Rho-associated Coiled-coil Kinase 1 Research Articles

Unraveling the mechanistic details of rho-associated coiled-coil kinase1 (ROCK1) inhibition: Implications from pharmacophore mapping and free energy analysis

Rho-associated coiled-coil kinases (ROCKs) are essential for various cellular processes and play a crucial role in neurological and inflammatory disorders. The two ROCK isoforms (ROCK1 and ROCK2) are involved in the pathogenesis of multiple diseases. Increased levels of ROCK1 are observed in patients with Alzheimer's disease (AD), making it a promising drug target. This work presents the mechanism of inhibitor binding at the ROCK1 active site using a combination of ligand and structure-based drug design techniques. Ligand-based pharmacophore studies were performed on previously reported ROCK1 inhibitors to unravel the 3D steric and electronic features essential for ROCK1 inhibition. Molecular docking and molecular dynamics-based free energy analysis studies were performed to recognize the amino acids assisting in inhibitor binding by stabilizing the identified pharmacophore features. The crucial 3D pharmacophore features of ROCK1 inhibitors included hydrophobic (HYD), hydrogen-bond acceptor (HBA), and hydrogen-bond donor (HBD) characteristics. These features were oriented next to the complementary amino acids in the ROCK1 active site, as evident from the detailed interaction analysis. The free energy calculations provided a quantifiable estimate of the favorable energetic contribution of Gly83, Val90, Lys105, Asp160, Leu205, and Asp216 to the total binding energy. These amino acids significantly contributed to the ROCK1-inhibitor complexes' overall binding free energy, indicating their importance in inhibitor binding.

Effect of cadmium on Rho GTPases signal transduction during osteoclast differentiation.

Osteoclasts are the key target cells for cadmium (Cd)-induced bone metabolism diseases, while Rho GTPases play an important role in osteoclast differentiation and bone resorption. To identify new therapeutic targets of Cd-induced bone diseases; we evaluated signal transduction through Rho GTPases during osteoclast differentiation under the influence of Cd. In osteoclastic precursor cells, 10nM Cd induced pseudopodia stretching, promoted cell migration, upregulated the levels of Cdc42, and RhoQ mRNAs and downstream Rho-associated coiled-coil kinase 1 (ROCK1) and ROCK2 proteins, and downregulated the actin-related protein 2/3 (ARP2/3) levels. Cd at 2 and 5μM shortened the pseudopodia, inhibited cell migration, and decreased ROCK1, ROCK2, and ARP2/3 protein levels; Cd at 5μM also reduced the mRNA expression levels of Rac1, Rac2, and RhoU mRNAs and decreased the level of phosphorylated (p)-cofilin. In osteoclasts, 10nM Cd induced the formation of sealing zones, slightly upregulated Cdc42 mRNA levels and ROCK2 and ARP2/3 protein levels and significantly reduced p-cofilin levels. Cd at 2μM and 5μM Cd blocked the fusion of precursor cells; and 5μM Cd downregulated the expression levels of RhoB, Rac1, Rac3, and RhoU mRNAs, and ROCK1, p-cofilin and ARP2/3 protein levels, significantly. In vivo, Cd (at 5 or 25 mg/L) increased the levels of key proteins RhoA, Rac1/2/3, Cdc42, and RhoU and their mRNAs in bone marrow cells. In summary, the results suggested that Cd affected the differentiation process of osteoclast and altered the expression of several Rho GTPases, which might be crucial targets of Cd during the differentiation of osteoclasts.

