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Background: Flow-mediated dilation (FMD) measures vascular endothelial function by evaluating the vasodilatory response of blood vessels to increased blood flow. Nevertheless, the association between FMD and stroke incidence in a general population remains unclear. This study investigated the association between vascular endothelial function and stroke incidence in the general Japanese population., Methods: Based on cohort data from the Tohoku Medical Megabank Community-based Cohort Study, participants aged ≥18 years were recruited from Iwate Prefecture, with the final sample comprising 2952 subjects., Results: The FMD level was 0.5%-27.1%, with a median of 5.0% (interquartile, 4.2%-11.3%). The mean follow-up period was 5.5 ± 1.8 years (range, 0.6-6.9 years). After dividing the participants into two subgroups according to the median FMD value, a multivariate Cox regression analysis adjusting for gender, age, smoking, alcohol consumption, systolic blood pressure, low-density lipoprotein cholesterol, estimated glomerular filtration rate, N-terminal pro-brain natriuretic peptide, high-sensitivity cardiac troponin T and hemoglobin A1c revealed that a lower FMD value was strongly associated with incidences of total stroke (hazard ratio[HR] = 2.13, 95% confidence interval[CI] = 1.48-3.07, p < 0.001), ischemic stroke (HR = 3.33, 95%CI = 2.00-5.52, p < 0.001), nonlacunar stroke (HR = 2.77, 95%CI = 1.49-5.16, p = 0.001), and lacunar stroke (HR = 5.12, 95%CI = 1.74-16.05, p = 0.003)., Conclusions: This study showed that a low FMD value might reflect vascular endothelial dysfunction and then was associated with ischemic stroke incidence in the general Japanese population, suggesting that FMD can be used as a tool to identify future stroke risk., (© 2023 The Authors.)

Elevated levels of circulating high-sensitivity cardiac troponin T (hs-cTnT) are associated with cardiovascular disease. This study aimed to examine whether hs-cTnT levels are associated with incident stroke in the elderly population. The Iwate Tohoku Medical Megabank Organization pooled participant data for a community-based cohort study (n = 15,063, 69.6 ± 3.4 years), with a mean follow-up period of 5.23 years for all-cause death and incident stroke. The follow-up revealed 316 incident strokes, including atherothrombotic (n = 98), cardioembolic (n = 54), lacunar (n = 63), hemorrhagic (n = 101), and 178 all-cause deaths. Participants were classified into quartiles according to hs-cTnT levels (Q1 ≦ 4 ng/L, Q2: 5-6 ng/L, Q3: 7-9 ng/L, and Q4 > 9 ng/L). After adjusting for sex, age, smoking, drinking, systolic blood pressure, estimated glomerular filtration rate, N-terminal pro-brain natriuretic peptide, hemoglobin A1c, and lipid profile, a Cox proportional hazard model showed that higher hs-cTnT levels were associated with ischemic stroke (Q1 vs. Q4, hazard ratio [HR] = 2.24, 95 % confidence interval [CI] = 1.12-4.51, p = 0.023). The incident of total stroke was not associated with hs-cTnT levels (Q1 vs. Q4, HR 1.39, 95 % CI = 0.89-1.74, p = 0.145). Numerical differences were highest regarding incident lacunar stroke subtypes; however, this association was not statistically significant. Higher hs-cTnT concentrations were associated with ischemic stroke in the elderly Japanese population., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Background: Bone metabolic dysregulation plays an important role in the pathogenesis of atherosclerosis; however, whether its markers contribute to coronary artery disease (CAD) risk in the general population remains unclear. Therefore, this study aimed to analyze the association between bone metabolic markers and CAD risk score in the general Japanese population., Methods: The Iwate Medical Megabank Organization collected individual participant data during a community-based cohort study in the Iwate prefecture (n = 5,095, age = 58.9 ± 12.4 years). Participants with osteoporosis, chronic kidney disease, malignant disease, or primary wasting disease were excluded from the study. The present study measured the levels of circulating bone metabolic markers, including total type I collagen N-terminal propeptide (TP1NP), bone-type alkaline phosphatase, cross-linked N-telopeptide of type 1 collagen (NTX), and intact parathyroid hormone. CAD risk and atherosclerosis were evaluated using the Suita score and brachial-ankle pulse wave velocity (baPWV) measurement, respectively., Results: Among the bone metabolic markers, TP1NP was strongly associated with a high Suita score (≥56 points) (OR = 0.77, 95% CI = 0.69-0.82, P < 0.001). When participants were divided into quartiles of TP1NP levels, the subgroup with the lowest TP1NP level was associated with a high Suita score (≥56 points) and high baPWV (>1,400 cm/s)., Conclusions: This study demonstrated that TP1NP levels decreased in participants with high Suita scores and high baPWV, suggesting that TP1NP downregulation may indicate future CAD risk and atherosclerosis progression in the general Japanese population., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Author(s).)

