Inhibition of human prostate smooth muscle contraction by the LIM kinase inhibitors, SR7826 and LIMKi3.

BACKGROUND AND PURPOSE: In men with benign prostatic hyperplasia, increased smooth muscle tone in the prostate may lead to bladder outlet obstruction and subsequent lower urinary tract symptoms. Consequently, medical treatment aims to inhibit prostate smooth muscle contraction. However, the efficacy of the treatment options available is limited, and improved understanding of mechanisms of prostate smooth muscle contraction and identification of new targets for medical intervention are mandatory. Several studies suggest that LIM kinases (LIMKs) promote smooth muscle contraction; however, this has not yet been examined. Here, we studied effects of the LIMK inhibitors on prostate smooth muscle contraction. EXPERIMENTAL APPROACH: Human prostate tissues were obtained from radical prostatectomy. Phosphorylation of cofilin, a LIMK substrate, was examined using a phospho-specific antibody. Smooth muscle contractions were studied in organ bath experiments. KEY RESULTS: Real-time PCR, Western blot and immunofluorescence suggested LIMKs are expressed in smooth muscle cells of prostate tissues. Two different LIMK inhibitors, SR7826 (1 µM) and LIMKi3 (1 µM), inhibited contractions of prostate strips, which were induced by electrical field stimulation, α1 -adrenoceptor agonists phenylephrine and methoxamine and the TXA2 analogue, U46619. LIMK inhibition in prostate tissues and cultured stromal cells (WPMY-1) was confirmed by cofilin phosphorylation, which was reduced by SR7826 and LIMKi3. In WPMY-1 cells, SR7826 and LIMKi3 caused breakdown of actin filaments and reduced viability. CONCLUSIONS AND IMPLICATIONS: Smooth muscle tone in the hyperplastic human prostate may underlie the effects of LIMKs, which promote contraction. Contraction of prostate strips can be inhibited by small molecule LIMK inhibitors.

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.

LIM kinase 1 interacts with myosin-9 and alpha-actinin-4 and promotes colorectal cancer progression.

BACKGROUND: LIM kinase 1 (LIMK1) is a key regulator of the cytoskeletal organisation involved in cell proliferation and migration. Even though LIMK1 is frequently dysregulated in epithelial cancers, the role and mechanisms of LIMK1 in colorectal cancer (CRC) remains unclear. METHODS: Immunohistochemical analysis was performed to examine the expression and clinical significance of LIMK1 in CRC samples. Loss- and gain-of-function assay was performed to investigate the effects of aberrant expression on cellular biological behaviour of CRC cells in vitro and in vivo. Immunoblotting and immunoprecipitation was used to screen LIMK1-related signalling pathways and downstream factors. RESULTS: In this study, our results showed that LIMK1 was upregulated in CRC tissues and localised in both the cytoplasm and the nucleus of CRC cells. Overexpression of LIMK1 in cytoplasmic and nuclear subcellular compartments was closely related to tumour metastasis and poor prognosis of CRC patients. Enhanced expression of cytoplasmic and nuclear LIMK1 significantly increased cell proliferation and migration by driving epithelial-mesenchymal transition and activating the PI3K/Akt signal pathway in vitro as well as promoting growth and metastasis of CRC xenografts, whereas opposite effects were achieved in LIMK1-silenced cells. Furthermore, we identified two tumour metastasis-associated proteins, MYH9 and ACTN4, as direct targets of LIMK1, which were required for a LIMK1-mediated aggressive phenotype. CONCLUSIONS: These findings indicate that LIMK1 plays a critical role in promoting CRC progression at subcellular level. Our findings provide new insights into the metastasis of CRC and advocate for the development of clinical intervention strategies against advanced CRC.

Overexpression of LIMK1 promotes migration ability of multidrug-resistant osteosarcoma cells.

