD31⁺/CD45⁻ cells for use as EC in vascular tissue engineering. To begin examining the mechanisms controlling cyclic strain-induced suppression of gene expression in CD31⁺/CD45⁻ cells, we depleted the heparan sulfate (HS) component of the glycocalyx, blocked integrins, and silenced the HS proteoglycan syndecan-4 in separate experiments. All of these treatments resulted in the reversal of cyclic strain-induced gene suppression. The current study and our previous work provide a deeper understanding of the mechanisms that balance the influence of cyclic strain and shear stress in endothelial cells.     

5′‐N‐ethylcarboxamide induces IL‐6 expression via MAPKs and NF‐κB activation through Akt,Ca2+/PKC,cAMP signaling pathways in mouse embryonic stem cells

Many studies suggest that adenosine modulates cell responses in a wide array of tissues through potent and selective regulation of cytokine production. This study examined the effects of adenosine on interleukin (IL)‐6 expression and its related signal pathways in mouse embryonic stem (ES) cells. In this study, the adenosine analogue 5′‐N‐ethylcarboxamide (NECA) increased IL‐6 protein expression level. Mouse ES cells expressed the A₁, A_2A, A_2B, and A₃ adenosine receptors (ARs), whose expression levels were increased by NECA and NECA‐induced increase of IL‐6 mRNA expression or secretion level was inhibited by the non‐specific AR inhibitor, caffeine. NECA increased Akt and protein kinase C (PKC) phosphorylation, intracellular Ca²⁺ and cyclic adenosine monophosphate (cAMP) levels, which were blocked by caffeine. On the other hand, NECA‐induced IL‐6 secretion was partially inhibited by Akt inhibitor, bisindolylmaleimide I (PKC inhibitor), SQ 22536 (adenylate cyclate inhibitor) and completely blocked by the 3 inhibitor combination treatment. In addition, NECA increased mitogen activated protein kinase' (MAPK) phosphorylation, which were partially inhibited by the Akt inhibitor, bisindolylmaleimide I, and SQ 22536 and completely blocked by the 3 inhibitor combination treatment. NECA‐induced increases of IL‐6 protein expression and secretion levels were inhibited by MAPK inhibition. NECA‐induced increase of nuclear factor (NF)‐κB phosphorylation was inhibited by MAPK inhibitors. NECA also increased cAMP response element‐binding protein (CREB) phosphorylation, which was blocked by MAPK or NF‐κB inhibitors. Indeed, NECA‐induced increase of IL‐6 protein expression and secretion was blocked by NF‐κB inhibitors. In conclusion, NECA stimulated IL‐6 expression via MAPK and NF‐κB activation through Akt, Ca²⁺/PKC, and cAMP signaling pathways in mouse ES cells. J. Cell. Physiol. 219: 752–759, 2009. © 2009 Wiley‐Liss, Inc.     

Role of PLD−PKCζ signaling axis in p47phox phosphorylation for activation of NADPH oxidase by angiotensin II in pulmonary artery smooth muscle cells

We sought to determine the mechanism by which angiotensin II (ANGII) stimulates NADPH oxidase‐mediated superoxide (O₂^.?) production in bovine pulmonary artery smooth muscle cells (BPASMCs). ANGII‐induced increase in phospholipase D (PLD) and NADPH oxidase activities were inhibited upon pretreatment of the cells with chemical and genetic inhibitors of PLD2, but not PLD1. Immunoblot study revealed that ANGII treatment of the cells markedly increases protein kinase C‐α (PKC‐α), ‐δ, ‐ε, and ‐ζ levels in the cell membrane. Pretreatment of the cells with chemical and genetic inhibitors of PKC‐ζ, but not PKC‐α, ‐δ, and ‐ε, attenuated ANGII‐induced increase in NADPH oxidase activity without a discernible change in PLD activity. Transfection of the cells with p47phox small interfering RNA inhibited ANGII‐induced increase in NADPH oxidase activity without a significant change in PLD activity. Pretreatment of the cells with the chemical and genetic inhibitors of PLD2 and PKC‐ζ inhibited ANGII‐induced p47phox phosphorylation and subsequently translocation from cytosol to the cell membrane, and also inhibited its association with p22phox (a component of membrane‐associated NADPH oxidase). Overall, PLD?PKCζ?p47phox signaling axis plays a crucial role in ANGII‐induced increase in NADPH oxidase‐mediated O₂^.? production in the cells.     

