Nonmuscle Myosin Heavy chain-B Research Articles

Alteration of the Expression and Functional Activities of Myosin II Isoforms in Enlarged Hyperplastic Prostates.

Benign prostatic hyperplasia (BPH) is a common pathologic process in aging men, and the contraction of the prostatic smooth muscles (SMs) in the stroma plays a vital role in this pathogenesis, leading to lower urinary tract symptoms (LUTSs). The isoforms of both the SM myosin (SMM) and non-muscle myosin (NMM) are associated with the contraction type of the prostatic SMs, but the mechanism has not been fully elucidated. We collected prostate tissues from 30 BPH patients receiving surgical treatments, and normal human prostate samples were obtained from 12 brain-dead men. A testosterone-induced (T-induced) rat model was built, and the epithelial hyperplastic prostates were harvested. Competitive RT-PCR was used to detect the expression of SMM isoforms. We investigated the contractility of human prostate strips in vitro in an organ bath. The results regarding the comparisons of SMM isoforms varied between rat models and human samples. In comparison with T-induced rats and controls, competitive RT-PCR failed to show any statistically significant difference regarding the compositions of SMM isoforms. For human prostates samples, BPH patients expressed more SM-1 isoforms (66.8% vs. 60.0%, p < 0.001) and myosin light chain-17b (MLC17b) (35.9% vs. 28.5%, p < 0.05) when compared to young donors. There was a significant decrease in prostate myosin heavy chain (MHC) expression in BPH patients, with a 66.4% decrease in MHC at the mRNA level and a 51.2% decrease at the protein level. The upregulated expression of non-muscle myosin heavy chain-B (NMMHC-B) was 1.6-fold at the mRNA level and 2.1-fold at the protein level. The organ bath study showed that isolated prostate strips from BPH patients produced slower tonic contraction compared to normal humans. In this study, we claim that in the enlarged prostates of patients undergoing surgeries, MHC expression significantly decreased compared to normal tissues, with elevated levels of SM-1, MLC17b, and NMMHC-B isoforms. Modifications in SMM and NMM might play a role in the tonic contractile properties of prostatic SMs and the development of LUTS/BPH. Understanding this mechanism might provide insights into the origins of LUTS/BPH and facilitate the identification of novel therapeutic targets.

Glibenclamide treatment modulates the expression and localization of myosin-IIB in diabetic rat brain

BackgroundMyosin-IIB is a non-muscle isoform in the brain with increased expression in the brains of diabetic rats. Chronic hyperglycemia caused by diabetes can impair learning and memory. Oral hypoglycemic agents such as glibenclamide have been used to control hyperglycemia. We report changes in the expression and distribution of myosin-IIB in the frontal cortex and hippocampus of diabetic rats treated with glibenclamide. MethodsThe brains were removed after 43days of treatment with glibenclamide (6mg/kg bw orally), homogenized and analyzed by Western blotting, qRT-PCR and immunohistochemistry. ResultsMyosin-IIB expression increased in the brains of diabetic rats. However, protein expression returned to control levels when treated with glibenclamide. In addition, the expression of MYH10 gene encoding non-muscle myosin heavy chain-B decreased in diabetic rats treated with glibenclamide. Moreover, we found weak myosin-IIB labeling in the hippocampus and frontal cortex of rats treated with glibenclamide. Therefore, the expression of myosin-IIB is affected by diabetes mellitus and may be modulated by glibenclamide treatment in rats. Structural changes in the hippocampus and prefrontal cortex are reversible, and glibenclamide treatment may reduce the patho-physiological changes in the brain. ConclusionsOur findings can contribute to the understanding of the regulation of myosins in the brains of diabetic rats.

