Dendritic spine loss caused by AlCl3 is associated with inhibition of the Rac 1/cofilin signaling pathway.

Aluminum (Al) has neurotoxicity that can result in cognitive dysfunction. Hippocampal dendritic spine loss is a pathological characteristic of cognitive dysfunction. Our previous study reported that Al exposure caused dendritic spine loss in the hippocampus, but the underlying mechanism remains unclear. In this study, rats were orally administered 50, 150 or 450 mg/kg of AlCl3 for 90 days. The dendritic spine density of the CA1 and DG regions was detected by Golgi-Cox staining. The F-actin/G-actin ratio, the expression of drebrin A and the components of the Rac 1/cofilin pathway were measured in the hippocampus. The results obtained showed that AlCl3 caused dendritic spine loss and decreased the F-actin/G-actin ratio. In addition, it was found that AlCl3 downregulated the expression of Rac 1, p-PAK, p-LIMK, p-cofilin and drebrin A and upregulated cofilin expression. Altogether, these results demonstrated that Al inactivated the Rac 1/cofilin pathway by inhibiting the phosphorylation of cofilin and the polymerization of F-actin, resulting in dendritic spine loss in the hippocampus.

LIMK/cofilin pathway and Slingshot are implicated in human colorectal cancer progression and chemoresistance.

Cofilin phospho-regulation is important for actin filament turnover and is implicated in cancer. Phosphorylation of cofilin is mediated by LIM kinases (LIMKs) and dephosphorylation by Slingshot phosphatases (SSH). LIMKs and SSH promote cancer cell invasion and metastasis and represent novel anti-cancer targets. However, little is known regarding LIMK/cofilin and SSH in human colorectal cancer (CRC). In this study, we aimed to address their expression and significance in human CRC. We evaluated expression of non-phosphorylated (active) and phosphorylated cofilin, LIMK1, LIMK2, and SSH1 by immunohistochemistry in 143 human CRC samples in relation to clinicopathologic parameters, response of metastatic disease to chemotherapy, and epithelial-mesenchymal transition (EMT) markers ß-catenin, E-cadherin, and ZEB. We show that active cofilin, LIMK1, LIMK2, and SSH1 are overexpressed in human CRC and are associated with tumor progression parameters. SSH1 is an independent predictor of lymph node metastasis by multivariate analysis. LIMK1 and SSH1 expression is also higher in non-responders to chemotherapy, and SSH1 is shown by multivariate analysis to independently predict response of metastatic disease to chemotherapy. Active cofilin, LIMK1, LIMK2, and SSH1 also correlated with the EMT markers examined. In addition, immunofluorescence analysis showed increased expression of active cofilin, LIMK1, LIMK2, and SSH1 in HT29 colon cancer cells resistant to 5-fluorouracil compared to parental HT29 cells. Our results suggest that F-actin regulators LIMK/cofilin pathway and SSH1 are associated with CRC progression and chemoresistance representing promising tumor biomarkers and therapeutic targets in CRC.

MicroRNA-138 inhibits migration and invasion of non-small cell lung cancer cells by targeting LIMK1.

MicroRNA (miR)-138 has previously been demonstrated to have a suppressive role in numerous types of human cancer, including non-small cell lung cancer (NSCLC). LIM domain kinase 1 (LIMK1) is a serine/threonine kinase that regulates actin polymerization via phosphorylation and inactivation of cofilin. Previous studies have reported that LIMK1 is associated with NSCLC; however, the underlying regulatory mechanism of LIMK1, and the association between LIMK1 and miR­138 in NSCLC cells, remains largely unknown. The present study aimed to reveal the regulatory roles of miR­138 and LIMK1 in NSCLC cell migration and invasion. Reverse transcription­quantitative polymerase chain reaction and western blot analysis were used to examine the mRNA and protein expression levels. Transwell and wound healing assays were conducted to determine cell invasion and migration. A luciferase reporter assay was used to determine the target association between miR­138 and LIMK1. The present study demonstrated that miR­138 was markedly downregulated in NSCLC tissues and cell lines, whereas the expression levels of LIMK1 were significantly upregulated. LIMK1 was further identified as a direct target of miR­138 in NSCLC H460 cells. Furthermore, overexpression of miR­138 significantly inhibited the protein expression of LIMK1, whereas knockdown of miR­138 upregulated the protein expression of LIMK1 in H460 cells. In addition, overexpression of miR­138 significantly inhibited the migration and invasion of NSCLC cells; however, overexpression of LIMK1 significantly promoted NSCLC cell migration and invasion. An investigation into the underlying molecular mechanism revealed that overexpression of miR­138 significantly decreased cofilin signaling activity, whereas knockdown of miR­138 notably enhanced cofilin signaling activity. In conclusion, the present study suggests that miR­138 may inhibit the migration and invasion of NSCLC cells by targeting the LIMK1/cofilin signaling pathway. Therefore, miR-138/LIMK1/cofilin may be considered a potential therapeutic target for the treatment of NSCLC.

