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Background: Endothelial-to-mesenchymal transition (EndMT) has been identified as a critical driver of vascular inflammation and atherosclerosis, and TGF-β (transforming growth factor β) is a key mediator of EndMT. Both EndMT and atherosclerosis are promoted by disturbed flow, whereas unidirectional laminar flow limits EndMT and is atheroprotective. How EndMT and endothelial TGF-β signaling are regulated by different flow patterns is, however, still poorly understood. Methods: Flow chamber experiments in vitro and endothelium-specific knockout mice were used to study the role of tenascin-X in the regulation of EndMT and atherosclerosis as well as the underlying mechanisms. Results: In human endothelial cells as well as in human and mouse aortae, unidirectional laminar flow but not disturbed flow strongly increased endothelial expression of the extracellular matrix protein TN-X (tenascin-X) in a KLF4 (Krüppel-like factor 4) dependent manner. Mice with endothelium-specific loss of TN-X (EC-Tnxb-KO) showed increased endothelial TGF-β signaling as well as increased endothelial expression of EndMT and inflammatory marker genes. When EC-Tnxb-KO mice were subjected to partial carotid artery ligation, we observed increased vascular remodeling. EC-Tnxb-KO mice crossed to low-density lipoprotein receptor-deficient mice showed advanced atherosclerotic lesions after being fed a high-fat diet. Treatment of EC-Tnxb-KO mice with an anti-TGF-beta antibody or additional endothelial loss of TGF-beta receptors 1 and 2 normalized endothelial TGF-beta signaling and prevented EndMT. In in vitro studies, we found that TN-X through its fibrinogen-like domain directly interacts with TGF-β and thereby interferes with its binding to the TGF-β receptor. Conclusions: In summary, we show that TN-X is a central mediator of flow-induced inhibition of EndMT, endothelial inflammation and atherogenesis, which functions by binding to and by blocking the activity of TGF-β. Our data identify a novel mechanism of flow-dependent regulation of vascular TGF-β, which holds promise for generating new strategies to prevent vascular inflammation and atherosclerosis.

Formation of NO by endothelial NOS (eNOS) is a central process in the homeostatic regulation of vascular functions including blood pressure regulation, and fluid shear stress exerted by the flowing blood is a main stimulus of eNOS activity. Previous work has identified several mechanosensing and -transducing processes in endothelial cells, which mediate this process and induce the stimulation of eNOS activity through phosphorylation of the enzyme via various kinases including AKT. How the initial mechanosensing and signaling processes are linked to eNOS phosphorylation is unclear. In human endothelial cells, we demonstrated that protein kinase N2 (PKN2), which is activated by flow through the mechanosensitive cation channel Piezo1 and Gq/G11-mediated signaling, as well as by Ca2+ and phosphoinositide-dependent protein kinase 1 (PDK1), plays a pivotal role in this process. Active PKN2 promoted the phosphorylation of human eNOS at serine 1177 and at a newly identified site, serine 1179. These phosphorylation events additively led to increased eNOS activity. PKN2-mediated eNOS phosphorylation at serine 1177 involved the phosphorylation of AKT synergistically with mTORC2-mediated AKT phosphorylation, whereas active PKN2 directly phosphorylated human eNOS at serine 1179. Mice with induced endothelium-specific deficiency of PKN2 showed strongly reduced flow-induced vasodilation and developed arterial hypertension accompanied by reduced eNOS activation. These results uncover a central mechanism that couples upstream mechanosignaling processes in endothelial cells to the regulation of eNOS-mediated NO formation, vascular tone, and blood pressure.

