Myosin Light Chain Protein Research Articles

Effects of high glucose on human umbilical vein endothelial cell permeability and myosin light chain phosphorylation.

BackgroundDiabetes mellitus is one of the most important risk factors for atherosclerosis. However, the mechanisms underlying high-glucose-induced atherosclerosis remain unclear. This study was designed to observe the effects of high-glucose stimulation on the permeability of cultured human umbilical vein endothelial cells (HUVECs), and to explore the effects of RhoA–Rho-associated protein kinase (ROCK) signal transduction pathway activation and myosin light chain (MLC) phosphorylation.MethodsHUVECs were cultured in conventional M199 medium to produce endothelial cell monolayers, and stimulated with high-glucose-M199 medium. The transmembrane transport of dextran and THP-1 cells and levels of MLC phosphorylation were measured. The effects of blocking the RhoA-ROCK pathway using dnRhoA or the ROCK inhibitor Y27632 on dextran and THP-1 transport and MLC phosphorylation were observed.ResultsTransendothelial migration of dextran and THP-1 cells were significantly increased by stimulation of HUVEC monolayers with high glucose (P < 0.05). This effect was attenuated by treatment with dnRhoA or Y27632.ConclusionHigh-glucose stimulation upregulated MLC phosphorylation and increased endothelial permeability by activating the RhoA-ROCK signaling pathway in HUVECs in vitro.

2D DIGE Does Not Reveal all: A Scotopic Report Suggests Differential Expression of a Single "Calponin Family Member" Protein for Tetany of Sphincters!

Using 2D differential gel electrophoresis (DIGE) and mass spectrometry (MS), a recent report by Rattan and Ali (2015) compared proteome expression between tonically contracted sphincteric smooth muscles of the internal anal sphincter (IAS), in comparison to the adjacent rectum [rectal smooth muscles (RSM)] that contracts in a phasic fashion. The study showed the differential expression of a single 23 kDa protein SM22, which was 1.87 fold, overexpressed in RSM in comparison to IAS. Earlier studies have shown differences in expression of different proteins like Rho-associated protein kinase II, myosin light chain kinase, myosin phosphatase, and protein kinase C between IAS and RSM. The currently employed methods, despite its high-throughput potential, failed to identify these well-characterized differences between phasic and tonic muscles. This calls into question the fidelity and validatory potential of the otherwise powerful technology of 2D DIGE/MS. These discrepancies, when redressed in future studies, will evolve this recent report as an important baseline study of “sphincter proteome.” Proteomics techniques are currently underutilized in examining pathophysiology of hypertensive/hypotensive disorders involving gastrointestinal sphincters, including achalasia, gastroesophageal reflux disease (GERD), spastic pylorus, seen during diabetes or chronic chemotherapy, intestinal pseudo-obstruction, and recto-anal incontinence. Global proteome mapping may provide instant snapshot of the complete repertoire of differential proteins, thus expediting to identify the molecular pathology of gastrointestinal motility disorders currently labeled “idiopathic” and facilitating practice of precision medicine.

Myofibrillogenesis regulator-1 attenuates hypoxia/reoxygenation-induced injury by repairing microfilaments in neonatal rat cardiomyocytes

Hypoxia/reoxygenation (H/R) injury is characterized by microfilament reorganization in cardiomyocytes. Previous studies have shown that myofibrillogenesis regulator-1 (MR-1) is expressed in the myocardium and promotes actin organization in cardiomyocytes. The purpose of this study was to investigate the role of MR-1 in attenuating hypoxia/reoxygenation injury in cardiomyocytes through promoting restoration of the microfilament. To address this aim, an H/R model of cultured neonatal cardiomyocytes was used to assess filamentous actin (F-actin) and α-actinin organization through immunofluorescence microscopy analysis. RT-PCR was used to detect mRNA levels of MR-1 and myosin regulatory light chain-2 (MLC-2). Western blot analysis was used to detect protein levels of MR-1 and filamentous actin/globular actin (F-/G-actin) as well as MLC-2 and myosin light chain kinase (MLCK) phosphorylation and protein expression. We also explored the effects of overexpressing or knocking down MR-1 on H/R injury and the MLCK/MLC-2/F-actin pathway. We found that H/R induced cardiomyocyte injury and disruption of F-actin and α-actinin with a decrease in the F-/G-actin ratio compared with controls. Compared with the H/R group, MR-1 overexpression attenuated H/R-induced injury and disruption of F-actin and α-actinin in cardiomyocytes with an increase in the F-/G-actin ratio. MR-1 overexpression also up-regulated H/R-induced MLCK and MLC-2 phosphorylation. However, MR-1 knockdown aggravated H/R injury by further disrupting F-actin and α-actinin, as well as decreasing the F-/G-actin ratio. MR-1 knockdown also down-regulated MLCK and MLC-2 phosphorylation induced by H/R injury. These findings suggest that MR-1 attenuates H/R-induced cardiomyocyte injury by promoting microfilament reorganization through the activation of the MLCK/MLC-2 pathway.