Long non-coding RNA Linc00205 promotes hepatoblastoma progression through regulating microRNA-154-3p/Rho-associated coiled-coil Kinase 1 axis via mitogen-activated protein kinase signaling

Hepatoblastoma (HB) is the most common pediatric liver tumor. The significant tumor heterogeneity of HB leads to varied prognoses among children with the disease. Recent studies have suggested that long non-coding RNAs (lncRNAs) can serve as novel therapies for HB treatment. Thus, in this study, we aimed to reveal the function and mechanism of the lncRNA Linc00205 in HB. Our results exhibited that, in both HB tissues and cell lines, levels of Linc00205 were significantly increased. In addition, knockdown of Linc00205 led to suppression of HB development. Moreover, we identified that Linc00205 was able to directly bind to miR-154-3p, thus isolating miR-154-3p from its target Rho-associated coiled-coil Kinase 1 (ROCK1). Further cellular behavioral experiments elucidated that the miR-154-3p inhibitor and ROCK1 overexpression were able to reverse the effect of downregulated Linc00205 on proliferation, migration, invasion, and apoptosis of HB cells by rescue assays via mitogen-activated protein kinase (MAPK) signaling. Our results demonstrated that Linc00205 enhanced HB progression by regulating ROCK1 expression via sponging miR-154-3p through MAPK signaling, which suggests a novel potential therapeutic target for HB.

Crosstalk between Drp1 phosphorylation sites during mitochondrial remodeling and their impact on metabolic adaptation

SummaryMitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases.

Computational study of paroxetine-like inhibitors reveals new molecular insight to inhibit GRK2 with selectivity over ROCK1

The G-protein coupled receptor kinase 2 (GRK2) regulates the desensitization of beta-adrenergic receptors (β-AR), and its overexpression has been implicated in heart failure. Hence, the inhibition of GRK2 is considered to be an important drug target for the treatment of heart failure. Due to the high sequence similarity of GRK2 with the A, G, and C family (AGC family) of kinases, the inhibition of GRK2 also leads to the inhibition of AGC kinases such as Rho-associated coiled-coil kinase 1 (ROCK1). Therefore, unraveling the mechanisms to selectively inhibit GRK2 poses an important challenge. We have performed molecular docking, three dimensional quantitative structure activity relationship (3D-QSAR), molecular dynamics (MD) simulation, and free energy calculations techniques on a series of 53 paroxetine-like compounds to understand the structural properties desirable for enhancing the inhibitory activity for GRK2 with selectivity over ROCK1. The formation of stable hydrogen bond interactions with the residues Phe202 and Lys220 of GRK2 seems to be important for selective inhibition of GRK2. Electropositive substituents at the piperidine ring and electronegative substituents near the amide linker between the benzene ring and pyrazole ring showed a higher inhibitory preference for GRK2 over ROCK1. This study may be used in designing more potent and selective GRK2 inhibitors for therapeutic intervention of heart failure.

Rho-associated coiled-coil kinase 1 activation mediates amyloid precursor protein site-specific Ser655 phosphorylation and triggers amyloid pathology.

Rho‐associated coiled‐coil kinase 1 (ROCK1) is proposed to be implicated in Aβ suppression; however, the role for ROCK1 in amyloidogenic metabolism of amyloid precursor protein (APP) to produce Aβ was unknown. In the present study, we showed that ROCK1 kinase activity and its APP binding were enhanced in AD brain, resulting in increased β‐secretase cleavage of APP. Furthermore, we firstly confirmed that APP served as a substrate for ROCK1 and its major phosphorylation site was located at Ser655. The increased level of APP Ser655 phosphorylation was observed in the brain of APP/PS1 mice and AD patients compared to controls. Moreover, blockade of APP Ser655 phosphorylation, or inhibition of ROCK1 activity with either shRNA knockdown or Y‐27632, ameliorated amyloid pathology and improved learning and memory in APP/PS1 mice. These findings suggest that activated ROCK1 targets APP Ser655 phosphorylation, which promotes amyloid processing and pathology. Inhibition of ROCK1 could be a potential therapeutic approach for AD.

Caveolin-1 knockdown increases the therapeutic sensitivity of lung cancer to cisplatin-induced apoptosis by repressing Parkin-related mitophagy and activating the ROCK1 pathway.

