Expression Of Myosin Light Chain-2 Research Articles

Engineered liposomes targeting hepatic stellate cells overcome pathological barriers and reverse liver fibrosis

Dual pathological barriers, including capillarized liver sinusoidal endothelial cells (LSECs) and deposited extracellular matrix (ECM), result in insufficient drug delivery, significantly compromising the anti-fibrosis efficacy. Additionally, excessive reactive oxygen species (ROS) in the hepatic microenvironment are crucial factors contributing to the progression of liver fibrosis. Hence, hyaluronic acid (HA) modified liposomes co-delivering all-trans retinoic acid (RA) and L-arginine (L-arg) were constructed to reverse hepatic fibrosis. By exhibiting exceptional responsiveness to the fibrotic microenvironment, our cleverly constructed liposomes efficiently disrupted the hepatic sinus pathological barrier, leading to enhanced accumulation of liposomes in activated hepatic stellate cells (HSCs) and subsequent induction of HSCs quiescence. Specially, excessive ROS in liver fibrosis promotes the conversion of loaded L-arg to nitric oxide (NO). The ensuing NO serves to reestablish the fenestrae structure of capillarized LSECs, thereby augmenting the likelihood of liposomes reaching the hepatic sinus space. Furthermore, subsequent oxidation of NO by ROS into peroxynitrite activates pro-matrix metalloproteinases into matrix metalloproteinases, which further disrupts the deposited ECM barrier. Consequently, this NO-induced cascade process greatly amplifies the accumulation of liposomes within activated HSCs. More importantly, the released RA could induce quiescence of activated HSCs by significantly downregulating the expression of myosin light chain-2, thereby effectively mitigating excessive collagen synthesis and ultimately leading to the reversal of liver fibrosis. Overall, this integrated systemic strategy has taken a significant step forward in advancing the treatment of liver fibrosis.

Low-Intensity Pulsed Ultrasound Improves the Functional Properties of Cardiac Mesoangioblasts.

Cell-based therapy is a promising approach for many diseases, including ischemic heart disease. Cardiac mesoangioblasts are committed vessel-associated progenitors that can restore to a significant, although partial, extent, heart structure and function in a murine model of myocardial infarction. Low-intensity pulsed ultrasound (LIPUS) is a non-invasive form of mechanical energy that can be delivered into biological tissues as acoustic pressure waves, and is widely used for clinical applications including bone fracture healing. We hypothesized that the positive effects of LIPUS on bone and soft tissue, such as increased cell differentiation and cytoskeleton reorganization, could be applied to increase the therapeutic potential of mesoangioblasts for heart repair. In this work, we show that LIPUS stimulation of cardiac mesoangioblasts isolated from mouse and human heart results in significant cellular modifications that provide beneficial effects to the cells, including increased malleability and improved motility. Additionally, LIPUS stimulation increased the number of binucleated cells and induced cardiac differentiation to an extent comparable with 5'-azacytidine treatment. Mechanistically, LIPUS stimulation activated the BMP-Smad signalling pathway and increased the expression of myosin light chain-2 together with upregulation of β1 integrin and RhoA, highlighting a potentially important role for cytoskeleton reorganization. Taken together, these results provide functional evidence that LIPUS might be a useful tool to explore in the field of heart cell therapy.

Myosin light chain 2 isoforms in gilthead sea bream (Sparus aurata L.): Molecular growth markers at early stages

