ROCK1 Deficiency Research Articles

ROCK1 deficiency preserves caveolar compartmentalization of signaling molecules and cell membrane integrity.

In this study, we investigated the roles of ROCK1 in regulating structural and functional features of caveolae located at the cell membrane of cardiomyocytes, adipocytes, and mouse embryonic fibroblasts (MEFs) as well as related physiopathological effects. Caveolae are small bulb-shaped cell membrane invaginations, and their roles have been associated with disease conditions. One of the unique features of caveolae is that they are physically linked to the actin cytoskeleton that is well known to be regulated by RhoA/ROCKs pathway. In cardiomyocytes, we observed that ROCK1 deficiency is coincident with an increased caveolar density, clusters, and caveolar proteins including caveolin-1 and -3. In the mouse cardiomyopathy model with transgenic overexpressing Gαq in myocardium, we demonstrated the reduced caveolar density at cell membrane and reduced caveolar protein contents. Interestingly, coexisting ROCK1 deficiency in cardiomyocytes can rescue these defects and preserve caveolar compartmentalization of β-adrenergic signaling molecules including β1-adrenergic receptor and type V/VI adenylyl cyclase. In cardiomyocytes and adipocytes, we detected that ROCK1 deficiency increased insulin signaling with increased insulin receptor activation in caveolae. In MEFs, we identified that ROCK1 deficiency increased caveolar and total levels of caveolin-1 and cell membrane repair ability after mechanical or chemical disruptions. Together, these results demonstrate that ROCK1 can regulate caveolae plasticity and multiple functions including compartmentalization of signaling molecules and cell membrane repair following membrane disruption by mechanical force and oxidative damage. These findings provide possible molecular insights into the beneficial effects of ROCK1 deletion/inhibition in cardiomyocytes, adipocytes, and MEFs under certain diseased conditions.

ROCK1 regulates insulin secretion from β-cells

ObjectiveThe endocrine pancreatic β-cells play a pivotal role in maintaining whole-body glucose homeostasis and its dysregulation is a consistent feature in all forms of diabetes. However, knowledge of intracellular regulators that modulate β-cell function remains incomplete. We investigated the physiological role of ROCK1 in the regulation of insulin secretion and glucose homeostasis. MethodsMice lacking ROCK1 in pancreatic β-cells (RIP-Cre; ROCK1loxP/loxP, β-ROCK1−/−) were studied. Glucose and insulin tolerance tests as well as glucose-stimulated insulin secretion (GSIS) were measured. An insulin secretion response to a direct glucose or pyruvate or pyruvate kinase (PK) activator stimulation in isolated islets from β-ROCK1−/− mice or β-cell lines with knockdown of ROCK1 was also evaluated. A proximity ligation assay was performed to determine the physical interactions between PK and ROCK1. ResultsMice with a deficiency of ROCK1 in pancreatic β-cells exhibited significantly increased blood glucose levels and reduced serum insulin without changes in body weight. Interestingly, β-ROCK1−/− mice displayed a progressive impairment of glucose tolerance while maintaining insulin sensitivity mostly due to impaired GSIS. Consistently, GSIS markedly decreased in ROCK1-deficient islets and ROCK1 knockdown INS-1 cells. Concurrently, ROCK1 blockade led to a significant decrease in intracellular calcium and ATP levels and oxygen consumption rates in isolated islets and INS-1 cells. Treatment of ROCK1-deficient islets or ROCK1 knockdown β-cells either with pyruvate or a PK activator rescued the impaired GSIS. Mechanistically, we observed that glucose stimulation in β-cells greatly enhanced ROCK1 binding to PK. ConclusionsOur findings demonstrate that β-cell ROCK1 is essential for glucose-stimulated insulin secretion and for glucose homeostasis and that ROCK1 acts as an upstream regulator of glycolytic pyruvate kinase signaling.

386-P: ROCK1 Regulation of AMPK in the Development of Diabetic Nephropathy

Rho-associated, coiled-coil-containing protein kinase (ROCK) is activated in the diabetic kidney. Studies over the past decade have illuminated ROCK signaling as an essential pathway in the pathogenesis of diabetic nephropathy. Accumulating evidence shows that renal dysfunction among patient with diabetes is associated with abnormal fatty acid oxidation in the kidney. However, the interaction of ROCK and fatty acid oxidation in diabetic kidneys remains unclear. Extracellular flux analyzer demonstrated that deficiency of ROCK1, one of the ROCK isoforms, improves TGF-β-induced mitochondrial dysfunction in primary cultured mesangial cells. ROCK1 deficiency rescued TGF-β-induced downregulation of fatty acid oxidation mediators such as peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) , and carnitine palmitoyltransferase 1A (CPT1A) . An investigation of mechanisms underlying this observation revealed activated ROCK1 axis through the phosphorylation of AMPK and increased expression of PGC-1α. Fluorescence histochemistry showed that ROCK1 deficiency suppresses ROS production induced by mitochondrial damage due to abnormal fatty acid metabolism. Consistent with this result, compared to wild-type mice, ROCK1 knockout mice exhibited reduced albuminuria and histological abnormalities along with the recovery of impaired fatty acid utilization and mitochondrial fragmentation. Furthermore, RNA-seq showed that ROCK1 deficiency in mesangial cells significantly altered the expression of genes involved in metabolic pathway. These observations indicate that ROCK1 is a key player in the development of diabetic renal injury. ROCK1 may be a potential therapeutic target for the treatment of diabetic nephropathy. Disclosure Y.Nagai: None. K.Matoba: None. K.Sekiguchi: None. Y.Takeda: None. Y.Kanazawa: None. D.Kawanami: None. K.Utsunomiya: None. R.Nishimura: Speaker's Bureau; Abbott Diabetes, Astellas Pharma Inc., AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Medtronic, Merck & Co., Inc., Novo Nordisk, Ono Pharmaceutical Co., Ltd., Sanofi, Taisho Pharmaceutical Holdings Co., Ltd., Takeda Pharmaceutical Company Limited, Teijin Pharma Limited, Terumo Corporation.

