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Supplementary Table S2

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The growth and progression of cancers are crucially regulated by the tumor microenvironment where tumor cells and stromal cells are mutually associated. In this study, we found that stomatin expression was markedly upregulated by the interaction between prostate cancer cells and stromal cells. Stomatin suppressed cancer cell proliferation and enhanced apoptosis in vitro and inhibited xenograft tumor growth in vivo. Stomatin inhibited Akt activation, which is mediated by phosphoinositide-dependent protein kinase 1 (PDPK1). PDPK1 protein stability was maintained by its binding to HSP90. Stomatin interacted with PDPK1 and interfered with the PDPK1–HSP90 complex formation, resulting in decreased PDPK1 expression. Knockdown of stomatin in cancer cells elevated Akt activation and promoted cell increase by promoting the interaction between PDPK1 and HSP90. Clinically, stomatin expression levels were significantly decreased in human prostate cancer samples with high Gleason scores, and lower expression of stomatin was associated with higher recurrence of prostate cancer after the operation. Collectively, these findings demonstrate the tumor-suppressive effect of stromal-induced stomatin on cancer cells.Significance:These findings reveal that interactions with stromal cells induce expression of stomatin in prostate cancer cells, which suppresses tumor growth via attenuation of the Akt signaling axis.

Transmembrane protein 168 (TMEM168) was found to be localized on the nuclear membrane. A heterozygous mutation (c.1616G>A, p. R539Q) in TMEM168 was identified in patients with Brugada syndrome. This mutation reduced expression of cardiomyocyte sodium channel Nav1.5 via Nedd4-2 E3 ubiquitin ligase-induced ubiquitination and degradation. However, the detailed molecular mechanism provoked by the TMEM168 mutant remains unclear. Here, we demonstrated that small heat shock protein αB-crystallin, which can bind to Nav1.5 and Nedd4-2 and interfere with the association of both proteins, was strongly recruited from the cell surface to the perinuclear region because of the much higher affinity of αB-crystallin with the TMEM168 mutant than with wild-type TMEM168. Following knockdown of αB-crystallin in HL-1 cardiomyocytes, the interaction of Nav1.5 with Nedd4-2 was increased, despite the reduced expression of Nav1.5. Moreover, reduction of Nav1.5 expression by αB-crystallin knockdown was rescued in the presence of a proteasome inhibitor MG-132, suggesting the importance of the αB-crystallin-modulated ubiquitin–proteasome system for the stability of Nav1.5 expression. Collectively, the balance of molecular interactions among Nav1.5, Nedd4-2 and αB-crystallin plays a role in the regulation of cardiomyocyte cell surface expression of Nav1.5, and the TMEM168 mutant disturbs this balance, resulting in a decrease in Nav1.5 expression.

Background: Glutamic acid, an amino acid that exhibits umami taste, is utilized in Japanese food and is abundant in human milk. We examined the influence of maternal habitual eating behavior on glutamic acid concentration in human milk. Research Aim: To determine the association between maternal dietary behaviors at the end of pregnancy and the 1st month postpartum and glutamic acid concentration in colostrum and mature milk. Method: This was a prospective, correlational, one-group longitudinal study. Women aged 20–30 years during the third trimester of pregnancy ( N = 30) consented to participate and completed the data collection. Dietary history questionnaires were used to measure food intake. Glutamic acid levels in whey from colostrum and mature milk and in plasma during late pregnancy and the first month postpartum were measured. Data were considered significant at p < .05. Basic statistics, correlation coefficients analysis, unpaired t test, and one-way analysis of variance were performed. Results: Glutamic acid concentrations in human milk and plasma were found to be significantly associated with the consumption of several different foods. There was no association between glutamic acid concentrations in human milk and plasma or between glutamic acid concentrations in colostrum and mature milk. The glutamic acid content of mature milk differed by physical activity level (mild and moderate) during the first month postpartum ( t [46] = 2.87, p < .01). Conclusion: There was no clear association between habitual dietary behavior and glutamic acid concentration in human milk. However, maternal factors other than diet may be important and require additional research.

The growth and progression of cancers are crucially regulated by the tumor microenvironment where tumor cells and stromal cells are mutually associated. In this study, we found that stomatin expression was markedly upregulated by the interaction between prostate cancer cells and stromal cells. Stomatin suppressed cancer cell proliferation and enhanced apoptosis in vitro and inhibited xenograft tumor growth in vivo. Stomatin inhibited Akt activation, which is mediated by phosphoinositide-dependent protein kinase 1 (PDPK1). PDPK1 protein stability was maintained by its binding to HSP90. Stomatin interacted with PDPK1 and interfered with the PDPK1–HSP90 complex formation, resulting in decreased PDPK1 expression. Knockdown of stomatin in cancer cells elevated Akt activation and promoted cell increase by promoting the interaction between PDPK1 and HSP90. Clinically, stomatin expression levels were significantly decreased in human prostate cancer samples with high Gleason scores, and lower expression of stomatin was associated with higher recurrence of prostate cancer after the operation. Collectively, these findings demonstrate the tumor-suppressive effect of stromal-induced stomatin on cancer cells. Significance: These findings reveal that interactions with stromal cells induce expression of stomatin in prostate cancer cells, which suppresses tumor growth via attenuation of the Akt signaling axis.

Perivascular adipose tissue (PVAT) secretes large amounts of inflammatory mediators and plays a certain role in atherosclerosis formation from the exterior of the vessel. In the present study, we examined the expression level of inflammation-related mediators using adipose tissue samples harvested from patients with and without coronary artery disease (CAD). The subjects were 23 patients who underwent elective coronary bypass surgery (CAD group) and 17 patients who underwent elective mitral valve surgery (non-CAD group) between January 2017 and March 2018. The adipose tissue was harvested from three sites: the ascending aorta (AO), subcutaneous fat (SC), and pericoronary artery (CO) for the measurement of the expression levels of interleukin (IL) -1β, IL-6, IL-10, tumor necrosis factor (TNF) -α, interferon (INF) -γ, and arginase (Arg) -1. In both the non-CAD and CAD groups, the expression levels of all mediators, except Agr-1, which showed a tendency to have higher levels in the SC than in the AO and CO, tended to upregulate in the AO than in the SC and CO. The CAD group had higher values of almost all mediators, except Arg-1. Most importantly, the expression levels of IL-1β, IL-6, and IL-10 in the coronary artery were significantly higher in the CAD group. The expression levels of inflammatory mediators in the pericoronary adipose tissue were significantly higher in the CAD than in the non-CAD group. The adipose tissue appears to influence atherosclerosis formation from the exterior of the coronary artery.

