Myosin Light Chain Dephosphorylation Research Articles

Dexamethasone restores TNFα-induced epithelial barrier dysfunction in primary rat alveolar epithelial cells.

Alveolar barrier dysfunction is one of the major pathophysiological changes in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). In ALI/ARDS, tumor necrosis factor-alpha (TNFα) disrupts the barriers of alveolar epithelium and endothelium. Glucocorticoids (GCs) exert anti-inflammatory effects and ameliorate pulmonary edema in ALI/ARDS. However, the involvement of GCs in the restoration of alveolar epithelial barrier dysfunction has not been extensively studied. Here, we elucidated that dexamethasone (Dex) restored TNFα-induced alveolar epithelial barrier dysfunction in vitro using primary rat alveolar epithelial cells isolated from Sprague-Dawley rats. Moreover, Dex promoted the alveolar epithelial cell barrier integrity by initiating GC receptor-mediated signaling via the downregulation of myosin light chain kinase (MLCK) expression and the dephosphorylation of myosin light chain (MLC) 2. Further investigation revealed that Dex enhanced the expression of zonula occludens-1 (ZO-1), a tight junction-related protein, at intercellular junction sites. These findings suggest that GCs strengthen the integrity of the alveolar epithelial barrier in ALI/ARDS via the GR-MLCK-pMLC2 axis.

Myosin light chain dephosphorylation by ppp1r12c promotes atrial hypocontractility in atrial fibrillation

Abstract Background Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls (P=0.02, n=12,12 in each group) with > 40% reduction in MLC2a phosphorylation (P=0.000001, n=12,12 in each group). PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF (P=0.03 and 0.007 respectively, n=8,8 in each group). In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls (P=5.0x10-6, n=12,8,12), with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls (P=0.02 and 0.04 respectively, n= 6,6,5). Conclusions AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key regulator of atrial contractility in AF.

Phosphoproteomic insight into the changes in structural proteins of muscle architecture associated with texture softening of grass carp (Ctenopharyngodon idella) fillets during chilling storage.

Texture is an important sensory attribute of fish affected by modifications of structural proteins in muscle architecture. To investigate the changes in protein phosphorylation during texture softening of fish, the proteins of grass carp muscle after chilling storage of 0day and 6days were compared by phosphoproteomics, and their association with texture was analyzed. Totally 1026 unique phosphopeptides on 656 phosphoproteins were identified as differential. They were mainly classified as intracellular myofibril and cytoskeleton, and extracellular matrix, of which the molecular function and biological process were binding into supramolecular assembly and myofilament contraction. The concomitant dephosphorylation of kinases and assembly regulators indicated dephosphorylation and disassembly tendency of sarcomeric architecture. Correlation analysis defined the relation between texture and dephosphorylation of myosin light chain, actin, collagen and cytoskeleton. This study revealed that protein phosphorylation may affect the texture of fish muscle through regulating sarcomeric assembly of structural proteins in muscle architecture.

Abstract EC.02: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atria Hypocontractility In Atrial Fibrillation

Background: Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, increases thromboembolic stroke risk five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function remain unknown. We tested the hypothesis that increased expression of PPP1R12C, the PP1 regulatory subunit targeting atrial myosin light chain 2 (MLC2a), causes hypophosphorylation of MLC2a and results in atrial hypocontractility. Methods: Right atrial appendage tissues were isolated from human AF patients versus sinus rhythm (SR) controls. Western blots, co-immunoprecipitation, and phosphorylation studies were performed to examine how the PP1c-PPP1R12C interaction causes MLC2a de-phosphorylation. In vitro studies of pharmacologic MRCK inhibitor (BDP5290) in atrial HL-1 cells were performed to evaluate PP1 holoenzyme activity on MLC2a. Cardiac-specific lentiviral PPP1R12C overexpression was performed in mice to evaluate atrial remodeling with atrial cell shortening assays, echocardiography, and AF inducibility with EP studies. Results: In human patients with AF, PPP1R12C expression was increased two-fold versus SR controls with > 40% reduction in MLC2a phosphorylation. 12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies utilizing drug BDP5290, which inhibits T560-PPP1R12C phosphorylation, demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Lenti-12C mice demonstrated a 150% increase in LA size versus controls, with reduced atrial strain and atrial ejection fraction. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusions: AF patients exhibit increased levels of PPP1R12C protein compared to controls. PPP1R12C overexpression in mice increases PP1c targeting to MLC2a and causes MLC2a dephosphorylation, which reduces atrial contractility and increases AF inducibility. These findings suggest that PP1 regulation of sarcomere function at MLC2a is a key regulator of atrial contractility in AF.