The purpose of this study was to investigate the association between xanthine oxidoreductase (XOR) activity and a high risk of cardiovascular disease (CVD) in a general Japanese population. The Iwate Tohoku Medical Megabank Organization pooled individual participant data from a general population-based cohort study in Iwate prefecture. The cardiovascular risk was calculated using the Framingham Risk Score (FRS). A total of 1605 of the 1631 participants (98.4%) had detectable XOR activity. Multiple regression analysis demonstrated that XOR activity was independently associated with body mass index (β = 0.26, p < 0.001), diabetes (β = 0.09, p < 0.001), dyslipidemia (β = 0.08, p = 0.001), and uric acid (β = 0.13, p < 0.001). Multivariate analysis showed that the highest quartile of XOR activity was associated with a high risk for CVD (FRS ≥ 15) after adjustment for baseline characteristics (OR 2.93, 95% CI 1.16-7.40). The area under the receiver operating characteristic curves of the FRS with XOR activity was 0.81 ( p = 0.008). XOR activity is associated with a high risk for CVD, suggesting that high XOR activity may indicate cardiovascular risk in a general Japanese population.

Background: We established a community-based cohort study to assess the long-term impact of the Great East Japan Earthquake on disaster victims and gene-environment interactions on the incidence of major diseases, such as cancer and cardiovascular diseases., Methods: We asked participants to join our cohort in the health check-up settings and assessment center based settings. Inclusion criteria were aged 20 years or over and living in Miyagi or Iwate Prefecture. We obtained information on lifestyle, effect of disaster, blood, and urine information (Type 1 survey), and some detailed measurements (Type 2 survey), such as carotid echography and calcaneal ultrasound bone mineral density. All participants agreed to measure genome information and to distribute their information widely., Results: As a result, 87,865 gave their informed consent to join our study. Participation rate at health check-up site was about 70%. The participants in the Type 1 survey were more likely to have psychological distress than those in the Type 2 survey, and women were more likely to have psychological distress than men. Additionally, coastal residents were more likely to have higher degrees of psychological distress than inland residents, regardless of sex., Conclusion: This cohort comprised a large sample size and it contains information on the natural disaster, genome information, and metabolome information. This cohort also had several detailed measurements. Using this cohort enabled us to clarify the long-term effect of the disaster and also to establish personalized prevention based on genome, metabolome, and other omics information.

The accuracy of previous genetic studies in predicting polygenic psychiatric phenotypes has been limited mainly due to the limited power in distinguishing truly susceptible variants from null variants and the resulting overfitting. A novel prediction algorithm, Smooth-Threshold Multivariate Genetic Prediction (STMGP), was applied to improve the genome-based prediction of psychiatric phenotypes by decreasing overfitting through selecting variants and building a penalized regression model. Prediction models were trained using a cohort of 3685 subjects in Miyagi prefecture and validated with an independently recruited cohort of 3048 subjects in Iwate prefecture in Japan. Genotyping was performed using HumanOmniExpressExome BeadChip Arrays. We used the target phenotype of depressive symptoms and simulated phenotypes with varying complexity and various effect-size distributions of risk alleles. The prediction accuracy and the degree of overfitting of STMGP were compared with those of state-of-the-art models (polygenic risk scores, genomic best linear-unbiased prediction, summary-data-based best linear-unbiased prediction, BayesR, and ridge regression). In the prediction of depressive symptoms, compared with the other models, STMGP showed the highest prediction accuracy with the lowest degree of overfitting, although there was no significant difference in prediction accuracy. Simulation studies suggested that STMGP has a better prediction accuracy for moderately polygenic phenotypes. Our investigations suggest the potential usefulness of STMGP for predicting polygenic psychiatric conditions while avoiding overfitting.

