LIM Kinase Research Articles

LIM kinases: cofilin and beyond.

LIM kinases are common downstream effectors of several signalization pathways and function as a signaling node that controls cytoskeleton dynamics through the phosphorylation of the cofilin family proteins. These last 10 years, several reports indicate that the functions of LIM kinases are more extended than initially described and, specifically, that LIM kinases also control microtubule dynamics, independently of their regulation of actin microfilament. In this review we analyze the data supporting these conclusions and the possible mechanisms that could be involved in the control of microtubules by LIM kinases. The demonstration that LIM kinases also control microtubule dynamics has pointed to new therapeutic opportunities. Consistently, several new LIM kinase inhibitors have been recently developed. We provide a comprehensive comparison of these inhibitors, of their chemical structure, their specificity, their cellular effects as well as their effects in animal models of various diseases including cancer.

PI3K Catalytic Isoform Alteration Promotes the LIMK1-related Metastasis Through the PAK1 or ROCK1/2 Activation in Cigarette Smoke-exposed Ovarian Cancer Cells

To investigate the molecular mechanisms by which long-term exposure to cigarette smoke extract (CSE) contributes to ovarian cancer metastasis. Western blot analysis for diverse p110 isoforms of phosphoinositide 3-kinase (PI3K)-related signaling pathway and epithelial-mesenchymal transition (EMT) markers was performed to analyze the underlying mechanisms. Migratory activity of CSE-exposed ovarian cancer cells was determined by transendothelial migration and invasion assay. After exposure to CSE for four weeks, CaOV3 (primary) and SKOV3 (metastatic) ovarian cancer cells showed enhanced mesenchymal characteristics and produced EMT-related cytokines [intwerleukin-8 (IL-8), vascular endothelial growth factor (VEGF) and transforming growth factor-beta 1 (TGF-β1)]. CSE exposure activated the Src-p110δ-p21 protein-activated kinase 1 (PAK1) in CaOV3 cells and the Lyn-p110β-Rho-associated kinases 1/2 (ROCK1/2) in SKOV3 cells, which led to the stimulation of LIM kinase 1 (LIMK1) phosphorylation and TGF-β1 release. LIMK1 knockdown efficiently blocked the migratory activity and suppressed the mesenchymal phenotypes of CSE-treated ovarian cancer cells. Reactive oxygen species (ROS) initiated the CSE-mediated EMT processes in ovarian cancer cells. Characterization of the p110 isotypes of PI3K is critical for regulating cancer metastasis; LIMK1 could be a common therapeutic target of ovarian cancer metastasis.

Downregulation of LIMK1\u2013ADF/cofilin by DADS inhibits the migration and invasion of colon cancer

This study aimed to explore whether the downregulation of LIM kinase 1 (LIMK1)-actin depolymerization factor (ADF, also known as destrin)/cofilin by diallyl disulfide (DADS) inhibited the migration and invasion of colon cancer. Previous studies have shown that silencing LIMK1 could significantly enhance the inhibitory effect of DADS on colon cancer cell migration and invasion, suggesting that LIMK1 was a target molecule of DADS, which needed further confirmation. This study reported that LIMK1 and destrin were highly expressed in colon cancer and associated with poor prognosis of patients with colon cancer. Also, the expression of LIMK1 was positively correlated with the expression of destrin. The overexpression of LIMK1 significantly promoted colon cancer cell migration and invasion. DADS obviously inhibited migration and invasion by suppressing the phosphorylation of ADF/cofilin via downregulation of LIMK1 in colon cancer cells. Furthermore, DADS-induced suppression of cell proliferation was enhanced and antagonized by the knockdown and overexpression of LIMK1 in vitro and in vivo, respectively. Similar results were observed for DADS-induced changes in the expression of vimentin, CD34, Ki-67, and E-cadherin in xenografted tumors. These results indicated that LIMK1 was a potential target molecule for the inhibitory effect of DADS on colon cancer cell migration and invasion.

