Protein kinase Calpha-RhoA cross-talk in CCL2-induced alterations in brain endothelial permeability

Monocyte chemoattractant protein-1 (MCP-1 or CCL2) regulates blood-brain barrier permeability by inducing morphological and biochemical alterations in the tight junction (TJ) complex between brain endothelial cells. The present study used cultured brain endothelial cells to examine the signaling networks involved in the redistribution of TJ proteins (occludin, ZO-1, ZO-2, claudin-5) by CCL2. The CCL2-induced alterations in the brain endothelial barrier were associated with de novo Ser/Thr phosphorylation of occludin, ZO-1, ZO-2, and claudin-5. The phosphorylated TJ proteins were redistributed/localized in Triton X-100-soluble as well as Triton X-100-insoluble cell fractions. Two protein kinase C (PKC) isoforms, PKCalpha and PKCzeta, had a significant impact on this event. Inhibition of their activity using dominant negative mutants PKCalpha-DN and PKCzeta-DN diminished CCL2 effects on brain endothelial permeability. Previous data indicate that Rho/Rho kinase signaling is involved in CCL2 regulation of brain endothelial permeability. The interactions between the PKC and Rho/Rho kinase pathways were therefore examined. Rho, PKCalpha, and PKCzeta activities were knocked down using dominant negative mutants (T17Rho, PKCalpha-DN, and PKCzeta-DN, respectively). PKCalpha and Rho, but not PKCzeta and Rho, interacted at the level of Rho, with PKCalpha being a downstream target for Rho. Double transfection experiments using dominant negative mutants confirmed that this interaction is critical for CCL2-induced redistribution of TJ proteins. Collectively these data suggest for the first time that CCL2 induces brain endothelial hyperpermeability via Rho/PKCalpha signal pathway interactions.     

The suppression of small GTPase rho signal transduction pathway inhibits angiogenesis in vitro and in vivo

Angiogenesis consists of multistep pathways such as the degradation of the matrix, proliferation of the endothelial cells, motility of the endothelial cells, formation of the cord structure and network formation of microvessels. The small GTPase Rho participates in cell motility through actin fiber polymerization. The role of the small GTPase Rho signal transduction pathway in regulating angiogenesis, however, is still unknown. In this study, we investigated the role of the small GTPase Rho signal transduction pathway in angiogenesis in vitro and in vivo using the exoenzyme, Clostridium botulinum C3 transferase, which specifically suppresses Rho and a compound, Y-27632, which suppresses p160ROCK (Rho-associated coiled-coil containing protein kinase). In this paper, we showed that the small GTPase Rho-p160ROCK signal transduction pathway played an important role in angiogenesis both in vitro and in vivo. These results suggest that inhibition of the small GTPase Rho signal transduction pathway by the p160ROCK inhibitor could be a possible new strategy for angiogenic diseases.     

Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.     

Acidosis Activation of the Proton-Sensing GPR4 Receptor Stimulates Vascular Endothelial Cell Inflammatory Responses Revealed by Transcriptome Analysis

Acidic tissue microenvironment commonly exists in inflammatory diseases, tumors, ischemic organs, sickle cell disease, and many other pathological conditions due to hypoxia, glycolytic cell metabolism and deficient blood perfusion. However, the molecular mechanisms by which cells sense and respond to the acidic microenvironment are not well understood. GPR4 is a proton-sensing receptor expressed in endothelial cells and other cell types. The receptor is fully activated by acidic extracellular pH but exhibits lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To delineate the function and signaling pathways of GPR4 activation by acidosis in endothelial cells, we compared the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4. The results demonstrated that acidosis activation of GPR4 in HUVEC substantially increased the expression of a number of inflammatory genes such as chemokines, cytokines, adhesion molecules, NF-κB pathway genes, and prostaglandin-endoperoxidase synthase 2 (PTGS2 or COX-2) and stress response genes such as ATF3 and DDIT3 (CHOP). Similar GPR4-mediated acidosis induction of the inflammatory genes was also noted in other types of endothelial cells including human lung microvascular endothelial cells and pulmonary artery endothelial cells. Further analyses indicated that the NF-κB pathway was important for the acidosis/GPR4-induced inflammatory gene expression. Moreover, acidosis activation of GPR4 increased the adhesion of HUVEC to U937 monocytic cells under a flow condition. Importantly, treatment with a recently identified GPR4 antagonist significantly reduced the acidosis/GPR4-mediated endothelial cell inflammatory response. Taken together, these results show that activation of GPR4 by acidosis stimulates the expression of a wide range of inflammatory genes in endothelial cells. Such inflammatory response can be suppressed by GPR4 small molecule inhibitors and hold potential therapeutic value.     