Abstract 13258: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Introduction: Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 6.1 million . AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light chain phosphorylation in AF. Objective We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12CPP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

Effect of α1D-adrenoceptor blocker for the reduction of ureteral contractions.

Urolithiasis is a common urinary tract disease with growing prevalence. Alpha1-adrenoceptors (α1-ARs) are abundant in ureteral smooth muscle, distributed with different α1-AR subtypes. α1D-AR is the most widely distributed in the ureter. However, the effect of α1D-AR blockade on ureteric contraction remains unknown. We dissected smooth muscle tissues (3 mm×3 mm) from the rat bladder and human ureter, tied silk strips on both tissue ends, and measured contraction in an organ bath chamber. Contraction activity in ureteral smooth muscle cells (USMCs) was immunocytochemically examined using primary rat and human USMC cultures. Using the organ bath system, we determined the inhibitory effects of silodosin, tamsulosin, and naftopidil. Naftopidil significantly decreased contractility of rat bladder tissue; similar results were observed in human ureteral tissue. To confirm ex vivo experimental results in vitro , we examined the phosphorylation of myosin light chain (MLC), a marker of contractility, in a primary human USMC culture. The examined drugs decreased phospho-MLC levels in human USMCs; however, naftopidil profoundly increased MLC dephosphorylation. We studied the effects of naftopidil, an α1D-AR inhibitor, on the ureter. Compared with alpha-blockers, naftopidil significantly relaxed ureteral smooth muscle. Therefore, naftopidil could be an effective therapy for patients with ureteral stones.

Deficiency of macrophage PHACTR1 impairs efferocytosis and promotes atherosclerotic plaque necrosis.

Efferocytosis, the process through which apoptotic cells (ACs) are cleared through actin-mediated engulfment by macrophages, prevents secondary necrosis, suppresses inflammation, and promotes resolution. Impaired efferocytosis drives the formation of clinically dangerous necrotic atherosclerotic plaques, the underlying etiology of coronary artery disease (CAD). An intron of the gene encoding PHACTR1 contains rs9349379 (A>G), a common variant associated with CAD. As PHACTR1 is an actin-binding protein, we reasoned that if the rs9349379 risk allele G causes lower PHACTR1 expression in macrophages, it might link the risk allele to CAD via impaired efferocytosis. We show here that rs9349379-G/G was associated with lower levels of PHACTR1 and impaired efferocytosis in human monocyte-derived macrophages and human atherosclerotic lesional macrophages compared with rs9349379-A/A. Silencing PHACTR1 in human and mouse macrophages compromised AC engulfment, and Western diet-fed Ldlr-/- mice in which hematopoietic Phactr1 was genetically targeted showed impaired lesional efferocytosis, increased plaque necrosis, and thinner fibrous caps - all signs of vulnerable plaques in humans. Mechanistically, PHACTR1 prevented dephosphorylation of myosin light chain (MLC), which was necessary for AC engulfment. In summary, rs9349379-G lowered PHACTR1, which, by lowering phospho-MLC, compromised efferocytosis. Thus, rs9349379-G may contribute to CAD risk, at least in part, by impairing atherosclerotic lesional macrophage efferocytosis.

Abstract P780: Myosin Light Chain Dephosphorylation by Ppp1r12c Promotes Atrial Hypocontractility in Atrial Fibrillation

Atrial fibrillation (AF) is the most common sustained arrhythmia, with an estimated prevalence in the U.S.of 6.1 million. AF increases the risk of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown. We have recently identified protein phosphatase 1 subunit 12c (PPP1R12C) as a key molecule targeting myosin light-chain phosphorylation in AF. Objective: We hypothesize that the overexpression of PPP1R12C causes hypophosphorylation of atrial myosin light-chain 2 (MLC2a), thereby decreasing atrial contractility in AF. Methods and Results: Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluate the role of the PP1c-PPP1R12C interaction in MLC2a de-phosphorylation, we utilized Western blots, co-immunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A), PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls. Conclusion: The overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, associated with a reduction in atrial contractility and an increase in AF inducibility. All these discoveries suggest that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.

