Temporal gradients in shear, but not spatial gradients, stimulate ERK1/2 activation in human endothelial cells

We have previously demonstrated temporal gradients in shear stress stimulate endothelial cell proliferation, whereas spatial gradients do not. In the present study, the extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway was investigated as a possible mediator for the promitogenic effect of temporal gradients. The sudden expansion flow chamber (SEFC) model was used to differentiate the effect of temporal gradients in shear from that of spatial gradients on ERK1/2 activation in human umbilical vein endothelial cells (HUVEC). ERK1/2 activation in the SEFC was not significantly different from control when HUVEC were exposed to spatial gradients alone. When a single temporal impulse was superimposed on spatial gradients, ERK1/2 activation was stimulated 330% (relative to spatial alone) within the region of spatial gradients. Inhibition of the ERK1/2 pathway with U-0126 abolished all effects of temporal gradients. To further separate temporal and spatial gradients, a conventional parallel plate flow chamber was utilized. Acute exposure to oscillations in flow at a frequency of 1 Hz stimulated ERK1/2 activation 620 +/- 88% relative to control, whereas a single impulse of flow increased ERK1/2 activation 166 +/- 19%. Flow without the temporal component did not significantly activate ERK1/2. These results suggest that the ERK1/2 pathway directly mediates the promitogenic effects of temporal gradients in shear stress.     

Regulation of connexin 43 gene expression by cyclical mechanical stretch in neonatal rat cardiomyocytes

Myocardial cells respond to changes in the mechanical forces imposed on them with changes in myocardial tension in the short term and with structural remodeling in the long term. Since these responses involve intercellular communication, we have investigated regulation of the gap junction proteins, connexin 43 (Cx43), connexin 40 (Cx40) and connexin 37 (Cx37), by cyclical mechanical stretch. Results were compared with parallel experiments on c-fos and GAPDH. Twenty percent stretch of cultured rat cardiomyocytes caused a 3-fold increase in Cx43 mRNA levels by 2 h. c-fos mRNA levels increased after 30 min of stretch, whereas Cx40, Cx37, and GADPH mRNA did not change. Protein levels of Cx43 increased by 4 h and remained elevated for 16 h. New protein synthesis was not a requirement for the stretch-induced rise in Cx43 expression, since mRNA levels were unaffected by treatment with cycloheximide. In addition, mechanical stretch induced alkalization of cardiomyocytes that was antagonized by inhibiting Na-H exchanger (NHE). Gap junction potential (Gj) was concomitantly elevated. Chemical closure of Cx channels by insulin was followed by inhibition of NHE. In conclusion, cyclical mechanical stretch caused increased expression of the gap junction protein Cx43 in cardiomyocytes and also the Gj. The augmentation of Cx43 mRNA expression and its functional status were associated with activation of NHE.     

KLF2-dependent, shear stress-induced expression of CD59: a novel cytoprotective mechanism against complement-mediated injury in the vasculature

Complement activation may predispose to vascular injury and atherogenesis. The atheroprotective actions of unidirectional laminar shear stress led us to explore its influence on endothelial cell expression of complement inhibitory proteins CD59 and decay-accelerating factor. Human umbilical vein and aortic endothelial cells were exposed to laminar shear stress (12 dynes/cm(2)) or disturbed flow (+/- 5 dynes/cm(2) at 1Hz) in a parallel plate flow chamber. Laminar shear induced a flow rate-dependent increase in steady-state CD59 mRNA, reaching 4-fold at 12 dynes/cm(2). Following 24-48 h of laminar shear stress, cell surface expression of CD59 was up-regulated by 100%, whereas decay-accelerating factor expression was unchanged. The increase in CD59 following laminar shear was functionally significant, reducing C9 deposition and complement-mediated lysis of flow-conditioned endothelial cells by 50%. Although CD59 induction was independent of PI3-K, ERK1/2 and nitric oxide, an RNA interference approach demonstrated dependence upon an ERK5/KLF2 signaling pathway. In contrast to laminar shear stress, disturbed flow failed to induce endothelial cell CD59 protein expression. Likewise, CD59 expression on vascular endothelium was significantly higher in atheroresistant regions of the murine aorta exposed to unidirectional laminar shear stress, when compared with atheroprone areas exposed to disturbed flow. We propose that up-regulation of CD59 via ERK5/KLF2 activation leads to endothelial resistance to complement-mediated injury and protects from atherogenesis in regions of laminar shear stress.     

Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin

Mechanosensing bone osteocytes express large amounts of connexin (Cx)43, the component of gap junctions; yet, gap junctions are only active at the small tips of their dendritic processes, suggesting another function for Cx43. Both primary osteocytes and the osteocyte-like MLO-Y4 cells respond to fluid flow shear stress by releasing intracellular prostaglandin E2 (PGE2). Cells plated at lower densities release more PGE2 than cells plated at higher densities. This response was significantly reduced by antisense to Cx43 and by the gap junction and hemichannel inhibitors 18 beta-glycyrrhetinic acid and carbenoxolone, even in cells without physical contact, suggesting the involvement of Cx43-hemichannels. Inhibitors of other channels, such as the purinergic receptor P2X7 and the prostaglandin transporter PGT, had no effect on PGE2 release. Cell surface biotinylation analysis showed that surface expression of Cx43 was increased by shear stress. Together, these results suggest fluid flow shear stress induces the translocation of Cx43 to the membrane surface and that unapposed hemichannels formed by Cx43 serve as a novel portal for the release of PGE2 in response to mechanical strain.     

Shear stress-induced upregulation of connexin 43 expression in endothelial cells on upstream surfaces of rat cardiac valves

Endothelial expression of the gap junction proteins, connexin (Cx) 37, Cx40, and Cx43, varies within the vascular network. While previous studies suggest that shear stress may upregulate Cx43, it is not well understood if shear stress affects the expression of all endothelial connexins and to what extent. Endothelial cells on the upstream and downstream surfaces of cardiac valves are subjected to considerably different intensities of shear stress. We therefore reasoned that we could determine the extent hemodynamic forces affect the expression of Cx37, Cx40, and Cx43 by comparing their immunohistochemical distribution on the upstream and downstream surfaces of rat cardiac valves. We found 70- to 200-fold greater expression of Cx43 in the endothelial cells on the upstream than on the downstream surfaces. However, Cx37 was expressed almost equally in the endothelial cells on upstream and downstream surfaces, and Cx40, a major connexin in most vascular endothelial cells, was not detected on either surface. In addition to the heterogeneity in Cx43 expression, endothelial cells on the upstream surface were 35% to 65% smaller than those on the corresponding downstream surface. These results suggest that shear stress may affect endothelial cell size and Cx43 expression but not Cx37 expression.     

Shear stress-induced upregulation of connexin 43 expression in endothelial cells on upstream surfaces of rat cardiac valves

Endothelial expression of the gap junction proteins, connexin (Cx) 37, Cx40, and Cx43, varies within the vascular network. While previous studies suggest that shear stress may upregulate Cx43, it is not well understood if shear stress affects the expression of all endothelial connexins and to what extent. Endothelial cells on the upstream and downstream surfaces of cardiac valves are subjected to considerably different intensities of shear stress. We therefore reasoned that we could determine the extent hemodynamic forces affect the expression of Cx37, Cx40, and Cx43 by comparing their immunohistochemical distribution on the upstream and downstream surfaces of rat cardiac valves. We found 70- to 200-fold greater expression of Cx43 in the endothelial cells on the upstream than on the downstream surfaces. However, Cx37 was expressed almost equally in the endothelial cells on upstream and downstream surfaces, and Cx40, a major connexin in most vascular endothelial cells, was not detected on either surface. In addition to the heterogeneity in Cx43 expression, endothelial cells on the upstream surface were 35% to 65% smaller than those on the corresponding downstream surface. These results suggest that shear stress may affect endothelial cell size and Cx43 expression but not Cx37 expression.     