Background: Epidemiological studies have shown that high circulating cystatin C is associated with a risk of cardiovascular disease (CVD) independent of creatinine-based renal function measurements. The present study investigated the comparison between the cystatin C-based estimated glomerular filtration rate (GFRcys) and creatinine-based GFR (GFRcr) to determine whether these measurements are associated with CV biomarkers and elevated CVD risk in a general Japanese population., Methods: The Iwate Tohoku Medical Megabank Organization pooled individual participant data from a general population-based cohort study in Iwate prefecture (n = 29,375). Chronic kidney disease (CKD) was estimated using the GFRcys, GFRcr and the urine albumin-to-creatinine ratio (UACR)., Results: The prevalence of CKD in the participants was found to be higher based on the GFRcr than the GFRcys. Multiple variable analyses after adjusting for baseline characteristics showed that high-sensitivity cardiac troponin T (hs-cTnT) and N-terminal pro-brain natriuretic peptide (NT-proBNP) were associated with the GFRcys. The area under the receiver operating characteristic (AUROC) curve for identifying individuals with a high Suita score was higher for the GFRcys (AUROC = 0.68) than it was for the GFRcr (AUROC = 0.64, P < 0.001). The GFRcys provided reclassification improvement for the CVD risk prediction model by the GFRcr (net reclassification improvement = 0.341; integrated discrimination improvement = 0.018, respectively, P < 0.001)., Conclusions: The GFRcys is more closely associated with CV biomarkers, including hs-cTnT and NT-proBNP levels, and a high Suita score than the GFRcr, and it provides additional value in the assessment of CVD risk using GFRcr.

PSD-Zip70 is a postsynaptic protein that regulates glutamatergic synapse formation and maturation by modulation of Rap2 activity. PSD-Zip70 knockout (PSD-Zip70KO) mice exhibit defective glutamatergic synaptic transmission in the prefrontal cortex (PFC) with aberrant Rap2 activation. As prefrontal dysfunction is implicated in the pathophysiology of stress-induced psychiatric diseases, we examined PSD-Zip70KO mice in a social defeat (SD) stress-induced mouse model of depression to investigate stress-induced alterations in synaptic function. Compared with wild-type (WT) mice, PSD-Zip70KO mice exhibited almost normal responses to SD stress in depression-related behaviors such as social activity, anhedonia, and depressive behavior. However, PSD-Zip70KO mice showed enhanced anxiety-like behavior irrespective of stress conditions. The density and size of dendritic spines of pyramidal neurons were reduced in the medial PFC (mPFC) in mice exposed to SD stress. Phosphorylation levels of the AMPA-type glutamate receptor (AMPA-R) GluA2 subunit at Ser880 were prominently elevated in mice exposed to SD stress, indicating internalization of surface-expressed AMPA-Rs and decreased postsynaptic responsiveness. Structural and functional impairments in postsynaptic responsiveness were associated with Rap2 GTPase activation in response to SD stress. Social stress-induced Rap2 activation was regulated by a PSD-Zip70-dependent pathway via interaction with SPAR/PDZ-GEF1. Notably, features such as Rap2 activation, dendritic spine shrinkage, and increased GluA2 phosphorylation were observed in the mPFC of PSD-Zip70KO mice even without SD stress. Together with our previous results, the present findings suggest that SD stress-induced postsynaptic hyporesponsiveness in glutamatergic synapses is mediated by PSD-Zip70-Rap2 signaling pathway and closely relates to anxiety-like behaviors., (Copyright © 2020 Mayanagi and Sobue.)

We launched an integrative multi-omics database, iMETHYL (http://imethyl.iwate-megabank.org). iMETHYL provides whole-DNA methylation (~24 million autosomal CpG sites), whole-genome (~9 million single-nucleotide variants), and whole-transcriptome (>14 000 genes) data for CD4 + T-lymphocytes, monocytes, and neutrophils collected from approximately 100 subjects. These data were obtained from whole-genome bisulfite sequencing, whole-genome sequencing, and whole-transcriptome sequencing, making iMETHYL a comprehensive database., Competing Interests: The authors declare no conflict of interest.