Heart failure is one of the leading causes of death worldwide. RhoA, a small GTPase, governs actin dynamics in various tissue and cell types, including cardiomyocytes; however, its involvement in cardiac function has not been fully elucidated. Here, we generated cardiomyocyte-specific RhoA conditional knockout (cKO) mice, which demonstrated a significantly shorter lifespan with left ventricular dilation and severely impaired ejection fraction. We found that the cardiac tissues of the cKO mice exhibited structural disorganization with fibrosis and also exhibited enhanced senescence compared with control mice. In addition, we show that cardiomyocyte mitochondria were structurally abnormal in the aged cKO hearts. Clearance of damaged mitochondria by mitophagy was remarkably inhibited in both cKO cardiomyocytes and RhoA-knockdown HL-1 cultured cardiomyocytes. In RhoA-depleted cardiomyocytes, we reveal that the expression of Parkin, an E3 ubiquitin ligase that plays a crucial role in mitophagy, was reduced, and expression of N-Myc, a negative regulator of Parkin, was increased. We further reveal that the RhoA-Rho kinase axis induced N-Myc phosphorylation, which led to N-Myc degradation and Parkin upregulation. Re-expression of Parkin in RhoA-depleted cardiomyocytes restored mitophagy, reduced mitochondrial damage, attenuated cardiomyocyte senescence, and rescued cardiac function both in vitro and in vivo. Finally, we found that patients with idiopathic dilated cardiomyopathy without causal mutations for dilated cardiomyopathy showed reduced cardiac expression of RhoA and Parkin. These results suggest that RhoA promotes Parkin-mediated mitophagy as an indispensable mechanism contributing to cardioprotection in the aging heart.

Brugada syndrome (BrS) is an inherited channelopathy responsible for almost 20% of sudden cardiac deaths in patients with nonstructural cardiac diseases. Approximately 70% of BrS patients, the causative gene mutation(s) remains unknown. In this study, we used whole exome sequencing to investigate candidate mutations in a family clinically diagnosed with BrS. A heterozygous 1616G>A substitution (R539Q mutation) was identified in the transmembrane protein 168 (TMEM168) gene of symptomatic individuals. Similar to endogenous TMEM168, both TMEM168 wild-type (WT) and mutant proteins that were ectopically induced in HL-1 cells showed nuclear membrane localization. A significant decrease in Na+ current and Nav 1.5 protein expression was observed in HL-1 cardiomyocytes expressing mutant TMEM168. Ventricular tachyarrhythmias and conduction disorders were induced in the heterozygous Tmem168 1616G>A knock-in mice by pharmacological stimulation, but not in WT mice. Na+ current was reduced in ventricular cardiomyocytes isolated from the Tmem168 knock-in heart, and Nav 1.5 expression was also impaired. This impairment was dependent on increased Nedd4-2 binding to Nav 1.5 and subsequent ubiquitination. Collectively, our results show an association between the TMEM168 1616G>A mutation and arrhythmogenesis in a family with BrS.

Anosmin-1 is a secreted glycoprotein encoded by the ANOS1 gene, and its loss of function causes Kallmann syndrome (KS), which is characterized by anosmia and hypogonadism due to olfactory bulb (OB) dysfunction. However, the physiological function of anosmin-1 remains to be elucidated. In KS, disordered angiogenesis is observed in OB, resulting in its hypoplasia. In this study, we examined the involvement of anosmin-1 in angiogenic processes. Anosmin-1 was detected on the vessel-like structure in OB of chick embryos, and promoted the outgrowth of vascular sprouts as shown by assays of OB tissue culture. Cell migration, proliferation, and tube formation of endothelial cells were induced by treatment with anosmin-1 as well as vascular endothelial growth factor-A (VEGF-A), and further enhanced by treatment with both of them. We newly identified that anosmin-1 activated VEGF receptor-2 (VEGFR2) by binding directly to it, and its downstream signaling molecules, phospholipase Cγ1 (PLCγ1) and protein kinase C (PKC). These results suggest that anosmin-1 plays a key role in the angiogenesis of developing OB through the VEGFR2–PLCγ1–PKC axis by enhancing the VEGF function.

Tumor growth and progression are complex processes mediated by mutual interactions between cancer cells and their surrounding stroma that include diverse cell types and acellular components, which form the tumor microenvironment. In this environment, direct intercellular communications play important roles in the regulation of the biological behaviors of tumors. However, the underlying molecular mechanisms are insufficiently defined. We used an in vitro coculture system to identify genes that were specifically expressed at higher levels in cancer cells associated with stromal cells. Major examples included epithelial membrane protein 1 (EMP1) and stomatin, which positively and negatively regulate tumor progression, respectively. EMP1 promotes tumor cell migration and metastasis via activation of the small GTPase Rac1, while stomatin strongly suppresses cell proliferation and induces apoptosis of cancer cells via inhibition of Akt signaling. Here we highlight important aspects of EMP1, stomatin, and their family members in cancer biology. Furthermore, we consider the molecules that participate in intercellular communications and signaling transduction between cancer cells and stromal cells, which may affect the phenotypes of cancer cells in the tumor microenvironment., Types of cell‐to‐cell contacts between cancer cells and stromal cells. The contacts regulate cancer cell behavior in the tumor microenvironment.

Diabetes mellitus (DM) causes injury to tissues and organs, including to the heart and kidney, resulting in increased morbidity and mortality. Thus, novel potential therapeutics are continuously required to minimize DM-related organ damage. We have previously shown that dipeptidyl peptidase III (DPPIII) has beneficial roles in a hypertensive mouse model, but it is unknown whether DPPIII has any effects on DM. In this study, we found that intravenous administration of recombinant DPPIII in diabetic db/db mice for eight weeks suppressed the DM-induced cardiac diastolic dysfunctions and renal injury without alteration of the blood glucose level. This treatment inhibited inflammatory cell infiltration and fibrosis in the heart, and blocked the increase in albuminuria by attenuating the disruption of the glomerular microvasculature and inhibiting the effacement of podocyte foot processes in the kidney. The beneficial role of DPPIII was, at least in part, mediated by the cleavage of a cytotoxic peptide, named Peptide 2, which was increased in db/db mice compared with normal mice. This peptide consisted of nine amino acids, was a digested fragment of complement component 3 (C3), and had an anaphylatoxin-like effect determined by the Miles assay and chemoattractant analysis. The effect was dependent on its interaction with the C3a receptor and protein kinase C-mediated RhoA activation downstream of the receptor in endothelial cells. In conclusion, DPPIII plays a protective role in the heart and kidney in a DM animal model through cleavage of a peptide that is a part of C3.