Is HSP70 upregulation crucial for cellular proliferative response in simulated microgravity?

Astronauts are susceptible to a variety of conditions such as motion sickness, muscular atrophy, bone demineralization and cardiovascular deconditioning. These findings suggest that the adaptation to the absence of gravity is due, at least in part, to the effects exerted by microgravity at the cellular level. Indeed, a number of studies have indicated that gravity affects mammalian cell growth and differentiation through the modulation of gene expression. We have characterized the behaviour of endothelial cells and of the human monocytic cell line U937 cultured in the NASA-developed bioreactor to simulate microgravity, the Rotating Wall Vessels (RWV). In simulated microgravity endothelial cells showed a different behavior which was dependent from the species and from the district of origin, while U937 in the RWV proliferated slower than the controls. All the effects we observed were promptly reversible upon return to normal culture conditions. It is noteworthy that all the cells which maintained the capability to proliferate in microgravity upregulated the stress protein HSP70. We therefore propose that only the cells which sense microgravity as a stressful condition and, consequently, overexpress HSP70 maintain their proliferative potential in simulated microgravity.     

Thrombin induces MCP-1 expression through Rho-kinase and subsequent p38MAPK/NF-κB signaling pathway activation in vascular endothelial cells

Thrombin has been shown to increase expression of chemokines such as monocyte chemoattractant protein 1 (MCP-1) in endothelial cells, leading to the development of atherosclerosis. However, the precise mechanism of this induction remains unknown. In the present study, we investigated whether the small G protein RhoA, and its effector, Rho-kinase are involved in MCP-1 induction by thrombin in endothelial cells. Y-27632, a specific Rho-kinase inhibitor, potently inhibited MCP-1 induction by thrombin. Y-27632 significantly decreased the chemotactic activity of thrombin-stimulated supernatants of endothelial cells on monocytes. Importantly, fasudil, a specific Rho-kinase inhibitor, attenuated MCP-1 gene expression in the aorta of db/db mice. Y-27632 attenuated thrombin-mediated phosphorylation of p38MAPK and p65, indicating that Rho-kinase mediates thrombin-induced MCP-1 expression through p38MAPK and NF-κB activation. Our findings demonstrate that the Rho/Rho-kinase signaling pathway plays a critical role in thrombin-mediated MCP-1 expression and function, and suggest that Rho/Rho-kinase may be an important target in the development of new therapeutic strategies for atherosclerosis.     

Lipopolysaccharide-induced expression of the competence gene KC in vascular endothelial cells is mediated through protein kinase C