Effects of mechanical strain on the function of Gap junctions in osteocytes are mediated through the prostaglandin EP2 receptor

Osteocytes embedded in the matrix of bone are thought to be mechanosensory cells that translate mechanical strain into biochemical signals that regulate bone modeling and remodeling. We have shown previously that fluid flow shear stress dramatically induces prostaglandin release and COX-2 mRNA expression in osteocyte-like MLO-Y4 cells, and that prostaglandin E2 (PGE2) released by these cells functions in an autocrine manner to regulate gap junction function and connexin 43 (Cx43) expression. Here we show that fluid flow regulates gap junctions through the PGE2 receptor EP2 activation of cAMP-dependent protein kinase A (PKA) signaling. The expression of the EP2 receptor, but not the subtypes EP1,EP3, and EP4, increased in response to fluid flow. Application of PGE2 or conditioned medium from fluid flow-treated cells to non-stressed MLO-Y4 cells increased expression of the EP2 receptor. The EP2 receptor antagonist, AH6809, suppressed the stimulatory effects of PGE2 and fluid flow-conditioned medium on the expression of the EP2 receptor, on Cx43 protein expression, and on gap junction-mediated intercellular coupling. In contrast, the EP2 receptor agonist butaprost, not the E1/E3 receptor agonist sulprostone, stimulated the expression of Cx43 and gap junction function. Fluid flow conditioned medium and PGE2 stimulated cAMP production and PKA activity suggesting that PGE2 released by mechanically stimulated cells is responsible for the activation of cAMP and PKA. The adenylate cyclase activators, forskolin and 8-bromo-cAMP, enhanced intercellular connectivity, the number of functional gap junctions, and Cx43 protein expression, whereas the PKA inhibitor, H89, inhibited the stimulatory effect of PGE2 on gap junctions. These studies suggest that the EP2 receptor mediates the effects of autocrine PGE2 on the osteocyte gap junction in response to fluid flow-induced shear stress. These data support the hypothesis that the EP2 receptor, cAMP, and PKA are critical components of the signaling cascade between mechanical strain and gap junction-mediated communication between osteocytes.     

Dexamethasone-induced insulin resistance is associated with increased connexin 36 mRNA and protein expression in pancreatic rat islets

Augmented glucose-stimulated insulin secretion (GSIS) is an adaptive mechanism exhibited by pancreatic islets from insulin-resistant animal models. Gap junction proteins have been proposed to contribute to islet function. As such, we investigated the expression of connexin 36 (Cx36), connexin 43 (Cx43), and the glucose transporter Glut2 at mRNA and protein levels in pancreatic islets of dexamethasone (DEX)-induced insulin-resistant rats. Study rats received daily injections of DEX (1 mg/kg body mass, i.p.) for 5 days, whereas control rats (CTL) received saline solution. DEX rats exhibited peripheral insulin resistance, as indicated by the significant postabsorptive insulin levels and by the constant rate for glucose disappearance (KITT). GSIS was significantly higher in DEX islets (1.8-fold in 16.7 mmol/L glucose vs. CTL, p < 0.05). A significant increase of 2.25-fold in islet area was observed in DEX vs. CTL islets (p < 0.05). Cx36 mRNA expression was significantly augmented, Cx43 diminished, and Glut2 mRNA was unaltered in islets of DEX vs. CTL (p < 0.05). Cx36 protein expression was 1.6-fold higher than that of CTL islets (p < 0.05). Glut2 protein expression was unaltered and Cx43 was not detected at the protein level. We conclude that DEX-induced insulin resistance is accompanied by increased GSIS and this may be associated with increase of Cx36 protein expression.     

Fluid shear stress remodels expression and function of junctional proteins in cultured bone cells

We tested the hypothesis that fluidshear stress () modifies the expression, function, and distributionof junctional proteins [connexin (Cx)43, Cx45, and zona occludens(ZO)-1] in cultured bone cells. Cell lines with osteoblastic (MC3T3-E1cells) and osteocytic (MLO-Y4 cells) phenotypes were exposed to-values of 5 or 20 dyn/cm² for 1-3 h.Immunostaining indicated that at 5 dyn/cm², thedistribution of Cx43, Cx45, and ZO-1 was moderately disrupted at cellmembranes; at 20 dyn/cm², disruption was more severe.Intercellular coupling was significantly decreased at both shear stresslevels. Western blots showed the downregulation of membrane-bound Cx43and ZO-1 and the upregulation of cytosolic Cx43 and Cx45 at differentlevels of shear stress. Similarly, Northern blots revealed thatexpression of Cx43, Cx45, and ZO-1 was selectively up- anddownregulated in response to different shear stress levels. Theseresults indicate that in cultured bone cells, fluid shear stressdisrupts junctional communication, rearranges junctional proteins, anddetermines de novo synthesis of specific connexins to an extent thatdepends on the magnitude of the shear stress. Such disconnection fromthe bone cell network may provide part of the signal whereby thedisconnected cells or the remaining network initiate focal bone remodeling.