Background and Purpose: The prediction of genetic predispositions to ischemic stroke (IS) may allow the identification of individuals at elevated risk and thereby prevent IS in clinical practice. Previously developed weighted multilocus genetic risk scores showed limited predictive ability for IS. Here, we investigated the predictive ability of a newer method, polygenic risk score (polyGRS), based on the idea that a few strong signals, as well as several weaker signals, can be collectively informative to determine IS risk., Methods: We genotyped 13 214 Japanese individuals with IS and 26 470 controls (derivation samples) and generated both multilocus genetic risk scores and polyGRS, using the same derivation data set. The predictive abilities of each scoring system were then assessed using 2 independent sets of Japanese samples (KyushuU and JPJM data sets)., Results: In both validation data sets, polyGRS was shown to be significantly associated with IS, but weighted multilocus genetic risk scores was not. Comparing the highest with the lowest polyGRS quintile, the odds ratios for IS were 1.75 (95% confidence interval, 1.33-2.31) and 1.99 (95% confidence interval, 1.19-3.33) in the KyushuU and JPJM samples, respectively. Using the KyushuU samples, the addition of polyGRS to a nongenetic risk model resulted in a significant improvement of the predictive ability (net reclassification improvement=0.151; P<0.001)., Conclusions: The polyGRS was shown to be superior to weighted multilocus genetic risk scores as an IS prediction model. Thus, together with the nongenetic risk factors, polyGRS will provide valuable information for individual risk assessment and management of modifiable risk factors., (© 2016 The Authors.)

The regulation of cell-substrate adhesion is tightly linked to the malignant phenotype of tumor cells and plays a role in their migration, invasion, and metastasis. Focal adhesions (FAs) are dynamic adhesion structures that anchor the cell to the extracellular matrix. Myocardin-related transcription factors (MRTFs), co-regulators of the serum response factor (SRF), regulate expression of a set of genes encoding actin cytoskeletal/FA-related proteins. Here we demonstrated that the forced expression of a constitutively active MRTF-A (CA-MRTF-A) in B16F10 melanoma cells induced the up-regulation of actin cytoskeletal and FA proteins, resulting in FA reorganization and the suppression of cell migration. Expression of CA-MRTF-A markedly increased phosphorylation of focal adhesion kinase (FAK) and paxillin, which are important components for FA dynamics. Notably, FAK activation was triggered by the clustering of up-regulated integrins. Our results revealed that the MRTF-SRF-dependent regulation of cell migration requires both the up-regulation of actin cytoskeletal/FA proteins and the integrin-mediated regulation of FA components via the FAK/Src pathway. We also demonstrated that activation of the MRTF-dependent transcription correlates FAK activation in various tumor cells. The elucidation of the correlation between MRTF and FAK activities would be an effective therapeutic target in focus of tumor cell migration.

The Great East Japan Earthquake (GEJE) and resulting tsunami of March 11, 2011 gave rise to devastating damage on the Pacific coast of the Tohoku region. The Tohoku Medical Megabank Project (TMM), which is being conducted by Tohoku University Tohoku Medical Megabank Organization (ToMMo) and Iwate Medical University Iwate Tohoku Medical Megabank Organization (IMM), has been launched to realize creative reconstruction and to solve medical problems in the aftermath of this disaster. We started two prospective cohort studies in Miyagi and Iwate Prefectures: a population-based adult cohort study, the TMM Community-Based Cohort Study (TMM CommCohort Study), which will recruit 80 000 participants, and a birth and three-generation cohort study, the TMM Birth and Three-Generation Cohort Study (TMM BirThree Cohort Study), which will recruit 70 000 participants, including fetuses and their parents, siblings, grandparents, and extended family members. The TMM CommCohort Study will recruit participants from 2013 to 2016 and follow them for at least 5 years. The TMM BirThree Cohort Study will recruit participants from 2013 to 2017 and follow them for at least 4 years. For children, the ToMMo Child Health Study, which adopted a cross-sectional design, was also started in November 2012 in Miyagi Prefecture. An integrated biobank will be constructed based on the two prospective cohort studies, and ToMMo and IMM will investigate the chronic medical impacts of the GEJE. The integrated biobank of TMM consists of health and clinical information, biospecimens, and genome and omics data. The biobank aims to establish a firm basis for personalized healthcare and medicine, mainly for diseases aggravated by the GEJE in the two prefectures. Biospecimens and related information in the biobank will be distributed to the research community. TMM itself will also undertake genomic and omics research. The aims of the genomic studies are: 1) to construct an integrated biobank; 2) to return genomic research results to the participants of the cohort studies, which will lead to the implementation of personalized healthcare and medicine in the affected areas in the near future; and 3) to contribute the development of personalized healthcare and medicine worldwide. Through the activities of TMM, we will clarify how to approach prolonged healthcare problems in areas damaged by large-scale disasters and how useful genomic information is for disease prevention.