MYPT1 O-GlcNAc modification regulates sphingosine-1-phosphate mediated contraction

Many intracellular proteins are modified by N-acetylglucosamine, a posttranslational modification termed O-GlcNAc. This modification is found on serine and threonine side-chains and has the potential to regulate signaling pathways through interplay with phosphorylation. Here, we discover and characterize one such example. We find that O-GlcNAc levels control the sensitivity of fibroblasts to actin contraction induced by the signaling lipid sphingosine-1-phosphate (S1P), culminating in the phosphorylation of myosin light chain (MLC) and cellular contraction. Specifically, O-GlcNAc modification of the phosphatase subunit MYPT1 inhibits this pathway by blocking MYPT1 phosphorylation, maintaining its activity and causing the dephosphorylation of MLC. Finally, we demonstrate that O-GlcNAc levels alter the sensitivity of primary human dermal fibroblasts in a collagen-matrix model of wound healing. Our findings have important implications for the role of O-GlcNAc in fibroblast motility and differentiation, particularly in diabetic wound healing.

MYOSIN LIGHT CHAIN DEPHOSPHORYLATION BY PPP1R12C PROMOTES ATRIAL HYPOCONTRACTILITY IN ATRIAL FIBRILLATION

Atrial fibrillation (AF) increases stroke risk five-fold. Atrial hypocontractility from atrial myosin light chain (MLC2a) dephosphorylation contributes to stroke risk in AF. Recent proteomic data has shown increased protein phosphatase 1 subunit 12C (PPP1R12C) targeting to MLC2a in AF. However, it

Abstract WMP39: Protein Phosphatase 1 Regulatory Subunit 12C Contributes to Atrial Myosin Light Chain Dephosphorylation in Atrial Fibrillation

Background: Atrial fibrillation (AF) increases stroke risk five-fold. Atrial hypocontractility from atrial myosin light chain (MLC2a) dephosphorylation contributes to stroke risk in AF. Recent proteomic data has shown increased protein phosphatase 1 subunit 12C (PPP1R12C) targeting to MLC2a in AF. However, it is unclear whether PPP1R12C causes MLC2a dephosphorylation in AF. Objective: Determine whether increased PPP1R12C expression causes MLC2a dephosphorylation and increases AF risk. Methods: Western blots and co-IPs were performed to evaluate the relationship among PPP1R12C, PP1c and MLC2a in human atrial tissues (AF vs SR). Mice with either a knockout (KO) or lentiviral (LV) cardiac overexpression of PPP1R12C were evaluated with invasive EP studies for AF inducibility vs WT controls. Results: In human AF, PPP1R12C was increased 4-fold ( P <0.005, n=6) with an 88% reduction in S-19-MLC2a phosphorylation ( P <0.05, n=4). PPP1R12C-PP1c and PPP1R12C-MLC2a binding was increased 2-fold in AF ( P <0.05, n=6). AF burden in LV-12C mice increased nearly tenfold vs. KO and WT mice ( P <0.05, n=6). Conclusion: In human AF, increased PPP1R12C expression is associated with reduced P-MLC2a through enhanced binding with the PP1c catalytic subunit. This dephosphorylation is a likely contributor to atrial hypocontractility and stroke risk in AF. Additionally, increased PPP1R12C expression in mice increases AF risk. Future studies will examine the effects of increased PPP1R12C expression upon atrial contractile function in mice.

Role of PI3K/Akt and MEK/ERK Signalling in cAMP/Epac-Mediated Endothelial Barrier Stabilisation.