     

Regulation of endothelial connexin40 expression by shear stress

Endothelial connexin (Cx)40 plays an important role in signal propagation along blood vessel walls, modulating vessel diameter and thereby blood flow. Blood flow, in turn, has been shown to alter endothelial Cx40 expression. However, the timing and shear stress dependence of this relationship have remained unclear, as have the signal transduction pathways involved and the functional implications. Therefore, the aim of this study was to quantify the effects of shear stress on endothelial Cx40 expression, to analyze the role of phosphoinositide 3-kinase (PI3K)/Akt signaling involved, and to assess the possible functional consequences for the adaptation of microvascular networks. First-passage human umbilical vein endothelial cells were exposed to defined shear stress conditions and analyzed for Cx40 using real-time RT-PCR and immunoblot analysis. Shear stress caused long-term induction of Cx40 protein expression, with two short-term mRNA peaks at 4 and 16 h, indicating the dynamic nature of the adaptation process. Maximum shear stress-dependent induction was observed at shear levels between 6 and 10 dyn/cm(2). Simulation of this pattern of shear-dependent Cx expression in a vascular adaptation model of a microvascular network led to an improved fit for the simulated results to experimental measurements. Cx40 expression was greatly reduced by inhibiting PI3K or Akt, with PI3K activity being required for basal Cx40 expression and Akt activity taking part in its shear stress-dependent induction.     

Tyrosine kinase activation is an immediate and essential step in hypotonic cell swelling-induced ERK activation and c-fos gene expression in cardiac myocytes.

Hypotonic stress causes rapid cell swelling and initiates various cellular adaptive processes. However, it is unknown how cells initially sense low osmolarity and convert it into intracellular signals. We investigated the signal transduction mechanism initiated by hypotonic cell swelling in cardiac myocytes using c-fos expression as a nuclear marker. Treatment of myocytes with hypotonic culture media rapidly induced c-fos expression, whereas hypertonic stress had no effect. Transfection of c-fos reporter gene constructs suggested that the hypotonic stress response element maps to the serum response element of the c-fos promoter. Hypotonic stress immediately (within 5 s) activated tyrosine kinase activity, while activation of ERK1/2 peaked at 5 min. Stress-activated kinase (JNK1) was modestly activated at 15 min, whereas HOG1 like kinase (p38) was not activated by hypotonic stress. Extensive pharmacological studies indicated that only tyrosine kinase inhibitors suppressed the hypotonic swelling-induced c-fos expression. The effect of hypotonic stress was mimicked by chlorpromazine, which is known to cause membrane deformation. These results suggest that the signaling mechanism of hypotonic stress is distinct from that of hyperosmolar stress in mammalian cells. Tyrosine kinase activation is the earliest detectable cell response and plays an essential role in hypotonic swelling-induced ERK1/2 activation and c-fos expression.     

Despite activation of EGF-receptor-ERK signaling pathway, epithelial proliferation is impaired in portal hypertensive gastric mucosa: relevance of MKP-1, c-fos, c-myc, and cyclin D1 expression.