Chemotherapy is the first-line treatment option for patients with lung cancer. However, therapeutic resistance occurs through an incompletely understood mechanism. Our research wants to investigate the influence of Caveolin-1 (Cav-1) on the therapeutic sensitivity of lung cancer in vitro. Results in this study demonstrated that Cav-1 levels were markedly inhibited in A549 lung cancer cells after exposure to cisplatin. Knockdown of caveolin further enhanced cisplatin-triggered cancer death in A549 cells. The functional investigation demonstrated that Cav-1 inhibition amplified the mitochondrial stress signaling induced by cisplatin, as evidenced by the mitochondrial reactive oxygen species burst, cellular metabolic disruption, mitochondrial membrane potential reduction, and mitochondrial caspase-9-related apoptosis activation. At the molecular level, cav-1 augmented cisplatin-mediated mitochondrial damage by inhibiting Parkin-related mitochondrial autophagy. Mitophagy activation effectively attenuated the promotive impact of Cav-1 knockdown on mitochondrial damage and cell death. Furthermore, our data indicated that Cav-1 affected Parkin-related mitophagy by activating the Rho-associated coiled-coil kinase 1 (ROCK1) pathway; inhibition of the ROCK1 axis prevented cav-1 knockdown-mediated cell death and mitochondrial damage. Taken together, our results provide ample data illuminate the necessary action exerted by Cav-1 on affecting cisplatin-related therapeutic resistance. Silencing of Cav-1 inhibited Parkin-related mitophagy, thus amplifying cisplatin-mediated mitochondrial apoptotic signaling. This finding identifies the Cav-1/ROCK1/Parkin/mitophagy axis as a potential target to overcome cisplatin-related resistance in lung cancer cells.

ARHGEF10L contributes to liver tumorigenesis through RhoA-ROCK1 signaling and the epithelial-mesenchymal transition

Aberrant activity of Rho small G-proteins and their regulators plays an important role in tumorigenesis. Rho guanine nucleotide exchange factor 10-Like (ARHGEF10L) is a member of the RhoGEF family that promotes the active GTP-bound state of Rho GTPases. This study used the Illumina GoldenGate microassay, Sequenom MassARRAY and TaqMan to analyze possible correlations between tag single nucleotide polymorphisms (tag SNPs) in the ARHGEF10L locus and various tumor risks. The genotyping analyses demonstrated a strong association of rs2244444 and rs12732894 with liver cancer. Western blotting and immunohistochemistry also revealed increased expression of ARHGEF10L in hepatocellular carcinoma tissues. Furthermore, increased cell proliferation, cell migration and RhoA activity; increased expression of Rho-associated coiled-coil kinase-1 (ROCK1), phospho- Ezrin/Radixin/Moesin (ERM), vimentin, N-cadherin and Slug, and decreased E-cadherin expression were detected in hepatocellular carcinoma cell Bel-7402 and HepG2 cells with transfection of ARHGEF10L-expressing plasmids. Opposite results were obtained in the two cell lines with transfection of anti-ARHGEF10L siRNA. Tumor-bearing mice were generated with Bel-7402 cells transfected with lentivirus vectors packaging short hairpin ARHGEF10L RNA. The xenograft tumors with the inhibited ARHGEF10L expression showed decreased tumor growth and expression of vimentin, N-cadherin and Slug. Additionally, decreased phospho-ERM expression was detected in Bel-7402 and HepG2 cells with transfection of anti-ROCK1 siRNA and increased expression of ROCK1 was detected in hepatocellular carcinoma tissues. E-cadherin, vimentin, N-cadherin and Slug are markers of the epithelial-to-mesenchymal transition (EMT). ROCK1, phospho-ERM and EMT have been reported to promote tumor cell proliferation, metastasis and angiogenesis. Our study suggests that increased expression of ARHGEF10L stimulates hepatocellular tumorigenesis by activating the RhoA-ROCK1- phospho ERM pathway and EMT.