Rho Kinase regulates neutrophil NET formation that is involved in UVB-induced skin inflammation.

Objective: Ultraviolet B (UVB) is an important trigger of skin inflammation and lupus with leukocyte recruitment to inflamed skin. We recently reported the involvement of neutrophil NETosis in UVB-induced skin inflammation, and that NETotic nuclear envelope rupture is driven by PKCα-mediated nuclear lamin B disassembly. To address the role of Actin cytoskeleton in NETosis, we investigated the effects of Rho kinase (ROCK) and its downstream actomyosin cytoskeletal networks on PKCα nuclear translocation and NET formation, as well as their involvement in UVB-induced skin inflammation.Methods: We studied the dynamic changes of ROCK and actomyosin cytoskeletal networks during NETosis induction and their involvement in PKCα nuclear translocation. Using mice with hematopoietic-specific ROCK1 deficiency, we investigated the effects of ROCK1 deficiency on NETosis, and its involvement in UVB-induced skin inflammation.Results: Our time course studies demonstrated the dynamic changes of actin polymerization and ROCK activation, support the role of actin cytoskeleton in nuclear translocation of cytosolic PKCα in early stage of NETosis induction. Inhibition of actin polymerization or key molecules of the ROCK/MLCK/myosin pathway decreased PKCα nuclear translocation and NET formation. Genetic deficiency of ROCK1, inhibited NETosis ex vivo and in vivo, decreased extracellular display of NET-associated IL-17A, TNFα, IFNγ, and IFNα in inflamed skin, which were correlated with the ameliorated skin inflammation in UVB-irradiated mice with hematopoietic-specific ROCK1 deficiency.Conclusions: ROCK regulated NETosis through modulation of PKCα nuclear translocation via actomyosin cytoskeletal networks in neutrophils. ROCK1 deficiency ameliorated UVB-induced skin inflammation by attenuation of NETosis and NET-associated cytokines.

394-P: ROCK1/AMPK Axis Regulates the Development of Diabetic Kidney Disease via Modulation of Fatty Acid Metabolism

The small GTPase Rho and its effector Rho-kinase (ROCK) are involved in the pathogenesis of diabetic kidney disease. Accumulating evidence shows that renal dysfunction in diabetic patient is associated with abnormal fatty acid oxidation in the kidney. However, the interaction of ROCK and fatty acid oxidation in diabetic kidneys remains unclear. Extracellular flux analyzer demonstrated that deficiency of ROCK1, one of the ROCK isoforms, improves TGF-β-induced mitochondrial dysfunction of cultured mesangial cells. ROCK1 deficiency rescued TGF-β-induced downregulation of fatty acid oxidation mediators such as peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), peroxisome proliferator-activated receptor α (PPARα), carnitine palmitoyltransferase 1A (CPT1A). An investigation of mechanisms underlying this observation revealed activated ROCK1 functions through the phosphorylation of AMPK and increased expression of PGC-1α. Furthermore, fluorescence histochemistry showed that ROCK1 deficiency suppresses ROS production as a result of mitochondrial damage due to abnormal fatty acid metabolism. Consistent with this result, ROCK1 knockout mice exhibited reduced albuminuria and histological abnormalities along with the recovery of impaired fatty acid utilization and mitochondrial fragmentation. These observations indicate that ROCK1 is a key player in the development of diabetic renal injury. Glomerular ROCK1 may be a potential therapeutic target for the treatment of diabetic kidney disease. Disclosure Y. Nagai: None. K. Matoba: Research Support; Self; Eli Lilly Japan K. K. K. Sekiguchi: None. R. Ukichi: None. Y. Takeda: None. T. Akamine: None. Y. Kanazawa: None. K. Utsunomiya: None. R. Nishimura: Speaker’s Bureau; Self; Abbott Japan Co., Ltd., Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly Japan K. K., Kissei Pharmaceutical Co., Ltd., Medtronic, MSD Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

1776-P: ROCK1 Deletion in Sim1-Expressing Neurons of the Hypothalamus Leads to Obesity Despite Hypophagia