Multidrug resistance (MDR) to chemotherapy is a major obstacle in the treatment of cancer and the resistance process is multifactorial. Studies on multidrug resistance mechanisms relied on the availability of cancer multidrug resistance cell lines that have been established. In this study we successfully established a multidrug resistance cell line MG63/VCR derived from human osteosarcoma cell line MG63 based on the induction by vincristine. MG63/VCR cells exhibited high resistance to vincristine and other anticancer drugs, accompanied by upregulated expression of MDR-associated genes MDR1, MRP1, and Bcl-2. Notably, we found that MG63/VCR cells exhibited higher migration ability compared to parental MG63 cells. Moreover, we demonstrated that LIMK1, a key regulator of actin cytoskeleton, was overexpressed at both mRNA and protein levels in MG63/VCR cells and the higher LIMK1 protein level was correlated with higher level of phosphorylated cofilin. In addition, knockdown of LIMK1 abolished the higher migration ability of MG63/ VCR cells. These results suggest that LIMK1 overexpression contributes to the invasion and metastasis of drug-resistant osteosarcoma and reveal LIMK as a novel therapeutic target for drug resistant osteosarcoma.

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).

Plexin C1, a receptor for semaphorin 7a, inactivates cofilin and is a potential tumor suppressor for melanoma progression.

Melanocytes are progenitor cells for melanoma, which arises through step-wise progression from dysplastic to invasive, to metastatic tumor. Our previous data showed that semaphorin 7A (Sema7A), a protein involved in axon guidance, stimulates melanocyte adhesion and dendricity through opposing actions of beta1-integrin and Plexin C1 receptors. We now show that Plexin C1 is diminished or absent in human melanoma cell lines; analysis of tissue microarrays of nevi, melanoma, and metastatic melanoma showed a decrease in Plexin C1 expression in metastatic melanoma, and an inverse correlation of Plexin C1 expression with depth of invasion. We examined the signaling intermediates of Sema7A and downstream targets of Plexin C1 in human melanocytes. Sema7A activated mitogen-activated protein kinase and inactivated cofilin, an actin-binding protein involved in cell migration. When Plexin C1 expression was silenced, Sema7A failed to phosphorylate cofilin, indicating that cofilin is downstream of Plexin C1. Further, Lim kinase II, a protein that phosphorylates cofilin, is upregulated by Sema7A in a Plexin C1-dependent manner. These data identify Plexin C1 as a potential tumor suppressor protein in melanoma progression, and suggest that loss of Plexin C1 expression may promote melanoma invasion and metastasis through loss of inhibitory signaling on cofilin activation.

Dynamic compression of chondrocytes induces a Rho kinase-dependent reorganization of the actin cytoskeleton.

The signal transduction mechanisms in chondrocytes that recognize applied forces and elicit the appropriate biochemical cellular responses are not well characterized. A current theory is that the actin cytoskeleton provides an intracellular framework onto which mechanosensation mechanisms are assembled. The actin cytoskeleton is linked to the extracellular matrix at multi-protein complexes called focal adhesions, and evidence exists that focal adhesions mediate the conversion of external physical forces into appropriate biochemical signal transduction events. The Rho GTPases affect the arrangement of actin cytoskeletal structures, and enhance the formation of focal adhesions, which link the cytoskeleton to the extracellular matrix. A major effector pathway downstream of Rho is the activation of Rho kinase (ROCK), which phosphorylates and activates Lim kinase, which in turn phosphorylates and inhibits the actin-depolymerizing protein cofilin. The objectives of this study were threefold: first, to quantify the actin reorganization in response to dynamic compression of agarose-embedded chondrocytes. Second, to test whether Rho kinase is required for the actin cytoskeletal reorganization induced by dynamic compression. Third, to test whether dynamic compression alters the intracellular localization of Rho kinase and actin remodeling proteins in chondrocytes. Dynamic compression of agarose-embedded chondrocytes induced actin cytoskeletal remodeling causing a significant increase in punctate F-actin structures. Rho kinase activity was required for these cytoskeletal changes. Dynamic compression increased the amount of phosphorylated Rho kinase. The chemokine CCL20 and inducible nitric oxide synthase (iNOS) were the most highly upregulated genes by dynamic compression and this response was reduced by the Rho kinase inhibitors. In conclusion, we show that dynamic compression induces changes in the actin cytoskeleton of agarose-embedded chondrocytes, and we establish methodology to quantify these changes. Furthermore, we show that Rho kinase activity is required for this actin reorganization and gene expression induced by dynamic compression.