Reversal of Fibrosis in Chronic Rejection in Mouse Airways through Combined Relaxin and Lysyl Oxidase Inhibitor Therapy

The leading cause of death in lung transplant recipients is the development of chronic rejection, signified by obliterative bronchiolitis, which is histologically characterized by a fibrotic process that leads to slow narrowing of the airways. The fibrosis occurs when activated fibroblasts deposit excess extracellular matrix (ECM), which compromises gas exchange. Accumulation of ECM proteins in chronic rejected allograft in lung transplant may increase the stiffness of tissue and result in further unbalance of protein turnover and fibrous obliteration of the airway. We hypothesized that relaxin, a hormone that helps pelvic and cervical expansion and relaxation during pregnancy, could ameliorate fibrosis in chronic rejection. Relaxin might prohibit fibroblast-to-myofibroblast differentiation and fibroblast contraction, thus inhibiting fibrosis. These effects can be amplified by combining with lysyl oxidase inhibitor, β-aminoproprilnitrile (BAPN), which functions by inhibiting the cross-linking of fiber-forming stiff collagen, which decreases the ECM stiffness. We test in vitro by transplanting tracheas from major histocompatibility complex-mismatched mice to C57BL/6 mice treated with recombinant relaxin combined with BAPN after chronic rejection. The combined treatment of relaxin and BAPN attenuates airway fibrosis, decreases cross-linked collagen deposition, and promotes re-epithelialization. Relaxin inhibits fibroblast-to-myofibroblast differentiation and procollagen deposition in fibroblasts plated on matrix with intermediate stiffness, but not high stiffness. Relaxin also decreases fibroblast contractility, evidenced by decreased myosin light chain protein phosphorylation, through up-regulating prostaglandin E2. The beneficial effects of combined treatment might result from the regulation of cell contractility by relaxin and the decreased ECM stiffness caused by inhibition of collagen cross-linking by BAPN.

Deficiency of the 15-kDa selenoprotein led to cytoskeleton remodeling and non-apoptotic membrane blebbing through a RhoA/ROCK pathway

The 15-kDa selenoprotein (Sep15) has been implicated in etiology of some types of cancer. Herein, inducible RNAi cell lines were established and cell morphology and motility were analyzed. The majority of Sep15-deficient cells (>95%) formed membrane blebs in a dynamic manner. Blebbing cells transformed cell morphology from a normal flat spindle shape to a spherical morphology. In blebbing cells, actin fibers moved to the cell periphery, covering and obscuring visualization of α-tubulin. Bleb formation was suppressed by the inhibitors of Rho-associated protein kinase (ROCK), RhoA or myosin light chain (MLC), restoring blebbing cells to wild-type morphology. RhoA activation and phosphorylation of myosin phosphatase target subunit 1 was induced by Sep15 knockdown. Sep15-deficient cells were non-apoptotic, and displayed a distinct relative localization of F-actin and α-tubulin from typical apoptotic blebbing cells. Our data suggest that Sep15 in Chang liver cells regulates the pathway that antagonizes RhoA/ROCK/MLC-dependent non-apoptotic bleb formation.

Skeletal muscle plasticity induced by seasonal acclimatization involves IGF1 signaling: implications in ribosomal biogenesis and protein synthesis.