Background and AimsActivation of the cAMP/Epac signalling stabilises endothelial barrier function. Moreover, its activation is accompanied by an activation of PI3K/Akt and MEK/ERK signalling in diverse cell types but their impact on endothelial barrier function is largely unknown. Here the role of PI3K/Akt and MEK/ERK signalling in cAMP/Epac-mediated endothelial barrier stabilisation was analysed.MethodsEndothelial barrier function was analysed in cultured human umbilical vein endothelial cells (HUVECs) by measuring flux of albumin. A modified cAMP analogue 8-pCPT-2′-O-Me-cAMP (Epac agonist) was used to specifically activate cAMP/Epac signalling.ResultsEpac agonist reduces the basal and attenuates thrombin-induced endothelial hyperpermeability accompanied by an activation of PI3K/Akt and MEK/ERK signalling. The qPCR data demonstrate HUVECs express PI3Kα, PI3Kβ, and PI3Kγ but not PI3Kδ isoforms. The western blot data demonstrate Epac agonist activates PI3Kα and PI3Kβ isoforms. Inhibition of MEK/ERK but not PI3K/Akt pathway potentiates the endothelial barrier protective effects of cAMP/Epac signalling. Inhibition of MEK/ERK signalling in the presence of Epac agonist induces a reorganisation of actin cytoskeleton to the cell periphery, enhanced VE-cadherin localisation at cell-cell junctions, and dephosphorylation of myosin light chains (MLC) but not inhibition of RhoA/Rock signalling. Moreover, Epac agonist promotes endothelial cell (EC) survival via reduction in activities of pro-apoptotic caspases in a PI3K/Akt and MEK/ERK signalling-dependent manner.ConclusionOur data demonstrate that the Epac agonist simultaneously activates diverse signalling pathways in ECs, which may have differential effects on endothelial barrier function. It activates PI3K/Akt and MEK/ERK signalling which mainly govern its pro-survival effects on ECs. Inhibition of MEK/ERK but not PI3K/Akt signalling enhances barrier stabilising and barrier protective effects of cAMP/Epac activation.Chemical Compounds Used In This Study8-pCPT-2′-O-Me-cAMP (PubChem CID: 9913268); Akt inhibitor VIII (PubChem CID: 10196499); AS-252424 (PubChem CID: 11630874); IC-87114 (PubChem CID: 9908783); PD 98059 (PubChem CID: 4713); PIK-75 (PubChem CID: 10275789); TGX-221 (PubChem CID: 9907093); Thrombin (PubChem CID: 90470996); U0126 (PubChem CID: 3006531); Wortmannin (PubChem CID: 312145).

Paying the Toll for Inflammation

Paying the Toll for Inflammation

Contribution of myofibril filament disassembly to textural deterioration of ice-stored grass carp fillet: Significance of endogenous proteolytic activity, loss of heat shock protein and dephosphorylation of myosin light chain

To investigate the underlying mechanism of softening of ice-stored grass carp fillet, changes in assembly structure of myofibrillar proteins and potential candidates for regulating this change including myosin regulatory chain phosphorylation, heat shock proteins (Hsp27, Hsp90, αB-crystallin and UNC45) and endogenous protease activity were studied. Comparison of SDS-PAGE pattern of myofibrillar proteins treated with EDC crosslinking showed that thin filament experienced rapid disassembly within initial 8 h, followed by depolymerization of thick filament consisting of myosin, which further exacerbated the myofibril disorganization of fillets. Pearson coefficient analysis showed that UNC45, Hsp90, Hsp27 and αB-crystallin concentration and cathepsin B, D, L activities were significantly correlated with dissociated MHC and actin. Therefore, the significant correlation between shear force and dissociated MHC and actin clearly demonstrated that post mortem disassembly of myofibril filaments, which was regulated by endogenous proteolytic activity and loss of Hsp, contributed to the softening of ice-stored grass carp fillets.