Nonmuscle Myosin II Is Required for Internalization of the Epidermal Growth Factor Receptor and Modulation of Downstream Signaling

Ligand-induced internalization of the epidermal growth factor receptor (EGFR) is an important process for regulating signal transduction, cellular dynamics, and cell-cell communication. Here, we demonstrate that nonmuscle myosin II (NM II) is required for the internalization of the EGFR and to trigger the EGFR-dependent activation of ERK and AKT. The EGFR was identified as a protein that interacts with NM II by co-immunoprecipitation and mass spectrometry analysis. This interaction requires both the regulatory light chain 20 (RLC20) of NM II and the kinase domain of the EGFR. Two paralogs of NM II, NM II-A, and NM II-B can act to internalize the EGFR, depending on the cell type and paralog content of the cell line. Loss (siRNA) or inhibition (25 μm blebbistatin) of NM II attenuates the internalization of the EGFR and impairs EGFR-dependent activation of ERK and AKT. Both internalization of the EGFR and downstream signaling to ERK and AKT can be partially restored in siRNA-treated cells by introduction of wild type (WT) GFP-NM II, but cannot be restored by motor mutant NM II. Taken together, these results suggest that NM II plays a role in the internalization of the EGFR and EGFR-mediated signaling pathways.

Kruppel-like Factor 4 is Required for the Expression of Vascular Smooth Muscle Cell Differentiation Marker Genes Induced by All-Trans Retinoic Acid

Krüppel-like factor 4 (KLF4) is involved in phenotypic modulation of vascular smooth muscle cells (VSMCs). All-trans retinoic acid (ATRA) inhibits VSMC proliferation and induces VSMC differentiation. However, the role of KLF4 in ATRA-elicited VSMC phenotypic modulation remains unclear. Here, we show that treatment of VSMCs with ATRA resulted in significant inhibition of proliferation and migration of VSMCs, as well as up-regulation of KLF4 and the VSMC differentiation marker genes SM22alpha and SM alpha-actin (alpha-SMA). At the same time, the KLF4 target gene p53 was up-regulated, while the VSMC dedifferentiation marker gene nonmuscle myosin heavy chain-B (SMemb) was down-regulated. We also show that overexpression of KLF4 in VSMCs increased the expression of p53, SM22alpha and alpha-SMA, but decreased the expression of SMemb and VSMC proliferation and migration. Silencing of KLF4 expression by KLF4-specific small interfering RNA (siRNA) abrogated the inducing effects of ATRA on p53, SM22alpha and alpha-SMA expression and neutralized the inhibitory effects of ATRA on SMemb expression and VSMC proliferation and migration. Thus, our data suggest that KLF4 is required for the expression of VSMC differentiation marker genes induced by ATRA and that this transcription factor is one of the key mediators of retinoid actions in VSMCs.

Spatiotemporal expression of smooth muscle markers in developing zebrafish gut

Smooth muscle is important for the contractility and elasticity of visceral organs. The zebrafish is an excellent model for understanding embryonic development, yet due to a lack of appropriate markers, visceral smooth muscle development remains poorly characterized. Here, we develop markers and trace the development of gut and swim bladder smooth muscle in embryonic and juvenile fish. The first smooth muscle marker we detect in the vicinity of the gut is the myoblast marker nonmuscle myosin heavy chain-b at 50 hours postfertilization (hpf), followed by the early smooth muscle markers SM22alpha-b, and alpha-smooth muscle actin at 56 and 60 hpf, respectively. Markers of more differentiated smooth muscle, smoothelin-b and cpi-17, appear by 3 days postfertilization (dpf). Tropomyosin, a relatively late marker, is first expressed at 4 dpf. We find that smooth muscle marker expression in the swim bladder follows the same sequence of marker expression as the gut, but markers have a temporal delay reflecting the later formation of swim bladder smooth muscle.

EARLY ACTIVATION OF INTERNAL MEDIAL SMOOTH MUSCLE CELLS IN THE RABBIT AORTA AFTER MECHANICAL INJURY: RELATIONSHIP WITH INTIMAL THICKENING AND PHARMACOLOGICAL APPLICATIONS