One of the most fundamental biological processes in living organisms that are affected by environmental fluctuations is growth. In fish, skeletal muscle accounts for the largest proportion of body mass, and the growth of this tissue is mainly controlled by the insulin-like growth factor (IGF) system. By using the carp (Cyprinus carpio), a fish that inhabits extreme conditions during winter and summer, we assessed the skeletal muscle plasticity induced by seasonal acclimatization and the relation of IGF signaling with protein synthesis and ribosomal biogenesis. The expression of igf1 in muscle decreased during winter in comparison with summer, whereas the expression for both paralogues of igf2 did not change significantly between seasons. The expression of igf1 receptor a (igf1ra), but not of igf1rb, was down-regulated in muscle during the winter as compared to the summer. A decrease in protein contents and protein phosphorylation for IGF signaling molecules in muscle was observed in winter-acclimatized carp. This was related with a decreased expression in muscle for markers of myogenesis (myoblast determination factor (myod), myogenic factor 5 (myf5), and myogenin (myog)); protein synthesis (myosin heavy chain (mhc) and myosin light chain (mlc3 and mlc1b)); and ribosomal biogenesis (pre-rRNA and ribosomal proteins). IGF signaling, and key markers of ribosomal biogenesis, protein synthesis, and myogenesis were affected by seasonal acclimatization, with differential regulation in gene expression and signaling pathway activation observed in muscle between both seasons. This suggests that these molecules are responsible for the muscle plasticity induced by seasonal acclimatization in carp.

MUSCLE DEVELOPMENT IN AMPHIOXUS: MORPHOLOGY, BIOCHEMISTRY, AND MOLECULAR BIOLOGY

ABSTRACT This review concerns the structure and biochemistry of muscle in amphioxus. Most work has focused on the segmented swimming (axial) muscles. These muscles derive from the medial wall of the somites, which arise as evaginations from the gut wall. The myotomal muscle cells of amphioxus, unlike those of vertebrates, never fuse, but remain mononucleate, contain only one myofibril, and span the entire length of the myotome. The muscle cells are very thin and lack a T-tubule system. There are two, maybe three, types of fibers. Innervation is via muscle tails, which contact the basal lamina of the nerve cord. The notochord is also composed of striated muscle cells, which similarly send muscle tails to the nerve cord. Less is known about the biochemistry of muscle. The notochord, like molluskan catch muscle, contains paramyosin. Among the muscle-specific proteins sequenced are alkali myosin light chain, troponin C and sarcoplasmic calcium-binding proteins, calcium-vector protein, and its target protein c...

A novel all-trans retinoid acid derivatives inhibits the migration of breast cancer cell lines MDA-MB-231 via myosin light chain kinase involving p38-MAPK pathway

ObjectiveTo explore the effect and its probable mechanism of a synthetic retinoid 4-amino-2-tri-fluoromethyl-phenyl ester (ATPR) on the migration of human breast cancer MDA-MB-231 cells. MethodsMTT assay was performed to measure the proliferation of MDA-MB-231 cells treated with different concentrations of all-trans retinoic acid (ATRA) and ATPR. The effect of ATPR and ML-7, a selective inhibitor of myosin light chain kinase (MLCK), and SB203580, an inhibitor of p38, on the migration of MDA-MB-231 cells were analyzed by wound healing assay. The expression of MLCK and phosphorylation of myosin light chain (MLC), ERK, JNK, p38 proteins were detected by western blot ResultsAfter the cells were treated by ATRA and ATPR, the proliferation and migration of breast cancer MDA-MB-231 cells were inhibited significantly. The IC 50 of ATRA and ATPR is 34.08 μmol/l and 18.06 μmol/l respectively. The relative migration rate of MDA-MB-231 cells treated with ATPR reached 50% at 48 h while the ATRA group is over 90%. The relative migration rate of ML-7 group and SB group had significant decrease compared with control group. The expression level of MLCK and phosphorylation of MLC of breast cancer cells was reduced when the cells were treated by ATPR with 48 h, the phosphorylation of ERK, JNK and p38 in breast cancer also reduced when cells were treated by ATPR with 2 h. In addition, ML-7 (50 μmol/l) could inhibit the phosphorylation of p38 and SB (50 μmol/l) could inhibit the expression of MLCK and phosphorylation of MLC. ConclusionsATPR had a better inhibition on the proliferation and the migration of breast cancer MDA-MB-231 cells than ATRA, and its probable mechanism was associated with the down regulation of expression of MLCK and phosphorylation of MLC protein involving p38-MAPK pathway.

CL316,243, a Selective β3‐Adrenoceptor Agonist Activates Protein Synthesis Through mTOR/p70S6K Signaling Pathway in L6 Cells.