Brugada syndrome (BrS) is diagnosed by a typical electrocardiography (ECG) with ST-segment elevation in precordial leads and tends to induce sudden cardiac death (SCD) due to ventricular tachycardia/fibrillation. About 20% of SCDs in non-structural cardiac diseases are considered to be caused by BrS. In patients with BrS, loss of function mutations in the Na+ channel is often observed, but the causative gene mutation is not detected for about 70% of BrS patients. Here, we investigated a family with clinically diagnosed BrS, in which no known gene mutations related to BrS had not been found, by whole exome sequencing. Novel heterozygous variant (c. 1616G>A, p. R539Q) in transmembrane protein 168 (TMEM168) was identified only in symptomatic family members. Similar to endogenous TMEM168, both wild-type and mutant TMEM168 localized at the nuclear membrane. Na+ current density in whole-cell patch-clamp recordings was significantly reduced in HL-1 cardiomyocytes transfected with TMEM168 R539Q mutant, compared with those with wild-type TMEM168. Next, heterozygous Tmem168 1616G>A knock-in mice were generated by the CRISPR/Cas9 genome editing technology. Although the knock-in mice had no abnormalities in ECG at the physiological state, the treatment with ajmaline caused various arrhythmias including ventricular tachycardia/fibrillation in the knock-in mice, but not in wild-type mice. Na+ current density and the parameters of action potentials were remarkably impaired in the cardiomyocytes of the knock-in mice. Optical mapping analysis in the whole heart showed the reduced left ventricular conduction velocity in the knock-in mice. The expression of Nav1.5, an α-subunit of the cardiac Na+ channel, was significantly decreased in the mutant TMEM168-transfected HL-1 cells and the knock-in hearts. We found that the decrease was caused by the enhanced ubiquitination of Nav1.5, which was mediated by increased binding of Nedd4–2 E3 ubiquitin ligase to Nav1.5 in the knock-in hearts. Co-immunoprecipitation experiments demonstrated that overactivity of Nedd4–2 is a result of Tmem168 mutant-mediated sequestration of a chaperon protein αB-crystallin, a Nav1.5-binding molecule that interferes with the interaction of Nedd4–2 with Nav1.5. These findings reveal the molecular mechanism of TMEM168 R539Q mutation-induced fatal ventricular arrhythmias in BrS. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): JSPS Grants-in-aid for Scientific Research

The clinical implications of alcohol consumption have been extensively examined; however, its effects on brain structures in apparently healthy community-dwellers remain unclear. Therefore, we investigated the relationship between alcohol consumption and brain gray matter volume (GMV) in community-dwelling Japanese men using voxel-based morphometry (VBM). We recruited cognitively intact Japanese men, aged 40-79 years, from a population-based cohort in Shiga, Japan. Brain magnetic resonance imaging was performed, on average, 2 years after demographic and medical information was obtained in 2010-2014. A multivariable linear regression analysis of 639 men was conducted to elucidate the relationship between the amount of alcohol consumed and GMV. VBM statistics were analyzed by threshold-free cluster enhancement with a family-wise error rate of, Research members：Minoru Horie, Yoshihisa Nakagawa, Takashi Yamamoto, Yasutaka Nakano, Emiko Ogawa, Hiroshi Maegawa, Katsutaro Morino, Itsuko Miyazawa, Yoshiyuki Watanabe, Kazuhiko Nozaki, Ikuo Tooyama, Akihiko Shiino, Akira Andoh, Teruhiko Tsuru, Hisakazu Ogita, Naomi Miyamatsu, Yasuyuki Nakamura, Aya Kadota, Keiko Kondo, Sayuki Torii, Takashi Kadowaki, Sayaka Kadowaki, Sentaro Suzuki, Takahiro Ito, Ayako Kunimura, Hiroyoshi Segawa, Akira Fujiyoshi, Aya Higashiyama, Tomonori Okamura, Koichiro Azuma, Tatsuya Sawamura, Michiya Igase, Yasuharu Tabara, Akira Sekikawa, Emma J M Barinas-Mitchell, Daniel Edmundowicz, Takayoshi Ohkubo, Atsushi Hozawa, Yoshitaka Murakam, Nagako Okuda, Hisatomi Arima, Atsushi Satoh, Yoshikuni Kita, Takashi Hisamatsu, Masahiko Yanagita, Robert D Abbott, Seiko Ohno, Naoyuki Takashima, Naoko Miyagawa, Maryam Zaid, Yoshino Saito

Tumor metastasis is the most common cause of cancer death. Elucidation of the mechanism of tumor metastasis is therefore important in the development of novel, effective anti-cancer therapies to reduce cancer mortality. Interaction between cancer cells and surrounding stromal cells in the tumor microenvironment is a key factor in tumor metastasis. Using a co-culture assay system with human prostate cancer LNCaP cells and primary human prostate stromal cells, we identified epithelial membrane protein 1 (EMP1) as a gene with elevated expression in the cancer cells. The orthotopic injection of LNCaP cells overexpressing EMP1 (EMP1-LNCaP cells) into the prostate of nude mice induced lymph node and lung metastases, while that of control LNCaP cells did not. EMP1-LNCaP cells had higher cell motility and Rac1 activity than control LNCaP cells. These results were also observed in other lines of cancer cells. We newly identified copine-III as an intracellular binding partner of EMP1. Knockdown of copine-III attenuated the increased cell motility and Rac1 activity in EMP1-LNCaP cells. Reduced cell motility and Rac1 activity following knockdown of copine-III in EMP1-LNCaP cells were recovered by re-expression of wild-type copine-III, but not of a copine-III mutant incapable of interacting with EMP1, suggesting the importance of the EMP1-copine-III interaction. Phosphorylated and activated Src and a Rac guanine nucleotide exchange factor Vav2 were found to be involved in the EMP1-induced enhancement of cell motility and Rac1 activation. Moreover, EMP1 was highly expressed in prostate cancer samples obtained from patients with higher Gleason score. These results demonstrate that upregulation of EMP1 significantly increases cancer cell migration that leads to tumor metastasis, suggesting that EMP1 may play an essential role as a positive regulator of tumor metastasis.

Background: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. Methods: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. Results: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5 ) to form the acetylcholine-activated potassium channel ( I KACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5 , suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of I KACh channel function by increasing the basal current, even in the absence of m 2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective I KACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. Conclusions: The I KACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant I KACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective I KACh channel blocker. Thus, the I KACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the I KACh channel.

