Stromal interaction molecule 1 (STIM1) induces calcium (Ca2+) entry by activating transient receptor potential canonical (TRPC) channel‐mediated store‐operated calcium (SOC) entry or ORAI1 mediated Ca2+ release‐activated Ca2+(CRAC) entry. However, mechanisms regulating STIM1 interaction with ORAI1/TRPC1 remains unclear. Myosin light chain kinase (MLCK)‐210, a Ca2+‐calmodulin dependent long isoform of MLCK, contains domains at N‐terminus which mediates protein‐protein interaction. We tested the hypothesis that MLCK through its N‐terminus interacts with STIM1, ORAI1 and TRPC1 leading to amplified and protracted increase in Ca2+ entry. We show that thapsigargin, which activates SOC as well as CRAC channels, induce markedly attenuated increase in Ca2+ entry in MLCK‐210−/− endothelial cells (ECs). Restoring N‐terminus but not C‐terminus of MLCK‐210 rescued Ca2+ entry in MLCK‐210−/− ECs. Over expression of N‐MLCK‐210 mutant in wild type ECs rapidly enhanced Ca2+ entry which had a steep slope quite distinct than vector expressing ECs. Interestingly, MLCK‐210 interacted with ORAI1, STIM1 and TRPC1 under basal condition and the interaction further increased following thapsigargin stimulation of ECs. These observations suggest that MLCK‐210 may facilitate STIM1 interaction with ORAI1 and TRPC1 to amplify and protract Ca2+entry in ECs

Pulmonary inflammation and severe edema developing post‐sepsis are the main hallmarks of Acute Lung Injury (ALI). We have shown that tamoxifen‐induced endothelial‐specific deletion of FAK led to disruption of lung vascular barrier and generation of pro‐inflammatory cytokines mimicking early phase of ALI. In this study, we addressed the role of microRNA (miR)‐150 in regulating lung inflammation downstream of FAK based on the tenet that the plasma level of mir‐150 were found to be decreased in septic patients. FAK deleted mice lungs showed markedly suppressed miR‐150 expression in miR‐microarray. We confirmed these findings in EC‐FAK−/− lungs and FAK‐depleted human endothelial cells (ECs) using mir‐150 specific primers. Furthermore, we show that in WT mice, miR‐150 expression decreased following LPS‐induced lung injury and subsequently was restored during resolution of lung inflammation. However, in miR‐150−/− mice, LPS persistently induced edema formation. These findings were recapitulated in WT mice where alteration of miR‐150 activity modulated edema formation and inflammation. We identified interleukin‐1R‐associated kinase‐2 (IRAK2), a critical mediator LPS‐induced inflammation, as a downstream effector of mir‐150. We showed augmented IRAK2 levels in EC‐FAK−/− and miR‐150−/− mice lungs as well as FAK knock down ECs. The IRAK2 3′UTR luciferase reporter assay confirmed it as a miR150 target. Our results demonstrate a critical role of miR‐150 downstream of FAK in dampening pulmonary inflammation by targeting IRAK2.

Sepsis‐induced acute lung injury (ALI) results from inflammatory cell infiltration and protein‐rich edema formation. Since focal adhesion kinase (FAK) influences adherens junction formation required for establishing the endothelial barrier, we generated tamoxifen‐inducible endothelial (EC) FAK deficient transgenic mice (EC‐FAK−/−) to address whether FAK controls pulmonary fluid balance and inflammation. EC‐FAK deletion severely disrupts the lung vasculature and promotes pro‐inflammatory cytokine generation such that EC‐FAK−/− mice have increased mortality to endotoxin (LPS). FAK null lung ECs showed actin stress fiber formation, adherens junction disassembly and increased pro‐inflammatory cytokine levels, naming EC‐FAK as a central regulator of lung fluid balance and inflammation. Mechanistically, loss of FAK markedly altered the balance of small GTPases RhoA and Rac1; RhoA activity was significantly increased whereas Rac1 was decreased in FAK null ECs. FAK deletion also amplified p38MAPK activity, which is known to generate cytokines downstream of toll‐like receptors. We confirmed these findings using siRNA‐mediated FAK suppression in human pulmonary ECs. Moreover, EC‐FAK levels were decreased in lungs of patients diagnosed with lung disease and in mice following sepsis. Thus, upregulating endothelial‐FAK provides an unexpected mechanism for preventing cytokine storm and ALI.