DNA synthesis of rat bone marrow mesenchymal stem cells through α1-adrenergic receptors

Multipotential bone marrow mesenchymal stem cells (BMSCs) are important in maintaining the microenvironment of the bone marrow (BM). Sympathetic nerves histologically innervate the BM; however, their role remains unclear. In this study, the effects of norepinephrine on DNA synthesis and the related signaling molecules involved in rBMSCs were examined.mRNA levels of the α1-adrenergic receptor subtypes increased following norepinephrine stimulation (10⁻⁵ M for 30 min). DNA synthesis increased in dose- and time-dependent manners as determined by [³H]thymidine incorporation. Intracellular Ca²⁺ concentration and translocation of protein kinase C from the cytosol to the membrane were also found to be elevated in rBMSCs. Phentolamine was able to suppress translocation of PKC. Norepinephrine also induced phosphorylation of ERK1/2, which was prevented by staurosporine treatment. Pretreatment with PD98059 inhibited ERK1/2 phosphorylation and DNA synthesis in rBMSCs.These findings indicate that norepinephrine stimulates DNA synthesis via α1-adrenergic receptors and downstream Ca²⁺/PKC and ERK1/2 activation in rBMSCs.     

PKCα promotes insulin secretion via TRPC1 phosphorylation in INS-1E cells

ABSTRACT

Protein kinase C (PKC) is a class of phospholipid-dependent serine/threonine kinases that contribute to cell survival, migration, and invasion. Previous studies demonstrated that PKC participates in insulin secretion. However, the role of PKC in glucose-stimulated insulin secretion (GSIS) remains unclear. Herein, we demonstrated that PKC is an important mediator of insulin secretion and revealed a close relationship between PKC activation and insulin secretion in INS-1E cells. Meanwhile, the presence of PKCα was found to induce TRPC1 phosphorylation in INS-1E cells. TRPC1 phosphorylation levels increased by activating PKCα activity. Inhibition of PKCα activity reduced TRPC1 phosphorylation. Finally, we showed that TRPC1 could reverse the decrease in intracellular Ca²⁺ levels and reduced insulin secretion induced by treatment with PKCα inhibitor under high glucose conditions. In conclusion, our findings indicated that TRPC1 and PKCα are involved in promoting insulin secretion and that PKCα promotes insulin secretion via TRPC1 phosphorylation in INS-1E cells.     

Strain activation of bovine aortic smooth muscle cell proliferation and alignment: Study of strain dependency and the role of protein kinase A and C signaling pathways

Smooth muscle cell (SMC) phenotype can be altered by physical forces as demonstrated by cyclic strain-induced changes in proliferation, orientation, and secretion of macromolecules. However, the magnitude of strain required and the intracellular coupling pathways remain ill defined. To examine the strain requirements for SMC proliferation, we selectively seeded bovine aortic SMC either on the center or periphery of silastic membranes which were deformed with 150 mm Hg vacuum (0–7% center; 7–24% periphery). SMC located in either the center or peripheral regions showed enhanced proliferation compared to cells grown under the absence of cyclic strain. Moreover, SMC located in the center region demonstrated significantly (P < 0.005) greater proliferation as compared to those in the periphery. In contrast, SMC exposed to high strain (7–24%) demonstrated alignment perpendicular to the strain gradient, whereas SMC in the center (0–7%) remained aligned randomly. To determine the mechanisms of these phenomena, we examined the effect of cyclic strain on bovine aortic SMC signaling pathways. We observed strain-induced stimulation of the cyclic AMP pathway including adenylate cyclase activity and cyclic AMP accumulation. In addition, exposure of SMC to cyclic strain caused a significant increase in protein kinase C (PKC) activity and enzyme translocation from the cytosol to a particulate fraction. Further study was conducted to examine the effect of strain magnitude on signaling, particularly protein kinase A (PKA) activity as well as cAMP response element (CRE) binding protein levels. We observed significantly (P < 0.05) greater PKA activity and CRE binding protein levels in SMC located in the center as compared to the peripheral region. However, inhibition of PKA (with 10 μM Rp-cAMP) or PKC (with 5–20 ng/ml staurosporine) failed to alter either the strain-induced increase in SMC proliferation or alignment. These data characterize the strain determinants for activation of SMC proliferation and alignment. Although strain activated both the AC/cAMP/PKA and the PKC pathways in SMC, singular inhibition of PKA and PKC failed to prevent strain-induced alignment and proliferation, suggesting either their lack of involvement or the multifactorial nature of these responses. J. Cell. Physiol. 170:228–234, 1997. © 1997 Wiley-Liss, Inc.     