HDAC7 in MLCP‐mediated endothelial barrier preservation

Acute lung injury (ALI) is an acute systemic inflammatory process characterized by pulmonary infiltrates hypoxemia and edema. ALI arises from a wide range of lung injuries such as toxins or inflammatory mediators. Despite the high level of mortality, to date no pharmacological therapies have been proven to improve ALI except from ventilation strategies. A deeper understanding of the pathogenic mechanisms involved remains a barrier to new and effective therapies that will reduce the vascular leak associated with ALI.A major cause of ALI is dysfunction of the pulmonary vascular endothelial barrier. Endothelial cells (EC) are a specialized cell type that line the lumen of the blood vessels serving every major organ system and provide a semi‐selective barrier between the blood and the interstitial space. Epigenetic mechanisms have emerged as a major regulator of enduring changes in cell behavior and the therapeutic potential of inhibiting histone deacetylases (HDACs) for the treatment of cardiovascular and inflammatory diseases is gaining increasing attention. In addition to histones, it has been shown that HDACs can deacetylate many non‐histone proteins to regulate cellular functions such as cytoskeletal acetylation and polymerization, and signal transduction. Further, it has been shown that HDAC inhibition is directly involved in EC barrier preservation, however, the molecular mechanisms of HDAC‐mediated EC barrier regulation remain largely unexplored.Our data indicate that the class IIa histone deacetylases comprising of HDACs 4, 5, 7, 9 are involved in the regulation of pulmonary vascular barrier in vitro and in lipopolysaccharide (LPS)‐induced murine model of ALI. These HDACs are known to function as transcriptional co‐repressors, however, they also are able to shuttle from nucleus to the cytoplasm thus becoming accessible for extra nuclear targets. We demonstrate that HDAC 7 is directly involved in LPS‐induced human lung microvascular EC (HLMVEC) barrier compromise. Further, barrier‐disruptive effects of LPS are accompanied by HDAC7 phosphorylation and its nuclear export. Conversely, agents that protect the EC barrier such as adenosine, decrease its phosphorylation. These data suggest the involvement of phosphatase activity in HDAC7‐mediated EC barrier regulation. Our results show that HDAC7 is a substrate for myosin light chain (MLC) phosphatase (MLCP) in HLMVEC. Conversely, down‐regulation of HDAC7 may affect MLCP activity. However, the physiological role of HDAC7/MLCP crosstalk in EC barrier regulation is currently unexplored. It is known that activation of MLCP leads to dephosphorylation of MLC thus opposing actomyosin contraction. Our newly published results suggest that MLCP activation may be involved in EC barrier preservation via the dephosphorylation of unconventional MLCP cytoskeletal targets such as moesin. Our results demonstrate a direct association between HDAC7 and moesin, suggesting a novel role of a HDAC7/MLCP/moesin signaling complex in the regulation of EC barrier. Collectively, these data demonstrate the involvement of HDAC7 activity in MLCP‐mediated EC barrier preservation and suggest, for the first time, the importance of HDAC7 nuclear export in pulmonary vascular injury induced by edemagenic agents such as LPS.Support or Funding InformationNIH; Contract grant number: HL101902This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis

VEGFR-2/Notch signalling regulates angiogenesis in part by driving the remodelling of endothelial cell junctions and by inducing cell migration. Here, we show that VEGF-induced polarized cell elongation increases cell perimeter and decreases the relative VE-cadherin concentration at junctions, triggering polarized formation of actin-driven junction-associated intermittent lamellipodia (JAIL) under control of the WASP/WAVE/ARP2/3 complex. JAIL allow formation of new VE-cadherin adhesion sites that are critical for cell migration and monolayer integrity. Whereas at the leading edge of the cell, large JAIL drive cell migration with supportive contraction, lateral junctions show small JAIL that allow relative cell movement. VEGFR-2 activation initiates cell elongation through dephosphorylation of junctional myosin light chain II, which leads to a local loss of tension to induce JAIL-mediated junctional remodelling. These events require both microtubules and polarized Rac activity. Together, we propose a model where polarized JAIL formation drives directed cell migration and junctional remodelling during sprouting angiogenesis.

Subcellular Spatial Control of Non-Muscle Myosin 2 Redistribution and Stress Fiber Strain by Molecular Tattoo