RETRACTED: MicroRNA-361-5p inhibits papillary thyroid carcinoma progression by targeting ROCK1

This article has been retracted: please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). This article has been retracted at the request of the Authors and Editor-in-Chief. The authors notified the journal and requested retraction of the article, stating: “We recently found that miR-361 did not affect the tumor growth in vivo. All the authors want to retract this paper. Furthermore, we apologize to the readership of the Journal for any inconvenience caused”. The journal was also alerted to an associated PubPeer post, in which images of xenograft tumors within Figure 6B, and images of colony formation assays within Figure 2C, appear to have been published in other articles, as detailed here: https://pubpeer.com/publications/0DA940799BE0D567BAC4659CDAB13F#. The journal requested the corresponding author provide a more detailed explanation to these concerns and associated raw data, but this request was not satisfactorily fulfilled. The Editor-in-Chief assessed the case and decided to retract the article.

MicroRNA-148a Acts as a Tumor Suppressor in Osteosarcoma via Targeting Rho-Associated Coiled-Coil Kinase.

MicroRNAs (miRs) have been demonstrated to be involved in the development and progression of osteosarcoma (OS), but the molecular mechanism still remains to be fully investigated. The present study investigated the function of miR-148a in OS, as well as its underlying mechanism. Our data showed that miR-148a was significantly downregulated in OS tissues compared to their matched adjacent normal tissues, and also in OS cell lines compared to normal human osteoblast cells. Low expression of miR-148a was significantly associated with tumor progression and a poor prognosis for OS patients. Rho-associated coiled-coil kinase 1 (ROCK1) was then identified as a target of miR-148a in Saos-2 and U2OS cells, and the expression of ROCK1 was significantly increased in OS tissues and cell lines. Moreover, the protein expression of ROCK1 was markedly reduced in miR-148a-overexpressing Saos-2 and U2OS cells, but significantly increased in miR-148a-downregulated Saos-2 and U2OS cells. Further investigation indicated that miR-148a had a suppressive effect on the proliferative, migratory, and invasive capacities of Saos-2 and U2OS cells. Moreover, overexpression of ROCK1 attenuated the inhibitory effects of miR-148a upregulation on the malignant phenotypes of Saos-2 and U2OS cells. In addition, overexpression of miR-148a significantly inhibited the tumor growth of U2OS cells in nude mice. Taken together, these data demonstrate that miR-148a acts as a tumor suppressor in OS, at least partly, via targeting ROCK1. Therefore, the miR-148a/ROCK1 axis may become a potential therapeutic target for OS.

Ror2-mediated alternative Wnt signaling regulates cell fate and adhesion during mammary tumor progression

Cellular heterogeneity is a common feature in breast cancer, yet an understanding of the coexistence and regulation of various tumor cell subpopulations remains a significant challenge in cancer biology. In the current study, we approached tumor cell heterogeneity from the perspective of Wnt pathway biology to address how different modes of Wnt signaling shape the behaviors of diverse cell populations within a heterogeneous tumor landscape. Using a syngeneic TP53-null mouse model of breast cancer, we identified distinctions in the topology of canonical Wnt β-catenin-dependent signaling activity and non-canonical β-catenin-independent Ror2-mediated Wnt signaling across subtypes and within tumor cell subpopulations in vivo. We further discovered an antagonistic role for Ror2 in regulating canonical Wnt/β-catenin activity in vivo, where lentiviral shRNA depletion of Ror2 expression augmented canonical Wnt/β-catenin signaling activity across multiple basal-like models. Depletion of Ror2 expression yielded distinct phenotypic outcomes and divergent alterations in gene expression programs among different tumors, despite all sharing basal-like features. Notably, we uncovered cell state plasticity and adhesion dynamics regulated by Ror2, which influenced Ras Homology Family Member A (RhoA) and Rho-Associated Coiled-Coil Kinase 1 (ROCK1) activity downstream of Dishevelled-2 (Dvl2). Collectively, these studies illustrate the integration and collaboration of Wnt pathways in basal-like breast cancer, where Ror2 provides a spatiotemporal function to regulate the balance of Wnt signaling and cellular heterogeneity during tumor progression.