Summary Vascular smooth muscle cells (VSMCs) represent the prevailing cell type of arterial vessels and are essential for blood vessel structure and homeostasis. They have substantial potential for phenotypic plasticity when exposed to various stimuli in their local microenvironment. How VSMCs maintain their differentiated contractile phenotype is still poorly understood. Here we demonstrate that the Hippo pathway effectors YAP and TAZ play a critical role in maintaining the differentiated contractile phenotype of VSMCs. In the absence of YAP/TAZ, VSMCs lose their differentiated phenotype and undergo osteogenic differentiation, which results in vascular calcification. Osteogenic transdifferentiation was accompanied by the upregulation of Wnt target genes. The absence of YAP/TAZ in VSMCs led to Disheveled 3 (DVL3) nuclear translocation and upregulation of osteogenesis-associated genes independent of canonical Wnt/β-catenin signaling activation. Our data indicate that cytoplasmic YAP/TAZ interact with DVL3 to avoid its nuclear translocation and osteogenic differentiation, thereby maintaining the differentiated phenotype of VSMCs., Graphical Abstract, Highlights • YAP/TAZ play an important role in maintaining vascular SMCs contractile phenotype • Loss of YAP/TAZ in vSMCs leads to reduced expression of smooth muscle marker genes • Loss of YAP/TAZ in vSMCs results in reduced artery contractility • Deficiency of YAP/TAZ in vSMCs leads to osteogenic transdifferentiation, Biological Sciences; Molecular Biology; Cell Biology; Developmental Biology; Transcriptomics

Macrophage inhibitory cytokine-1 (MIC-1) is a cytokine with pleotropic actions and its expression is markedly increased by inflammation and cardiac injury and in cancers. In particular, MIC-1 production after cardiac ischemia injury is associated with enhanced cardiac angiogenesis as well as myocardial protection. However, it remains uncertain whether MIC-1 itself has proangiogenic activity. In this study, we tried to determine the precise role of MIC-1 in physiological and pathological angiogenesis. Human microvessel endothelial cells responded to MIC-1 with enhanced angiogenic behaviors. Employing various angiogenesis assays, MIC-1 was found to promote vessel formation and development with a potency similar to that of vascular endothelial growth factor (VEGF). MIC-1 transgenic (Tg) mice also displayed enhanced neovascularization in both developing embryos and neonatal mouse retinas, compared with wild-type mice. Furthermore, endothelial cells (ECs) isolated from MIC-1 Tg mouse lung exhibited higher angiogenic potential than ECs from wild-type lung. MIC-1-induced angiogenesis was also observed in the recovery or healing processes of injuries such as hindlimb ischemia and skin wounds in mice. However, unlike VEGF, MIC-1 induced neither endothelial inflammation nor increased vascular permeability. In ECs, the MIC-1 signal exerted proangiogenic actions via the MEK/extracellular signal-regulated kinase- and phosphatidylinositol 3-kinase/Akt-dependent pathways. Notably, these MIC-1 signaling events in ECs were abrogated by small interfering RNA-mediated knockdown of GFRAL, suggesting that GFRAL is an EC receptor for MIC-1. In summary, we here show a novel role of MIC-1 as a potent EC activator, which promotes both normal and injury-related angiogenesis.

Myogenic vasoconstriction is an autoregulatory function of small arteries. Recently, G-protein-coupled receptors have been involved in myogenic vasoconstriction, but the downstream signalling mechanisms and the in-vivo-function of this myogenic autoregulation are poorly understood. Here, we show that small arteries from mice with smooth muscle-specific loss of G12/G13 or the Rho guanine nucleotide exchange factor ARHGEF12 have lost myogenic vasoconstriction. This defect was accompanied by loss of RhoA activation, while vessels showed normal increases in intracellular [Ca2+]. In the absence of myogenic vasoconstriction, perfusion of peripheral organs was increased, systemic vascular resistance was reduced and cardiac output and left ventricular mass were increased. In addition, animals with defective myogenic vasoconstriction showed aggravated hypotension in response to endotoxin. We conclude that G12/G13- and Rho-mediated signaling plays a key role in myogenic vasoconstriction and that myogenic tone is required to maintain local and systemic vascular resistance under physiological and pathological condition.