The existence of β3‐adrenergic receptors (AR) in skeletal muscle has been demonstrated by the detection of the β3‐mRNA and protein and by the selective effects β3‐AR agonists on metabolic oxidation and glucose utilization. However, the role of β3‐AR on protein metabolism in skeletal muscle remains still unclear. To determine whether β3‐AR regulates protein translation we evaluated in L6 skeletal muscle cells the effect of CL316,243, a selective β3‐AR agonist, on the rate of protein synthesis and on the activation of the Akt/mTOR/ p70S6K signaling pathway which is known to support the protein synthesis requirement during muscle growth. When L6 cells were incubated in the presence of CL 316,243, we observed a net increase of [35S] methionine incorporation into muscle cell proteins such myosin heavy chain and myosin light chain at 24 hr. The anabolic effect of CL 316,243 was associated with an increase of p70S6K which was inhibited by the β3‐AR antagonist, SR 59230A, but not by the β2‐AR antagonist ICI 118551. In addition, the increase in p70S6K levels induced by CL 316,243 was inhibited by wortmannin, a PI3K inhibitor, and by rapamycin, mTOR inhibitor suggesting that β3‐AR regulates protein synthesis via PI3K/Akt/ p70S6K signaling pathway. This finding unveils a new cellular mechanism by which β‐AR system exerts anabolic actions on skeletal protein metabolism and therefore regulates skeletal muscle mass.

Protective Effects of the mTOR Inhibitor Everolimus on Cytoskeletal Injury in Human Podocytes Are Mediated by RhoA Signaling

Podocytes are highly differentiated kidney cells playing an important role in maintaining the glomerular filtration barrier. Particularly, the integrity of the actin cytoskeleton is crucial as cytoskeletal damage associated with foot process effacement and loss of slit diaphragms constitutes a major aspect of proteinuria. Previously, the mammalian target of rapamycin (mTOR) was linked to actin regulation and aberrant activity of the kinase was associated with renal disease. In this study, actin-related effects of mTOR inhibition by the immunosuppressant everolimus (EV) were investigated in human podocytes using an in vitro model of puromycin aminonucleoside (PAN) induced proteinuria. EV substantially recovered aberrant podocyte behavior by re-establishing a stationary phenotype with decreased migration efficiency, enhanced cell adhesion and recovery of actin stress fibers. Biochemical studies revealed substantial increase in the activity of RhoA and the effector pathway Rho-associated protein kinase (ROCK) and myosin light chain (MLC) by EV, all known regulators of stress fiber generation. Taken together, we show for the first time cytoskeleton stabilizing effects of the mTOR inhibitor EV and establish RhoA signaling as a key mediator in this process.

Tissue expression of PPAR-alpha isoforms in Scophthalmus maximus and transcriptional response of target genes in the heart after exposure to WY-14643

Peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of lipid and carbohydrate metabolism and can be activated either by natural ligands as fatty acids or by synthetic ligands including several environmental chemicals. In this study, two PPARα isoforms (α1 and α2) were analyzed in turbot (Scophthalmus maximus) for a different tissue distribution. PPARα1 was ubiquitously expressed, while the PPARα2 was predominantly expressed in the heart. Following this result, turbot juveniles were exposed by injection to a synthetic selective PPARα agonist, WY-14643, for 14 days. Suppression subtractive hybridization (SSH) was performed with pools of heart samples of control and exposed fish to get insights into PPARα-regulated genes in the heart of juvenile turbot. Four genes were positively identified in the forward-subtracted and 12 genes in the reverse-subtracted cDNA SSH library, corresponding to the down-regulated and up-regulated genes in response to the WY-14643 treatment, respectively. The confirmation of these results in individual samples of juvenile turbot exposed to WY-14643 revealed a statistically significant mRNA induction of two cardiac muscle proteins (myosin light chain 2 and tropomyosin 4), which were shown to be involved in heart contraction and heartbeat regulation in other teleost species. Herewith, we showed for the first time that PPARα2 is predominantly expressed in the heart and that a PPARα agonist can induce the mRNA expression of cardiac muscle proteins in teleosts.

Heavy and light roles: myosin in the morphogenesis of the heart

Myosin is an essential component of cardiac muscle, from the onset of cardiogenesis through to the adult heart. Although traditionally known for its role in energy transduction and force development, recent studies suggest that both myosin heavy-chain and myosin light-chain proteins are required for a correctly formed heart. Myosins are structural proteins that are not only expressed from early stages of heart development, but when mutated in humans they may give rise to congenital heart defects. This review will discuss the roles of myosin, specifically with regards to the developing heart. The expression of each myosin protein will be described, and the effects that altering expression has on the heart in embryogenesis in different animal models will be discussed. The human molecular genetics of the myosins will also be reviewed.