Diallyl disulfide suppresses epithelial-mesenchymal transition, invasion and proliferation by downregulation of LIMK1 in gastric cancer.

Diallyl disulfide (DADS) has been shown to have multi-targeted antitumor activities. We have previously discovered that it has a repressive effect on LIM kinase-1 (LIMK1) expression in gastric cancer MGC803 cells. This suggests that DADS may inhibit epithelial-mesenchymal transition (EMT) by downregulating LIMK1, resulting in the inhibition of invasion and growth in gastric cancer. In this study, we reveal that LIMK1 expression is correlated with tumor differentiation, invasion depth, clinical stage, lymph node metastasis, and poor prognosis. DADS downregulated the Rac1-Pak1/Rock1-LIMK1 pathway in MGC803 cells, as shown by decreased p-LIMK1 and p-cofilin1 levels, and suppressed cell migration and invasion. Knockdown and overexpression experiments performed in vitro demonstrated that downregulating LIMK1 with DADS resulted in restrained EMT that was coupled with decreased matrix metalloproteinase-9 (MMP-9) and increased tissue inhibitor of metalloproteinase-3 (TIMP-3) expression. In in vitro and in vivo experiments, the DADS-induced suppression of cell proliferation was enhanced and antagonized by the knockdown and overexpression of LIMK1, 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 indicate that downregulation of LIMK1 by DADS could explain the inhibition of EMT, invasion and proliferation in gastric cancer cells.

Transcriptional profiling of GBM invasion genes identifies effective inhibitors of the LIM kinase-Cofilin pathway.

Malignant gliomas are highly proliferative and invasive neoplasms where total surgical resection is often impossible and effective local radiation therapy difficult. Consequently, there is a need to develop a greater understanding of the molecular events driving invasion and to identify novel treatment targets. Using microarray analysis comparing normal brain samples and mesenchymal glioblastoma multiforme (GBM), we identified over 140 significant genes involved in cell migration and invasion. The cofilin (CFL) pathway, which disassembles actin filaments, was highly up-regulated compared to normal brain. Up-regulation of LIM domain kinase 1 and 2 (LIMK1/2), that phosphorylates and inactivates cofilin, was confirmed in an additional independent data set comparing normal brain to GBM. We identified and utilized two small molecule inhibitors BMS-5 and Cucurbitacin I directed against the cofilin regulating kinases, LIMK1 and LIMK2, to target this pathway. Significant decreases in cell viability were observed in glioma cells treated with BMS-5 and Cucurbitacin I, while no cytotoxic effects were seen in normal astrocytes that lack LIMK. BMS-5 and Cucurbitacin I promoted increased adhesion in GBM cells, and decreased migration and invasion. Collectively, these data suggest that use of LIMK inhibitors may provide a novel way to target the invasive machinery in GBM.

Adaptor protein LRAP25 mediates myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) regulation of LIMK1 protein in lamellipodial F-actin dynamics.

Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) has been shown to localize to the lamella of mammalian cells through its interaction with an adaptor protein, leucine repeat adaptor protein 35a (LRAP35a), which links it with myosin 18A (MYO18A) for activation of the lamellar actomyosin network essential for cell migration. Here, we report the identification of another adaptor protein LRAP25 that mediates MRCK association with LIM kinase 1 (LIMK1). The lamellipodium-localized LRAP25-MRCK complex is essential for the regulation of local LIMK1 and its downstream F-actin regulatory factor cofilin. Functionally, inhibition of either MRCK or LRAP25 resulted in a marked suppression of LIMK1 activity and down-regulation of cofilin phosphorylation in response to aluminum fluoride induction in B16-F1 cells, which eventually resulted in deregulation of lamellipodial F-actin and reorganization of cytoskeletal structures causing defects in cell polarization and motility. These biochemical and functional characterizations thus underline the functional relevance of the LRAP25-MRCK complex in LIMK1-cofilin signaling and the importance of LRAP adaptors as key determinants of MRCK cellular localization and downstream specificities.