Plumbagin reduces osteopontin-induced invasion through inhibiting the Rho-associated kinase signaling pathway in A549 cells and suppresses osteopontin-induced lung metastasis in BalB/c mice

Lung cancer is the second most commonly diagnosed cancer and the leading cause of cancer deaths in both men and women in the United States. It has been recently demonstrated that osteopontin (OPN) effectively inhibits cofilin activity through the focal adhesion kinase (FAK)/AKT/Rho-associated kinase (ROCK) pathway to induce the invasion of human non-small cell lung cancer (NSCLC) cells. Plumbagin was isolated from the roots of the medicinal plant Plumbago zeylanica L. and has been reported to possess anticancer activities. However, the molecular mechanisms by which plumbagin inhibits the invasion of cancer cells is still unclear. In this study, the anti-invasive and anti-metastatic mechanisms of plumbagin were investigated in OPN-treated NSCLC A549 cells. OPN effectively induced the motility and invasion of NSCLC A549 cells and H1299 cells, which was strongly suppressed by plumbagin with no evidence of cytotoxicity. In addition, lamellipodia formation at the leading edge of cells by OPN was dramatically decreased in plumbagin-treated cells. Plumbagin caused an effective inhibition in OPN-induced the expression of ROCK1 as well as the phosphorylation of LIM kinase 1 and 2 (LIMK1/2), and cofilin. OPN-induced the phosphorylation of FAK and AKT was impaired without affecting their total forms by plumbagin treatment. OPN facilitated metastatic lung colonization, which was effectively suppressed in plumbagin-treated mice. Taken together, these results suggest that plumbagin reduces OPN-induced the invasion of NSCLC A549 cells, which resulted from inhibiting the ROCK pathway mediated by the FAK/AKT pathway and suppresses lung metastasis in vivo.

108 - Inhibition of prostate smooth muscle contraction by the LIM kinase inhibitor, SR-7826: A new anticontractile strategy and implications for a role of LIM kinases in the control of prostate smooth muscle tone

108 - Inhibition of prostate smooth muscle contraction by the LIM kinase inhibitor, SR-7826: A new anticontractile strategy and implications for a role of LIM kinases in the control of prostate smooth muscle tone

Regulation of RhoA activity by the cellular prion protein

The cellular prion protein (PrPC) is a highly conserved glycosylphosphatidylinositol (GPI)-anchored membrane protein that is involved in the signal transduction during the initial phase of neurite outgrowth. The Ras homolog gene family member A (RhoA) is a small GTPase that is known to have an essential role in regulating the development, differentiation, survival, and death of neurons in the central nervous system. Although recent studies have shown the dysregulation of RhoA in a variety of neurodegenerative diseases, the role of RhoA in prion pathogenesis remains unclear. Here, we investigated the regulation of RhoA-mediated signaling by PrPC using both in vitro and in vivo models and found that overexpression of PrPC significantly induced RhoA inactivation and RhoA phosphorylation in hippocampal neuronal cells and in the brains of transgenic mice. Using siRNA-mediated depletion of endogenous PrPC and overexpression of disease-associated mutants of PrPC, we confirmed that PrPC induced RhoA inactivation, which accompanied RhoA phosphorylation but reduced the phosphorylation levels of LIM kinase (LIMK), leading to cofilin activation. In addition, PrPC colocalized with RhoA, and the overexpression of PrPC significantly increased neurite outgrowth in nerve growth factor-treated PC12 cells through RhoA inactivation. However, the disease-associated mutants of PrPC decreased neurite outgrowth compared with wild-type PrPC. Moreover, inhibition of Rho-associated kinase (ROCK) substantially facilitated neurite outgrowth in NGF-treated PC12 cells, similar to the effect induced by PrPC. Interestingly, we found that the induction of RhoA inactivation occurred through the interaction of PrPC with RhoA and that PrPC enhanced the interaction between RhoA and p190RhoGAP (a GTPase-activating protein). These findings suggest that the interactions of PrPC with RhoA and p190RhoGAP contribute to neurite outgrowth by controlling RhoA inactivation and RhoA-mediated signaling and that disease-associated mutations of PrPC impair RhoA inactivation, which in turn leads to prion-related neurodegeneration.