To present the characteristics and pathology of a patient with congenital achromatopsia. The patient was a 22-year-old Japanese woman who was 8 years old when she first visited our clinic. Comprehensive ophthalmic examinations including visual acuity measurements, perimetry, optical coherence tomography (OCT), fundus autofluorescence (FAF) imaging, electroretinography (ERG), and color vision tests were performed. Her genomic DNA was used as the template for the amplification of exons of five candidate genes for achromatopsia; CNGA3, CNGB3, GNAT2, PDE6C, and PDE6H, and the amplified products were sequenced. A missense mutation, found in the CNGA3, was studied both electrophysiologically and biochemically. Her phenotype was typical of congenital complete achromatopsia. She was followed for 14 years, and her vision and fundus findings were stable. However, the scotopic ERG b-waves at age 22 were smaller than those at age 8, and her FAF images showed increased autofluorescence in both maculae. Genetic examinations revealed combined heterozygous mutations of c.997_998delGA and p.M424V in the CNGA3 gene. The homomeric channel consisting of the CNGA3 subunit with the p.M424V mutation had a weak cGMP-activated current in patch-clamp recordings. In heterologous expression analyses, the expression at the cell surface of the mutant CNGA3 subunit was about 28 % of the wild type. The two novel mutations found in the CNGA3 gene, c.997_998delGA and p.M424V, can cause complete achromatopsia. The vision of the patient was stationary until the third decade of life although the FAF was altered at the age of 22 years.

Summary SGLT2 inhibitors offer strong renoprotection in subjects with diabetic kidney disease (DKD). But the mechanism for such protection is not clear. Here, we report that in damaged proximal tubules of high-fat diet-fed ApoE-knockout mice, a model of non-proteinuric DKD, ATP production shifted from lipolysis to ketolysis dependent due to hyperactivation of the mechanistic target of rapamycin complex 1 (mTORC1). We further found that empagliflozin raised endogenous ketone body (KB) levels, and thus its use or treatment with 1,3-butanediol, a KB precursor, prevented decreases in renal ATP levels and organ damage in the mice. The renoprotective effect of empagliflozin was abolished by gene deletion of Hmgcs2, a rate-limiting enzyme of ketogenesis. Furthermore, KBs attenuated mTORC1-associated podocyte damage and proteinuria in diabetic db/db mice. Our findings show that SGLT2 inhibition-associated renoprotection is mediated by an elevation of KBs that in turn corrects mTORC1 hyperactivation that occurs in non-proteinuric and proteinuric DKD.

Blue cone monochromacy (BCM) is characterized by loss of function of both OPN1LW (the first) and OPN1MW (the downstream) genes on the X chromosome. The purpose of this study was to investigate the first and downstream genes in the OPN1LW/OPN1MW array in four unrelated Japanese males with BCM. In Case 1, only one gene was present. Abnormalities were found in the promoter, which had a mixed unique profile of first and downstream gene promoters and a −71A > C substitution. As the promoter was active in the reporter assay, the cause of BCM remains unclear. In Case 2, the same novel mutation, M273K, was present in exon 5 of both genes in a two-gene array. The mutant pigments showed no absorbance at any of the wavelengths tested, suggesting that the mutation causes pigment dysfunction. Case 3 had a large deletion including the locus control region and entire first gene. Case 4 also had a large deletion involving exons 2–6 of the first gene. As an intact LCR was present upstream and one apparently normal downstream gene was present, BCM in Case 4 was not ascribed solely to the deletion. The deletions in Cases 3 and 4 were considered to have been caused by non-homologous recombination.

Vascular endothelial growth factors (VEGFs) include five molecules (VEGF-A, -B, -C, -D, and placental growth factor), and have various roles that crucially regulate cellular functions in many kinds of cells and tissues. Intracellular signal transduction induced by VEGFs has been extensively studied and is usually initiated by their binding to two classes of transmembrane receptors: receptor tyrosine kinase VEGF receptors (VEGF receptor-1, -2 and -3) and neuropilins (NRP1 and NRP2). In addition to many established results reported by other research groups, we have previously identified small G proteins, especially Ras homologue gene (Rho) and Ras-related protein (Rap), as important mediators of VEGF-A-stimulated signaling in cancer cells as well as endothelial cells. This review article describes the VEGF-A-induced signaling pathways underlying diverse cellular functions, including cell proliferation, migration, and angiogenesis, and the involvement of Rho, Rap, and their related molecules in these pathways.

Dysregulation of cell metabolism is a hallmark of cancer. The mevalonate pathway in lipid metabolism has been implicated as a potential target of cancer therapy for hepatocellular carcinoma (HCC). The role of the Forkhead Box M1 (FoxM1) transcription factor in HCC development has been well documented, however, its involvement in cancer metabolism of HCC has not been fully determined. Here, we hypothesized that FoxM1 is involved in the mevalonate pathway of cholesterol biosynthesis in HCC. Inhibition of the mevalonate pathway by statins, inhibitors of 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR), resulted in reduced expression of FoxM1 and increased cell death in human hepatoma cells. Re-exposure of mevalonate, a product of HMGCR, restored these effects. Likewise, knockdown of HMGCR reduced FoxM1 expression, indicating that FoxM1 expression was regulated by the mevalonate pathway in HCC. Mechanistically, protein geranylgeranylation was found to be responsible for FoxM1 expression and geranylgeranylated proteins, including RhoA, Rac1 or Cdc42, were shown to be involved in this process. In surgically resected human HCC tissues, the gene expression of FoxM1 had a positive correlation with that of the mevalonate pathway-related genes, such as HMGCR or sterol regulatory element-binding protein 2 (SREBP2). Furthermore, the gene expression of FoxM1 along with that of HMGCR or SREBP2 defined prognosis of HCC patients, suggesting the clinical significance of the mevalonate-FoxM1 pathway in human HCC. Our data indicate that FoxM1 links the mevalonate pathway to oncogenic signals in HCC. Thus, we propose a novel therapeutic approach to inhibit FoxM1 by targeting the mevalonate pathway for HCC.