Acute lung injury (ALI), characterized by capillary barrier dysfunction and increased reactive oxygen species (ROS) generation in the pulmonary vasculature, is associated with high morbidity/mortality and has no effective therapy. The role of endothelium-derived ROS and mechanisms to prevent ROS in the pulmonary vasculature remain enigmatic in the pathogenesis of ALI. Since focal adhesion kinase (FAK) maintains cell-matrix and inter-endothelial adhesion, we generated endothelial (EC) FAK deficient transgenic adult mice (EC-FAK−/−) to test that FAK controls vessel integrity and ROS production to prevent ALI. We show loss of EC-FAK causes destruction of alveolar structures, interstitial and alveolar edema, and leukocyte seepage into the interstitium and alveolar space. EC-FAK deletion augmented NADPH oxidase and peroxidase activities in lung endothelium causing increased ROS levels and EC apoptosis. Nox2 and Nox4 are the major NADPH oxidases in ECs. Impairment of FAK resulted in increased Nox2 protein expression whereas Nox4 levels remained unchanged suggesting increased ROS production in EC-FAK null lungs was due to hyperactivation of Nox2. These findings were recapitulated in FAK depleted human pulmonary ECs. EC-FAK null mice show increased mortality following sub-lethal dose of i.p. LPS. Our results reveal a novel role of FAK in suppressing Nox2 expression and ROS generation thereby preventing ALI.

Focal adhesion kinase (FAK) maintains basal endothelial barrier function and limits increases in endothelial permeability by inflammatory mediators. We found that endothelial specific deletion of FAK in mice markedly increased leukocyte infiltration and pulmonary inflammation indicative of acute lung injury (ALI). The involvement of microRNAs in human diseases is fast getting recognized and they represent a novel class of targets for therapeutic and diagnostic development. We therefore hypothesized a possible role of microRNAs in anti‐inflammatory response downstream of FAK. MicroRNA profiling from endothelial cell specific FAK null lungs (EC‐FAK) showed significant down regulation of miR‐150. A similar down‐regulation of miR‐150 expression was observed upon knockdown of FAK by siRNA and also by using miR‐150 inhibitor in human pulmonary arterial endothelial cells (HPAEC). Target prediction identified interleukin‐1R‐associated kinase‐2 (IRAK2) as one of the top and noteworthy candidates of miR‐150. IRAK2 has recently emerged as a critical mediator of lipopolysaccharide (LPS) and Interleukin‐1 (IL‐1) induced inflammation. We showed that IRAK2 expression is increased markedly at both mRNA and protein levels in FAK knock down HPAEC or EC‐FAK null lungs. Inhibition of miR‐150 also induced IRAK2 expression. Our results indicate that FAK induces miR‐150 expression to suppress IRAK2 expression and ensuing pulmonary inflammation.

Ligands by binding to G protein coupled receptors (GPCRs) stimulate dissociation of heterotrimeric G proteins into Gα and Gβγ subunits. Released Gα and Gβγ subunits induce discrete signaling cues that differentially regulate focal adhesion kinase (FAK) activity and endothelial barrier function. Activation of G proteins downstream of receptors such as protease activated receptor 1 (PAR1) and histamine receptors rapidly increases endothelial permeability which reverses naturally within the following 1-2 h. However, activation of G proteins coupled to the sphingosine-1-phosphate receptor 1 (S1P1) signal cues that enhance basal barrier endothelial function and restore endothelial barrier function following the increase in endothelial permeability by edemagenic agents. Intriguingly, both PAR1 and S1P1 activation stimulates FAK activity, which associates with alteration in endothelial barrier function by these agonists. In this review, we focus on the role of the G protein subunits downstream of PAR1 and S1P1 in regulating FAK activity and endothelial barrier function.