1. Smooth muscle cells (SMC) participate in both inflammatory and dedifferentiation processes during atherosclerosis, as well as during mechanical injury following angioplasty. In the latter, we studied medial SMC differentiation and inflammation processes implicated early after de-endothelialization in relation to mechanical stresses. We hypothesized that activation of a subpopulation of SMC within the media plays a crucial role in the early phase of neointimal formation. 2. For this purpose, we used a rabbit model of balloon injury to study activation and differentiation of medial SMC in the early time after denudation and just before neointima thickening. Inflammation was evaluated by the expression of vascular cell adhesion molecule (VCAM)-1, integrin alpha4beta1 and nuclear factor (NF)-kB. Myosin isoforms and 2P1A2 antigen, a membrane protein expressed by rabbit dedifferentiated SMC, were used as markers of differentiation. 3. On day 2 after de-endothelialization, VCAM-1, alpha4beta1 and NF-kB were coexpressed by a well-defined subpopulation of SMC of the internal part of the media, in the vicinity of the blood stream. At the same time, the majority of SMC throughout the media expressed non-muscle myosin heavy chain-B (nm-MHC-B) and 2P1A2 antigen. On day 7, when intimal thickening appeared, SMC of the media were no longer activated, whereas some intimal SMC expressed the activation markers. Thus, after de-endothelialization, early dedifferentiation occurs in most of the medial SMC, whereas activation concerned only a subpopulation of SMC located in the internal media. Using the T-type voltage-operated calcium channel blocker mibefradil (0.1-1 micromol/L) in SMC culture, we showed that this agent exhibited an antiproliferative effect in a dose-dependent manner only on undifferentiated cells. 4. In conclusion, the results suggest that the activated SMC represent cells that are potentially able to migrate and participate in the intimal thickening process. Thus, the medial SMC inflammatory process, without any contribution of inflammatory cells, may represent a major mechanism underlying the development of intimal thickening following mechanical stress. In humans, inhibition of T-type calcium channels may be a tool to prevent the early proliferation step leading to neointimal formation.

Increased expression of non-muscle myosin heavy chain-B in connective tissue cells of hypertrophic rat urinary bladder.

Expression of the non-muscle myosin heavy chain-B (NM-MHC-B, also denoted as the embryonic smooth muscle myosin heavy chain, SMemb) was examined in rat urinary bladder during growth in response to a partial urinary outflow obstruction. Following obstruction, the weight of the urinary bladder increased more than five-fold within 10 days. Immunohistochemistry with a polyclonal antiserum against the C-terminal sequence of NM-MHC-B revealed very few NM-MHC-B immunoreactive cells in the control urinary bladders. In hypertrophic bladders, the number of NM-MHC-B immunoreactive cells markedly increased. The majority of such cells were found in the interstitium surrounding smooth muscle bundles and also in the subserosal and submucosal layers. Western blot analysis showed that the NM-MHC-B expression was transient; the content of NM-MHC-B immunoreactive material had doubled 10 days after obstruction and then declined towards the control level after 6 weeks. Immunohistochemistry revealed co-localization of NM-MHC-B and vimentin within the same cells. NM-MHC-B did not co-localize with smooth muscle actin, suggesting that the source of NM-MHC-B is not a de-differentiated smooth muscle cell or myofibroblast but a non-muscle cell possibly reacting to tissue distension or stress. The NM-MHC-B-positive cells could have a role in the production of extracellular matrix and growth factors or be involved in modulation of spontaneous contractile activity.

Inducible expression of basic transcription factor binding protein 2 plays a potential role in the development of the allograft vascular disease

A number of studies have demonstrated that various genes are differentially expressed between proliferating and quiescent vascular smooth muscle cells (SMCs). However, transcription factors which play an important role in this process remain largely unknown. We have recently identified basic transcription factor binding protein 2 (BTEB2) as a transcription factor regulating the expression of the SMemb gene, which is involved in the phenotypic modulation of vascular SMCs. SMemb is a nonmuscle myosin heavy chain-B isoform that is expressed in proliferating SMCs. It is also expressed in the thickened intima and media of cardiac allograft coronary arteries. The aim of this study was to investigate the expression of BTEB2 in allograft vascular disease.