MiR-199a/b-5p inhibits hepatocellular carcinoma progression by post-transcriptionally suppressing ROCK1.

In this study, we explored the actions of miR-199a/b-5p during hepatocellular carcinoma (HCC) progression and its potential target genes. Through heatmap miRNA expression analysis of 15 matched HCC tumor and adjacent non-tumor liver tissues from the TCGA database, we detected 19 mRNAs that were upregulated and 13 that were downregulated specifically in HCC. Among these, miR-199 family members were downregulated in HCC tumors and cell lines, as compared to controls. Low miR-199a/b-5p expression was also associated with poor overall survival of HCC patients. miR-199a/b-5p overexpression in HCC cell lines inhibited cell proliferation, migration and invasion, both in vitro and in vivo. In addition, miR199-a/b-5p post-transcriptionally suppressed Rho-associated coiled-coil kinase 1 (ROCK1). This in turn led to inhibition of ROCK1/MLC and PI3K/Akt signaling, which is necessary for HCC proliferation and metastasis. These results indicate that miR-199a/b acts as tumor suppressors in HCC and represent promising therapeutic targets.

Dasatinib inhibits actin fiber reorganization and promotes endothelial cell permeability through RhoA-ROCK pathway.

Treatment with dasatinib, a tyrosine kinase inhibitor, is associated with edema, pleural effusion, and pulmonary edema. We investigated the effect of dasatinib on the barrier function of human microvascular endothelial cells‐1 (HMEC‐1) in vitro and in vivo. The permeability of HMEC‐1 to fluorescein isothiocyante (FITC)‐dextran increased in Transwell chambers within 5 min following the addition of therapeutic concentrations of dasatinib. The change in permeability was associated with increased activation of RhoA GTPase and its effector Rho‐associated coiled‐coil kinase 1(ROCK1). RhoA inhibitor C3 transferase almost completely inhibited dasatinib‐induced increase in permeability. Under similar conditions, imatinib had no effect on permeability or activation of RhoA. Since integrin‐induced cell spreading suppresses RhoA activation, we examined the effect of dasatinib on cell spreading on fibronectin substrate. Dasatinib impaired endothelial cell spreading in a concentration‐dependent manner and induced disorganization of actin fibers. Tyrosine kinases play an essential role in transmitting signals from integrins to RhoA and we examined tyrosine phosphorylation of several cytoskeletal proteins. Dasatinib markedly inhibited tyrosine phosphorylation of p130 Crk‐associated substrate (p130cas), paxillin and vinculin. These results suggest that the inhibition of tyrosine phosphorylation of the focal adhesion plaque components by dasatinib may alter the assembly of actin fibers resulting in the activation of RhoA/ROCK pathway. Consistent with these findings, dasatinib‐induced increase in the permeability was blocked by ROCK inhibitor y27632. In vivo administration of y27632, significantly inhibited the dasatinib‐induced extravasation of Evans blue in mice and dasatinib‐induced increase in microvascular permeability was attenuated in ROCK1‐deficient mice. These findings suggest that ROCK inhibitors could serve as therapeutic modalities to ameliorate the dasatinib‐induced pulmonary changes.

ROCK1 Is Associated with Alzheimer's Disease-Specific Plaques, as well as Enhances Autophagosome Formation But not Autophagic Aβ Clearance.