The KC gene is a cell cycle-dependent competence gene originally identified in platelet-derived growth factor-stimulated BALB/c-3T3 cells. This gene is also induced in murine peritoneal macrophages in response to activation stimuli. We have examined the expression of the KC gene in cultured porcine aortic endothelial cells following treatment with bacterial lipopolysaccharide (LPS) as a first step in defining the early molecular events involved in endothelial cell stimulation by physiologically relevant modulators. LPS markedly elevated the steady-state level of KC mRNA in confluent endothelial cells; maximum induction of KC occurred in the cells following exposure to 10 ng/ml LPS for 2 h. LPS did not increase the growth fraction of the cells, nor was the KC mRNA level changed in dense endothelial cells stimulated to enter the cell cycle with epidermal growth factor. However, KC mRNA expression was elevated by addition of serum to starved, subconfluent endothelial cell cultures. Treatment of endothelial cells with phorbol myristate acetate (PMA) and 1-oleoyl-2-acetyl-glycerol (OAG) also induced KC gene expression. A maximum response was obtained with 10 nM PMA, the effect decreasing with higher levels of the phorbol ester. The calcium ionophore A23187 exhibited little stimulatory activity alone; however, the ionophore did cause a doubling in the PMA-stimulated KC expression. The increased expression of KC induced by LPS and PMA was inhibited by the presence of 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine (H7), a protein kinase C inhibitor, but not by HA1004 (an H7 analogue with little protein kinase C inhibitory activity). No cytotoxicity was observed in inhibitor or LPS-treated endothelial cell cultures. These results demonstrate that KC gene expression is stimulated by LPS in vascular endothelial cells in a proliferation-independent process. Second, unlike LPS-induced KC expression in macrophages and platelet-derived growth factor-induced KC expression in 3T3 cells, LPS induction of KC in endothelial cells appears to require activation of protein kinase C.     

RhoA exerts a permissive effect on volume-regulated anion channels in vascular endothelial cells

Cell swelling triggers in most cell typesan outwardly rectifying anion current,I_Cl,swell, via volume-regulated anion channels (VRACs). We have previously demonstrated in calf pulmonary artery endothelial (CPAE) cells that inhibition of the Rho/Rho kinase/myosin light chain phosphorylation pathway reduces the swelling-dependent activation of I_Cl,swell. However, theseexperiments did not allow us to discriminate between a direct activatorrole or a permissive effect. We now show that the Rho pathway did notaffect VRAC activity if this pathway was activated by transfecting CPAEcells with constitutively active isoforms of G (a Rho activatingheterotrimeric G protein subunit), Rho, or Rho kinase. Furthermore,biochemical and morphological analysis failed to demonstrate activationof the Rho pathway during hypotonic cell swelling. Finally,manipulating the Rho pathway with either guanosine5'-O-(3-thiotriphosphate) or C3 exoenzyme had no effect onVRACs in caveolin-1-expressing Caco-2 cells. We conclude that the Rhopathway exerts a permissive effect on VRACs in CPAE cells, i.e.,swelling-induced opening of VRACs requires a functional Rho pathway,but not an activation of the Rho pathway.

     

AKAP12 in astrocytes induces barrier functions in human endothelial cells through protein kinase Czeta

Interactions between astrocytes and blood vessels are essential for the formation and maintenance of the blood-neural barrier (BNB). Astrocyte-derived A-kinase anchor protein 12 (AKAP12) influences BNB formation, but the mechanism of regulation of BNB functions by AKAP12 is not fully understood. We have defined a new pathway of barriergenesis in human retina microvascular endothelial cells (HRMECs) involving astrocytic AKAP12. Treatment of HRMECs with conditioned media from AKAP12-overexpressing astrocytes reduced phosphorylation of protein kinase Czeta (PKCzeta), decreased the levels of vascular endothelial growth factor (VEGF) mRNA and protein, and increased thrombospondin-1 (TSP-1) levels, which led to antiangiogenesis and barriergenesis. Transfection of a small interference RNA targeting PKCzeta decreased VEGF levels and increased TSP-1 levels in HRMECs. Rho is a putative downstream signal of PKCzeta, and inhibition of Rho kinase with a specific inhibitor, Y27632, decreased VEGF levels and increased TSP-1 levels. We therefore suggest that AKAP12 in astrocytes differentially regulates the expression of VEGF and TSP-1 via the inhibition of PKCzeta phosphorylation and Rho kinase activity in HRMECs.     

