Masato Maeda

The ezrin/radixin/moesin (ERM) proteins are involved in actin filament/plasma membrane interaction that is regulated by Rho. We examined whether ERM proteins are directly phosphorylated by Rho-associated kinase (Rho-kinase), a direct target of Rho. Recombinant full-length and COOH-terminal half radixin were incubated with constitutively active catalytic domain of Rho-kinase, and approximately 30 and approximately 100% of these molecules, respectively, were phosphorylated mainly at the COOH-terminal threonine (T564). Next, to detect Rho-kinase-dependent phosphorylation of ERM proteins in vivo, we raised a mAb that recognized the T564-phosphorylated radixin as well as ezrin and moesin phosphorylated at the corresponding threonine residue (T567 and T558, respectively). Immunoblotting of serum-starved Swiss 3T3 cells with this mAb revealed that after LPA stimulation ERM proteins were rapidly phosphorylated at T567 (ezrin), T564 (radixin), and T558 (moesin) in a Rho-dependent manner and then dephosphorylated within 2 min. Furthermore, the T564 phosphorylation of recombinant COOH-terminal half radixin did not affect its ability to bind to actin filaments in vitro but significantly suppressed its direct interaction with the NH2-terminal half of radixin. These observations indicate that the Rho-kinase-dependent phosphorylation interferes with the intramolecular and/ or intermolecular head-to-tail association of ERM proteins, which is an important mechanism of regulation of their activity as actin filament/plasma membrane cross-linkers.

BACKGROUND: Fabry's disease is considered very rare. Left ventricular hypertrophy is one of the common manifestations in adults with classic hemizygous disease. Recently, several cases of an atypical variant of hemizygous Fabry's disease, with manifestations limited to the heart, have been reported. Therefore, we assessed the incidence of hemizygosity for Fabry's disease among male patients with left ventricular hypertrophy. METHODS: We measured plasma alpha-galactosidase activity in 230 consecutive male patients with left ventricular hypertrophy. Clinical manifestations were assessed, endomyocardial biopsies were performed, and the patients were screened for mutations in the alpha-galactosidase gene. RESULTS: Seven of the 230 patients with left ventricular hypertrophy (3 percent) had low plasma alpha-galactosidase activity (0.4 to 1.2 nmol per hour per milliliter; 4 to 14 percent of the mean value in normal controls). These seven unrelated patients, ranging in age from 55 to 72 years, did not have angiokeratoma, acroparesthesias, hypohidrosis, or corneal opacities, which are typical manifestations of Fabry's disease. Endomyocardial biopsy was performed in five patients and revealed marked sarcoplasmic vacuolization in all five. Samples from four patients were examined by electron microscopy and revealed typical lysosomal inclusions with a concentric lamellar configuration in all four. Two patients had novel missense mutations in exon 1 (Ala20Pro) and exon 6 (Met296lle). The remaining five had no mutations in the coding region of the alpha-galactosidase gene, but the amounts of the alpha-galactosidase messenger RNA were markedly lower than normal. CONCLUSIONS: Seven unrelated patients with atypical variants of hemizygous Fabry's disease were found among 230 men with left ventricular hypertrophy (3 percent). Fabry's disease should be considered as a cause of unexplained left ventricular hypertrophy.

The cadherin molecules at adherens junctions have multiple isoforms. Cadherin isoform switching (cadherin switching) occurs during normal developmental processes to allow cell types to segregate from one another. Tumor cells often recapitulate this activity and the result is an aggressive tumor cell that gains the ability to leave the site of the tumor and metastasize. At present, we understand some of the mechanisms that promote cadherin switching and some of the pathways downstream of this process that influence cell behavior. Specific cadherin family members influence growth-factor-receptor signaling and Rho GTPases to promote cell motility and invasion. In addition, p120-catenin probably plays multiple roles in cadherin switching, regulating Rho GTPases and stabilizing cadherins.

Epithelium-to-mesenchyme transitions (EMTs) are characterized by morphological and behavioral changes in cells. During an EMT, E-cadherin is downregulated while N-cadherin is upregulated. The goal of this study was to understand the role cadherin switching plays in EMT using a classical model system: transforming growth factor beta1 (TGF-beta1)-mediated EMT in mammary epithelial cells. We showed that stress fibers and focal adhesions are increased, and cell-cell junctions are decreased in response to TGF-beta1. Moreover, these changes were reversible upon removal of TGF-beta1. Downregulation of E-cadherin and upregulation of N-cadherin were both transcriptional. Neither experimental knockdown nor experimental overexpression of N-cadherin interfered with the morphological changes. In addition, the morphological changes associated with EMT preceded the downregulation of E-cadherin. Interestingly, TGF-beta1-induced motility in N-cadherin-knockdown cells was significantly reduced. Together, these data suggest that cadherin switching is necessary for increased motility but is not required for the morphological changes that accompany EMT.

Epithelial-to-mesenchymal transition (EMT) is a fundamental biological process whereby epithelial cells lose their polarity and undergo a transition to a mesenchymal phenotype. When cancer cells invade adjacent tissues, they use a mechanism akin to EMT, and understanding the molecular mechanisms that drive this transition will facilitate studies into new targets for prevention of metastasis. Extracellular stimuli, such as growth factors, and their cytosolic effectors cooperate to promote EMT. In highly fibrotic cancers like lung cancer, it is thought that extracellular matrix molecules, including collagen, can initiate signals that promote EMT. Here, we present data showing that collagen I induces EMT in non-small cell lung cancer cell lines, which is prevented by blocking transforming growth factor (TGF)-beta3 signaling. In addition, we show that collagen I-induced EMT is prevented by inhibitors of phosphoinositide 3-kinase and extracellular signal-related kinase signaling, which promotes transcription of TGF-beta3 mRNA in these cells. Thus, our data are consistent with the hypothesis that collagen I induces EMT in lung cancer cells by activating autocrine TGF-beta3 signaling. Epidermal growth factor also seems to initiate EMT via a TGF-dependent mechanism.