Signal mingle: Micropatterns of BMP-2 and fibronectin on soft biopolymeric films regulate myoblast shape and SMAD signaling

In vivo, bone morphogenetic protein 2 (BMP-2) exists both in solution and bound to the extracellular matrix (ECM). While these two modes of presentation are known to influence cell behavior distinctly, their role in the niche microenvironment and their functional relevance in the genesis of a biological response has sparsely been investigated at a cellular level. Here we used the natural affinity of BMP-2 for fibronectin (FN) to engineer cell-sized micropatterns of BMP-2. This technique allowed the simultaneous control of the spatial presentation of fibronectin-bound BMP-2 and cell spreading. These micropatterns induced a specific actin and adhesion organization around the nucleus, and triggered the phosphorylation and nuclear translocation of SMAD1/5/8 in C2C12 myoblasts and mesenchymal stem cells, an early indicator of their osteoblastic trans-differentiation. We found that cell spreading itself potentiated a BMP-2-dependent phosphorylation of SMAD1/5/8. Finally, we demonstrated that FN/BMP-2-mediated early SMAD signaling depended on LIM kinase 2 and ROCK, rather than myosin II activation. Altogether, our results show that FN/BMP-2 micropatterns are a useful tool to study the mechanisms underlying BMP-2-mediated mechanotransduction. More broadly, our approach could be adapted to other combinations of ECM proteins and growth factors, opening an exciting avenue to recreate tissue-specific niches in vitro.

Effects of Hippocampal LIMK Inhibition on Memory Acquisition, Consolidation, Retrieval, Reconsolidation, and Extinction.

Long-lasting changes in dendritic spines provide a physical correlate for memory formation and persistence. LIM kinase (LIMK) plays a critical role in orchestrating dendritic actin dynamics during memory processing, since it is the convergent downstream target of both the Rac1/PAK and RhoA/ROCK pathways that in turn induce cofilin phosphorylation and prevent depolymerization of actin filaments. Here, using a potent LIMK inhibitor (BMS-5), we investigated the role of LIMK activity in the dorsal hippocampus during contextual fear memory in rats. We first found that post-training administration of BMS-5 impaired memory consolidation in a dose-dependent manner. Inhibiting LIMK before training also disrupted memory acquisition. We then demonstrated that hippocampal LIMK activity seems to be critical for memory retrieval and reconsolidation, since both processes were impaired by BMS-5 treatment. Contextual fear memory extinction, however, was not sensitive to the same treatment. In conclusion, our findings demonstrate that hippocampal LIMK activity plays an important role in memory acquisition, consolidation, retrieval, and reconsolidation during contextual fear conditioning.

Effects of vitrification and a Rho-associated coiled-coil containing protein kinase 1 inhibitor on the meiotic and developmental competence of feline oocytes.

Oocyte cryopreservation is the technique of choice for the long-term storage of female gametes. However, it induces an irreversible loss of oocyte viability and function. We examined the effects of vitrification and a Rho-associated coiled-coil containing protein kinase 1 (ROCK1) inhibitor (ROCKi) on the meiotic and developmental competence of feline oocytes. We examined the expression of LIM kinase (LIMK) 1 and 2, with and without ROCKi treatment. Cumulus oocyte complexes (COCs) were matured in vitro with 0, 10, 20, and 40 µM ROCKi. The oocytes were subsequently assessed for maturation rate and embryo development following in vitro fertilization. We repeated the COC experiment, but vitrified and warmed the COCs prior to culture. We detected LIMK1 and LIMK2 expression in feline oocytes, which could be downregulated by ROCKi treatment. The ROCKi at 10 µM affected neither meiotic nor developmental competence (P > 0.05, versus control). However, high concentrations of ROCKi during maturation induced meiotic arrest at metaphase I. Appropriate concentrations of ROCKi significantly improved the normal fertilization rate of vitrified warmed oocytes (49.4 ± 3.4%) compared with that of the control (42.8 ± 8.6%, P < 0.05). The ROCKi also significantly improved the embryo cleavage rate (36.1 ± 3.8%) as compared with the non-treated control (27.4 ± 2.5%, P < 0.05). Thus, this study revealed that the main mediators of the ROCK cascade (LIM kinases) are expressed in feline oocytes. The ROCKi (10 µM) did not compromise the meiotic or developmental competence of feline oocytes. In addition, 10 µM ROCKi improved the cytoplasmic maturation of vitrified–warmed oocytes as indicated by their fertilization competence.