The paraventricular nucleus of the hypothalamus (PVN) is a key site in the brain for regulating energy balance. ROCK1 has also been shown to be a major regulator of energy balance and metabolism in the hypothalamus, but its role in the PVN is still unclear. To explore this role, mice selectively lacking ROCK1 in single-minded homolog 1 gene (Sim1) neurons (Sim1-ROCK1-/-), which are densely expressed in the PVN and in parts of the amygdala, were studied. ROCK1 deficiency in Sim1 expressing neurons leads to obesity on regular chow in male and female mice after 11-12 weeks. This effect is due to increased fat mass as assessed by EchoMRI and direct dissection of fat depots. Serum levels of insulin, leptin and free fatty acid in Sim1-ROCK1-/- mice were markedly increased compared to control mice. Surprisingly, daily food intake in Sim1-ROCK1-/- mice were significantly decreased compared with control mice. Moreover, the ability of melanocortin to decrease food intake was impaired when ROCK1 was deleted from Sim1-expressing neurons. No differences in O2 consumption and CO2 production measured by the Comprehensive Lab Animal Monitoring System (CLAMS) were found between obese and lean mice with or without ROCK1 deletion. These data identify ROCK1 as an important mediator of energy balance in the PVN, which leads to a paradoxical obese/hypophagic phenotype. The underlying mechanisms for the divergence in obesity, energy expenditure and caloric intake are being further explored in our lab. Disclosure H. Lee: None. J. Han: None. A.G. Uner: None. S.J. Choi: None. R.M. Pereira: None. X. Sun: None. H. Kim: None. Y. Kim: None.

Different roles of myocardial ROCK1 and ROCK2 in cardiac dysfunction and postcapillary pulmonary hypertension in mice

Although postcapillary pulmonary hypertension (PH) is an important prognostic factor for patients with heart failure (HF), its pathogenesis remains to be fully elucidated. To elucidate the different roles of Rho-kinase isoforms, ROCK1 and ROCK2, in cardiomyocytes in response to chronic pressure overload, we performed transverse aortic constriction (TAC) in cardiac-specific ROCK1-deficient (cROCK1-/-) and ROCK2-deficient (cROCK2-/-) mice. Cardiomyocyte-specific ROCK1 deficiency promoted pressure-overload-induced cardiac dysfunction and postcapillary PH, whereas cardiomyocyte-specific ROCK2 deficiency showed opposite results. Histological analysis showed that pressure-overload-induced cardiac hypertrophy and fibrosis were enhanced in cROCK1-/- mice compared with controls, whereas cardiac hypertrophy was attenuated in cROCK2-/- mice after TAC. Consistently, the levels of oxidative stress were up-regulated in cROCK1-/- hearts and down-regulated in cROCK2-/- hearts compared with controls after TAC. Furthermore, cyclophilin A (CyPA) and basigin (Bsg), both of which augment oxidative stress, enhanced cardiac dysfunction and postcapillary PH in cROCK1-/- mice, whereas their expressions were significantly lower in cROCK2-/- mice. In clinical studies, plasma levels of CyPA were significantly increased in HF patients and were higher in patients with postcapillary PH compared with those without it. Finally, high-throughput screening demonstrated that celastrol, an antioxidant and antiinflammatory agent, reduced the expressions of CyPA and Bsg in the heart and the lung, ameliorating cardiac dysfunction and postcapillary PH induced by TAC. Thus, by differentially affecting CyPA and Bsg expressions, ROCK1 protects and ROCK2 jeopardizes the heart from pressure-overload HF with postcapillary PH, for which celastrol may be a promising agent.

Disruption of ROCK1 gene restores autophagic flux and mitigates doxorubicin-induced cardiotoxicity.

Doxorubicin is among the essential medicines with a wide antitumor spectrum, but its clinical application is limited by its cardiotoxicity. We recently discovered that ROCK1 is a key molecule in mediating cardiac remodeling in response to various stresses. To determine the roles of ROCK1 in doxorubicin cardiotoxicity, we gave three doses of doxorubicin injections to wild type (WT) and ROCK1−/− mice with one week intervals between treatments, the cumulative dose being 24 mg/kg. ROCK1−/− mice exhibited preserved cardiac function, reduced apoptosis, autophagy and fibrosis compared to the WT mice. To further determine the cellular mechanisms, we have examined the role of ROCK1 in cardiomyocytes using cardiomyocyte-specific knockout mice, MHC-Cre/ROCK1fl/fl, which partially reproduced the cardioprotective characteristics of ROCK1−/− mice, indicating that ROCK1 in both cardiomyocytes and non-cardiomyocytes mediates doxorubicin cardiotoxicity. To elucidate the molecular mechanisms, a detailed time course study after a single doxorubicin injection at 10 mg/kg was performed in ROCK1−/− and MHC-Cre/ROCK1fl/fl mice. The molecular analysis revealed that both ROCK1−/− and MHC-Cre/ROCK1fl/fl hearts exhibited significant reduction of doxorubicin-induced early responses including increased apoptotic (Bax) and autophagic (p62/SQSTM1 and LC3-II) markers, associated with reduced Beclin 1 phosphorylation on Thr119, supporting reduced Beclin 1-mediated autophagy initiation due to increased association of Beclin 1 with Bcl 2 or Bcl-XL in these hearts compared to the WT or ROCK1fl/fl mice. These results support that ROCK1 deficiency is cardioprotective against doxorubicin-induced cardiotoxicity at least in part through reducing Beclin 1-mediated autophagy initiation in cardiomyocytes and restoring autophagic flux to ameliorate doxorubicin cardiotoxicity.