Background Pressure overload induces cardiac hypertrophy, which often ends in heart failure. Afadin is an adaptor protein that is ubiquitously expressed and, in the heart, it localizes at intercalated disks. The current study aimed to examine the afadin-mediated cardiac phenotype in mice exposed to different types of pressure overload: transverse aortic constriction (TAC) burden and angiotensin II (Ang II) stimulation.Methods and Results:Conditional knockout mice with selective deletion of afadin (afadin cKO) in cardiomyocytes were generated. TAC-operated and Ang II-infused mice at 4 weeks had a similar degree of pressure overload and cardiac hypertrophy in the heart. In afadin cKO mice, TAC operation caused progressive left ventricular dysfunction and heart failure, while Ang II infusion did not deteriorate cardiac function. Furthermore, TAC operation produced more fibrosis and apoptosis in the heart than Ang II infusion, and the expression of growth differentiation factor 15, which can promote apoptosis, in the afadin cKO heart was higher in TAC-operated mice than Ang II-infused ones. Conclusions In the 2 pressure overload models, myocardial afadin is involved in mechanical stress-induced, but not pharmacological Ang II-related, compensated cardiac hypertrophy.

Dipeptidyl peptidases (DPPs) belong to one of the protease families classified under EC 3.4.14 in the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology. DPPs family consists of eight members in the mammalian species. They play a role in oligopeptide N-terminal processing and degradation of bioactive peptides. Over the past 20 years, most of the studies have been focused on DPP 4 that has important roles in metabolism and immunity. A large number of pharmacological inhibitors against DPP 4 have been tested rigorously and some of them are now used in the treatment of type 2 diabetes and obesity. In addition, current researches cast a spotlight on other physiological and pathological functions of DPP family members such as DPP 3 for the purpose of investigating their application as novel therapeutic compounds. In this review, we provide an update about the pathophysiological functions of DPPs, and discuss the future potential of the DPP family as pharmacological and therapeutic agents and targets.

Adhesive intercellular connections at cardiomyocyte intercalated disks (IDs) support contractile force and maintain structural integrity of the heart muscle. Disturbances of the proteins at IDs deteriorate cardiac function and morphology. An adaptor protein afadin, one of the components of adherens junctions, is expressed ubiquitously including IDs. At present, the precise role of afadin in cardiac physiology or disease is unknown. To explore this, we generated conditional knockout (cKO) mice with cardiomyocyte-targeted deletion of afadin. Afadin cKO mice were born according to the expected Mendelian ratio and have no detectable changes in cardiac phenotype. On the other hand, chronic pressure overload induced by transverse aortic constriction (TAC) caused systolic dysfunction, enhanced fibrogenesis and apoptosis in afadin cKO mice. Afadin deletion increased macrophage infiltration and monocyte chemoattractant protein-1 expression, and suppressed transforming growth factor (TGF) β receptor signaling early after TAC procedure. Afadin also associated with TGFβ receptor I at IDs. Pharmacological antagonist of TGFβ receptor I (SB431542) augmented mononuclear infiltration and fibrosis in the hearts of TAC-operated control mice. In conclusion, afadin is a critical molecule for cardiac protection against chronic pressure overload. The beneficial effects are likely to be a result from modulation of TGFβ receptor signaling pathways by afadin.

Aim Mutations in lipoprotein-associated phospholipase A2 (Lp-PLA2) are related to atherosclerosis. However, the molecular effects of Lp-PLA2 on atherosclerosis have not been fully investigated. Therefore, this study attempted to elucidate this issue. Methods Monocytes were isolated from randomly selected healthy male volunteers according to each Lp-PLA2 genotype (wild-type Lp-PLA2 [Lp-PLA2 (V/V)], the heterozygous V279F mutation [LpPLA2 (V/F)] and the homozygous V279F mutation [Lp-PLA2 (F/F)]) and differentiated into macrophages. The level of apoptosis in the macrophages following incubation without serum was measured using the annexin V/propidium iodide double staining method, and the underlying mechanisms were further examined using a culture cell line. Results The average plasma Lp-PLA2 concentration [Lp-PLA2 (V/V): 129.4 ng/mL, Lp-PLA2 (V/F): 70.7 ng/mL, Lp-PLA2 (F/F): 0.4 ng/mL] and activity [Lp-PLA2 (V/V): 164.3 nmol/min/mL, LpPLA2 (V/F): 100.9 nmol/min/mL, Lp-PLA2 (F/F): 11.6 nmol/min/mL] were significantly different between each genotype, although the basic clinical characteristics were similar. The percentage of apoptotic cells was significantly higher among the Lp-PLA2 (F/F) macrophages compared with that observed in the Lp-PLA2 (V/V) macrophages. This induction of apoptosis was independent of the actions of acetylated low-density lipoproteins. In addition, the transfection of the expression plasmid of V279F mutant Lp-PLA2 into Cos-7 cells or monocyte/macrophage-like U937 cells promoted apoptosis. The knockdown of Lp-PLA2 also increased the number of apoptotic cells. Among the cells expressing mutant Lp-PLA2, the caspase-7 activity was increased, while the activated Akt level was decreased. Conclusions The V279F mutation of Lp-PLA2 positively regulates the induction of apoptosis in macrophages and Cos-7 cells. An increase in the caspase-7 activity and a reduction in the activated Akt level are likely to be involved in this phenomenon.

The members of the family of epithelial membrane proteins (EMPs), EMP1, EMP2, and EMP3, possess four putative transmembrane domain structures and are composed of approximately 160 amino acid residues. EMPs are encoded by the growth arrest-specific 3 (GAS3)/peripheral myelin protein 22 kDa (PMP22) gene family. The GAS3/PMP22 family members play roles in cell migration, growth, and differentiation. Evidence indicates an association of these molecules with cancer progression and metastasis. Each EMP has pro- and anti-metastatic functions that are likely involved in the complex mechanisms of cancer progression. We have recently demonstrated that the upregulation of EMP1 expression facilitates cancer cell migration and invasion through the activation of a small GTPase, Rac1. The inoculation of prostate cancer cells overexpressing EMP1 into nude mice leads to metastasis to the lymph nodes and lungs, indicating that EMP1 contributes to metastasis. Pro-metastatic properties of EMP2 and EMP3 have also been proposed. Thus, targeting EMPs may provide new insights into their clinical utility. Here, we highlight the important aspects of EMPs in cancer biology, particularly invasiveness and metastasis, and describe recent therapeutic approaches.