Serum Proteomic Signature of Human Chagasic Patients for the Identification of Novel Potential Protein Biomarkers of Disease

Chagas disease is initiated upon infection by Trypanosoma cruzi. Among the health consequences is a decline in heart function, and the pathophysiological mechanisms underlying this manifestation are not well understood. To explore the possible mechanisms, we employed IgY LC10 affinity chromatography in conjunction with ProteomeLab PF2D and two-dimensional gel electrophoresis to resolve the proteome signature of high and low abundance serum proteins in chagasic patients. MALDI-TOF MS/MS analysis yielded 80 and 14 differentially expressed proteins associated with cardiomyopathy of chagasic and other etiologies, respectively. The extent of oxidative stress-induced carbonyl modifications of the differentially expressed proteins (n = 26) was increased and coupled with a depression of antioxidant proteins. Functional annotation of the top networks developed by ingenuity pathway analysis of proteome database identified dysregulation of inflammation/acute phase response signaling and lipid metabolism relevant to production of prostaglandins and arachidonic acid in chagasic patients. Overlay of the major networks identified prothrombin and plasminogen at a nodal position with connectivity to proteome signature indicative of heart disease (i.e., thrombosis, angiogenesis, vasodilatation of blood vessels or the aorta, and increased permeability of blood vessel and endothelial tubes), and inflammatory responses (e.g., platelet aggregation, complement activation, and phagocyte activation and migration). The detection of cardiac proteins (myosin light chain 2 and myosin heavy chain 11) and increased levels of vinculin and plasminogen provided a comprehensive set of biomarkers of cardiac muscle injury and development of clinical Chagas disease in human patients. These results provide an impetus for biomarker validation in large cohorts of clinically characterized chagasic patients.

Abstract 2290: MiR-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-beta signalling pathway

Abstract MicroRNAs are post-transcriptional gene regulators that play a ubiquitous role in cellular functioning. Aberrant expression of miRNAs has been correlated with many cancer types, including neuroblastoma. Neuroblastoma is an often fatal pediatric cancer derived from precursor cells of the sympathetic nervous system. The single most important indicator of unfavourable metastatic disease is amplification of the MYCN oncogenic transcription factor. MiR-335 is significantly down-regulated in MYCN amplified tumors (Bray et al Plos One 2009; 4:e7850) suggesting a possible tumor suppressive function. This study aims to determine if miR-335 functions as a tumor suppressor in neuroblastoma and to elucidate the mechanism by which it elicits this effect. Ectopic up-regulation of miR-335 significantly reduced the motile and invasive capacity of neuroblastoma cell lines. Inversely, down-regulation of miR-335 significantly increased their migratory and invasive potential. mRNA expression profiling arrays in conjunction with the miRNA target prediction algorithm TargetScan identified potential targets of miR-335. Three of these candidate genes, ROCK1, MAPK1 and LRG1, were selected for further investigation because of their known or predicted involvement in cell migration and invasion. Down-regulation of these genes by miR-335 was validated at mRNA and protein levels and direct targeting by miR-335 was confirmed by luciferase reporter assays. siRNA-mediated inhibition of each of these three genes recapitulated the diminished cell migration and invasion associated with miR-335 up-regulation. ROCK1 and MAPK1 are members of two of the major branches of the oncogenic non-canonical TGF-beta signalling pathway, whose signals converge to tightly regulate the phosphorylation or activation status of the motor protein myosin light chain (MLC). LRG1 is a putative TGF-beta pathway member that has been proposed to bind to TGF-beta receptor II. We demonstrate that siRNA mediated inhibition of ROCK1, MAPK1 and LRG1 leads to a significant reduction in phosphorylated levels of MLC protein. Furthermore, we demonstrate that up-regulation of miR-335 inhibits phosphorylation or activation of MLC through targeting of these three genes. Finally, we demonstrate using a MYCN repressible cell line and MYCN ChIP-chip experiments that miR-335 is directly repressed by the oncogenic transcription factor MYCN. Therefore, we conclude that miR-335 is directly repressed by MYCN in MYCN amplified neuroblastoma tumors leading to the increased expression of the oncogenic non-canonical TGF-beta pathway members ROCK1, MAPK1 and LRG1 which function to enhance the metastatic propensity of the tumor cells. 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 2290. doi:1538-7445.AM2012-2290