The cellular distribution of the motor protein non-muscle myosin 2 (NM2) leads to different forms of intracellular strain driving cell motility, cytokinesis and morphogenetic processes including axonal growth and retraction. However, it remains elusive how these cellular processes are governed by the dynamic changes in NM2 localization and supramolecular assembly. To address this problem, we determined the effect of different types of NM2 inhibition on the dynamics of load-bearing stress fibers and unloaded inner cytoplasmatic NM2 structures in live HeLa cells. We followed NM2 redistribution via FRAP, applied also in combination with our recently developed optopharmacological tool, Molecular Tattoo, which allows subcellular confinement of drug effects via 2-photon induced photocrosslinking to targets. We found that the Rho-kinase inhibitor, Y-27632, dramatically accelerates NM2 redistribution and induces stress fiber dissolution, due to NM2 filament disassembly resulting from myosin light chain dephosphorylation. When NM2 was inhibited by para-nitroblebbistatin (pNBleb) or locally by tattooed azidoblebbistatin, in the stress fibers a significant acceleration and suppression of NM2 redistribution was detected at moderate and high inhibitor concentrations, respectively. The observed effects were local and specific for load-bearing peripheral stress fibers, implying the role of mechanical load in NM2 redistribution. Furthermore, in these tests stress fibers remained intact, contrary to that seen upon Rho-kinase inhibition. These results highlight that variations in the localization and/or pharmacological mechanism of NM2 inhibition produce distinct effects on intracellular strain and cellular morphogenesis.Supported by Hungarian Research and Innovation Fund (VKSZ_14-1-2015-0052).

Enzymatic changes in myosin regulatory proteins may explain vasoplegia in terminally ill patients with sepsis.

The current study was conducted with the hypothesis that failure of maintenance of the vascular tone may be central to failure of the peripheral circulation and spiralling down of blood pressure in sepsis. Namely, we examined the balance between expression of myosin light chain (MLC) phosphatase and kinase, enzymes that regulate MLCs dephosphorylation and phosphorylation with a direct effect on pharmacomechanical coupling for smooth muscle relaxation and contraction respectively. Mechanical recordings and enzyme immunoassays of vascular smooth muscle lysates were used as the major methods to examine arterial biopsy samples from terminally ill sepsis patients. The results of the present study provide evidence that genomic alteration of expression of key regulatory proteins in vascular smooth muscles may be responsible for the relentless downhill course in sepsis. Down-regulation of myosin light chain kinase (MLCK) and up-regulation of MLCK may explain the loss of tone and failure to mount contractile response invivo during circulation. The mechanical studies demonstrated the inability of the arteries to develop tone when stimulated by phenylephrine invitro. The results of our study provide indirect hint that control of inflammation is a major therapeutic approach in sepsis, and may facilitate to ameliorate the progressive cardiovascular collapse.

Abstract B55: Chemokine biased agonists regulate pancreatic cancer migration and metastasis through bioenergetic signaling

