Masayoshi Uehata

Calcium sensitization of smooth muscle mediated by a Rho-associated protein kinase in hypertension.

Abnormal smooth-muscle contractility may be a major cause of disease states such as hypertension, and a smooth-muscle relaxant that modulates this process would be useful therapeutically. Smooth-muscle contraction is regulated by the cytosolic Ca2+ concentration and by the Ca2+ sensitivity of myofilaments: the former activates myosin light-chain kinase and the latter is achieved partly by inhibition of myosin phosphatase. The small GTPase Rho and its target, Rho-associated kinase, participate in this latter mechanism in vitro, but their participation has not been demonstrated in intact muscles. Here we show that a pyridine derivative, Y-27632, selectively inhibits smooth-muscle contraction by inhibiting Ca2+ sensitization. We identified the Y-27632 target as a Rho-associated protein kinase, p160ROCK. Y-27632 consistently suppresses Rho-induced, p160ROCK-mediated formation of stress fibres in cultured cells and dramatically corrects hypertension in several hypertensive rat models. Our findings indicate that p160ROCK-mediated Ca2+ sensitization is involved in the pathophysiology of hypertension and suggest that compounds that inhibit this process might be useful therapeutically.

An essential part for Rho-associated kinase in the transcellular invasion of tumor cells

Adhesion of tumor cells to host cell layers and subsequent transcellular migration are pivotal steps in cancer invasion and metastasis. The small GTPase Rho controls cell adhesion and motility through reorganization of the actin cytoskeleton and regulation of actomyosin contractility. Cultured rat MM1 hepatoma cells migrate through a mesothelial cell monolayer in vitro in a serum–dependent, Rho–mediated manner. Among several proteins isolated as putative target molecules of Rho, the ROCK (ROK) family of Rho–associated serine–threonine protein kinases are thought to participate in the induction of focal adhesions and stress fibers in cultured cells, and to mediate calcium sensitization of smooth muscle contraction by enhancing phosphorylation of the regulatory light chain of myosin. Transfection of MM1 cells with cDNA encoding a dominant active mutant of ROCK conferred invasive activity independently of serum and Rho. In contrast, expression of a dominant negative, kinase–defective ROCK mutant substantially attenuated the invasive phenotype. A specific ROCK inhibitor (Y–27632; ref. 11) blocked both Rho–mediated activation of actomyosin and invasive activity of these cells. Furthermore, continuous delivery of this inhibitor using osmotic pumps considerably reduced the dissemination of MM1 cells implanted into the peritoneal cavity of syngeneic rats. These results indicate that ROCK plays an essential part in tumor cell invasion, and demonstrate its potential as a therapeutic target for the prevention of cancer invasion and metastasis.

Use and properties of ROCK-specific inhibitor Y-27632

Publisher Summary This chapter discusses the use and properties of Rho kinase (ROCK)-specific inhibitor Y-27632. The chapter describes three examples of in vitro applications of Y-27632 in tissues and cultured cells. Y-27632 and related compounds specifically bind to the ROCK family of kinases and inhibit their kinase activity. ROCK family of kinases is involved in the increase of actomyosin-based contractility through phosphorylation and down regulation of myosin phosphatase.Y-27632 inhibits ROCK by competing with ATP for binding to the catalytic site. The use of Y-27632 as a smooth muscle relaxant is also discussed in the chapter. The chapter explains the inhibition of lysophosphatidic acid (LPA)-induced neurite retraction in N1E-115 neuroblastoma cells and specificity of Y-27632 actions. The chapter concludes with a discussion of in viv o applications of Y-27632.

Wf-536 prevents tumor metastasis by inhibiting both tumor motility and angiogenic actions.

The signaling pathway of Rho and Rho-associated coiled-coil forming protein kinase (ROCK) is involved in tumor metastasis. In the present study, we investigated the suppressive effect of a novel inhibitor of ROCK, Wf-536 [(+)-(R)-4-(1-Aminoethyl)-N-(4-pyridyl) benzamide monohydrochloride], on spontaneous tumor metastasis in vivo and analyzed its action on tumor cell motility and angiogenesis to clarify its action mechanism. Wf-536 (0.3-3 mg/kg/day) was found to inhibit Lewis lung carcinoma (LLC) metastasis and LLC-induced angiogenesis in orally treated mice; in vitro, it inhibited both invasion and migration by LLC cells and invasion, migration, and formation of capillary-like tubes on Matrigel by endothelial cells, without cytotoxicity or anti-proliferative action in either cell type. We conclude that Wf-536 has tumor anti-metastatic activity which may depend on inhibition of tumor motility and angiogenesis. The findings support its further clinical development as an anti-metastatic agent.