Bone Marrow-Derived Human Mesenchymal Stem Cells Express Cardiomyogenic Proteins But Do Not Exhibit Functional Cardiomyogenic Differentiation Potential

Despite their paracrine activites, cardiomyogenic differentiation of bone marrow (BM)-derived mesenchymal stem cells (MSCs) is thought to contribute to cardiac regeneration. To systematically evaluate the role of differentiation in MSC-mediated cardiac regeneration, the cardiomyogenic differentiation potential of human MSCs (hMSCs) and murine MSCs (mMSCs) was investigated in vitro and in vivo by inducing cardiomyogenic and noncardiomyogenic differentiation. Untreated hMSCs showed upregulation of cardiac tropopin I, cardiac actin, and myosin light chain mRNA and protein, and treatment of hMSCs with various cardiomyogenic differentiation media led to an enhanced expression of cardiomyogenic genes and proteins; however, no functional cardiomyogenic differentiation of hMSCs was observed. Moreover, co-culturing of hMSCs with cardiomyocytes derived from murine pluripotent cells (mcP19) or with murine fetal cardiomyocytes (mfCMCs) did not result in functional cardiomyogenic differentiation of hMSCs. Despite direct contact to beating mfCMCs, hMSCs could be effectively differentiated into cells of only the adipogenic and osteogenic lineage. After intramyocardial transplantation into a mouse model of myocardial infarction, Sca-1(+) mMSCs migrated to the infarcted area and survived at least 14 days but showed inconsistent evidence of functional cardiomyogenic differentiation. Neither in vitro treatment nor intramyocardial transplantation of MSCs reliably generated MSC-derived cardiomyocytes, indicating that functional cardiomyogenic differentiation of BM-derived MSCs is a rare event and, therefore, may not be the main contributor to cardiac regeneration.

MiRNA-335 suppresses neuroblastoma cell invasiveness by direct targeting of multiple genes from the non-canonical TGF-β signalling pathway

Transforming growth factor-β (TGF-β) signaling regulates many diverse cellular activities through both canonical (SMAD-dependent) and non-canonical branches, which includes the mitogen-activated protein kinase (MAPK), Rho-like guanosine triphosphatase and phosphatidylinositol-3-kinase/AKT pathways. Here, we demonstrate that miR-335 directly targets and downregulates genes in the TGF-β non-canonical pathways, including the Rho-associated coiled-coil containing protein (ROCK1) and MAPK1, resulting in reduced phosphorylation of downstream pathway members. Specifically, inhibition of ROCK1 and MAPK1 reduces phosphorylation levels of the motor protein myosin light chain (MLC) leading to a significant inhibition of the invasive and migratory potential of neuroblastoma cells. Additionally, miR-335 targets the leucine-rich alpha-2-glycoprotein 1 (LRG1) messenger RNA, which similarly results in a significant reduction in the phosphorylation status of MLC and a decrease in neuroblastoma cell migration and invasion. Thus, we link LRG1 to the migratory machinery of the cell, altering its activity presumably by exerting its effect within the non-canonical TGF-β pathway. Moreover, we demonstrate that the MYCN transcription factor, whose coding sequence is highly amplified in a particularly clinically aggressive neuroblastoma tumor subtype, directly binds to a region immediately upstream of the miR-335 transcriptional start site, resulting in transcriptional repression. We conclude that MYCN contributes to neuroblastoma cell migration and invasion, by directly downregulating miR-335, resulting in the upregulation of the TGF-β signaling pathway members ROCK1, MAPK1 and putative member LRG1, which positively promote this process. Our results provide novel insight into the direct regulation of TGF-β non-canonical signaling by miR-335, which in turn is downregulated by MYCN.