Rho GAPs and GEFs

Within blood vessels, endothelial cell–cell and cell–matrix adhesions are crucial to preserve barrier function, and these adhesions are tightly controlled during vascular development, angiogenesis, and transendothelial migration of inflammatory cells. Endothelial cellular signaling that occurs via the family of Rho GTPases coordinates these cell adhesion structures through cytoskeletal remodelling. In turn, Rho GTPases are regulated by GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors (GEFs). To understand how endothelial cells initiate changes in the activity of Rho GTPases, and thereby regulate cell adhesion, we will discuss the role of Rho GAPs and GEFs in vascular biology. Many potentially important Rho regulators have not been studied in detail in endothelial cells. We therefore will first overview which GAPs and GEFs are highly expressed in endothelium, based on comparative gene expression analysis of human endothelial cells compared with other tissue cell types. Subsequently, we discuss the relevance of Rho GAPs and GEFs for endothelial cell adhesion in vascular homeostasis and disease.     

Cross-cascade activation of ERKs and ternary complex factors by Rho family proteins.

Mitogens promote cell growth through integrated signal transduction networks that alter cellular metabolism, gene expression and cytoskeletal organization. Many such signals are propagated through activation of MAP kinase cascades partly regulated by upstream small GTP-binding proteins. Interactions among cascades are suspected but not defined. Here we show that Rho family small G proteins such as Rac1 and Cdc42hs, which activate the JNK/SAPK pathway, cooperate with Raf-1 to activate the ERK pathway. This causes activation of ternary complex factors (TCFs), which regulate c-fos gene expression through the serum response element. Examination of ERK pathway kinases shows that neither MEK1 nor Ras will synergize with Rho-type proteins, and that only MEK1 is fully activated, indicating that MEKs are a focal point for cross-cascade regulation. Rho family proteins utilize PAKs for this effect, as expression of an active PAK1 mutant can substitute for Rho family small G proteins, and expression of an interfering PAK1 mutant blocks Rho-type protein stimulation of ERKs. PAK1 phosphorylates MEK1 on Ser298, a site important for binding of Raf-1 to MEK1 in vivo. Expression of interfering PAK1 also reduces stimulation of TCF function by serum growth factors, while expression of active PAK1 enhances EGF-stimulated MEK1 activity. This demonstrates interaction among MAP kinase pathway elements not previously recognized and suggests an explanation for the cooperative effect of Raf-1 and Rho family proteins on cellular transformation.     

RNA-binding protein Khd1 and Ccr4 deadenylase play overlapping roles in the cell wall integrity pathway in Saccharomyces cerevisiae

The Saccharomyces cerevisiae RNA-binding protein Khd1/Hek2 associates with hundreds of potential mRNA targets preferentially, including the mRNAs encoding proteins localized to the cell wall and plasma membrane. We have previously revealed that Khd1 positively regulates expression of MTL1 mRNA encoding a membrane sensor in the cell wall integrity (CWI) pathway. However, a khd1Δ mutation has no detectable phenotype on cell wall synthesis. Here we show that the khd1Δ mutation causes a severe cell lysis when combined with the deletion of the CCR4 gene encoding a cytoplasmic deadenylase. We identified the ROM2 mRNA, encoding a guanine nucleotide exchange factor (GEF) for Rho1, as a target for Khd1 and Ccr4. The ROM2 mRNA level was decreased in the khd1Δ ccr4Δ mutant, and ROM2 overexpression suppressed the cell lysis of the khd1Δ ccr4Δ mutant. We also found that Ccr4 negatively regulates expression of the LRG1 mRNA encoding a GTPase-activating protein (GAP) for Rho1. The LRG1 mRNA level was increased in the ccr4Δ and khd1Δ ccr4Δ mutants, and deletion of LRG1 suppressed the cell lysis of the khd1Δ ccr4Δ mutant. Our results presented here suggest that Khd1 and Ccr4 modulate a signal from Rho1 in the CWI pathway by regulating the expression of RhoGEF and RhoGAP.     