ROCK1 via LIM kinase regulates growth, maturation and actin based functions in mast cells.

Understanding mast cell development is essential due to their critical role in regulating immunity and autoimmune diseases. Here, we show how Rho kinases (ROCK) regulate mast cell development and can function as therapeutic targets for treating allergic diseases. Rock1 deficiency results in delayed maturation of bone marrow derived mast cells (BMMCs) in response to IL-3 stimulation and reduced growth in response to stem cell factor (SCF) stimulation. Further, integrin-mediated adhesion and migration, and IgE-mediated degranulation are all impaired in Rock1-deficient BMMCs. To understand the mechanism behind altered mast cell development in Rock1−/− BMMCs, we analyzed the activation of ROCK and its downstream targets including LIM kinase (LIMK). We observed reduced activation of ROCK, LIMK, AKT and ERK1/2 in Rock1-deficient BMMCs in response to SCF stimulation. Further, loss of either Limk1 or Limk2 also demonstrated altered BMMC maturation and growth; combined deletion of both Limk1 and Limk2 resulted in further reduction in BMMC maturation and growth. In passive cutaneous anaphylaxis model, deficiency of Rock1 or treatment with ROCK inhibitor Fasudil protected mice against IgE-mediated challenge. Our results identify ROCK/LIMK pathway as a novel therapeutic target for treating allergic diseases involving mast cells.

Dissecting the Mechanisms of Doxorubicin and Oxidative Stress-Induced Cytotoxicity: The Involvement of Actin Cytoskeleton and ROCK1.

We have recently reported that ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has superior anti-apoptotic and pro-survival effects than antioxidants against doxorubicin, a chemotherapeutic drug. Although oxidative stress is the most widely accepted mechanism, our studies suggest that ROCK1-dependent actin cytoskeleton remodeling plays a more important role in mediating doxorubicin cytotoxicity on MEFs. To further explore the contributions of ROCK1-dependent actin cytoskeleton remodeling in response to stress, this study investigates the mechanistic differences between the cytotoxic effects of doxorubicin versus hydrogen peroxide (H2O2), with a focus on cytoskeleton alterations, apoptosis and necrosis induction. We found that both types of stress induce caspase activation but with different temporal patterns and magnitudes in MEFs: H2O2 induces the maximal levels (2 to 4-fold) of activation of caspases 3, 8, and 9 within 4 h, while doxorubicin induces much higher maximal levels (15 to 25-fold) of caspases activation at later time points (16–24 h). In addition, necrosis induced by H2O2 reaches maximal levels within 4 h while doxorubicin-induced necrosis largely occurs at 16–24 h secondary to apoptosis. Moreover, both types of stress induce actin cytoskeleton remodeling but with different characteristics: H2O2 induces disruption of stress fibers associated with cytosolic translocation of phosphorylated myosin light chain (p-MLC) from stress fibers, while doxorubicin induces cortical F-actin formation associated with cortical translocation of p-MLC from central stress fibers. Furthermore, N-acetylcysteine (an antioxidant) is a potent suppressor for H2O2-induced cytotoxic effects including caspase activation, necrosis, and cell detachment, but shows a much reduced inhibition on doxorubicin-induced changes. On the other hand, ROCK1 deficiency is a more potent suppressor for the cytotoxic effects induced by doxorubicin than by H2O2. These results support the notion that doxorubicin induces caspase activation, necrosis, and actin cytoskeleton alterations largely through ROCK1-dependent and oxidative stress-independent pathways.

ROCK1 deficiency enhances protective effects of antioxidants against apoptosis and cell detachment.

We have recently reported that the homologous Rho kinases, ROCK1 and ROCK2, play different roles in regulating stress-induced stress fiber disassembly and cell detachment, and the ROCK1 deficiency in mouse embryonic fibroblasts (MEF) has remarkable anti-apoptotic, anti-detachment and pro-survival effects against doxorubicin, a chemotherapeutic drug. This study investigated the roles of ROCK isoforms in doxorubicin-induced reactive oxygen species (ROS) generation which is believed to be the major mechanism underlying its cytotoxicity to normal cells, and especially to cardiomyocytes. Different antioxidants have been shown to provide a protective role reported in numerous experimental studies, but clinical trials of antioxidant therapy showed insufficient benefit against the cardiac side effect. We found that both ROCK1−/− and ROCK2−/− MEFs exhibited reduced ROS production in response to doxorubicin treatment. Interestingly, only ROCK1 deficiency, but not ROCK2 deficiency, significantly enhanced the protective effects of antioxidants against doxorubicin-induced cytotoxicity. First, ROCK1 deficiency and N-acetylcysteine (an anti-oxidant) treatment synergistically reduced ROS levels, caspase activation and cell detachment. In addition, the reduction of ROS generation in ROCK1−/− MEFs in response to doxorubicin treatment was in part through inhibiting NADPH oxidase activity. Furthermore, ROCK1 deficiency enhanced the inhibitory effects of diphenyleneiodonium (an inhibitor of NADPH oxidase) on ROS generation and caspase 3 activation induced by doxorubicin. Finally, ROCK1 deficiency had greater protective effects than antioxidant treatment, especially on reducing actin cytoskeleton remodeling. ROCK1 deficiency not only reduced actomyosin contraction but also preserved central stress fiber stability, whereas antioxidant treatment only reduced actomyosin contraction without preserving central stress fibers. These results reveal a novel strategy to enhance the protective effect of antioxidant therapy by targeting the ROCK1 pathway to stabilize the actin cytoskeleton and boost the inhibitory effects on ROS production, apoptosis and cell detachment.