Alzheimer’s disease (AD) is the most prevalent form of late-life dementia in the population, characterized by amyloid plaque formation and increased tau deposition, which is modulated by Rho-associated coiled-coil kinase 1 (ROCK1). In this study, we further analyze whether ROCK1 regulates the metabolism of amyloid precursor protein (APP). We show that ROCK1 is colocalized with mature amyloid-β (Aβ) plaques in patients with AD, in that ROCK1 enhances the amyloidogenic pathway, and that ROCK1 mediated autophagy enhances the intracellular buildup of Aβ in a cell model of AD (confirmed by increased ROCK1 and decreased Beclin 1 protein levels, with neuronal autophagosome accumulation in prefrontal cortex of AD APP/PS1 mouse model). In vitro over-expression of ROCK1 leads to a decrease in Aβ secretion and an increase in the expression of autophagy-related molecules. ROCK1 interacts with Beclin1, an autophagy initiator, and enhances the intracellular accumulation of Aβ. Reciprocally, overexpression of APP/Aβ promotes ROCK1 expression. Our data suggest ROCK1 participates in regulating Aβ secretion, APP shedding and autophagosome accumulation, and that ROCK1, rather than other kinases, is more likely to be a targetable enzyme for AD therapy.

Structure-Based Design, Synthesis, and Biological Evaluation of Highly Selective and Potent G Protein-Coupled Receptor Kinase 2 Inhibitors.

G protein-coupled receptors (GPCRs) are central to many physiological processes. Regulation of this superfamily of receptors is controlled by GPCR kinases (GRKs), some of which have been implicated in heart failure. GSK180736A, developed as a Rho-associated coiled-coil kinase 1 (ROCK1) inhibitor, was identified as an inhibitor of GRK2 and co-crystallized in the active site. Guided by its binding pose overlaid with the binding pose of a known potent GRK2 inhibitor, Takeda103A, a library of hybrid inhibitors was developed. This campaign produced several compounds possessing high potency and selectivity for GRK2 over other GRK subfamilies, PKA, and ROCK1. The most selective compound, 12n (CCG-224406), had an IC50 for GRK2 of 130 nM, >700-fold selectivity over other GRK subfamilies, and no detectable inhibition of ROCK1. Four of the new inhibitors were crystallized with GRK2 to give molecular insights into the binding and kinase selectivity of this class of inhibitors.

MiR-494-3p induced by compressive force inhibits cell proliferation in MC3T3-E1 cells

Mechanical stimuli regulate fundamental cell processes such as proliferation, differentiation, and morphogenesis. We attempted to identify microRNA (miRNA) whose expression is changed during compressive treatment in MC3T3-E1, a pre-osteoblastic cell line. Microarray analysis followed by reverse transcription-quantitative polymerase chain reaction revealed that compressive force at 294Pa for 24h in MC3T3-E1 cells increased levels of miR-494-3p, miR-146a-5p, miR-210-3p, and miR-1247-3p. Among these miRNAs, miR-494-3p was found to inhibit cell proliferation in MC3T3-E1 cells. Furthermore, cells subjected to compressive force showed slower cell growth compared with control cells. Levels of mRNA for fibroblast growth factor receptor 2 (FGFR2) and Rho-associated coiled-coil kinase 1 (ROCK1), which were predicted to be targets of miR-494-3p, were decreased by compressive force or overexpression of miR-494-3p mimics in MC3T3-E1 cells. Furthermore, binding sites of miR-494-3p within 3'-untranslated regions of Fgfr2 and Rock1 were determined using luciferase reporter assay. In conclusion, compressive force affected expressions of several miRNAs including miR-494-3p in MC3T3-E1 cells. Compressive force might inhibit cell proliferation in osteoblasts by up-regulating miR-494-3p followed by FGFR2 and ROCK1 gene repressions.