The production of high quality juveniles at hatchery stages is important to ensure high growth potential during the on-growing phase. In the present paper, molecular growth markers of muscle development and growth in gilthead sea bream were validated in developmental stages/sizes that exhibit high accelerated growth rate, and rearing practices that are characterized by considerable size disparity. In gilthead sea bream the skeletal myosin light chain 2 (MLC2) is a structural molecule of the muscle that exists in two isoforms, A and B, which are known to mark hyperplasia and hypertrophy, respectively. The expression levels of the two isoforms and the robustness of its correlation with length growth were validated in a) larvae and juveniles of gilthead sea bream originating from the same broodstock and raised under intensive vs semi-intensive conditions, and b) in juvenile gilthead sea bream reared under commercial aquaculture practices and subjected to size grading with sorters of increasing diameter. A replacement of MLC2A by MLC2B was observed at metamorphosis in agreement with previous results. Larvae reared at the semi-intensive system outperformed in length growth and MLC2 expression those reared intensively and an earlier and faster replacement of MLC2A by MLC2B was observed at metamorphosis. Similarly, the ratio of MLC2A/MLC2B expression exhibited a tight, significantly negative correlation to total length of juveniles, independent of age. Overall, comparison of groups of gilthead sea bream of different origin rose under different conditions and of different developmental stages (larvae vs juveniles) provided significant evidence for the robustness of MLC2 expression as a growth marker. • The expression of myosin light chain 2 (MLC2) isoforms was correlated to length. • Groups of larvae and juveniles exhibiting different growth were studied. • Individuals of same age, yet different length were compared for MLC2 expression. • MLC2A/MLC2B expression was significantly and negatively correlated to total length. • This correlation was independent of age.

Increase in cell motility by carbon ion irradiation via the Rho signaling pathway and its inhibition by the ROCK inhibitor Y-27632 in lung adenocarcinoma A549 cells

This study aimed to investigate the effect of carbon ion (C-ion) irradiation on cell motility through the ras homolog gene family member (Rho) signaling pathway in the human lung adenocarcinoma cell line A549. Cell motility was assessed by a wound-healing assay, and the formation of cell protrusions was evaluated by F-actin staining. Cell viability was examined by the WST-1 assay. The expression of myosin light chain 2 (MLC2) and the phosphorylation of MLC2 at Ser19 (P-MLC2-S19) were analyzed by Western blot. At 48 h after irradiation, the wound-healing assay demonstrated that migration was significantly greater in cells irradiated with C-ion (2 or 8 Gy) than in unirradiated cells. Similarly, F-actin staining showed that the formation of protrusions was significantly increased in cells irradiated with C-ion (2 or 8 Gy) compared with unirradiated cells. The observed increase in cell motility due to C-ion irradiation was similar to that observed due to X-ray irradiation. Western-blot analysis showed that C-ion irradiation (8 Gy) increased P-MLC2-S19 expression compared with in unirradiated controls, while total MLC2 expression was unchanged. Exposure to a non-toxic concentration of Y-27632, a specific inhibitor of Rho-associated coiled-coil-forming protein kinase (ROCK), reduced the expression of P-MLC2-S19 after C-ion irradiation (8 Gy), resulting in a significant reduction in migration. These data suggest that C-ion irradiation increases cell motility in A549 cells via the Rho signaling pathway and that ROCK inhibition reduces that effect.

Mycophenolic acid mediated disruption of the intestinal epithelial tight junctions

Gastrointestinal toxicity is a common adverse effect of mycophenolic acid (MPA) treatment in organ transplant patients, through poorly understood mechanisms. Phosphorylation of myosin light chain 2 (MLC2) is associated with epithelial tight junction (TJ) modulation which leads to defective epithelial barrier function, and has been implicated in GI diseases. The aim of this study was to investigate whether MPA could induce epithelial barrier permeability via MLC2 regulation. Caco-2 monolayers were exposed to therapeutic concentrations of MPA, and MLC2 and myosin light chain kinase (MLCK) expression were analyzed using PCR and immunoblotting. Epithelial cell permeability was assessed by measuring transepithelial resistance (TER) and the flux of paracellular permeability marker FITC-dextran across the epithelial monolayers. MPA increased the expression of MLC2 and MLCK at both the transcriptional and translational levels. In addition, the amount of phosphorylated MLC2 was increased after MPA treatment. Confocal immunofluorescence analysis showed redistribution of TJ proteins (ZO-1 and occludin) after MPA treatment. This MPA mediated TJ disruption was not due to apoptosis or cell death. Additionally ML-7, a specific inhibitor of MLCK was able to reverse both the MPA mediated decrease in TER and the increase in FITC-dextran influx, suggesting a modulating role of MPA on epithelial barrier permeability via MLCK activity. These results suggest that MPA induced alterations in MLC2 phosphorylation and may have a role in the patho-physiology of intestinal epithelial barrier disruption and may be responsible for the adverse effects (GI toxicity) of MPA on the intestine.