Rho Associated Coiled-Coil Kinase-1 Regulates Collagen-Induced Phosphatidylserine Exposure In Platelets

IntroductionPlatelet activation results in a loss of membrane phospholipid asymmetry with concomitant exposure of procoagulant phosphatidylserine (PS) at the outer leaflet of membrane bilayer. PS provides binding sites for the assembly of prothrombinase and the tenase complex on activated platelets. In spite of its central role in thrombosis and hemostasis, the signal transduction mechanisms leading to PS exposure are not well characterized. Previously, we have shown that ROCK inhibitor, Y-27632, induces increased PS exposure following activation of platelets. However, Y-27632 does not distinguish between ROCK-1 and ROCK-2. Here, we studied activation induced PS exposure in ROCK-1 deficient platelets. MethodsPlatelets were isolated from ROCK-1 deficient and control mice. PS expression was measured on a flow cytometer using FITC-lactadherin (5 μg/ml). Platelet aggregation was carried out by standard turbidometric methods. Platelet procoagulant activity was measured in a prothrombinase assay consisting of prothrombin (1μM), factor Va (10 nM), factor Xa (10 pM), calcium (1.5 mM), collagen (10 μg/ml), and platelets (1x106/ml). Thrombin formation was determined by measuring absorbance at 405 nm in a microplate reader Calcium mobilization was measured by incubating ROCK-1 and WT platelets with Fura 2 AM and measuring the Fura 2 fluorescence after collagen treatment. An in vivo light/dye-induced endothelial injury/thrombosis model was used to assess the occlusion in cremasteric venules of mice. Phosphorylation status was measured by Western blot analysis of platelet lysate before and after activation. ResultsCollagen induced significantly more PS exposure in ROCK-1 deficient platelets compared to platelets from wild type littermates. ROCK-1 deficient platelets are more procoagulant as it generates more thrombin compared to control platelets. ROCK-1 deficient mice have a shorter occlusion time in a light/dye induced endothelial injury/thrombosis model in cremasteric venules compared to its littermates (3.16 ± 1.33 min versus 6.6 ± 2.6 min; p = 0.01). Since PS exposure is associated with increase in cytosolic calcium level, we measured calcium concentration in WT and ROCK-1 deficient platelets following collagen stimulation. There was no significant difference in calcium levels in collagen stimulated ROCK-1 and WT platelets. Hence, it is unlikely that the increased PS exposure in ROCK-1 deficient platelets is due to changes in calcium mobilization. Besides calcium, actin cytoskeleton rearrangement also plays a major role in PS exposure. ROCK1 modulates cytoskeleton rearrangement is through LIM kinase-1 (LMK-1). ROCK-1 mediated activation of LIMK-1 catalyses phosphorylation of cofilin-1, an actin severing protein. Phosphorylation inactivates actin severing activity of cofilin-1. Hence, ROCK-1 deficiency should lead to activation of cofilin-1. Indeed, collagen stimulated ROCK-1 deficient platelets showed a major reduction in phosphorylation of cofilin-1. Increased activity of cofilin-1 in ROCK-1 deficient platelets is further supported by the fact that F- actin content is significantly reduced in ROCK-1 deficient platelets. ConclusionWe postulate that cofilin-1-induced F-actin severing and subsequent cytoskeletal rearrangement plays an important role in collagen-induced PS exposure in platelets. Disclosures:No relevant conflicts of interest to declare.

ROCK1 functions as a critical regulator of stress erythropoiesis and survival by regulating p53

AbstractErythropoiesis is a dynamic, multistep process whereby hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Although several downstream signaling molecules have been identified that regulate steady-state erythropoiesis, the major regulators under conditions of stress remain poorly defined. Rho kinases (ROCKs) belong to a family of serine/threonine kinases. Using gene-targeted ROCK1-deficient mice, we show that lack of ROCK1 in phenylhydrazine-induced oxidative stress model results in enhanced recovery from hemolytic anemia as well as enhanced splenic stress erythropoiesis compared with control mice. Deficiency of ROCK1 also results in enhanced survival, whereas wild-type mice die rapidly in response to stress. Enhanced survivability of ROCK1-deficient mice is associated with reduced level of reactive oxygen species. BM transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous. We show that ROCK1 binds to p53 and regulates its stability and expression. In the absence of ROCK1, p53 phosphorylation and expression is significantly reduced. Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia.