VEGF selectively induces Down syndrome critical region 1 gene expression in endothelial cells: a mechanism for feedback regulation of angiogenesis?

The Down syndrome critical region 1 (DSCR1) gene (also known as MCIP1, Adapt78) encodes a regulatory protein that binds to calcineurin catalytic A subunit and acts as a regulator of the calcineurin-mediated signaling pathway. We show in this study that DSCR1 is greatly induced in endothelial cells in response to VEGF, TNF-alpha, and A23187 treatment, and that this up-regulation is inhibited by inhibitors of the calcineurin-NFAT (nuclear factor of activated T cells) signaling pathway as well as by PKC inhibition and a Ca(2+) chelator. We hypothesized that the up-regulation of DSCR1 gene expression in endothelial cells could act as an endogenous feedback inhibitor for angiogenesis by regulating the calcineurin-NFAT signaling pathway. Our transient transfection analyses confirm that the overexpression of DSCR1 abrogates the up-regulation of reporter gene expression driven by both the cyclooxygenase 2 and DSCR1 promoters in response to stimulators. Our results indicate that DSCR1 up-regulation may represent a potential molecular mechanism underlying the regulation of angiogenic genes activated by the calcineurin-NFAT signaling pathway in endothelial cells.     

Mechanosensing role of caveolae and caveolar constituents in human endothelial cells

A variety of evidence suggests that endothelial cell functions are impaired in altered gravity conditions. Nevertheless, the effects of hypergravity on endothelial cell physiology remain unclear. In this study we cultured primary human endothelial cells under mild hypergravity conditions for 24-48 h, then we evaluated the changes in cell cycle progression, caveolin1 gene expression and in the caveolae status by confocal microscopy. Moreover, we analyzed the activity of enzymes known to be resident in caveolae such as endothelial nitric oxide synthase (eNOS), cycloxygenase 2 (COX-2), and prostacyclin synthase (PGIS). Finally, we performed a three-dimensional in vitro collagen gel test to evaluate the modification of the angiogenic responses. Results indicate that hypergravity shifts endothelial cells to G(0)/G(1) phase of cell cycle, reducing S phase, increasing caveolin1 gene expression and causing an increased distribution of caveolae in the cell interior. Hypergravity also increases COX-2 expression, nitric oxide (NO) and prostacyclin (PGI2) production, and inhibits angiogenesis as evaluated by 3-D collagen gel test, through a pathway not involving apoptosis. Thus, endothelial cell caveolae may be responsible for adaptation of endothelium to hypergravity and the mechanism of adaptation involves an increased caveolin1 gene expression coupled to upregulation of vasodilators as NO and PGI2.     

Overexpression of Axl reverses endothelial cells dysfunction in high glucose and hypoxia

The receptor tyrosine kinase Axl is involved in diabetic vascular disease. This study aims to investigate the effect of high glucose on endothelial cells injury and Axl expression in hypoxia condition in vitro, and we present details of the mechanism associated with overexpression of Axl rescue the high glucose injury. Our results showed that high glucose impaired both human umbilical vein endothelial cells (HUVECs) and EAhy926 cells angiogenesis in hypoxia condition. In addition, high glucose inhibits Axl and hypoxia-inducible factor 1-α (HIF-1α) protein expression in hypoxia condition. Axl overexpression significantly reversed endothelial cells dysfunction in high glucose/hypoxia. Furthermore, Axl overexpression in EAhy926 cells increases HIF-1α protein synthesis through PI3K/Akt/mTOR/p70 ^S6K signal pathway but not Mek/Erk in high glucose/hypoxia condition. This study demonstrates that high glucose can alter Axl signaling and HIF-1α in hypoxia condition. Overexpression of Axl may rescue endothelial cells dysfunction and HIF-1α expression through its downstream signals in high glucose/hypoxia.