Hypertension is a primary risk factor for cardiovascular diseases including myocardial infarction and stroke. Major determinants of blood pressure are vasodilatory factors such as nitric oxide (NO) released from the endothelium under the influence of fluid shear stress exerted by the flowing blood. Several endothelial signaling processes mediating fluid shear stress–induced formation and release of vasodilatory factors have been described. It is, however, still poorly understood how fluid shear stress induces these endothelial responses. Here we show that the endothelial mechanosensitive cation channel PIEZO1 mediated fluid shear stress–induced release of adrenomedullin, which in turn activated its G(s)-coupled receptor. The subsequent increase in cAMP levels promoted the phosphorylation of endothelial NO synthase (eNOS) at serine 633 through protein kinase A (PKA), leading to the activation of the enzyme. This G(s)/PKA-mediated pathway synergized with the AKT-mediated pathways leading to eNOS phosphorylation at serine 1177. Mice with endothelium-specific deficiency of adrenomedullin, the adrenomedullin receptor, or Gα(s) showed reduced flow-induced eNOS activation and vasodilation and developed hypertension. Our data identify fluid shear stress–induced PIEZO1 activation as a central regulator of endothelial adrenomedullin release and establish the adrenomedullin receptor and subsequent G(s)-mediated formation of cAMP as a critical endothelial mechanosignaling pathway regulating basal endothelial NO formation, vascular tone, and blood pressure.

// Jaeseob Lee 1, * , Hee-Jung Byun 1, * , Moon-Sung Lee 2 , Young-June Jin 1 , Dooil Jeoung 3 , Young-Myeong Kim 4 , Hansoo Lee 1, 2 1 Department of Biological Sciences, College of Natural Sciences, Kangwon National University, Chunchon, Kangwon-do 200-701, Republic of Korea 2 BIT Medical Convergence Graduate Program, Kangwon National University, Chunchon, Kangwon-do 200-701, Republic of Korea 3 Department of Biochemistry, College of Natural Sciences, Kangwon National University, Chunchon, Kangwon-do 200-701, Republic of Korea 4 Department of Molecular and Cellular Biochemistry, School of Medicine, Kangwon National University, Chunchon, Kangwon-do 200-701, Republic of Korea * These authors have contributed equally to this work Correspondence to: Hansoo Lee, email: hslee@kangwon.ac.kr Keywords: CD82, EMT, fibronectin matrix, integrin signaling, cancer invasion Received: July 22, 2016 Accepted: November 14, 2016 Published: December 01, 2016 ABSTRACT The transmembrane protein CD82/KAI1 suppresses the metastatic potential of various cancer cell types. Moreover, decrease or loss of CD82 expression is closely associated with malignancy and poor prognosis in many human cancers including prostate cancer. Despite intense scrutiny, the mechanisms underlying the metastasis-suppressing role of CD82 are still not fully understood. Here, we found that a fibronectin matrix induced mesenchymal phenotypes in human prostate cancer cells with no or low CD82 expression levels. However, high CD82 expression rendered prostate cancer cells to have intensified epithelial characteristics upon fibronectin engagement, along with decreased cell motility and invasiveness. The CD82 function of inhibiting fibronectin-induced epithelial-to-mesenchymal transition (EMT) was dependent not only on CD82 interactions with fibronectin-binding α 3 β 1 /α 5 β 1 integrins but also on the integrin-mediated intracellular signaling events. Notably, CD82 attenuated the FAK-Src and ILK pathways downstream of the fibronectin-receptor integrins. Immunofluorescence staining of human prostate cancer tissue specimens illustrated a negative association of CD82 with EMT-related gene expression as well as prostate malignancy. Altogether, these results suggest that CD82 suppresses EMT in prostate cancer cells adhered to the fibronectin matrix by repressing adhesion signaling through lateral interactions with the associated α 3 β 1 and α 5 β 1 integrins, leading to reduced cell migration and invasive capacities.