Legumain (EC 3.4.22.34) is an asparaginyl endopeptidase. Strong legumain activity was observed in the mouse kidney, and legumain was found to be highly expressed in tumors. We previously reported that bovine kidney annexin A2 was co-purified with legumain and that legumain cleaved the N-terminal region of annexin A2 at an Asn residue in vitro and in vivo. In this study, we found a p53-binding site in intron 1 of the human legumain gene using computational analysis. To determine whether transcription of the legumain gene is regulated by p53, HCT116 cells were transfected with p53 siRNA and the effect of knockdown of p53 expression on legumain expression was examined. The results showed that expression levels of both legumain mRNA and protein were decreased in the siRNA-treated cells. Furthermore, enzyme activity of legumain was also increased by doxorubicin and its activity was reduced by knockdown of p53 in HCT116 cells. These results suggest that legumain expression and its enzyme activity are regulated by p53.

Legumain (EC 3.4.22.34) is an asparaginyl endopeptidase. Strong legumain activity was observed in the mouse kidney, and legumain was highly expressed in tumors. We previously reported that bovine kidney annexin A2 was co-purified with legumain and that legumain cleaved the N-terminal region of annexin A2 at an Asn residue in vitro. In this study, to determine whether annexin A2 is cleaved by legumain in vivo, siRNA-lipoplex targeting mouse legumain was injected into mouse tail veins. Mouse kidneys were then isolated and the effect of knockdown of legumain expression on annexin A2 cleavage was examined. The results showed that both legumain mRNA and protein expression levels were decreased in the siRNA-treated mouse kidneys and that legumain activity toward a synthetic substrate, Z-Ala-Ala-Asn-MCA, was decreased by about 40% in the kidney but not in the liver or spleen. Furthermore, cleavage of annexin A2 at the N-terminal region was decreased in the mouse kidney that had been treated with the legumain siRNA-lipoplex. These results suggest that legumain siRNA was delivered to the kidney by using LipoTrust and that the reduced legumain expression inhibited legumain-induced degradation of annexin A2 in vivo. (C) 2012 Elsevier Inc. All rights reserved.

Background:Smoking is an overwhelming, but preventable, risk factor for cardiovascular diseases (CVD), although smoking prevalence remains high in developed and developing countries in East Asia., Methods and results:In a population-based sample of 1019 Japanese men aged 40 to 79 years, without CVD, we examined cross-sectional associations of smoking status, cumulative pack-years, daily consumption, and time since cessation, with subclinical atherosclerosis at 4 anatomically distinct vascular beds, including coronary artery calcification, carotid intima-media thickness (CIMT) and plaque, aortic artery calcification (AoAC), and ankle-brachial index. Current, former, and never smoking were present in 32.3%, 50.0%, and 17.7%, respectively. Compared to never smokers, current smokers had significantly higher risks of subclinical atherosclerosis in all 4 circulations (eg, odds ratios for coronary artery calcification >0, 1.79 [95% CIs, 1.16-2.79]; CIMT >1.0 mm, 1.88 [1.02-3.47]; AoAC >0, 4.29 [2.30-7.97]; and ankle-brachial index 1.0 mm, 1.94 [1.13-3.34]; and AoAC >0, 2.55 [1.45-4.49]). Dose-response relationships of pack-years and daily consumption, particularly with CIMT, carotid plaque, AoAC, and ankle-brachial index, were observed among both current and former smokers, and even a small amount of pack-years or daily consumption among current smokers was associated with coronary artery calcification and AoAC, whereas time since cessation among former smokers was linearly associated with lower burdens of all atherosclerotic indices., Conclusions:Cigarette smoking was strongly associated with subclinical atherosclerosis in multiple vascular beds in Japanese men, and these associations attenuated with time since cessation.

Dipeptidyl peptidase III (DPP III) cleaves dipeptide residues from the N terminus of polypeptides ranging from 3 to 10 amino acids in length and is implicated in pathophysiological processes through the breakdown of certain oligopeptides or their fragments. In this study, we newly identified the biochemical properties of DPP III for angiotensin II (Ang II), which consists of 8 amino acids. DPP III quickly and effectively digested Ang II with K m = 3.7×10 −6 mol/L. In the in vivo experiments, DPP III remarkably reduced blood pressure in Ang II–infused hypertensive mice without alteration of heart rate. DPP III did not affect hemodynamics in noradrenalin-induced hypertensive mice or normotensive mice, suggesting specificity for Ang II. When DPP III was intravenously injected every other day for 4 weeks after Ang II osmotic minipump implantation in mice, Ang II–induced cardiac fibrosis and hypertrophy were significantly attenuated. This DPP III effect was at least similar to that caused by an angiotensin receptor blocker candesartan. Furthermore, administration of DPP III dramatically reduced the increase in urine albumin excretion and kidney injury and inflammation markers caused by Ang II infusion. Both DPP III and candesartan administration showed slight additive inhibition in the albumin excretion. These results reveal a novel potential use of DPP III in the treatment of hypertension and its protective effects on hypertension-sensitive organs, such as the heart and kidneys.

Novel mutations in the L visual pigment gene found in Japanese men with protan color-vision defect having a normal order L/M gene array

High molecular weight kininogen (HK) is a plasma glycoprotein with multiple functions, including the regulation of coagulation. We previously demonstrated that domain 5 (D5 H ), a functional domain of HK, and its derived peptides played an important role in the vitronectin-mediated suppression of cancer cell adhesion and invasion. However, the underlying mechanisms of the D5 H -mediated suppressive effects remain to be elucidated. Here, we showed that D5 H and its derivatives inhibited the collagen-mediated cell adhesion and invasion of human osteosarcoma MG63 cells. Using purified D5 H fused to glutathione- S -transferase (GST) and D5 H -derived peptides for column chromatography, an actin-binding protein, α-actinin-4, was identified as a binding protein of D5 H with high-affinity for P-5m, a core octapeptide of D5 H . Immunofluorescence microscopy demonstrated that D5 H co-localized with α-actinin-4 inside MG63 cells. In addition, exogenous GST-D5 H added to the culture media was transported into MG63 cells, although GST alone as a control was not. As α-actinin-4 regulates actin polymerization necessary for cell adhesion and is related to the integrin-dependent attachment of cells to the extracellular matrix, our results suggest that D5 H may modulate cell adhesion and invasion together with actinin-4.