ROCK1 Functions As a Critical Regulator of Stress Erythropoiesis and Survival by Regulating p53

Abstract 916Erythropoiesis is a dynamic, multistep process in which hematopoietic stem cells differentiate toward a progressively committed erythroid lineage through intermediate progenitors. Under normal physiologic condition, erythropoiesis takes place primarily in the bone marrow of humans and mice. Efficient stress erythropoiesis is crucial to survival and recovery from various pathophysiologic conditions including blood loss, anemia, and therapeutic procedures used in the treatment of hematological malignancies such as chemotherapy and stem cell transplantation. Impaired stress erythropoiesis can be fatal under these conditions. In order to develop improved strategies to stimulate stress erythropoiesis in patients with such conditions, it is critical to identify the molecular mechanisms that regulate this process. While many of the downstream signaling molecules and pathways have been elucidated in steady state erythroid cell development, the major regulators under stress conditions remain to be defined. The small families of Rho GTPases and their downstream effectors, Rho kinases have been implicated in regulating various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells and inflammatory cells. Rho kinases (ROCK1 and ROCK2) belong to a family of serine/threonine kinases, and their physiologic role in erythropoiesis is not known. Utilizing mice deficient in the expression of ROCK1, we demonstrate no significant difference with respect to erythroid parameters in peripheral blood under steady-state conditions (n=19 mice for each genotype). The frequency of Ter119+, CD71+ or double-positive erythroid cells in the bone marrow and spleen of ROCK1-deficient mice was comparable to wild type (WT) controls at basal levels (n=7). In response to myelotoxic stress, ROCK1 deficient Ter119 positive cells demonstrated enhanced survival and recovery compared to WT controls (n=3–6, *p< 0.05). Further, using a phenylhydrazine (PHZ)-induced murine model of hemolytic anemia, we demonstrate that ROCK1-deficient mice exhibit increased red blood cells and enhanced hematocrits relative to WT mice (n=6, *p< 0.05). In addition, ROCK1−/− mice support efficient erythro-splenomegaly, and exhibit a threefold increase in splenic weight after PHZ injection compared to WT controls (n=6, *p< 0.05). Flow cytometric analysis revealed increased frequency of Ter119/CD71 double positive erythroid progenitor pool in ROCK1−/− spleens after PHZ treatment compared to WT (n=6, *p< 0.05). Furthermore, histopathological analysis of WT and ROCK1−/− spleens following PHZ treatment revealed a defined population of white pulp and red pulp in control spleens but enhanced extramedullary erythropoiesis in ROCK1−/− spleens (n=3, *p< 0.05). In vitro colony forming assays showed that ROCK1-deficient splenocytes generated more erythroid colony forming units (CFU-Es) and eventually generated more erythroid-burst forming units (BFU-Es) compared to WT splenocytes in response to different concentrations of EPO and combination of EPO and SCF (n=3, *p< 0.05). Deficiency of ROCK1 also resulted in enhanced survival of mice treated with PHZ compared to controls (n=17 mice per group, *p< 0.05). The enhanced survival of ROCK1-deficient mice in response to PHZ was associated with reduced reactive oxygen species (ROS) levels compared to WT (n=6 mice per group, *p< 0.05). Bone marrow transplantation studies revealed that enhanced stress erythropoiesis in ROCK1-deficient mice is stem cell autonomous (n=6 mice per genotype, *p< 0.05). Remarkably, the red cell phenotype observed in ROCK1−/− mice is similar to that reported in mice deficient in p53. We show that ROCK1 binds p53 directly and regulates its stability and expression (n=3). In absence of ROCK1, p53 phosphorylation and expression is reduced in ROCK1−/− erythroblasts (n=3). Our findings reveal that ROCK1 functions as a physiologic regulator of p53 under conditions of erythroid stress. These findings are expected to offer new perspectives on stress erythropoiesis and may provide a potential therapeutic target in human disease characterized by anemia. Disclosures:No relevant conflicts of interest to declare.

ROCK1 functions as a suppressor of inflammatory cell migration by regulating PTEN phosphorylation and stability

AbstractRho kinases belong to a family of serine/threonine kinases whose role in recruitment and migration of inflammatory cells is poorly understood. We show that deficiency of ROCK1 results in increased recruitment and migration of macrophages and neutrophils in vitro and in vivo. Enhanced migration resulting from ROCK1 deficiency is observed despite normal expression of ROCK2 and a significant reduction in overall ROCK activity. ROCK1 directly binds PTEN in response to receptor activation and is essential for PTEN phosphorylation and stability. In the absence of ROCK1, PTEN phosphorylation, stability, and its activity are significantly impaired. Consequently, increased activation of downstream targets of PTEN, including PIP3, AKT, GSK-3β, and cyclin D1, is observed. Our results reveal ROCK1 as a physiologic regulator of PTEN whose function is to repress excessive recruitment of macrophages and neutrophils during acute inflammation.

ROCK1 Functions as a Suppressor of Inflammatory Cell Migration by Regulating PTEN Phosphorylation and Stability.