Portal hypertensive (PHT) gastric mucosa has increased susceptibility to injury and impaired mucosal healing. Our previous study demonstrated increased ERK activation and MAP kinase phosphatase-1 (MKP-1) overexpression in PHT gastric mucosa. However, it remains unknown which tyrosine kinase receptors are involved in ERK activation and whether ERK activation results in increased cell proliferation. We examined whether EGF receptor (EGF-R) is involved in ERK activation and whether ERK activation triggers epithelial proliferation in PHT gastric mucosa. In gastric mucosa of PHT and sham-operated (SO) rats we studied: (1) EGF-R mRNA and protein expression as well as phosphorylation and membrane protein tyrosine kinase (PTK) activity; (2) ERK2 phosphorylation and activity; (3) MKP-1 mRNA and protein; (4) c-fos, c-myc and cyclin D1 mRNAs, and gastric epithelial proliferation. In PHT gastric mucosa: (1) EGF-R mRNA, protein and phosphorylation and membrane PTK activity were all significantly increased by 38%, 49%, 43% and 49%, respectively; (2) ERK2 phosphorylation and activity were significantly increased by 40% and 50 %, respectively; (3) MKP-1 mRNA and protein expression were significantly increased by 27% and 34%, respectively. In contrast, (4) c-fos, c-myc, and cyclin D1 mRNAs expression were all significantly decreased in PHT gastric mucosa by 36%, 33%, and 49%, respectively, and cell proliferation was significantly lower that in SO rats (11% in PHT vs. 18% in SO). These results suggest that in PHT gastric mucosa, ERK activation is mediated through EGF-R upregulation, but the gastric epithelial proliferation is impaired, possibly by MKP-1 overexpression, leading to reduction of c-fos, c-myc and cyclin D1.     

Heat shock triggers MAPK activation and MKP-1 induction in Leydig testicular cells

Testicular function is highly dependent on temperature control. In Leydig testicular cells, the signaling pathway activated by heat stress is poorly defined. Here we describe the molecular events triggered by heat shock (HS, 10 min, 45 degrees C) in MA-10 cells. HS produced a rapid and transient activation of ERK1/2 and JNK kinases, and also increased MAP kinase phosphatase-1 (MKP-1) protein and mRNA levels. The effect of HS on MKP-1 messenger reached significance at 15 min, peaked (3.5-fold) at 60 min, and was partially dependent on ERK1/2 activity. The temporal profiles of MKP-1 protein levels and MAPKs phospho-dephosphorylation suggest that MKP-1 induction could contribute to ERK1/2 and JNK inactivation after HS. In summary, this study indicates that the response to heat stress in Leydig cells includes the activation of MAPKs related to cell survival (ERK1/2) and death (JNK), and the induction of a MAPK activity inhibitory loop.     

Role of [Ca2+]i in induction of c-fos, c-jun, and c-myc mRNA in rat PTE after oxidative stress.

Oxidative stress plays an important role in various types of cell injury and tumor promotion. Cells respond to oxidative stress in many ways including changes in membrane organization, ion movements, and altered gene expression, all of which contribute to the subsequent fate of affected cells. In this study, we investigated the expression of the proto-oncogenes c-fos, c-myc, and c-jun, which play a key role in proliferation and differentiation, using primary cultures of rat proximal tubular epithelium exposed to oxidative stress generated by the xanthine/xanthine oxidase system. This system generates superoxide and H2O2 in the extracellular space stimulating the release of active oxygen species from inflammatory cells. c-fos mRNA was expressed within 15 min, peaked at 30 min, and returned to constitutive levels by 3 h. c-jun mRNA began to rise after 30 min, peaked at 120 min, and remained above the constitutive levels up to 180 min. c-myc mRNA expression was less affected by the treatment, with levels increasing gradually over the 180 min period. The expression of c-fos was inhibited by superoxide dismutase but not by catalase and was super-induced by cycloheximide. H2O2 alone did not induce any c-fos mRNA in this system. Chelation of extracellular ionized calcium by EGTA or of intracellular ionized calcium by Quin 2/AM resulted in a marked decrease of c-fos expression. Two protein kinase C inhibitors, H-7 and staurosporine, partly diminished the expression of c-fos, whereas a third, 2-aminopurine, which has a broader spectrum of inhibiting protein kinases, almost completely abolished it. A poly ADP-ribosylation inhibitor, 3-aminobenzamide, had no effect on c-fos expression in this system. Our results show that oxidative stress provokes sequential expression of c-fos, c-jun, and c-myc, mRNA in this order. This c-fos expression appears to be largely controlled by calcium ion movement, which could include protein kinase C activation. Another protein kinase or kinases also appear to play an important role.