Endothelial cell specific deletion of FAK leads to embryonic lethality due to impaired vascular formation. Since FAK maintains basal endothelial barrier function and limits increases in its permeability by inflammatory mediators we generated tamoxifen‐inducible, Cre‐driven endothelial specific FAK null adult mice (i‐EC‐FAK−/−) to investigate the role of endothelial FAK in regulating lung vascular integrity. Control or i‐EC‐FAK mice were given 2mg tamoxifen for 5 d and lungs were harvested after 7 d to examine histology and microvessel permeability. Specific FAK deletion was confirmed using immunohistochemistry and confocal imaging of lung sections. Compared to control, i‐EC‐FAK lungs showed destruction of alveolar structures, interstitial and alveolar edema, and leukocyte seepage into the lung interstitium, indicative of acute lung injury (ALI). Arterial blood gas analysis revealed similar P02 and PC02 levels but the hemoglobin levels were significantly lower in i‐EC‐FAK mice, supporting leaky blood vessels and internal bleeding. Next, we investigated the effect of vascular dysfunction on the physical ability of i‐EC‐FAK mice by exposing them to a forced swim test. In contrast to control mice which swim rapidly, i‐EC‐FAK mice only float, suggesting exhaustion. Our results reveal that loss of endothelial FAK leads to ALI, identifying FAK as a novel therapeutic target to prevent ALI.

We have shown that focal adhesion kinase (FAK), a determinant of cell‐matrix adhesion, maintains endothelial barrier function and limits the increase in endothelial permeability induced by inflammatory mediators. We conditionally depleted endothelial FAK (CD‐FAK) using Tie2‐Cre in FAK‐floxed mice to investigate the specific role of FAK in regulating lung vascular permeability (LVP). LVP was determined by measuring lung wet/dry ratio and Evans blue albumin accumulation in lung parenchyma. We show CD‐FAK mice have increased basal LVP, and activation of thrombin receptor PAR1 further augmented this increase. Sphingosine‐1‐phosphate (S1P), by binding to S1P receptor‐1 (S1P1), strengthens the endothelial barrier via Rac1 mediated adherens junction (AJ) assembly. We assessed the effect of FAK knockdown in S1P1 signaling. Depletion of FAK prevented S1P1‐mediated Rac1 activation, AJ assembly, and endothelial barrier strengthening. S1P could only partially reverse PAR1‐induced increase in LVP in CD‐FAK mice. Since S1P1 contains tyrosine residues, we examined if FAK regulated S1P1 signaling by post‐translationally modifying S1P1. We show that S1P stimulated the interaction between S1P1 and FAK, and that S1P induced tyrosine phosphorylation of S1P1 in a FAK‐dependent manner. These findings identify S1P1 as a novel effector of FAK, which upon activation by FAK, may serve as a mechanism to limit basal LVP.