We have analyzed L/M visual pigment gene arrays in 119 Japanese men with protanopia color vision defect and found that five had a normal gene order of L-M. Among the five men, two (identified as A376 and A642) had apparently normal L genes. To clarify their L gene defect, the whole L or M gene from A376 and control subjects was cloned in an expression vector. Total RNA extracted from the transfected HEK293 cells was analyzed by Northern blot and reverse transcription-polymerase chain reaction. The product from the cloned L gene of A376 was smaller than the normal control due to the absence of exon 3. To investigate such exon-skipping at splicing, minigenes of exon 3 accompanying introns 2 and 3 were prepared from A376, A642, and control subjects. The minigenes of A376 (L) and A642 (L) showed the product lacking exon 3 only, while the minigene of normal control N44 (L) showed the product retaining exon 3 only. Exchanging of introns 2 and 3 between the A376 (L) and N44 (L) minigenes showed that the skipping of exon 3 was caused by the exon itself. Seven differences in exon 3 between A376 (L) and N44 (L) were all within already-known polymorphisms as follows: G(151-3), C(153-1), G(155-3), A(171-1), T(171-3), G(178-1) and G(180-1) in A376 (L) and A642 (L), and A(151-3), A(153-1), C(155-3), G(171-1), G(171-3), A(178-1) and T(180-1) in N44 (L). An in vitro mutagenesis experiment with these nucleotides in the minigenes showed that exon 3 was completely skipped at splicing only in the haplotype observed in A376 (L) and A642 (L). These results suggest that complete skipping of exon 3 at splicing, due to the unique haplotype of the exon, causes loss of expression of L-opsin in these men.

Cell movement is an important cellular function not only in physiological but also in pathological conditions. Although numerous studies have been conducted to reveal the mechanism of cell movement, the full picture has yet to be depicted, likely due to the complex features of cell movement. We show here that the scaffold protein afadin dilute domain-interacting protein (ADIP), an afadin-binding protein, is involved in the regulation of cell movement. ADIP localized at the leading edge of moving cells in response to platelet-derived growth factor (PDGF) and was required for the formation of the leading edge and the promotion of cell movement. Impaired cell movement observed in ADIP knockdown cells was not rescued by expression of an ADIP mutant that is incapable of binding to afadin, leading to the notion that the function of ADIP in moving cells depends on its interaction with afadin. Knockdown of ADIP as well as knockdown of afadin inhibited the activation of the small G protein Rac, which is important for the formation of the leading edge and the promotion of cell movement. Furthermore, ADIP interacted with Vav2, a GDP/GTP exchange factor for Rac, in a Src phosphorylation-dependent manner, suggesting that ADIP mediates the activation of Rac through Vav2. These results indicate that ADIP plays an essential role in PDGF-induced cell movement by interacting with afadin and Vav2 and regulating the activation of Rac.

The nectin cell adhesion molecules interact in trans with each other through their extracellular regions and with afadin through their cytoplasmic tails, forming adherens junctions in cooperation with cadherins. In a single cell, Necl-5 (nectin-like molecule-5) localizes at the leading edge and regulates directional cell movement in response to a chemoattractant. In such a single cell, afadin also localizes at the leading edge without interacting with nectins or Necl-5. It remains unknown how the nectin-nectin and nectin-afadin interactions are initiated when moving cells contact each other to initiate the formation of adherens junctions. We show here that the Necl-5-nectin interaction induced by cell-cell contact enhances the nectin-afadin interaction. This interaction then enhances the nectin-nectin interaction, which further enhances the nectin-afadin interaction in a positive feedback manner. Thus, the Necl-5-nectin, nectin-nectin, and nectin-afadin interactions cooperatively increase the clustering of the nectin-afadin complex at the cell-cell contact sites, promoting the formation of the nectin-based cell-cell adhesion.

Rationale : Angiogenesis contributes to physiological and pathological conditions, including atherosclerosis. The Rap1 small G protein regulates vascular integrity and angiogenesis. However, little is known about the effectors of Rap1 involved in angiogenesis. It is not known whether afadin, an adherens junction protein that connects immunoglobulin-like adhesion molecule nectins to the actin cytoskeleton and binds activated Rap1, plays a role in angiogenesis. Objective : We investigated the role of endothelial afadin in angiogenesis and attempted to clarify the underlying molecular mechanism. Methods and Results : Treatment of human umbilical vein endothelial cells (HUVECs) with vascular endothelial growth factor (VEGF) and sphingosine 1-phosphate (S1P) induced the activation of Rap1. Activated Rap1 regulated intracellular localization of afadin. Knockdown of Rap1 or afadin by small interfering RNA inhibited the VEGF- and S1P-induced capillary-like network formation, migration, and proliferation, and increased the serum deprivation-induced apoptosis of HUVECs. Knockdown of Rap1 or afadin decreased the accumulation of adherens and tight junction proteins to the cell–cell contact sites. Rap1 regulated the interaction between afadin and phosphatidylinositol 3-kinase (PI3K), recruitment of the afadin–PI3K complex to the leading edge, and the activation of Akt, indicating the involvement of Rap1 and afadin in the PI3K–Akt signaling pathway. Binding of afadin to Rap1 regulated the activity of Rap1 in a positive-feedback manner. In vivo, conditional deletion of afadin in mouse vascular endothelium using a Cre-loxP system impaired the VEGF- and S1P-induced angiogenesis. Conclusions : These results demonstrate a novel molecular mechanism by which Rap1 and afadin regulate the VEGF- and S1P-induced angiogenesis.

Nectin-like molecule-5 (Necl-5) is an immunoglobulin (Ig)-like molecule that is up-regulated in many types of cancer cells. It was shown experimentally that Necl-5 enhances cell migration, proliferation, and metastasis, but its clinical significance has not been documented. The aim of this study was to observe the expression of Necl-5 in surgically resected primary lung adenocarcinomas and to investigate its clinical significance. A total of 63 surgically resected primary pulmonary adenocarcinoma tissues were investigated by immunohistochemistry for the expression of Necl-5. The relationship between expression of Necl-5 and clinicopathological features was analyzed, and the influence of Necl-5 expression on outcomes in these patients was assessed. A strong expression of Necl-5 by cancer cells was observed in 43 of the 63 tumors. The overexpression of Necl-5 by cancer cells was significantly associated with lymph node metastasis (P = 0.0398), TNM staging (P = 0.0367), and the bronchioloalveolar carcinoma ratio of tumors (P = 0.0423). Furthermore, the disease-free survival rate in patients with positive Necl-5 overexpression was significantly lower than that in patients with negative Necl-5 overexpression (P = 0.0004). Multivariate survival analysis revealed Necl-5 expression to be an independent risk factor for an unfavorable outcome (P = 0.0294). Additionally, an analysis including only the stage I cases revealed that the disease-free survival rate of the Necl-5-positive group was significantly lower than that of the Necl-5-negative group (P = 0.0192). These results indicate that Necl-5 plays a role in mediating tumor cell invasion and that the overexpression of Necl-5 in cancer cells has clinical significance for prognostic evaluation of patients with primary pulmonary adenocarcinoma. (Cancer Sci 2010; 101: 1326–1330)