Impaired alveolar formation and maintenance are features of many pulmonary diseases that are associated with significant morbidity and mortality. In a forward genetic screen for modulators of mouse lung development, we identified the non-muscle myosin II heavy chain gene, Myh10. Myh10 mutant pups exhibit cyanosis and respiratory distress, and die shortly after birth from differentiation defects in alveolar epithelium and mesenchyme. From omics analyses and follow up studies, we find decreased Thrombospondin expression accompanied with increased matrix metalloproteinase activity in both mutant lungs and cultured mutant fibroblasts, as well as disrupted extracellular matrix (ECM) remodeling. Loss of Myh10 specifically in mesenchymal cells results in ECM deposition defects and alveolar simplification. Notably, MYH10 expression is downregulated in the lung of emphysema patients. Altogether, our findings reveal critical roles for Myh10 in alveologenesis at least in part via the regulation of ECM remodeling, which may contribute to the pathogenesis of emphysema., Abnormal alveolar development and homeostasis are common features of pulmonary disease. Here the authors show that Myh10 expression is reduced in emphysema patients, and that Myh10 loss of function impairs alveolar formation and lung morphogenesis via upregulation of matrix metalloproteinase activity and altered matrix remodeling.

Macrophage inhibitory cytokine-1 (MIC-1) is highly associated with malignant human cancers and has been suggested to be involved in tumor angiogenesis. In the present study, we examined the effect of MIC-1 on endothelial cell proliferation to confirm the angiogenesis-promoting role of MIC-1. MIC-1 treatment accelerated progression of the G(1) stage in the cell cycle of human umbilical vein endothelial cells (HUVECs), leading to an increased cell proliferation rate. MIC-1 augmented the levels of cyclins D1 and E without altering the levels of cyclin-dependent kinase (CDK) inhibitors, thereby increasing protein kinase activity of CDKs and subsequent phosphorylation of the Rb protein followed by nuclear translocation of E2F. MIC-1-induced expression of cyclins D1 and E was mediated by AP-1 and E2F-1 transcription factors, and among the AP-1 members, c-Jun and JunD appeared to participate in MIC-1-dependent transcription of the cyclin D1 gene. Additionally, the PI3K/Akt, JNK, and ERK pathways were found to mediate MIC-1-induced cyclin D1 expression in HUVECs. Importantly, lung endothelial cells isolated from MIC-1 transgenic mouse displayed a higher proliferation rate and cyclin D1 and E levels relative to their wild-type counterparts. These results suggest that MIC-1 secreted from cancer cells stimulates endothelial cell proliferation by enhancing AP-1- and E2F-dependent expression of G(1) cyclins via PI3K/Akt, JNK, and ERK signaling pathways, potentially leading to enhanced tumor angiogenesis.

The activity of matrix metalloproteinases (MMPs), which selectively degrades the extracellular matrix (ECM), is critical in angiogenesis. Conversely, changes in ECM composition/structure alter the expression and activity of MMPs in various cell types. In the present study, we examined whether changes in ECM composition affect MMPs expression/activity of endothelial cells and thereby alter the surrounding ECM structure. Among the ECM molecules examined, fibronectin (FN) and vitronectin (VN) increased the expression and activity of MMP-9 in human umbilical vein endothelial cells (HUVECs). Both α(5)β(1) and α(v)β(3) integrins were involved in FN-induced MMP-9 expression. Also, FN-induced MMP-9 expression was found to be mediated by AP-1 transcription factors, including c-Jun, JunB, and JunD. Inhibitors or siRNAs specific to AP-1 activating signal transducers, including FAK-Src, PI3K/Akt, ERK, and JNK, abolished both FN-induced AP-1 activation and MMP-9 expression. VN-induced AP-1 activation and MMP-9 expression were also mediated by these AP-1 activating signal transducers in addition to p38 MAPK. Moreover, treatment with FN or VN resulted in increased degradation of collagen on HUVEC culture plates. Taken together, our data suggest that both fibronectin and vitronectin induce MMP-9 expression via the AP-1-activating signaling pathways in endothelial cells, and thereby stimulate degradation of surrounding collagen, leading to alterations in ECM structure and potentially the promotion of angiogenesis.