We showed that focal adhesion kinase (FAK) is required to reverse the thrombin‐induced increase in endothelial permeability. Fyn is a non‐receptor Src family tyrosine kinase, which phosphorylates FAK. We investigated the role of Fyn in regulating lung microvascular permeability using Fyn deficient mice. First, we determined whether activation of PAR‐1 by specific activating peptide (TFLLRN), alters FAK activity in lungs. Lungs of WT or Fyn−/− mice received i.v. injection of either control or PAR‐1 activating peptide were homogenized for determination of FAK phosphorylation. We observed that PAR‐1 agonist peptide increased FAK phosphorylation at Y397 and Y576 in WT mice lungs, but failed to induce FAK phosphorylation in lungs of Fyn−/− mice. Next, we determined microvessel filtration coefficient (Kf,c) in lungs isolated from WT and Fyn−/− mice. We observed that basal Kf,,c was significantly higher in Fyn−/− lungs than WT lungs. We also determined Evans blue albumin extravasation and lung wet‐ to dry weight ratio following PAR‐1 activation to assess the role of Fyn in regulating lung edema formation. Injection of PAR‐1 peptide produced significantly greater increase in lung vascular permeability in Fyn−/− mice, indicating that Fyn limits the PAR‐1‐induced pulmonary edema. We next addressed whether rescuing FAK activation could restore lung vascular permeability in Fyn−/− mice. Liposomes conjugated with FAK double phospho‐mimicing mutant (Y397D/Y576D) were injected in Fyn−/− mice and lung vascular permeability was determined after confirmation of FAK mutant expression. We show that rescuing FAK phosphorylation markedly suppressed PAR‐1 induced lung vascular permeability in Fyn−/− mice. These studies identify Fyn as the critical tyrosine kinase that by inducing FAK activation maintains lung vascular barrier function.

The inflammatory mediator thrombin proteolytically activates protease-activated receptor (PAR1) eliciting a transient, but reversible increase in vascular permeability. PAR1-induced dissociation of Gα subunit from heterotrimeric Gq and G12/G13 proteins is known to signal the increase in endothelial permeability. However, the role of released Gβγ is unknown. We now show that impairment of Gβγ function does not affect the permeability increase induced by PAR1, but prevents reannealing of adherens junctions (AJ), thereby persistently elevating endothelial permeability. We observed that in the naive endothelium Gβ1, the predominant Gβ isoform is sequestered by receptor for activated C kinase 1 (RACK1). Thrombin induced dissociation of Gβ1 from RACK1, resulting in Gβ1 interaction with Fyn and focal adhesion kinase (FAK) required for FAK activation. RACK1 depletion triggered Gβ1 activation of FAK and endothelial barrier recovery, whereas Fyn knockdown interrupted with Gβ1-induced barrier recovery indicating RACK1 negatively regulates Gβ1-Fyn signaling. Activated FAK associated with AJ and stimulated AJ reassembly in a Fyn-dependent manner. Fyn deletion prevented FAK activation and augmented lung vascular permeability increase induced by PAR1 agonist. Rescuing FAK activation in fyn−/− mice attenuated the rise in lung vascular permeability. Our results demonstrate that Gβ1-mediated Fyn activation integrates FAK with AJ, preventing persistent endothelial barrier leakiness.

Dynamic interactions between adherens junctions and cell‐matrix adhesions mediated by actin cytoskeleton regulate endothelial barrier function. Barrier‐enhancing agonist, sphingosine‐1‐phosphate (S1P), by binding its S1P receptor1 (S1P‐1) on the endothelial cell surface enhance endothelial monolayer integrity by Rac1‐mediated stabilization of intercellular junctions and by promoting cell‐matrix adhesions at focal adhesions complexes (FACs). Focal adhesion kinase (FAK) regulates the cell‐matrix adhesive contact at focal adhesion sites. We address the possibility that S1P via FAK‐induces Rac1 activation which in turn signals lamellopodia formation to promote cell‐cell interactions and endothelial barrier enhancement. We show that S1P induces FAK activation, as evidenced by phosphorylation on tyrosine residues 397 and 576 in endothelial cells, in association with enhancement of endothelial barrier function. In YFP‐actin mutant transducing cells S1P induced robust lamellopodia formation, consistent with the role of Rac1 in strengthening intercellular junctions. Inhibition of FAK by transduction of dominant negative FAK mutant (FRNK) markedly suppressed basal Rac1 activity. FRNK expression also increased basal endothelial permeability as measured by determining transendothelial monolayer resistance and S1P could not restored it to the level seen in control cells. These findings suggest that S1P induced FAK activation may play an important role in Rac1 mediated endothelial barrier enhancement.