Abstract 240Neutrophils and macrophages are major cellular components of the innate immune response and are recruited rapidly in large numbers to sites of infection. The small family of Rho GTPases and its downstream effectors, Rho kinases (Rho-associated, coiled-coil containing protein kinase) have been implicated in regulating various cellular functions including actin cytoskeleton organization, cell adhesion, and cell motility in non-hematopoietic cells. Rho kinases (ROCK1 and ROCK2) belong to a family of serine/threonine kinases whose role in inflammation is not known. Here we show that deficiency of ROCK1 but not ROCK2 results in increased recruitment of macrophages (3.2 fold, n=8, *p<0.01) and neutrophils (3.4 fold, n=5 *p<0.05) compared to WT controls in an in vivo model of aseptic peritonitis. In vitro, deficiency of ROCK1 in bone marrow derived macrophages shows a significant increase in haptotactic transwell migration in response to M-CSF as well as MCP-1 on fibronectin as well as an increase in migration towards the wounded area in a wound healing assay compared to controls (∼3 fold, n=3, *p<0.005). Consistently, deficiency of ROCK1 in bone marrow derived neutrophils also shows a ∼2.63 fold increase in migration in response to fMLP compared to WT bone marrow derived neutrophils (BMNs) in a chemotactic migration assay. ROCK1 deficient macrophages also demonstrate a ∼2.5 fold increase in adhesion on fibronectin (n=3, *p<0.002). The enhanced migration and adhesion in ROCK1−/− macrophages was observed in spite of comparable expression of F4/80 (WT; 85.63% vs. ROCK1−/−; 88.68%, n=4), α4β1 and α5β1 integrins (WT; 67.49% & 88.2% vs. ROCK1−/−; 71.82 % & 87.09%, n=4), while no significant difference in the phagocytosis of sheep red blood cells was observed between WT and ROCK1−/− macrophages (Phagocytic index: WT; 98% vs. ROCK1−/− 97%, n=3, p>.05). Close examination of the cytoskeleton of ROCK1 deficient macrophages using confocal microscopy revealed more F-actin content on the entire cell surface compared to wildtype controls. Consistently, flow cytometric analysis using Alexa 488-phalloidin staining revealed abundance of F-actin in ROCK1−/− macrophages compared to WT controls (WT; 46.19% vs. ROCK1−/−; 65.23%, n=3, *p<0.05). Furthermore, immunofluorescence imaging of podosomes carried out using anti-vinculin antibody revealed more pronounced and increased podosomes in ROCK1 deficient macrophages compared to WT controls (n=3, *p<0.05). Biochemical analysis of ROCK1−/− macrophages revealed that the enhanced recruitment of ROCK1 deficient macrophages and neutrophils was apparent in spite of normal expression of ROCK2 in ROCK1−/− cells and a 60% reduction in overall ROCK activity. Interestingly, although both ROCK1 and ROCK2 co-immunoprecipitate with PTEN in response to cytokine induced stimulation, only ROCK1 appeared to be essential for PTEN phosphorylation, activation and stability. In the absence of ROCK1, PTEN phosphorylation, its activity and stability were significantly impaired in spite of the presence of ROCK2 (n=3, *p<0.05). Consequently, an increase in the activation of downstream targets of PTEN including AKT, GSK-3β and cyclinD1 was observed in ROCK1 deficient macrophages relative to controls (n=3). Taken together, these studies reveal a biochemical pathway involving ROCK1 and PTEN which is involved in the recruitment of macrophages and neutrophils during acute inflammation. Thus, ROCK1 likely functions as a physiologic regulator of PTEN whose function is to repress excessive recruitment of macrophages and neutrophils during acute inflammation. Disclosures:No relevant conflicts of interest to declare.

Targeted Disruption of ROCK1 Causes Insulin Resistance in Vivo

Insulin signaling is essential for normal glucose homeostasis. Rho-kinase (ROCK) isoforms have been shown to participate in insulin signaling and glucose metabolism in cultured cell lines. To investigate the physiological role of ROCK1 in the regulation of whole body glucose homeostasis and insulin sensitivity in vivo, we studied mice with global disruption of ROCK1. Here we show that, at 16-18 weeks of age, ROCK1-deficient mice exhibited insulin resistance, as revealed by the failure of blood glucose levels to decrease after insulin injection. However, glucose tolerance was normal in the absence of ROCK1. These effects were independent of changes in adiposity. Interestingly, ROCK1 gene ablation caused a significant increase in glucose-induced insulin secretion, leading to hyperinsulinemia. To determine the mechanism(s) by which deletion of ROCK1 causes insulin resistance, we measured the ability of insulin to activate phosphatidylinositol 3-kinase and multiple distal pathways in skeletal muscle. Insulin-stimulated phosphatidylinositol 3-kinase activity associated with IRS-1 or phospho-tyrosine was also reduced approximately 40% without any alteration in tyrosine phosphorylation of insulin receptor in skeletal muscle. Concurrently, serine phosphorylation of IRS-1 at serine 632/635, which is phosphorylated by ROCK in vitro, was also impaired in these mice. Insulin-induced phosphorylation of Akt, AS160, S6K, and S6 was also decreased in skeletal muscle. These data suggest that ROCK1 deficiency causes systemic insulin resistance by impairing insulin signaling in skeletal muscle. Thus, our results identify ROCK1 as a novel regulator of glucose homeostasis and insulin sensitivity in vivo, which could lead to new treatment approaches for obesity and type 2 diabetes.