Targeting LIM kinases in taxane resistant tumors

Targeting LIM kinases in taxane resistant tumors

Abstract 1171: LIMK1 functions as a modulator of expression and targeting of CXCR4 in prostate cancer cells through a negative feedback loop

Abstract Increased activation of the GPCR CXCR4/CXCL12 signaling axis is commonly noted in metastatic prostate cancer and shows an association with poor prognosis. CXCR4, and its ligand, CXCL12/SDF-1a, are recognized as powerful mediators for tumor microenvironment remodeling and chemoprotection. LIM kinase 1 (LIMK1), a serine/threonine kinase, is an actin and microtubule modulatory protein that is also overexpressed in a variety of cancers including prostate cancer. Earlier, we showed that LIMK1 is involved in cell invasion and inhibition of LIMK1 expression reverted the invasive phenotype of PC3 prostate cancer cells. Activation of CXCR4/CXCL12 axis promotes activation of LIMK1 through RhoGTPAse pathway and promotes docetaxel resistance. Here we show that LIMK1 expression and functions have a positive correlation with CXCR4 expression and subcellular localization. We used metastatic prostate cancer cell lines PC3 and M12 and the non-tumorigenic prostate cell lines BPH and P69 to monitor expression and membrane localization of CXCR4 using western blots, flow cytometry and immunofluorescence analysis. Our analysis showed that cells with higher expression of LIMK1 (PC3) overexpress CXCR4, and that cell lines with low levels of LIMK1 (BPH and P69) express relatively lower amounts of CXCR4. Flow cytometric analysis showed a stronger surface staining of CXCR4 in the LIMK1 overexpressing PC3 cells compared to BPH cells. Next, we studied the effect of shRNA-mediated inhibition of LIMK1 expression on CXCR4 expression in PC3 or M12 cells. Western blot and immunofluorescence analysis showed noticeable reduction in CXCR4 levels, and its surface localization, as noted by flow cytometry, in LIMK1 shRNA transfected PC3 and M12 cells. To assess if the kinase activity of LIMK1 is essential for CXCR4 expression and surface localization, we treated these cells with LIMK1 specific kinase inhibitor BMS-5 and determined expression of CXCR4. Western blots and immunofluorescence analysis showed reduced expression of CXCR4 in treated cells compared to vehicle treated cells. Our observation indicates an indirect association between LIMK1 and CXCR4 expression and shows for the first time that expression of LIMK1 is required for the maintenance of CXCR4 expression. Our study demonstrates a novel positive feed back regulatory mechanism between LIMK1 and CXCR4 and suggest a possible combination treatment option through inhibition of LIMK1 expression and/or its kinase activity for improving chemosensitivity of prostate cancer cells with overactive CXCR4/CXCL12 axis. Citation Format: Richard Ottman, Lisa Ritchey, Ryan Herchan, Alexia Bossan, Ratna Chakrabarti. LIMK1 functions as a modulator of expression and targeting of CXCR4 in prostate cancer cells through a negative feedback loop. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1171.

Rho-associated protein kinase 1 (ROCK1) is increased in Alzheimer's disease and ROCK1 depletion reduces amyloid-β levels in brain.