The tetraspanin membrane protein CD151 has been suggested to regulate cancer invasion and metastasis by initiating signaling events. The CD151-mediated signaling pathways involved in this regulation remain to be revealed. In this study, we found that stable transfection of CD151 into MelJuSo human melanoma cells lacking CD151 expression significantly increased cell motility, matrix metalloproteinase-9 (MMP-9) expression, and invasiveness. The enhancement of cell motility and MMP-9 expression by CD151 overexpression was abrogated by inhibitors and small interfering RNAs targeted to focal adhesion kinase (FAK), Src, p38 MAPK, and JNK, suggesting an essential role of these signaling components in CD151 signaling pathways. Also, CD151-induced MMP-9 expression was shown to be mediated by c-Jun binding to AP-1 sites in the MMP-9 gene promoter, indicating AP-1 activation by CD151 signaling pathways. Meanwhile, CD151 was found to be associated with alpha(3)beta(1) and alpha(6)beta(1) integrins in MelJuSo cells, and activation of associated integrins was a prerequisite for CD151-stimulated MMP-9 expression and activation of FAK, Src, p38 MAPK, JNK, and c-Jun. Furthermore, CD151 on one cell was shown to bind to neighboring cells expressing CD151, suggesting that CD151 is a homophilic interacting protein. The homophilic interactions of CD151 increased motility and MMP-9 expression of CD151-transfected MelJuSo cells, along with FAK-, Src-, p38 MAPK-, and JNK-mediated activation of c-Jun in an adhesion-dependent manner. Furthermore, C8161 melanoma cells with endogenous CD151 were also shown to respond to homophilic CD151 interactions for the induction of adhesion-dependent activation of FAK, Src, and c-Jun. These results suggest that homophilic interactions of CD151 stimulate integrin-dependent signaling to c-Jun through FAK-Src-MAPKs pathways in human melanoma cells, leading to enhanced cell motility and MMP-9 expression.

Despite the importance of multiple tetraspanin proteins in cancer invasion and metastasis, little is known about the role and significance of tetraspanin CD81 in these processes. In the present study, we examined CD81 effects on melanoma cell invasiveness and metastasis. Transfection of CD81 into melanoma cells lacking endogenous CD81 expression significantly enhanced the migrating, invasive, and metastatic abilities of melanoma cells. Interestingly, membrane type 1 matrix metalloproteinase (MT1-MMP) expression was found in CD81-expressing melanoma cells but not in CD81-deficient cells. siRNA knockdown of CD81 in melanoma cells with endogenous CD81 demonstrated decreased MT1-MMP levels and cell motility. Notably, CD81-induced cell migration was abrogated by antibody blocking and siRNA knockdown of MT1-MMP, indicating that MT1-MMP is responsible for CD81-stimulated melanoma cell migration. Promoter analysis revealed an essential role of the Sp1 transcription factor in CD81-induced MT1-MMP transcription. We also demonstrate that the Sp1-activating Akt pathway is involved in adhesion-dependent CD81 signaling to induce MT1-MMP expression and cell motility. Importantly, human skin cancer tissue specimens displayed a positive correlation of CD81 with MT1-MMP expression levels and a close association of CD81 with malignant melanomas. Taken together, these results strongly suggest that CD81 stimulates melanoma cell motility by inducing MT1-MMP expression through the Akt-dependent Sp1 activation signaling pathway, leading to increased melanoma invasion and metastasis.

The CD99 gene encodes two distinct transmembrane proteins by alternative splicing of its transcript. To examine the effects of two CD99 isoforms on the invasive phenotypes of breast cancer cells, MDA-MB-231 and MCF-7 human breast cancer cell lines were stably transfected with CD99 cDNAs encoding the major wild-type form (type I) or a minor splice variant (type II). As a result, expression of CD99 type II, but not type I, markedly elevated the motility, binding to fibronectin, MMP-9 expression, and invasiveness of MDA-MB-231 and MCF-7 breast cancer cells. In MDA-MB-435 breast cancer cells expressing both CD99 type I and type II, invasion-related cellular activities were inhibited by the transfection of small interfering RNA (siRNA) targeted to CD99 type II. Meanwhile, CD99 type II-induced MMP-9 expression in MDA-MB-231 cells was shown to be mediated by the binding of AP-1 factors to the MMP-9 gene promoter. Gel shift assay revealed that ligation of CD99 type II with antibody resulted in the binding of JunD to the AP-1 site of the MMP-9 promoter region. Initiation of CD99 type II signaling by antibody ligation increased expression of JunD and FosB AP-1 factors, along with phosphorylation of Src, Akt, p38 MAPK, ERK, and JNK. Knockdown of JunD and FosB by siRNA transfection abolished the positive effects of CD99 type II on the motility and MMP-9 expression of MDA-MB-231 cells. Increased expression of JunD and FosB as well as elevated cell motility and MMP-9 expression by CD99 type II ligation were also abrogated by inhibitors, dominant-negative forms, and siRNAs for Akt1, ERK1/2, and JNK1 but not for p38 MAPK. These results suggest that expression of a splice variant of CD99 contributes to the invasive ability of human breast cancer cells by up-regulating AP-1-mediated gene expression through the Akt-dependent ERK and JNK signaling pathways.