n-3 PUFAs are essential for neuronal development and brain function. However, the molecular mechanisms underlying their biological effects remain unclear. Here we examined the mechanistic action of docosahexaenoic acid (DHA), the most abundant n-3 polyunsaturated fatty acids in the brain. We found that DHA treatment of cortical neurons resulted in enhanced axon outgrowth that was due to increased axon elongation rates. DHA-mediated axon outgrowth was accompanied by the translational up-regulation of Tau and collapsin response mediator protein 2 (CRMP2), two important axon-related proteins, and the activation of Akt and p70 S6 kinase. Consistent with these findings, rapamycin, a potent inhibitor of mammalian target of rapamycin (mTOR), prevented DHA-mediated axon outgrowth and up-regulation of Tau and CRMP2. In addition, DHA-dependent activation of the Akt-mTOR-S6K pathway enhanced 5'-terminal oligopyrimidine tract-dependent translation of Tau and CRMP2. Therefore, our results revealed an important role for the Akt-mTOR-S6K pathway in DHA-mediated neuronal development., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Differences in DNA collection protocols may be a potential confounder in epigenome-wide association studies (EWAS) using a large number of blood specimens from multiple biobanks and/or cohorts. Here we show that pre-analytical procedures involved in DNA collection can induce systematic bias in the DNA methylation profiles of blood cells that can be adjusted by cell-type composition variables. In Experiment 1, whole blood from 16 volunteers was collected to examine the effect of a 24 h storage period at 4°C on DNA methylation profiles as measured using the Infinium HumanMethylation450 BeadChip array. Our statistical analysis showed that the P-value distribution of more than 450,000 CpG sites was similar to the theoretical distribution (in quantile-quantile plot, λ = 1.03) when comparing two control replicates, which was remarkably deviated from the theoretical distribution (λ = 1.50) when comparing control and storage conditions. We then considered cell-type composition as a possible cause of the observed bias in DNA methylation profiles and found that the bias associated with the cold storage condition was largely decreased (λ adjusted = 1.14) by taking into account a cell-type composition variable. As such, we compared four respective sample collection protocols used in large-scale Japanese biobanks or cohorts as well as two control replicates. Systematic biases in DNA methylation profiles were observed between control and three of four protocols without adjustment of cell-type composition (λ = 1.12-1.45) and no remarkable biases were seen after adjusting for cell-type composition in all four protocols (λ adjusted = 1.00-1.17). These results revealed important implications for comparing DNA methylation profiles between blood specimens from different sources and may lead to discovery of disease-associated DNA methylation markers and the development of DNA methylation profile-based predictive risk models.

Dysregulation of synapse formation and plasticity is closely related to the pathophysiology of psychiatric and neurodevelopmental disorders. The prefrontal cortex (PFC) is particularly important for executive functions such as working memory, cognition, and emotional control, which are impaired in the disorders. PSD-Zip70 (Lzts1/FEZ1) is a postsynaptic density (PSD) protein predominantly expressed in the frontal cortex, olfactory bulb, striatum, and hippocampus. Here we found that PSD-Zip70 knock-out (PSD-Zip70KO) mice exhibit working memory and cognitive defects, and enhanced anxiety-like behaviors. These abnormal behaviors are caused by impaired glutamatergic synapse transmission accompanied by tiny-headed immature dendritic spines in the PFC, due to aberrant Rap2 activation, which has roles in synapse formation and plasticity. PSD-Zip70 modulates the Rap2 activity by interacting with SPAR (spine-associated RapGAP) and PDZ-GEF1 (RapGEF) in the postsynapse. Furthermore, suppression of the aberrant Rap2 activation in the PFC rescued the behavioral defects in PSD-Zip70KO mice. Our data demonstrate a critical role for PSD-Zip70 in Rap2-dependent spine synapse development in the PFC and underscore the importance of this regulation in PFC-dependent behaviors., Significance Statement: PSD-Zip70 deficiency causes behavioral defects in working memory and cognition, and enhanced anxiety due to prefrontal hypofunction. This study revealed that PSD-Zip70 plays essential roles in glutamatergic synapse maturation via modulation of the Rap2 activity in the PFC. PSD-Zip70 interacts with both SPAR (spine-associated RapGAP) and PDZ-GEF1 (RapGEF) and modulates the Rap2 activity in postsynaptic sites. Our results provide a novel Rap2-specific regulatory mechanism in synaptic maturation involving PSD-Zip70., (Copyright © 2015 the authors 0270-6474/15/3514327-14$15.00/0.)