Proteomic analysis of metabolic, cytoskeletal and stress response proteins in human heart failure

Human heart failure is a complex syndrome and a primary cause of morbidity and mortality in the world. However, the molecular pathways involved in the remodelling process are poorly understood. In this study, we performed exhaustive global proteomic surveys of cardiac ventricle isolated from failing and non-failing human hearts, and determined the regulatory pathway to uncover the mechanism underlying heart failure. Two-dimensional gel electrophoresis (2-DE) coupled with tandem mass spectrometry was used to identify differentially expressed proteins in specimens from failing (n = 9) and non-failing (n = 6) human hearts. A total of 25 proteins with at least 1.5-fold change in the failing heart were identified; 15 proteins were up-regulated and 10 proteins were down-regulated. The altered proteins belong to three broad functional categories: (i) metabolic [e.g. NADH dehydrogenase (ubiquinone), dihydrolipoamide dehydrogenase, and the cytochrome c oxidase subunit]; (ii) cytoskeletal (e.g. myosin light chain proteins, troponin I type 3 and transthyretin) and (iii) stress response (e.g. αB-crystallin, HSP27 and HSP20). The marked differences in the expression of selected proteins, including HSP27 and HSP20, were further confirmed by Western blot. Thus, we carried out full-scale screening of the protein changes in human heart failure and profiled proteins that may be critical in cardiac dysfunction for future mapping.

Anthrax lethal toxin-mediated disruption of endothelial VE-cadherin is attenuated by inhibition of the Rho-associated kinase pathway.

Systemic anthrax disease is characterized by vascular leakage pathologies. We previously reported that anthrax lethal toxin (LT) induces human endothelial barrier dysfunction in a cell death-independent manner with actin stress fiber formation and disruption of adherens junctions (AJs). In the present study, we further characterize the molecular changes in the AJ complex and investigate whether AJ structure and barrier function can be preserved by modulating key cytoskeletal signaling pathways. Here, we show that LT reduces total VE-cadherin protein and gene expression but the expression of the key linker protein beta-catenin remained unchanged. The changes in VE-cadherin expression correlated temporally with the appearance of actin stress fibers and a two-fold increase in phosphorylation of the stress fiber-associated protein myosin light chain (p-MLC) and cleavage of Rho-associated kinase-1 (ROCK-1). Co-treatment with ROCK inhibitors (H-1152 and Y27632), but not an inhibitor of MLC kinase (ML-7), blocked LT-induced p-MLC enhancement and stress fiber formation. This was accompanied by the restoration of VE-cadherin expression and membrane localization, and attenuation of the LT-induced increase in monolayer permeability to albumin. Together, these findings suggest the ROCK pathway may be a relevant target for countering LT-mediated endothelial barrier dysfunction.

Dynamic Regulation of Vascular Myosin Light Chain (MYL9) with Injury and Aging

BackgroundAging-associated changes in the cardiovascular system increase the risk for disease development and lead to profound alterations in vascular reactivity and stiffness. Elucidating the molecular response of arteries to injury and age will help understand the exaggerated remodeling of aging vessels.Methodology/Principal FindingsWe studied the gene expression profile in a model of mechanical vascular injury in the iliac artery of aging (22 months old) and young rats (4 months old). We investigated aging-related variations in gene expression at 30 min, 3 d and 7 d post injury. We found that the Myosin Light Chain gene (MYL9) was the only gene differentially expressed in the aged versus young injured arteries at all time points studied, peaking at day 3 after injury (4.6 fold upregulation (p<0.05) in the smooth muscle cell layers. We confirmed this finding on an aging aortic microarray experiment available through NCBI's GEO database. We found that Myl9 was consistently upregulated with age in healthy rat aortas. To determine the arterial localization of Myl9 with age and injury, we performed immunohistochemistry for Myl9 in rat iliac arteries and found that in healthy and injured (30 days post injury) arteries, Myl9 expression increased with age in the endothelial layers.Conclusions/SignificanceThe consistent upregulation of the myosin light chain protein (Myl9) with age and injury in arterial tissue draws attention to the increased vascular permeability and to the age-caused predisposition to arterial constriction after balloon angioplasty.

Signaling pathways in cerebral vasospasm

AbstractCerebral vasospasm is a deadly complication following the rupture of intracranial aneurysms. One new development in the experimental treatment of cerebral vasospasm is the looming target of signaling pathways. The pathogenesis of cerebral vasospasm involves multiple signaling pathways in proliferation, inflammation, cell death, smooth muscle phenotype changes, vascular remodeling, and contraction. A review of all of these areas is beyond the scope of this article, and as such, three systems that mediate these vascular responses have been selected: the tyrosine kinase-MAP kinase pathway, the sphingosine-1-Rho myosin light chain kinase pathway and protein kinase C.