PICK1 links Argonaute 2 to endosomes in neuronal dendrites and regulates miRNA activity.

MicroRNAs fine-tune gene expression by inhibiting the translation of mRNA targets. Argonaute (Ago) proteins are critical mediators of microRNA-induced post-transcriptional silencing and have been shown to associate with endosomal compartments, but the molecular mechanisms that underlie this process are unclear, especially in neurons. Here, we report a novel interaction between Ago2 and the BAR-domain protein, PICK1. We show that PICK1 promotes Ago2 localization at endosomal compartments in neuronal dendrites and inhibits Ago2 function in translational repression following neuronal stimulation. We propose that PICK1 provides a link between activity-dependent endosomal trafficking and local regulation of translation in neurons.

MiR-27b targets LIMK1 to inhibit growth and invasion of NSCLC cells.

Non-small cell lung cancer (NSCLC), which accounts for ~80 % of lung cancer cases, is one of the most common causes for cancer-related death. microRNAs (miRNAs) have been found to play critical roles in the development and progression of NSCLC. miR-27b has recently been reported as a tumor suppressor in several cancers, but its role in NSCLC remains poorly understood. In this study, we found that miR-27b was remarkably decreased in both NSCLC tissues and cell lines. Moreover, overexpression of miR-27b significantly suppressed NSCLC cells proliferation and invasion. LIM kinase 1 (LIMK1), an essential protein for malignant transformation, was found to be a target of miR-27b. Ectopic expression of LIMK1 dramatically dampened mir-27b action of cancer inhibition. Finally, LIMK1 was found to be negatively correlated with miR-27b in NSCLC patients. Our results demonstrated a tumor-suppressive role of miR-27b in NSCLC, suggesting a potential therapeutic target for NSCLC.

Renal expression of arachidonic acid metabolizing enzymes and RhoA/Rho kinases in fructose insulin resistant hypertensive rats.

Fructose feeding has been shown to induce insulin resistance and hypertension. Renal protein expression for the cytochrome P (CYP) 450 arachidonic acid metabolizing enzymes has been shown to be altered in other models of diet-induced hypertension. Of special interest is CYP4A, which produces the potent vasoconstrictor, 20-hydroxyeicosatetraenoic acid and CYP2C, which catalyzes the formation of the potent dilators epoxyeicosatrienoic acids as well as soluble epoxide hydrolase (sEH) which metabolizes the latter to dihydroxyeicosatrienoic acids. The RhoA/Rho kinase (ROCK) signaling pathway is downstream of arachidonic acid and is reported to mediate metabolic-cardio-renal dysfunctions in some experimental models of insulin resistance and diabetes. The aim of the present study was to determine the expression of CYP4A, CYP2C23, CYP2C11, sEH, RhoA, ROCK-1, ROCK-2, and phospho-Lin-11/Isl-1/Mec-3 kinase (LIMK) in kidneys of fructose-fed (F) rats. Male Wistar rats were fed a high fructose diet for 8 weeks. Body weight, systolic blood pressure, insulin sensitivity, and renal expression of the aforementioned proteins were assessed. No change was observed in the body weight of F rats; however, euglycemia and hyperinsulinemia implicating impaired glucose tolerance and significant elevation in systolic blood pressure were observed. Renal expression of CYP4A and CYP2C23 was significantly increased while that of CYP2C11 and sEH was not changed in F rats. Equal expression for RhoA in both control and F rats and an enhanced level of ROCK-1 and ROCK-2 constitutively activate 130 kDa cleavage fragments as well as phospho-LIMK. These data suggest that the kidneys could be actively participating in the pathogenesis of insulin resistance-induced hypertension through the arachidonic acid CYP 450-RhoA/Rho kinase pathway(s).