Alzheimer's disease (AD) is the leading cause of dementia and mitigating amyloid-β (Aβ) levels may serve as a rational therapeutic avenue to slow AD progression. Pharmacologic inhibition of the Rho-associated protein kinases (ROCK1 and ROCK2) is proposed to curb Aβ levels, and mechanisms that underlie ROCK2's effects on Aβ production are defined. How ROCK1 affects Aβ generation remains a critical barrier. Here, we report that ROCK1 protein levels were elevated in mild cognitive impairment due to AD (MCI) and AD brains compared to controls. Aβ42 oligomers marginally increased ROCK1 and ROCK2 protein levels in neurons but strongly induced phosphorylation of Lim kinase 1 (LIMK1), suggesting that Aβ42 activates ROCKs. RNAi depletion of ROCK1 or ROCK2 suppressed endogenous Aβ40 production in neurons, and Aβ40 levels were reduced in brains of ROCK1 heterozygous knock-out mice compared to wild-type littermate controls. ROCK1 knockdown decreased amyloid precursor protein (APP), and treatment with bafilomycin accumulated APP levels in neurons depleted of ROCK1. These observations suggest that reduction of ROCK1 diminishes Aβ levels by enhancing APP protein degradation. Collectively, these findings support the hypothesis that both ROCK1 and ROCK2 are therapeutic targets to combat Aβ production in AD. Mitigating amyloid-β (Aβ) levels is a rational strategy for Alzheimer's disease (AD) treatment, however, therapeutic targets with clinically available drugs are lacking. We hypothesize that Aβ accumulation in mild cognitive impairment because of AD (MCI) and AD activates the RhoA/ROCK pathway which in turn fuels production of Aβ. Escalation of this cycle over the course of many years may contribute to the buildup of amyloid pathology in MCI and/or AD.

LIM Kinase Inhibitor Pyr1 Reduces the Growth and Metastatic Load of Breast Cancers.

LIM kinases (LIMK) are emerging targets for cancer therapy, and they function as network hubs to coordinate actin and microtubule dynamics. When LIMKs are inhibited, actin microfilaments are disorganized and microtubules are stabilized. Owing to their stabilizing effect on microtubules, LIMK inhibitors may provide a therapeutic strategy to treat taxane-resistant cancers. In this study, we investigated the effect of LIMK inhibition on breast tumor development and on paclitaxel-resistant tumors, using a novel selective LIMK inhibitor termed Pyr1. Treatment of breast cancer cells, including paclitaxel-resistant cells, blocked their invasion and proliferation in vitro and their growth in vivo in tumor xenograft assays. The tumor-invasive properties of Pyr1 were investigated in vivo by intravital microscopy of tumor xenografts. A striking change of cell morphology was observed with a rounded phenotype arising in a subpopulation of cells, while other cells remained elongated. Notably, although Pyr1 decreased the motility of elongated cells, it increased the motility of rounded cells in the tumor. Pyr1 administration prevented the growth of metastasis but not their spread. Overall, our results provided a preclinical proof of concept concerning how a small-molecule inhibitor of LIMK may offer a strategy to treat taxane-resistant breast tumors and metastases. Cancer Res; 76(12); 3541-52. ©2016 AACR.

Wnt5A/Ryk signaling critically affects barrier function in human vascular endothelial cells

ABSTRACTSatisfactory therapeutic strategies for septic shock are still missing. Previously we found elevated levels of Wnt5A in patients with severe sepsis and septic shock. Wnt5A is released by activated macrophages but knowledge of its effects in the vascular system remains scant. Here we investigate the response of human coronary artery endothelial cells (HCAEC) to Wnt5A. We used a genome-wide differential expression approach to define novel targets regulated by Wnt5A. Gene ontology analysis of expression profiles revealed clusters of genes involved in actin cytoskeleton remodeling as the predominant targets of Wnt5A. Wnt5A targeted Rho-associated protein serine/threonine kinase (ROCK), leading to phosphorylation of LIM kinase-2 (LIMK2) and inactivation of the actin depolymerization factor cofilin-1 (CFL1). Functional experiments recording cytoskeletal rearrangements in living cells showed that Wnt5A enhanced stress fiber formation as a consequence of reduced actin depolymerization. The antagonist Wnt inhibitory factor 1 (WIF1) that specifically interferes with the WIF domain of Ryk receptors prevented actin polymerization. Wnt5A disrupted β-catenin and VE-cadherin adherens junctions forming inter-endothelial gaps. Functional experiments targeting the endothelial monolayer integrity and live recording of trans-endothelial resistance revealed enhanced permeability of Wnt5A-treated HCAEC. Ryk silencing completely prevented Wnt5A-induced endothelial hyperpermeability. Wnt5A decreased wound healing capacity of HCAEC monolayers; this was restored by the ROCK inhibitor Y-27632. Here we show that Wnt5A acts on the vascular endothelium causing enhanced permeability through Ryk interaction and downstream ROCK/LIMK2/CFL1 signaling. Wnt5A/Ryk signaling might provide novel therapeutic strategies to prevent capillary leakage in systemic inflammation and septic shock.