Abstract Across all stages of initial diagnosis, the majority of patients with pancreatic ductal adenocarcinoma (PDAC) eventually succumb to metastatic disease. Yet, there are no drugs approved for disruption of the specific mechanisms behind PDAC metastasis. As soluble/secreted proteins, chemokines provide a unique opportunity to manipulate cancer cell movement and metastasis. Our prior reports show that chemokines such as CXCL12 regulate movement in a biphasic concentration dependent fashion, wherein low doses drive migration and higher doses are cytostatic. This biphasic migration response reflects the dose dependent oligomerization of the ligand, with monomeric CXCL12 as pro-migratory and dimeric CXCL12 as anti-migratory. Previously, we demonstrated that the CXCL12 gene is silenced in human PDAC tumor cells and, when re-expressed, is a tumor suppressor decreasing growth and metastasis. Herein, we examine how CXCL12 inhibits PDAC malignancy and explore CXCL12 use as a biologic therapy for PDAC metastasis. Established and patient-derived PDAC cells along with tumor cells derived from the B6-KRasLSL.G12D/+-p53R172H/+-PdxCretg/+ (KPC) model were used to test the hypothesis that CXCL12 biphasic chemotactic signaling reflects biased agonist regulation of bioenergetic signaling. In transwell assays, human PDAC cells responded to recombinant wild type CXCL12 (L12WT) in a biphasic manner, with 10 nM L12WT as pro-migratory and concentrations ≥ 100 nM as cytostatic. As predicted by the biased agonist model, migratory and cytostatic doses of L12WT stimulated equivalent intracellular calcium mobilization. Though calcium signaling has previously been associated with activity of AMP-Kinase (AMPK), a known regulator of metabolic stress, no prior studies have established a link between bioenergetics and chemotactic migration. Given CXCL12 dual regulation of growth and migration, we next tested the hypothesis that CXCL12 alters bioenergetic metabolism using a Seahorse XF analyzer. In MiaPaCa2 cells, while cell bioenergetics were unchanged by migratory doses, cytostatic doses of L12WT significantly decreased both ATP-linked oxidative phosphorylation and reserve glycolysis capacity. In parallel, cytostatic doses of L12WT significantly increased levels of phosphorylated AMPK in human and murine PDAC cells. L12WT-induced AMPK activity was calcium/calmodulin-kinase II dependent. Anti-motile effects of CXCL12 were dependent on AMPK signaling as the specific inhibitor Compound C completely prevented the cytostasis induced by L12WT. Subsequent experiments established that cytostatic doses of L12WT drove inhibitory phosphorylation of MYPT1, preventing dephosphorylation of myosin light chain (MLC). Thus, use of the AMPK inhibitor reversed the CXCL12 biphasic phenomenon by restoring balance to MLC dephosphorylation-phosphorylation cycling, allowing PDAC cells to once again migrate. CXCL12 biphasic migration was mimicked with locked mutants of CXCL12, with monomer (L121) as pro-migratory but equal levels of dimer (L122) as anti-migratory. Stimulation of PDAC cells with L122 lead to more sustained AMPK activity when compared with L121. Finally, in mouse orthotopic xenografts of two different PDAC cell lines, intraperitoneal administration of 5 μM L12WT significantly decreased PDAC metastasis to the liver and lung. Together, these data demonstrate for the first time that the cytostatic component of CXCL12 function is AMPK dependent. Given the ability of CXCL12 to regulate bioenergetic migratory signaling, our data show the promise of recombinant CXCL12 and biased agonist variants, as a biologic therapy for abrogating PDAC metastasis. Citation Format: Ishan Roy, Donna McAllister, Egal Gorse, Kate Dixon, Gang Cheng, Anthony Getschman, Dannielle Engle, Susan Tsai, David Tuveson, Douglas Evans, Brian Volkman, Balaraman Kalyanaraman, Michael Dwinell. Chemokine biased agonists regulate pancreatic cancer migration and metastasis through bioenergetic signaling. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Innovations in Research and Treatment; May 18-21, 2014; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2015;75(13 Suppl):Abstract nr B55.

Prostaglandin E2‐Dependent Blockade of Actomyosin and Stress Fibre Formation Is Mediated Through S1379 Phosphorylation of ROCK2

Prostaglandin E2 is a pleiotropic bioactive lipid that controls cytoskeletal alterations, although the precise G-protein coupled EP receptor signalling mechanisms remain ill defined. We adopted a phosphoproteomic approach to characterize post-receptor downstream signalling substrates using antibodies that selectively recognize and immunoprecipitate phosphorylated substrates of a number of kinases. Using human synovial fibroblasts in monolayer cell culture, PGE2 induced rapid and sustained changes in cellular morphology and reduction in cytoplasmic volume that were associated with disassembly of the phalloidin-stained stress fibres as judged by light and confocal microscopy. Furthermore, PGE2 induced a rapid dephosphorylation of myosin light chain II (MLC) at S19 under basal or cytokine-induced conditions that was linked to an activation of myosin light chain phosphatase. The use of specific synthetic EP agonists suggested that the response was mediated by EP2 receptors, as other EP agonists did not manifest the same effect on MLC phosphorylation. In addition, PGE2 induced sustained Y118 dephosphorylation of phospho-paxillin and loss of focal adhesions as observed by confocal microscopy and Western analysis. Phosphoproteomic analysis of PGE2 /GPCR/PKA phosphosubstrates identified a unique, non-redundant, phosphorylated (>30-fold) site on rho-associated coiled coil-containing kinase 2 (ROCK2) at S1379. Analysis of ROCK2 mutant behaviour (e.g. S1379A) in overexpression studies revealed that PGE2 -dependent phosphorylation of ROCK2 resulted in the inhibition of the kinase, since induced MLC phosphorylation was no longer blocked by PGE2 nor could PGE2 induce disassembly of stress fibres. Thus, PGE2 -dependent blockade of actomyosin fibre formation, characteristic of myofibroblasts, may be mediated through specific ROCK2 S1379 phosphorylation.