Cofilin-1 – Induced Actin Reorganization and Phosphatidylserine Exposure in Platelets

Background: Platelet activation leads to the transbilayer movement of phosphatidylserine (PS) from inner to the outer leaflet of membrane bilayer. Exteriorization of PS promotes platelet procoagulant activity by promoting the assembly of tenase and the prothrombinase complex on platelet membrane. In a previous study, we observed that Rho-associated coiled-coil kinase-1 (ROCK1) deficiency in murine platelets or ROCK inhibition by Y-27632 in human platelets resulted in increase in PS exposure and platelet procoagulant activity. ROCK1 deficient platelets had a marked decrease in phosphorylation of cofilin-1. Cofilin-1 decreases actin filament length by increasing the rate of dissociation of actin monomers and its activity is abolished by phosphorylation. These studies suggested a role for cofilin-1-induced actin reorganization in collagen-induced PS exposure. Cofilin-1 activity is also modulated by its interactions with phosphatidylinositol 4, 5-bisphosphate (PIP2) and a cofilin-1 binding protein, Wdr1, which enhances capacity of cofilin-1 to accelerate depolymerization by capping their barbed ends. Here, we studied the role of cofilin-1 phosphorylation and its interaction with PIP2 and Wdr1 in activation induced PS exposure in platelets.Materials and Methods: We isolated platelet membrane, cytosol and cytoskeleton through differential centrifugation from resting and collagen-stimulated platelets and assessed the relative abundance of cofilin-1 and phosphocofilin-1 by mobility shift in phosphate-affinity polyacrylamide gel electrophoresis. This method allows simultaneous detection of relative proportions of phosphoproteins and its nonphospho counterparts. For cofilin-1 and Wdr-1 distribution, platelets were immobilized on a polylysine-coated cover slip or on a collagen-coated cover slip, fixed, permeabilized and immunostained with appropriate antibody. In addition, PS exposure (FITC lactadherin binding), F-actin (Alexa Fluor 488-phalloidin) and calcium concentration (Fura-2AM fluorescence) were also measured.Results: Immunofluorescence images show that in resting platelets, cofilin-1 is present in distinct patches in the plasma membrane and following activation with collagen, cofilin-1 is redistributed in a discrete granular pattern throughout the cytoplasm. In parallel, we also studied the relative distribution and the phosphorylation status of cofilin-1 in various subcellular fractions of platelets. In resting platelets, cofilin-1 is present in the plasma membrane and in the cytosol but absent in the cytoskeleton. Following activation with collagen, cofilin-1 moves to the cytoskeleton with a concomitant decrease in the plasma membrane fraction. These results suggest that active cofilin-1 is incorporated into reorganizing actin cytoskeleton during platelet activation. In ROCK1-deficient mice, there is increased cofilin-1 in cytoskeletal fraction, which also correlates with increase in PS exposure. Blocking PIP2 hydrolysis by U73122 prevents cofilin-1 translocation and PS exposure. A cell permeable cofilin-1 peptide containing the actin binding site of cofilin-1 (amino acids Trp104 to Met115) at the carboxy terminus (that competitively inhibits cofilin-1 binding to F-actin), blocks cofilin-1 translocation and decreases PS exposure. Platelets from a Wdr1-deficient mice which express less than 2% of wild-type protein in platelets, showed impaired collagen-induced PS exposure despite equivalent increase in intra platelet calcium compared to wild-type platelets.Conclusion: Our results show cofilin-1 trafficking from membranre to cytoskeleton and subsequent actin reorganization precedes PS exposure during platelet activation. In ROCK1-deficient platelets, there is increased cofilin-1 activity due to decreased serine-3 phosphorylation and it is associated with increased cofilin-1 translocation to actin filaments and enhanced PS exposure. In contrast, inhibiting cofilin-1 translocation, either by inhibiting PIP2 hydrolysis or by competitive inhibition by a cell permeable peptide, prevents cofilin-1 trafficking and decreases PS exposure. Furthermore, in Wdr1-deficient mouse platelets, which have diminished cofilin-1 activity, have impaired PS exposure during platelet activation. DisclosuresNo relevant conflicts of interest to declare.