IL-4 Causes Hyperpermeability of Vascular Endothelial Cells through Wnt5A Signaling.

Microvascular leakage due to endothelial barrier dysfunction is a prominent feature of T helper 2 (Th2) cytokine mediated allergic inflammation. Interleukin-4 (IL-4) is a potent Th2 cytokine, known to impair the barrier function of endothelial cells. However, the effectors mediating IL-4 induced cytoskeleton remodeling and consequent endothelial barrier dysfunction remain poorly defined. Here we have used whole genome transcriptome profiling and gene ontology analyses to identify the genes and processes regulated by IL-4 signaling in human coronary artery endothelial cells (HCAEC). The study revealed Wnt5A as an effector that can mediate actin cytoskeleton remodeling in IL-4 activated HCAEC through the regulation of LIM kinase (LIMK) and Cofilin (CFL). Following IL-4 treatment, LIMK and CFL were phosphorylated, thereby indicating the possibility of actin stress fiber formation. Imaging of actin showed the formation of stress fibers in IL-4 treated live HCAEC. Stress fiber formation was notably decreased in the presence of Wnt inhibitory factor 1 (WIF1). Non-invasive impedance measurements demonstrated that IL-4 increased the permeability and impaired the barrier function of HCAEC monolayers. Silencing Wnt5A significantly reduced permeability and improved the barrier function of HCAEC monolayers upon IL-4 treatment. Our study identifies Wnt5A as a novel marker of IL-4 activated vascular endothelium and demonstrates a critical role for Wnt5A in mediating IL-4 induced endothelial barrier dysfunction. Wnt5A could be a potential therapeutic target for reducing microvascular leakage and edema formation in Th2 driven inflammatory diseases.

Structural Basis for Noncanonical Substrate Recognition of Cofilin/ADF Proteins by LIM Kinases

Cofilin/actin-depolymerizing factor (ADF) proteins are critical nodes that relay signals from protein kinase cascades to the actin cytoskeleton, in particular through site-specific phosphorylation at residue Ser3. This is important for regulation of the roles ofcofilin in severing and stabilizing actin filaments. Consequently, cofilin/ADF Ser3 phosphorylation is tightly controlled as an almost exclusive substrate for LIM kinases. Here we determine the LIMK1:cofilin-1 co-crystal structure. We find an interface that is distinct from canonical kinase-substrate interactions. We validate this previously unobserved mechanism for high-fidelity kinase-substrate recognition by invitro kinase assays, examination of cofilin phosphorylation in mammalian cells, and functional analysis in S.cerevisiae. The interface is conserved across all LIM kinases. Remarkably, we also observe both pre- and postphosphotransfer states in the same crystal lattice. This study therefore provides a molecular understanding of how kinase-substrate recognition acts as a gatekeeper to regulate actin cytoskeletal dynamics.

LIMK Regulates Tumor-Cell Invasion and Matrix Degradation Through Tyrosine Phosphorylation of MT1-MMP.