Tetraspanin CD82 suppresses cancer metastasis by inhibiting cell motility and invasiveness. In an effect to reveal the mechanism underlying the metastasis-suppressing role of CD82, we here examined the effect of CD82 on epithelial-mesenchymal transition (EMT), a critical event of cancer cells that promotes migration and invasion. In human prostate cancer cell lines, an increase in CD82 expression levels by exogenous gene delivery elevated E-cadherin levels, but decreased the levels of mesenchymal cell proteins including Snail, Twist, vimentin, N-cadhherin, and α-smooth muscle actin, suggesting EMT-suppressing role of CD82. Also, increased CD82 expression resulted in decreased motility and invasiveness of prostate cancer cells. In contrast, CD82 knockdown decreased E-cadherin levels, but increased the levels of mesenchymal cell proteins, together with cell morphology change to fibroblast-like shape. CD82 also interfered with TGF-β1- and Wnt-induced EMT. Additionally, CD82 decreased expression of β-catenin target genes such as c-myc and cyclin D1 by inhibiting nuclear translocation of β-catenin. Notably, the negative effect of CD82 on EMT was apparent on cells adhered to fibronectin. Fibronectin-binding α3β1/α5β1 integrins were involved in CD82-mediated inhibition of EMT. Importantly, human prostate cancer tissue specimens displayed a positive correlation of CD82 with E-cadherin expression levels, along with a negative correlation of CD82 with vimentin levels. Moreover, both CD82 and E-cadherin levels were inversely correlated with the malignant progression stages of human prostate cancers. Taken together, these results strongly suggest that CD82 suppresses motility and invasiveness of prostate cancer cells by inhibiting EMT in an α3β1/α5β1 integrin-dependent manner. Citation Format: Hee-Jung Byun, Yong-Sun Lee, Young-June Jin, Jae-Sub Lee, Young-Myeong Kim, Hansoo Lee. Metastasis suppressor CD82 functions as a negative regulator in the adhesion-dependent epithelial-to-mesenchymal transition in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1051. doi:10.1158/1538-7445.AM2014-1051

Macrophage inhibitory cytokine-1 (MIC-1) is highly associated with malignant human cancers and has been suggested to be involved in tumor angiogenesis. In the present study, we examined the effect of MIC-1 on proliferation of endothelial cells. MIC-1 treatment accelerated progression of the G1 stage in cell cycle of human umbilical vein endothelial cells, together with increased cell proliferation. MIC-1 augmented the levels of cyclin D1 and E without affecting the levels of CDK inhibitors, thereby increasing activity of CDK, which led to Rb phosphorylation and subsequent E2F-1 nuclear translocation. MIC-1-induced expression of cyclins D1 and E was found to be mediated by AP-1 and E2F-1 transcription factors, and among the AP-1 members, c-Jun and JunD appeared to participate in MIC-1-mediated up-regulation of cyclin D1. Additionally, PI3K/Akt and ERK were found to mediate MIC-1-induced cyclin D1 expression. Importantly, lung endothelial cells of MIC-1 transgenic mouse exhibited increased cell proliferation rate and cyclin D1 and E levels as compared to the wild-type mouse counterparts. These results suggest that MIC-1 secreted from cancer cells stimulates endothelial cell proliferation by up-regulating of the G1 cyclins through the PI3K/Akt- and ERK-dependent AP-1 and E2F activation signaling pathways, potentially leading to the promotion of tumor angiogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3041. doi:1538-7445.AM2012-3041