Abstract 270: Rnd3/RhoE Haploinsufficient Mice are Hypersensitive to Pressure Overload and Develop Apoptotic Cardiomyopathy with Heart Failure

Rationale: Rho family guanosine triphosphatase (GTPase) 3 (Rnd3, also called RhoE), a member of the small Rho GTPase family, has been suggested to regulate cell actin cytoskeleton dynamics, cell migration, and apoptosis through the Rho kinase-dependent signaling pathway. The biological function of Rnd3 in the heart is unknown. The downregulation of small GTPase Rnd3 transcripts was found in patients with end-stage heart failure. The pathological significance of Rnd3 loss in the transition to heart failure remains unexplored. Objective: To investigate the functional consequence of Rnd3 downregulation and the associated molecular mechanism. Methods and Results: we generated Rnd3 +/- haploinsufficient mice to mimic the downregulation of Rnd3 observed in the failing human heart. Rnd3 +/- mice were viable; however, the mice developed heart failure after pressure overload by transverse aortic constriction (TAC). Remarkable apoptosis, increased caspase-3 activity, and elevated Rho kinase activity were detected in the Rnd3 +/- haploinsufficient animal hearts. Pharmacological inhibition of Rho kinase by Fasudil treatment partially improved Rnd3 +/- mouse cardiac functions and attenuated myocardial apoptosis. To determine if Rho associated coiled-coil kinase 1 (ROCK1) was responsible for Rnd3 deficiency-mediated apoptotic cardiomyopathy, we established a double knockout mouse line, the Rnd3 haploinsufficient mice with ROCK1-null background (Rnd3 +/-/ROCK1-/- ). Again, genetic deletion of ROCK1 partially but not completely rescued Rnd3 deficiency-mediated heart failure phenotype. Conclusion: Downregulation of Rnd3 correlates with cardiac loss of function as in heart failure patients. Hyperactivation of Rho kinase activity is responsible in part for the apoptotic cardiomyopathy development. Further investigation of ROCK1-independent mechanisms in Rnd3-mediated cardiac remodeling should be the focus for future study.

Rnd3 haploinsufficient mice are predisposed to hemodynamic stress and develop apoptotic cardiomyopathy with heart failure.

Rho family guanosine triphosphatase (GTPase) 3 (Rnd3), a member of the small Rho GTPase family, has been suggested to regulate cell actin cytoskeleton dynamics, cell migration, and apoptosis through the Rho kinase-dependent signaling pathway. The biological function of Rnd3 in the heart is unknown. The downregulation of small GTPase Rnd3 transcripts was found in patients with end-stage heart failure. The pathological significance of Rnd3 loss in the transition to heart failure remains unexplored. To investigate the functional consequence of Rnd3 downregulation and the associated molecular mechanism, we generated Rnd3+/− haploinsufficient mice to mimic the downregulation of Rnd3 observed in the failing human heart. Rnd3+/− mice were viable; however, the mice developed heart failure after pressure overload by transverse aortic constriction (TAC). Remarkable apoptosis, increased caspase-3 activity, and elevated Rho kinase activity were detected in the Rnd3+/− haploinsufficient animal hearts. Pharmacological inhibition of Rho kinase by fasudil treatment partially improved Rnd3+/− mouse cardiac functions and attenuated myocardial apoptosis. To determine if Rho-associated coiled-coil kinase 1 (ROCK1) was responsible for Rnd3 deficiency-mediated apoptotic cardiomyopathy, we established a double-knockout mouse line, the Rnd3 haploinsufficient mice with ROCK1-null background (Rnd3+/−/ROCK1−/−). Again, genetic deletion of ROCK1 partially but not completely rescued Rnd3 deficiency-mediated heart failure phenotype. These data suggest that downregulation of Rnd3 correlates with cardiac loss of function as in heart failure patients. Animals with Rnd3 haploinsufficiency are predisposed to hemodynamic stress. Hyperactivation of Rho kinase activity is responsible in part for the apoptotic cardiomyopathy development. Further investigation of ROCK1-independent mechanisms in Rnd3-mediated cardiac remodeling should be the focus for future study.