Abstract 1318: 17β-estradiol inhibits metastatic granulosa cell migration by repressing ERK1/2 pathway via an extra-nuclear mechanism of action.

Abstract Granulosa cell tumor (GCT) is a rare but severe form of ovarian tumor that is characterized by its tendency to recur years after surgical ablation. Because there is no efficient curative treatment beyond surgery, ∼20% of adult patients die of the consequences of their tumor. However, very little is known of the molecular etiology of this pathology. Although GCTs express the estrogen receptors (ER) ERα and ERβ, the effect of 17β-estradiol (E2) in disease progression remains elusive. The purpose of our study was to evaluate the potential role of E2 in GCT pathogenesis and to determine its underlying mechanisms of actions. To address this issue, molecular studies were performed using two human ER-positive GCT-derived cell lines, i.e., COV434 and KGN cells established from a primary tumor and from a metastatic recurrence, respectively. In both cell lines, E2 treatment (1 to 100 nM) up to 72 hours did not affect cell growth, as assessed by MTT assay and automatic cell counting. Importantly, horizontal migration assay showed that a 24-hour E2 treatment (10 nM) significantly inhibited KGN cell migration by ∼25%, in terms of migration distance and migrating cells. In contrast, no effect was observed in the primary tumor-derived COV434 cell line. Western blot studies revealed that in KGN, but not in COV434 cells, E2 treatment (10 nM) led to a rapid (as early as 5 minutes) and durable (up to 24 hours) decrease by ∼30% in the phosphorylation levels of ERK1/2, while it had no effect on ERK1/2 abundance. This was accompanied by the down-regulation of the expression of myosin light chain-2 (MLC-2), which is involved in stress fiber contraction during cell migration. ERK1/2 inhibition by the MEK1/2 inhibitor U0126 mimicked E2 actions, suggesting that the effect of E2 in KGN cells mostly results from the alteration of ERK1/2 pathway. To identify the ER involved in E2 action in KGN cells, a pharmacological approach with ER agonists was used. As none of the ER agonists could mimic E2 action, we investigated the possibility that the G protein coupled estrogen receptor (GPER or GPR30), which belongs to the G protein coupled receptor family, was involved. GPER expression was confirmed by RT-PCR and Western blot assays. Treatment of KGN cells with the GPER agonist G-1 (50 nM) rapidly and durably inhibited ERK1/2 pathway and cell migration. Pre-treatment with the GPER antagonist G15 (200 nM) completely prevented the E2-induced repression of ERK1/2 activity and inhibition of cell migration. Altogether, our study reveals that E2 would prevent metastasis spreading by inhibiting ERK1/2 activity to down-regulate MLC2 expression through a GPER-dependent mechanism. It, thus, brings evidence that extra-nuclear actions of E2 can be inhibitory to cancer pathways. Further, our observations may have translational implications, as they suggest the potential benefit of selective GPER agonists for patients with recurrent GCT. Citation Format: Charlotte M. François, Richard Wargnier, Joëlle Cohen-Tannoudji, Celine J. Guigon. 17β-estradiol inhibits metastatic granulosa cell migration by repressing ERK1/2 pathway via an extra-nuclear mechanism of action. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1318. doi:10.1158/1538-7445.AM2013-1318

An autocrine role for leptin in mediating the cardiomyocyte hypertrophic effects of angiotensin II and endothelin-1