Glucocorticoids (GCs) mediate the effects of stress to cause structural plasticity in brain regions such as the hippocampus, including simplification of dendrites and shrinkage of dendritic spines. However, the molecular mechanics linking stress and GCs to these effects remain largely unclear. Here, we demonstrated that corticosterone (CORT) reduces the expression levels of caldesmon (CaD), causing dendritic spines to become vulnerable. CaD regulates cell motility by modulating the actin-myosin system and actin filament stability. In cultured rat hippocampal neurons, CaD localized to dendritic spines by binding to filamentous actin (F-actin), and CaD expression levels increased during spine development. CaD stabilized the F-actin dynamics in spines, thereby enlarging the spine heads, whereas CaD knockdown decreased the spine-head size via destabilization of the F-actin dynamics. CaD was also required for chemical LTP-induced actin stabilization. The CaD expression levels were markedly decreased by exposure to CORT mediated by suppression of serum response factor-dependent transcription. High CORT levels reduced both the spine-head size and F-actin stability similarly to CaD knockdown, and overexpressing CaD abolished the detrimental effect of CORT on dendritic spine development. These results indicate that CaD enlarges the spine-head size by stabilizing F-actin dynamics, and that CaD is a critical target in the GC-induced detrimental effects on dendritic spine development.

The actin cytoskeleton plays a key role in regulating cell motility. Caldesmon (CaD) is an actin-linked regulatory protein found in smooth muscle and non-muscle cells that is conserved among a variety of vertebrates. It binds and stabilizes actin filaments, as well as regulating actin-myosin interaction in a calcium (Ca2+)/calmodulin (CaM)- and/or phosphorylation-dependent manner. CaD function is regulated qualitatively by Ca2+/CaM and by its phosphorylation state and quantitatively at the mRNA level, by three different transcriptional regulation of the CALD1 gene. CaD has numerous functions in cell motility, such as migration, invasion, and proliferation, exerted via the reorganization of the actin cytoskeleton. Here we will outline recent findings regarding CaD's structural features and functions.

The glucocorticoids (GCs), the most downstream effectors of the hypothalamic-pituitary-adrenal (HPA) axis, are the main mediators of stress response. Stress-triggered GCs as well as acute and chronic GC treatment can impair the structural plasticity and function of the brain. The exposure of perinatal animals and humans to excess stress or GCs can affect the brain development, resulting in altered behaviors in the adult offspring of animals and an increased risk of psychiatric disorders in humans. Despite the numerous studies documenting these effects, the underlying mechanism remains unclear. In this commentary we will focus on the effect of excess GCs on cortical development. We have recently showed that excess-GC-dependent retardation of the radial migration of neural progenitor cells (NPCs) is caused by the dysregulation of actin-myosin interaction via upregulation of caldesmon (CaD), an actin-linked regulatory protein. The elucidation of the molecular mechanisms that underlie the detrimental action of GCs on cortical development will expand our understanding of how stress/GCs alter the formation of neural networks and affect behaviors later in life.

Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. We noticed that the expression of myocardin was markedly downregulated in human uterine leiomyosarcoma cells. Restoration of myocardin expression induced the reexpression of smooth muscle marker proteins and the formation of well-developed actin fibers. A concomitant increase in the expression of a cyclin-dependent kinase inhibitor, p21, led to significantly reduced cell proliferation, via p21's inhibition of the G(1)-S transition. A p21 promoter-reporter assay showed that myocardin markedly increased p21's promoter activity. Furthermore, a serum response factor (SRF)-binding cis-element CArG box in the p21 promoter region was required for this myocardin effect. Chromatin immunoprecipitation and DNA-protein binding assays showed that myocardin indirectly bound to the CArG box in the p21 promoter through the interaction with SRF. Furthermore, immunohistochemistry revealed that the levels of myocardin and p21 were both lower in leiomyosarcoma samples than in normal smooth muscle tissue. Taken together, our results indicate that the downregulation of myocardin expression facilitates cell cycle progression via the reduction of p21 expression in human leimyosarcomas and suggest that myocardin could be a useful therapeutic target for this disease.