Tight junctions (TJs) and adherens junctions (AJs) are major junctional apparatuses in epithelial cells. Claudins and junctional adhesion molecules (JAMs) are major cell adhesion molecules (CAMs) at TJs, whereas cadherins and nectins are major CAMs at AJs. Claudins and JAMs are associated with ZO proteins, whereas cadherins are associated with beta- and alpha-catenins, and nectins are associated with afadin. We previously showed that nectins first form cell-cell adhesions where the cadherin-catenin complex is recruited to form AJs, followed by the recruitment of the JAM-ZO and claudin-ZO complexes to the apical side of AJs to form TJs. It is not fully understood how TJ components are recruited to the apical side of AJs. We studied the roles of afadin and ZO-1 in the formation of TJs in Madin-Darby canine kidney (MDCK) cells. Before the formation of TJs, ZO-1 interacted with afadin through the two proline-rich regions of afadin and the SH3 domain of ZO-1. During and after the formation of TJs, ZO-1 dissociated from afadin and associated with JAM-A. Knockdown of afadin impaired the formation of both AJs and TJs in MDCK cells, whereas knockdown of ZO-1 impaired the formation of TJs, but not AJs. Re-expression of full-length afadin restored the formation of both AJs and TJs in afadin-knockdown MDCK cells, whereas re-expression of afadin-DeltaPR1-2, which is incapable of binding to ZO-1, restored the formation of AJs, but not TJs. These results indicate that the transient interaction of afadin with ZO-1 is necessary for the formation of TJs in MDCK cells.

In the hippocampus, synapses are formed between mossy fiber terminals and CA3 pyramidal cell dendrites and comprise highly developed synaptic junctions (SJs) and puncta adherentia junctions (PAJs). Dynamic remodeling of synapses in the hippocampus is implicated in learning and memory. Components of both the nectin–afadin and cadherin–catenin cell adhesion systems exclusively accumulate at PAJs. We investigated the role of afadin at synapses in mice in which the afadin gene was conditionally inactivated in hippocampal neurons. In these mutant mice, the signals for not only nectins, but also N-cadherin and β-catenin, were hardly detected in the CA3 area, in addition to loss of the signal for afadin, resulting in disruption of PAJs. Ultrastructural analysis revealed an increase in the number of perforated synapses, suggesting the instability of SJs. These results indicate that afadin is involved not only in the assembly of nectins and cadherins at synapses, but also in synaptic remodeling.

In response to chemoattractants, migrating cells form protrusions, such as lamellipodia and filopodia, and structures, such as ruffles over lamellipodia, focal complexes and focal adhesions at leading edges. The formation of these leading edge structures is essential for directional cell movement. Nectin-like molecule-5 (Necl-5) interacts in cis with PDGF receptor and integrin alpha(v)beta(3), and enhances the activation of signalling molecules associated with these transmembrane proteins, which results in the formation of leading edge structures and enhancement of directional cell movement. When migrating cells come into contact with each other, cell-cell adhesion is initiated, resulting in reduced cell velocity. Necl-5 first interacts in trans with nectin-3. This interaction is transient and induces down-regulation of Necl-5 expression at the cell surface, resulting in reduced cell movement. Cell proliferation is also suppressed by the down-regulation of Necl-5, because the inhibitory effect of Necl-5 on Sprouty2, a negative regulator of the Ras signalling, is diminished. PDGF receptor and integrin alpha(v)beta(3), which have interacted with Necl-5, then form a complex with nectin, which initiates cell-cell adhesion and recruits cadherin to the nectin-based cell-cell adhesion sites to form stable adherens junctions. The formation of adherens junctions stops cell movement, in part through inactivation of integrin alpha(v)beta(3) caused by the trans-interaction of nectin. Thus, nectin and Necl-5 play key roles in the regulation of cell movement and proliferation.

Integrin plays an essential role in the formation of cell-matrix junctions and is also involved in the fundamental cellular functions. In the process of the formation of cell-cell junctions, an immunoglobulin-like cell-cell adhesion molecule nectin initially trans-interacts together and promotes the formation of adherens junctions (AJs) cooperatively with another cell-cell adhesion molecule cadherin. The activation of integrin alpha(v)beta(3) is critically necessary for this nectin-induced formation of AJs. However, after the establishment of AJs, integrin alpha(v)beta(3) becomes inactive and retains the association with nectin at AJs. The molecular mechanism of this dynamic regulation of integrin alpha(v)beta(3) during the formation of AJs remains unclear. We found here that the expression of phosphatidylinositol-phosphate kinase type Igamma90 (PIPKIgamma90), which is involved in the regulation of integrin activation, in Madin-Darby canine kidney cells, preferentially reversed the inactivation of integrin alpha(v)beta(3) at cell-cell adhesion sites and partially disrupted E-cadherin-based AJs. The activation of PIPKIgamma is correlated with its phosphorylation state. The tyrosine phosphatase protein-tyrosine phosphatase mu (PTPmu) effectively dephosphorylated PIPKIgamma and thus canceled the PIPKIgamma-dependent activation of integrin alpha(v)beta(3) by blocking the interaction of integrin alpha(v)beta(3) with talin. Moreover, PTPmu associated with nectin, and its phosphatase activity was enhanced by the trans-interaction of nectin, leading to the decrease in PIPKIgamma90 phosphorylation. Therefore, the trans-interaction of nectin essentially functions in the inactivation of integrin at AJs through the PTPmu-induced inactivation of PIPKIgamma.

Afadin directly links nectin, an immunoglobulin-like cell–cell adhesion molecule, to actin filaments (F-actin) at adherens junctions (AJs). The nectin–afadin complex is important for the formation of not only AJs but also tight junctions (TJs) in epithelial cells. Studies using afadin-knockout mice have revealed that afadin is indispensable for embryonic development by organizing the formation of cell–cell junctions. However, the molecular mechanism of cell–cell junction disorganization during embryonic development in afadin-knockout mice is poorly understood. To address this, we took advantage of embryoid bodies (EBs) as a model system. The formation of cell–cell junctions including AJs and TJs was impaired in afadin-null EBs. The proper accumulation of the Par complex and the activation of Cdc42 and atypical PKC (aPKC), which are crucial for the formation of cell polarity, were also inhibited by knockout of afadin. In addition, the disruption of afadin caused the abnormal deposition of laminin and the dislocalization of its receptors integrin α6 and integrin β1. These results indicate that afadin organizes the formation of cell–cell junctions by regulating cell polarization in early embryonic development.