Leptin is a 16 kDa product of the obesity gene secreted primarily by adipocytes. We recently identified cardiomyocytes as a target for the direct hypertrophic effects of leptin and suggested that leptin may be a biological link between obesity and cardiovascular pathologies. Activation of the renin–angiotensin and endothelin systems is associated with development of cardiovascular diseases and plasma renin levels are elevated in obese individuals. We therefore determined possible interaction between these factors in mediating hypertrophy in cultured neonatal rat ventricular myocytes. Treatment for 24 h with leptin (3.1 nM), angiotensin II (100 nM) or endothelin-1 (ET-1, 10 nM) significantly increased cell area by 37%, 36% and 35%, respectively and significantly increased gene expression of myosin light chain-2 and α-skeletal actin as well as leucine incorporation. The hypertrophic effects of all three agents were prevented by leptin and a leptin triple mutant receptor antagonist whereas the AT1 receptor blocker (Sar1-lle8)-Ang II or the ETA receptor blocker BQ123 was ineffective against leptin-induced hypertrophy. Both angiotensin II and ET-1 significantly increased leptin levels in the culture medium by fivefold. Moreover, both angiotensin II and ET-1 increased the gene expression of the short form (OBRa) by 180% and long form (OBRb) of leptin receptors by 200%, and this increase was abolished by both leptin receptor and leptin antibodies and leptin triple mutant. Although both angiotensin II and ET-1 increased phosphorylation of MAPK (p38, ERK1/2 and JNK) and NF-κB, the ability of leptin blockade to attenuate the hypertrophic responses was generally dissociated from these effects suggesting an alternate, yet to be identified cellular pathway mediating this role of leptin. Our studies therefore suggest a novel autocrine function for leptin in mediating the hypertrophic effects of both angiotensin II and ET-1 in cardiac myocytes.

Inhibition of phenylephrine induced hypertrophy in rat neonatal cardiomyocytes by the mitochondrial K ATP channel opener diazoxide

The effect of the putative mitochondrial K ATP channel opener diazoxide (100 μM) was studied in terms of its ability to modulate the hypertrophic effect of 24 h treatment with the α 1 adrenoceptor agonist phenylephrine (PE; 10 μM) in cultured neonatal rat ventricular myocytes. PE on its own significantly increased cell size by 40%, 3H leucine incorporation by 37% and produced more than a threefold elevation in both atrial natriuretic peptide and myosin light chain-2 expression. These effects were nearly completely prevented by diazoxide although the inhibitory effect of this agent was generally mitigated by the mitochondrial K ATP channel antagonists 5-hydroxydecanoic acid (100 μM) and glibenclamide (50 μM). Although PE produced an early threefold elevation in MAP kinase activation this was generally unaffected by diazoxide. PE also produced a greater than threefold increase in Na–H exchanger isoform 1 (NHE-1) expression which, was prevented by diazoxide treatment. Our study therefore, demonstrates a potential antihypertrophic influence of mitochondrial K ATP channel activation which, is related to diminished NHE-1 expression. Mitochondrial K ATP channel activation could represent an effective approach to minimize the myocardial hypertrophic process.

The obesity-associated peptide leptin induces hypertrophy in neonatal rat ventricular myocytes.

One of the major manifestations of obesity is increased production of the adipocyte-derived 16-kDa peptide leptin, which is also elevated in heart disease, including congestive heart failure. However, whether leptin can directly alter the cardiac phenotype is not known. We therefore studied the effect of leptin as a potential hypertrophic factor in cultured myocytes from 1- to 4-day-old neonatal rat heart ventricles. Using RT-PCR, we demonstrate that these cells express the short-form (OB-Ra) leptin receptor. Twenty-four hours of exposure to leptin (0.31 to 31.3 nmol/L) produces a significantly increased cell surface area that peaked at 0.63 nmol/L. Subsequent experiments were done with 3.1 nmol/L leptin, which significantly increased cell area by 42%, protein synthesis by 32%, and alpha-skeletal actin and myosin light chain-2 expression by 250% and 300%, respectively. These events occurred in the absence of any increased cell death. Hypertrophy was preceded by rapid activation of the mitogen-activated protein kinase system including p38 and p44/42 as early as 5 minutes after leptin addition, whereas hypertrophy was inhibited by the p38 inhibitor SB203580 but not by the p44/42 inhibitor PD98059. Our results demonstrate a direct hypertrophic effect of leptin and may offer a biological link between hypertrophy and hyperleptinemic conditions such as obesity.

Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle. Isolation of a cDNA clone for repressor protein Nished.

The expression of the cardiac myosin light chain 2 (MLC2) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (-360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to DeltaCSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor "runt," a known repressor of gap and pair-rule gene expression in Drosophila.

Heart-specific targeting of firefly luciferase by the myosin light chain-2 promoter and developmental regulation in transgenic mice.

Based on hybridization studies indicating constitutive expression levels of the endogenous myosin light chain-2 (MLC-2) gene in embryonic, fetal, and adult myocardium, a model system for selective targeting of genes to the heart of transgenic mice has been developed. A 2.1-kb DNA fragment of the 5' flanking region of the rat cardiac MLC-2 gene was fused to the firefly luciferase reporter gene and introduced into fertilized mouse oocytes. In four independent transgenic mouse lines, the expression of the MLC-2-luciferase fusion gene was found exclusively in heart muscle. In contrast to the endogenous MLC-2 gene, no luciferase activity was detectable in slow-twitch skeletal muscle or any other tissue of transgenic mice. This result suggests that the 2.1-kb DNA fragment of the 5' flanking region of the cardiac MLC-2 gene contains the regulatory elements required for selective gene expression in cardiac myocytes in vivo. In contrast to the endogenous steady-state MLC-2 expression during development, transgenic luciferase activity was 10-fold higher during embryogenesis, when formation of the ventricular loop and septum takes place. The enhanced luciferase activity in early heart development may suggest a growth-dependent control mechanism, involving either transcriptional or posttranscriptional regulation. In conclusion, this model system with the 2.1-kb ventricle-specific MLC-2 promoter sequence should facilitate the overexpression of gene products in the developing and mature heart muscle and further elucidate molecular mechanisms of myocardial diseases such as cardiomyopathies.

Transcriptional activation of the cardiac myosin light chain 2 and atrial natriuretic factor genes by protein kinase C in neonatal rat ventricular myocytes.

A cultured myocardial cell model was used to examine the role of protein kinase C-dependent pathways in the transcriptional activation of two cardiac muscle genes [myosin light chain 2 (MLC-2) and atrial natriuretic factor (ANF)] during alpha-adrenergic receptor-mediated hypertrophy. Phorbol ester (phorbol 12-myristate 13-acetate) and the alpha-adrenergic agonist phenylephrine both activate protein kinase C (PKC) and induce 4- to 5-fold increases in the expression of MLC-2 and ANF promoter/luciferase reporter genes with little effect on Rous sarcoma virus/luciferase or minimal prolactin promoter/luciferase genes. To further assess the role of PKC in cardiac gene regulation, PKC expression vectors encoding constitutively activated PKC-alpha or PKC-beta, or a catalytically inactive PKC, were transiently cotransfected with the cardiac promoter/luciferase constructs. Cotransfection of either activated PKC-alpha or PKC-beta cDNA induces the expression of MLC-2 and ANF promoter/luciferase genes and of a reporter gene responsive to the transcription factor AP-1. The Rous sarcoma virus/luciferase and minimal prolactin promoter/luciferase genes are not concomitantly induced by cotransfectin with the PKC genes, indicating specificity of the transcriptional effect. The finding that activated PKC increases cardiac gene transcription suggests that activation of this enzyme may be a proximal signal for coregulation of two cardiac genes, MLC-2 and ANF, during the course of myocardial cell hypertrophy.