During their metastatic spread, cancer cells need to remodel the extracellular matrix in order to migrate through stromal compartments adjacent to the primary tumor. Dissemination of breast carcinoma cells is mediated by membrane type 1-matrix metalloproteinase (MT1-MMP/MMP14), the main invadopodial matrix degradative component. Here, we identify MT1-MMP as a novel interacting partner of dual-specificity LIM Kinase-1 and -2 (LIMK1/2), and provide several evidence for phosphorylation of tyrosine Y573 in the cytoplasmic domain of MT1-MMP by LIMK. Phosphorylation of Y573 influences association of F-actin binding protein cortactin to MT1-MMP-positive endosomes and invadopodia formation and matrix degradation. Moreover, we show that LIMK1 regulates cortactin association to MT1-MMP-positive endosomes, while LIMK2 controls invadopodia-associated cortactin. In turn, LIMK1 and LIMK2 are required for MT1-MMP-dependent matrix degradation and cell invasion in a three-dimensional type I collagen environment. This novel link between LIMK1/2 and MT1-MMP may have important consequences for therapeutic control of breast cancer cell invasion.

Mitochondrial Translocation of High Mobility Group Box 1 Facilitates LIM Kinase 2-Mediated Programmed Necrotic Neuronal Death

High mobility group box 1 (HMGB1) acts a signaling molecule regulating a wide range of inflammatory responses in extracellular space. HMGB1 also stabilizes nucleosomal structure and facilitates gene transcription. Under pathophysiological conditions, nuclear HMGB1 is immediately transported to the cytoplasm through chromosome region maintenance 1 (CRM1). Recently, we have reported that up-regulation of LIM kinase 2 (LIMK2) expression induces HMGB1 export from neuronal nuclei during status epilepticus (SE)-induced programmed neuronal necrosis in the rat hippocampus. Thus, we investigated whether HMGB1 involves LIMK2-mediated programmed neuronal necrosis, but such role is not reported. In the present study, SE was induced by pilocarpine in rats that were intracerebroventricularly infused with saline, control siRNA, LIMK2 siRNA or leptomycin B (LMB, a CRM1 inhibitor) prior to SE induction. Thereafter, we performed Fluoro-Jade B staining, western blots and immunohistochemical studies. LIMK2 knockdown effectively attenuated SE-induced neuronal death and HMGB1 import into mitochondria accompanied by inhibiting nuclear HMGB1 release and abnormal mitochondrial elongation. LMB alleviated SE-induced neuronal death and nuclear HMGB1 release. However, LMB did not prevent mitochondrial elongation induced by SE, but inhibited the HMGB1 import into mitochondria. The efficacy of LMB was less effective to attenuate SE-induced neuronal death than that of LIMK2 siRNA. These findings indicate that nuclear HMGB1 release and the subsequent mitochondrial import may facilitate and deteriorate programmed necrotic neuronal deaths. The present data suggest that the nuclear HMGB1 release via CRM1 may be a potential therapeutic target for the programmed necrotic neuronal death induced by SE.

Evidence for elevated (LIMK2 and CFL1) and suppressed (ICAM1, EZR, MAP2K2, and NOS3) gene expressions in metabolic syndrome.

The metabolic syndrome (MetS) is a common multicomponent condition including abdominal obesity, dyslipidemia, hypertension, and hyperglycaemia. The aim of this study was to investigate the associations of the expression of a panel of signalling genes with the MetS in a Turkish population. A total of 54 MetS patients and 42 healthy controls with similar age and sex were included to this study. mRNA from blood samples was extracted, and real-time polymerase chain reaction was performed for gene expressions using a BioMark 96.96 dynamic array system. We observed marked increases in LIM kinase 2 (LIMK2) and cofilin 1 (CFL1) gene expressions in MetS patients. However, there were significant decreases in intercellular adhesion molecules 1 (ICAM1), ezrin (EZR), mitogen-activated protein kinase kinase 2 (MAP2K2), and nitric oxide synthase 3 (NOS3) gene expressions in MetS patients. Additionally, no marked changes were noted in other 15 genes studied. This is the first study to provide evidence that activation of LIMK2/CFL1 pathway may play an important role in MetS.

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.