Mammalian target of rapamycin (mTOR) is an important regulator of neuronal development and functions. Although it was reported recently that mTOR signaling is critical for neuronal polarity, the underlying mechanism remains unclear. Here, we describe the molecular pathway of mTOR-dependent axon specification, in which the collapsing response mediator protein 2 (CRMP2) and Tau are major downstream targets. The activity of mTOR effector 70-kDa ribosomal protein S6 kinase (p70S6K) specifically increases in the axon during neuronal polarity formation. The mTOR inhibitor rapamycin suppresses the translation of some neuronal polarity proteins, including CRMP2 and Tau, thereby inhibiting axon formation. In contrast, constitutively active p70S6K up-regulates the translation of these molecules, thus inducing multiple axons. Exogenous CRMP2 and Tau facilitate axon formation, even in the presence of rapamycin. In the 5'-untranslated region of Tau and CRMP2 mRNAs, we identified a 5'-terminal oligopyrimidine tract, which mediates mTOR-governed protein synthesis. The 5'-terminal oligopyrimidine tract sequences of CRMP2 and Tau mRNAs strongly contribute to the up-regulation of their translation in the axon in response to the axonal activation of the mTOR-p70S6K pathway. Taken together, we conclude that the local translation of CRMP2 and Tau, regulated by mTOR-p70S6K, is critical for the specification of neuronal polarity.

During cortical development, newly generated neurons migrate radially toward their final positions. Although several candidate genes essential for this radial migration have been reported, the signaling pathways regulating it are largely unclear. Here we studied the role of phosphatidylinositol (PI) 3-kinase and its downstream signaling molecules in the radial migration of cortical neurons in vivo and in vitro. The expression of constitutively active and dominant-negative PI 3-kinases markedly inhibited radial migration. In the neocortical slice culture, a PI 3-kinase inhibitor suppressed the formation of GTP-bound Rac1 and Cdc42 and radial migration. Constitutively active and dominant-negative forms of Rac1 and Cdc42 but not Akt also significantly inhibited radial migration. In migrating neurons, wild-type Rac1 and Cdc42 showed different localizations; Rac1 localized to the plasma membrane and Cdc42 to the perinuclear region on the side of the leading processes. These results suggest that both the PI 3-kinase/Rac1 and Cdc42 pathways are involved in the radial migration of cortical neurons and that they have different roles.

Background: The coordinated activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) is critical for the induction of vascular and visceral smooth muscle cell (SMC) dedifferentiation. We previously reported that on the forced activation of both MAPKs, visceral SMCs secrete a non-heparin-binding protein factor(s) that is involved in the dedifferentiation of neighboring SMCs. In this study, we sought to identify the dedifferentiation factor(s) derived from vascular SMCs (VSMCs)., Methods and Results: We fractionated the VSMC dedifferentiation factor(s) in the conditioned medium obtained from differentiated VSMCs in which both ERK and p38MAPK were forcedly activated and identified epiregulin as a major autocrine/paracrine factor for VSMC dedifferentiation. The epiregulin-induced VSMC dedifferentiation was mediated through the coordinated activation of ERK and p38MAPK. Unsaturated lysophosphatidic acid and platelet-derived growth factor-BB, which are potent VSMC dedifferentiation factors, rapidly upregulated epiregulin mRNA expression in an ERK- and p38MAPK-dependent manner. Reverse transcriptase-polymerase chain reaction and/or immunohistological analyses revealed the restricted expression of epiregulin in human atherosclerotic and balloon-injured rat arteries, in which the phenotypic modulation of medial VSMCs occurred in vivo., Conclusions: Epiregulin is released from VSMCs primed by atherogenic factors and acts as a major autocrine/paracrine factor for VSMC dedifferentiation. It may be involved in the progression of vascular remodeling such as atherosclerosis.