Deficiency of ROCK1 in bone marrow‐derived cells protects against atherosclerosis in LDLR −/− mice

Rho kinases (ROCKs) are serine-threonine protein kinases that regulate the actin cytoskeleton. Recent studies suggest that ROCKs also play an important role in cardiovascular disease. However, the isoform- and tissue-specific role of ROCKs in mediating this process is unknown. Using homologous recombination, we generated mutant mice harboring alleles with homozygous deletion of ROCK1 (ROCK1(-/-)). Most ROCK1(-/-) mice die perinatally. However, a few ROCK1(-/-) mice survive to adulthood, are phenotypically normal, and have no apparent compensatory changes in ROCK2. Using these ROCK1(-/-) mice, we show that ROCK1 in bone marrow-derived macrophages is critical to the development of atherosclerosis, in part, by mediating foam cell formation and macrophage chemotaxis. Lipid accumulation and atherosclerotic lesions were reduced in atherosclerosis-prone LDLR(-/-) mice, whose bone marrows have been replaced with bone marrows derived from ROCK1(-/-) mice. Bone marrow-derived ROCK1-deficient macrophages exhibited impaired chemotaxis to monocyte chemotactic protein-1 and showed reduced ability to take up lipids and to develop into foam cells when exposed to modified low-density lipoprotein. These findings indicate that ROCK1 in bone marrow-derived cells is a critical mediator of atherogenesis and suggest that macrophage ROCK1 may be an important therapeutic target for vascular inflammation and atherosclerosis.

Abstract 306: Deficiency of ROCK1 in Macrophages Protects Against Atherosclerosis

Background: Rho kinases (ROCKs) are serine-threonine protein kinases that regulate various cellular functions. There is increasing evidence that the RhoA/ROCK pathway plays an important pathophysiological role in cardiovascular diseases. However, direct evidence of which ROCK isoforms or target tissues are involved in the atherogenic process is still lacking. Objective: The aim of this study was to determine the effect of ROCK1 deficiency on atherogenesis and how ROCK1 affects key atherosclerosis-related macrophage function such as lipid uptake and chemotaxis. Methods: We utilized ROCK1 −/− mice and the atherosclerosis-prone apolipoprotein E knockout (apoE −/− ) mice or low-density lipoprotein receptor knockout (LDLR −/− ) mice to investigate the role of ROCK1 in the pathogenesis of atherosclerotic plaque formation. Bone marrow-derived macrophages from ROCK1 −/− and ROCK1 +/+ mice were used to investigate acetylated (Ac)LDL-mediated foam cell formation and chemotaxis. Results: Compared to atherosclerosis-prone apoE −/− mice, apoE −/− ROCK1 +/− mice had substantially less fatty streaks foam cells and atherosclerosis (77.0 ± 12.9 × 10 3 μm 2 versus 166.4 ± 14.6 × 10 3 μm 2 , P &lt; 0.01). Atherosclerotic lesions were reduced also in LDLR −/− mice, whose bone marrow were replaced with bone marrow derived from ROCK1 −/− mice compared to ROCK1 +/+ recipients (181.5 ± 15.6 × 10 3 μm 2 versus 448.5 ± 33.3 × 10 3 μm 2 , P &lt; 0.05). Bone marrow-derived ROCK1-deficient macrophages exhibited impaired chemotaxis to monocyte chemotactic protein-1 and showed reduced ability to take up lipids and to develop into foam cells when exposed to modified low density lipoprotein. Conclusion: These findings indicate that ROCK1 in macrophages is a critical mediator of foam cell formation, macrophage chemotaxis and atherogenesis, and suggest that macrophage ROCK1 may be an important therapeutic target for vascular inflammation and atherosclerosis.

Abstract 1224: Rock1 Deletion Prevents Cardiac Dilation And Improves Contractile Function In Pathological Cardiac Hypertrophy

The development of left ventricular cardiomyocyte hypertrophy in response to increased hemodynamic load and neurohormonal stress is initially a compensatory response. However, persistent stress eventually leads to dilated heart failure, which is a common cause of heart failure in human hypertensive and valvular heart disease. We have recently reported that ROCK1 homozygous knockout mice exhibited reduced cardiac fibrosis and cardiomyocyte apoptosis, while displayed a preserved compensatory hypertrophic response to pressure overload. Here, we tested effects of ROCK1 deficiency on cardiac hypertrophy, dilation, and dysfunction by using the transgenic Gαq mice which represent a well-characterized and highly relevant genetic mouse model of pathological hypertrophy and heart failure. We have shown that ROCK1 deletion prevented left ventricular dilation and contractile dysfunction in Gαq mice under basal condition. ROCK1 deletion also partially rescued bradycardia and improved contractile response to β-adrenergic stimulation in Gαq mice. Although the development of cardiomyocyte hypertrophy was not affected, ROCK1 deletion in Gαq mice resulted in a concentric hypertrophic phenotype associated with reduced induction of hypertrophic markers. Finally, ROCK1 deletion prevented down-regulation of type V adenylyl cyclase expression, which is a critical molecular defect contributing to the impaired β-adrenergic signaling in Gαq mice. The present study establishes for the first time a role for ROCK1 in cardiac dilation and contractile dysfunction.