WF-536 INHIBITS METASTATIC INVASION BY ENHANCING THE HOST CELL BARRIER and INHIBITING TUMOUR CELL MOTILITY

1. Rho‐associated coiled‐coil forming protein serine/threonine kinase (ROCK) is involved in the development of tumour metastasis. Wf‐536, (+)‐(R)‐4‐(1‐Aminoethyl)‐N‐(4‐pyridyl) benzamide monohydrochloride, a novel inhibitor of ROCK, inhibits tumour metastasis in some animal models. To metastasise, tumour cells have to disturb the tight intercellular junctions and the basement membrane matrix of the host tissue, which, respectively, create an intercellular barrier and the extracellular membrane. To clarify the mechanism of Wf‐536 in inhibition of tumour metastasis, we analysed the effect of Wf‐536 on the transition of tumour cells through the host cell layer and the basement membrane in in vitro systems.

Direct in Vitro and in Vivo Evidence for Interaction between Hsp47 Protein and Collagen Triple Helix

Background: Procollagen needs the collagen-specific molecular chaperone Hsp47 for correct folding. Results: Hsp47 binds to the triple-helix form of collagen model peptides in vitro and in vivo, but not to the monomer form. Conclusion: Hsp47 functions for triple-helix collagen as a molecular chaperone in the endoplasmic reticulum. Significance: This study provides the basis of the functional mechanism of Hsp47 in collagen molecular maturation. Hsp47 (heat shock protein 47), a collagen-specific molecular chaperone, is essential for the maturation of various types of procollagens. Previous studies have suggested that Hsp47 may preferentially recognize the triple-helix form of procollagen rather than unfolded procollagen chains in the endoplasmic reticulum. However, the underlying mechanism has remained unclear because of limitations in the available methods for detecting in vitro and in vivo interactions between Hsp47 and collagen. In this study, we established novel methods for this purpose by adopting a time-resolved FRET technique in vitro and a bimolecular fluorescence complementation technique in vivo. Using these methods, we provide direct evidence that Hsp47 binds to collagen triple helices but not to the monomer form in vitro. We also demonstrate that Hsp47 binds a collagen model peptide in the trimer conformation in vivo. Hsp47 did not bind collagen peptides that had been modified to block their ability to form triple helices in vivo. These results conclusively indicate that Hsp47 recognizes the triple-helix form of procollagen in vitro and in vivo.

Small guanosine triphosphatase Rho/Rho-associated kinase as a novel regulator of intracellular redistribution of lysosomes in invasive tumor cells.

Abstract. To investigate the role of RhoA on the intracellular membrane dynamics of lysosomes in rat hepatoma cells (MM1), we analyzed the localization of lysosomal aspartic proteinase cathepsin D by confocal immunofluorescence microscopy in the dominant active RhoA-transfected cells. Here we show that the transfection of the dominant active form of human small guanosine triphosphatase (GTPase) RhoA in MM1 cells, a highly invasive cell line, causes the redistribution and spreading of small punctate structures stained for cathepsin D throughout the cytoplasm. We found that the microtubule organization was markedly different in the two cell lines: uniformly developed and polymerized microtubule filaments were seen in the mock transfectants; however, the dynamic organization of microtubules was less pronounced in the active RhoA transfectants. Furthermore, we found for the first time that a selective inhibitor of Rho-associated kinase (p160ROCK), Y-27632, impeded the subcellular spreading of cathepsin D staining and promoted reclustering of cathepsin D toward the perinuclear region in the active RhoA-transfected cells. To our knowledge, this is the first indication that the RhoA/ROCK-mediated signaling pathway is involved in the intracellular membrane dynamics of lysosomes by regulating the cytoskeletal microtubule organization as well as the actin cytoskeletons.

A quantitative affinity-based technique for the identification of potential lead compounds

Here, we describe the development and validation of a quantitative analytical method for rapid evaluation of protein-compound interactions. The method uses size-exclusion chromatography in a 96-well format with liquid chromatography/mass spectrometry (qSEC-LC/MS) by which the amount of a compound that was originally in complex with a target protein is determined as an indicator of the binding affinity. Proof of concept of this new analytical approach was performed using a thrombin-inhibitor model. The results showed that the qSEC-LC/MS could be developed into an effective affinity-based analytical technique, despite a few limitations such as difficulty in determining the K(d) value accurately.