Background: We previously identified unsaturated (16:1, 18:1, and 18:2) but not saturated (12:0, 14:0, 16:0, and 18:0) lysophosphatidic acids (LPAs) as potent factors for vascular smooth muscle cell (VSMC) dedifferentiation. Unsaturated LPAs strongly induce VSMC dedifferentiation via the coordinated activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK), resulting in the proliferation and migration of dedifferentiated VSMCs. Here, we investigated the effects of 18:1 and 18:0 LPAs (as representative unsaturated and saturated LPAs, respectively) on the vasculature in vivo., Methods and Results: Rat common carotid arteries (CCAs) were treated transiently with 18:1 or 18:0 LPA and then examined by histological and biochemical analyses. The 18:1 but not 18:0 LPA potently induced vascular remodeling that was composed primarily of neointima. The incorporation of [3H]18:1 LPA into the CCAs revealed that a sufficient amount of unmetabolized [3H]18:1 LPA to induce VSMC dedifferentiation was present in the vascular wall. The 18:1 LPA-induced neointimal formation in vivo was also dependent on the coordinated activation of ERK and p38MAPK. Unlike balloon-injured CCAs, the 18:1 LPA-treated CCAs showed a histological similarity to human atherosclerotic arteries., Conclusions: This is the first report demonstrating a role for a naturally occurring unsaturated LPA in inducing vascular remodeling in vivo and provides a novel animal model for neointimal formation.

The postsynaptic site of the excitatory synapse, which is composed of the postsynaptic density (PSD) attached to the postsynaptic membrane, is a center for synaptic plasticity. To reveal the molecular organization and functional regulation of the postsynaptic site, we cloned a 70 kDa protein that is concentrated in PSDs using a monoclonal antibody against the PSD. This protein, named PSD-Zip70, is highly homologous to the human FEZ1/LZTS1 gene product. PSD-Zip70 contains an N-myristoylation consensus sequence, a polybasic cluster in the N-terminal region and four leucine-zipper motifs in the C-terminal region. Light and electron microscopy showed that this protein was localized to the dendritic spines, especially in the PSD and the postsynaptic membrane. Fractionation of the synaptic plasma membrane demonstrated that PSD-Zip70 was localized to the PSD and the dendritic raft. In Madin-Darby canine kidney (MDCK) cells, exogenous PSD-Zip70 was targeted to the apical plasma membrane of microvilli, and its N-myristoylation was necessary for this targeting. In hippocampal neurons, N-myristoylation was also required for the membrane localization and the C-terminal region was critically involved in the synaptic targeting. These results suggest that PSD-Zip70 may be involved in the dynamic properties of the structure and function of the postsynaptic site.

The channel activity of NMDA receptors is regulated by phosphorylation by protein kinases and by interaction with other proteins. Recombinant NR1/NR2A subtype NMDA receptor channels are potentiated by the protein tyrosine kinase Src, an effect which is mediated by a reduction in the high-affinity, voltage-independent Zn(2+) inhibition. However, it has been reported that Src-induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn(2+) inhibition. The post-synaptic density protein PSD-95 interacts with the C-terminus of NR2 subunits of the NMDA receptor. Here we demonstrate that PSD-95 eliminates the Src-induced potentiation of NR1/NR2A channels expressed in oocytes and reduces the sensitivity of the channels to Zn(2+). Our results reveal that the absence of Src-induced potentiation of PSD-95-coupled NR1/NR2A channels is not to due to the reduced sensitivity of these channels to Zn(2+). These results indicate that PSD-95 functionally modulates NR1/NR2A channels and explain why Src-induced potentiation of NMDA receptor currents in hippocampus neurons is not mediated by a reduction in Zn(2+) inhibition.

Serum response factor and the (CC(A/T)(6)GG) (CArG) box interact to promote the transcription of c-fos and muscle genes; this tissue-specific activity may require co-regulators for serum response factor. The alpha(1) integrin promoter contains two cis-elements besides the CArG box: a TAAT sequence, a consensus binding site for homeoproteins, and a GATA-binding box. As a candidate TAAT-binding factor, we cloned an NK family homeobox gene, Nkx-3.2, which is expressed mainly in smooth muscle tissues and skeletal structures. Nkx-3.2, serum response factor, and GATA-6 were co-expressed only in the medial smooth muscle layer of arteries. These three transcription factors formed a complex with their corresponding cis-elements and cooperatively transactivated smooth muscle genes, including alpha(1) integrin, SM22alpha, and caldesmon. Cardiac muscle-specific members of the NK and GATA families exist, and the triad of Nkx-2.5, serum response factor, and GATA-4 also transactivated the cardiac atrial natriuretic factor gene, which contains a CArG-like box, a GATA-binding box, and an NK-binding element. Our findings demonstrate that smooth and cardiac muscle have a shared transcriptional machinery and that the GATA and NK families confer muscle specificity on the serum response factor/CArG interaction.