Stretch-induced phosphorylation of focal adhesion kinase in endothelial cells: role of mitochondrial oxidants

Mechanical stretch activates a number of signaling pathways in endothelial cells, and it elicits a variety of functional responses including increases in the phosphorylation of focal adhesion kinase (FAK), a nonreceptor tyrosine kinase involved in integrin-mediated signal transduction. Stretch also triggers an increase in the generation of reactive oxygen species (ROS), which may function as second messengers in the signal transduction cascades that activate cellular responses to strain. Mitochondria represent an important source of ROS in the cell, and these organelles may release ROS in response to strain by virtue of their attachment to cytoskeletal proteins. We therefore tested whether cyclic stretch increases FAK phosphorylation at Tyr397 through a mitochondrial ROS signaling pathway in bovine pulmonary artery endothelial cells (BPAEC). Oxidant signaling, measured using 2'7'-dichlorofluorescin (DCFH), increased 152 +/- 16% during 1.5 h of cyclic strain relative to unstrained controls. The mitochondrial inhibitors diphenylene iodonium (5 microM) or rotenone (2 microM) attenuated this increase, whereas L-nitroarginine (100 microM), allopurinol (100 microM), or apocynin (30 microM) had no effect. The antioxidants ebselen (5 microM) and dithiodidiethyldithiocarbamate (1 mM) inhibited the strain-induced increase in oxidant signaling, but Hb (5 microM) had no effect. These results indicate that strain induces oxidant release from mitochondria. Treatment with cytochalasin D (5 microM) abrogated strain-induced DCFH oxidation in BPAEC, indicating that actin filaments were required for stretch-induced mitochondrial ROS generation. Cyclic strain increased FAK phosphorylation at Tyr397, but this was abolished by mitochondrial inhibitors as well as by antioxidants. Strain-induced FAK phosphorylation was abrogated by inhibition of protein kinase C (PKC) with Ro-31-8220 or G?-6976. These findings indicate that mitochondrial oxidants generated in response to endothelial strain trigger FAK phosphorylation through a signaling pathway that involves PKC.     

Strain-induced vascular endothelial cell proliferation requires PI3K-dependent mTOR-4E-BP1 signal pathway

The aim of this study was to determine whether the phosphatidylinositol 3-kinase (PI3K)-dependent mammalian target of rapamycin (mTOR)-eukaryotic initiation factor 4E binding protein 1 (4E-BP1) signal pathway and S6 kinase (S6K), the major element of the mTOR pathway, play a role in the enhanced vascular endothelial cell (EC) proliferation induced by cyclic strain. Bovine aortic ECs were subjected to an average of 10% strain at a rate of 60 cycles/min for < or =24 h. Cyclic strain-induced EC proliferation was reduced by pretreatment with rapamycin but not the MEK1 inhibitor PD-98059. The PI3K inhibitors wortmannin and LY-294002 also attenuated strain-induced EC proliferation and strain-induced activation of S6K. Rapamycin but not PD-98059 prevented strain-induced S6K activation, and PD-98059 but not rapamycin prevented strain-induced activation of extracellular signal-regulated kinases 1 and 2. Cyclic strain also activated 4E-BP1, which could be inhibited by PI3K inhibitors. These data suggest that the PI3K-dependent S6K-mTOR-4E-BP1 signal pathway may be critically involved in strain-induced bovine aortic EC proliferation.