Suppression of the tumorigenicity of mutant p53-transformed rat embryo fibroblasts through expression of a newly cloned rat nonmuscle myosin heavy chain-B

In our previous study, a rat homolog of human nonmuscle myosin heavy chain-B (nmMHC-B) was identified by mRNA differential display comparing of transformed against nontransformed Rat 6 cells overexpressing mutant p53val135 gene. The nmMHC-B was found to be expressed in normal Rat 6 embryo fibroblast cell line, but markedly suppressed in the mutant p53val135-transformed Rat 6 cells. To examine the possible involvement of nmMHC-B in cell transformation, we first cloned and sequenced the full length cDNA of rat nmMHC-B, which was then cloned into an ecdysone-expression vector. The resulting construct was introduced into the T2 cell line, a mutant p53val135-transformed Rat 6 cells lacking the expression of the endogenous nmMHC-B. The clonal transfectants, expressing muristerone A-induced nmMHC-B, displayed a slightly flatter morphology and reached to a lower saturation density compared to the parental transformed cells. Reconstitution of actin filamental bundles was also clearly seen in cells overexpressing the nmMHC-B. In soft agar assays, nmMHC-B transfectants formed fewer and substantially smaller colonies than the parental cells in response to muristerone A induction. Moreover, it was strikingly effective in suppressing the tumorigenicity of the T2 cells when tested in nude mice. Thus, the nmMHC-B, known as a component of the cytoskeletal network, may act as a tumor suppressor gene. Our current finding may reveal a novel role of nmMHC-B in regulating cell growth and cell signaling in nonmuscle cells. Oncogene (2001) 20, 58 - 68.

Molecular Cloning and Functional Analysis of the Promoter Region of Rat Nonmuscle Myosin Heavy Chain-B Gene

Rat nonmuscle myosin heavy chain-B (r-nmMHC-B) mRNA was previously found downregulated in Rat 6 fibroblasts transformed by mutant p53val135 [J. W. P. Yam, J. Y. Zheng, and W. L. W. Hsiao (1987) Biochem. Biophys. Res. Commun. 266, 472–480]. Overexpression of exogenous r-nmMHC-B could partially reverse the transforming phenotypes both in vitro and in vivo. The downregulation of r-nmMHC-B was also observed in Rat 6 transformed by c-H-ras and v-myc oncogenes. We cloned a 5.2-kb r-nmMHC-B promoter region. Sequence analysis of −1248 to +1 revealed no TATA box, but did show that it contained CAAT boxes, E12/E47, MyoD, MEF, E2F, CREB, and SP1 binding sites. Based on transient reporter assays, the promoter/enhancer activities were unusually extended to the entire 5.2 kb region in normal Rat 6 cultures, but markedly suppressed in p53val135-, and c-H-ras-transformed cells. The activity detected by the reporter assay corresponded to levels of mRNA as analyzed previously by Northern blots in each respective cell line. Thus, the switch-off of the r-nmMHC-B in the transformed cells is very likely controlled by upstream transcriptional factors, which might have been altered in the course of neoplastic transformation.

Identification and Characterization of Genes Whose Expressions Are Altered in Rat 6 Fibroblasts Transformed by Mutant p53val135

The wild-type tumor suppressor gene p53 is known as a transcription factor in activating or suppressing target genes that encode proteins in regulating genome stability, DNA damage, cell arrest, and apoptosis. However, the role of mutant p53 in the process of cell transformation is still unclear. Our recent work indicated that overexpression of mutant p53val135 induced high incidence of spontaneous transformation in prolonged cultures of Rat 6 fibroblasts. In order to identify genes related to neoplastic transformation induced by the mutant p53, the p53val135-overexpressor R6#13-8 and its derived spontaneously transformed cell line T2 were analyzed by mRNA differential display. In a systematic screening with 80 primer sets of RT-PCR reactions, three genes were found to be differentially expressed between R6#13-8 and T2 cells. Two genes, identified as homologues of the growth factor inducible immediate-early gene Cyr61 and the human nonmuscle myosin heavy chain-B, were down-regulated in T2 cells. Interestingly, both genes were also suppressed in Rat 6 cells transformed by c-H-ras and v-myc, but not by v-src genes. The third gene is a homologue of the frizzled related protein, a gene family that acts, in some cases, as an antagonist to the Wnt signaling pathway. It is intriguing that the rat homologue of the frizzled related protein was only expressed in p53val135-overexpressing cells, but not in the parental Rat 6 cells. However, the same gene was also highly expressed in ras-transformed Rat 6 cells, and moderately expressed in v-src-transformed Rat 6 cells. This is the first study in which the association of mutant p53 to these three genes is revealed. Our current report may provide new clues to the role of mutant p53 in the process of cell transformation.

Nonmuscle myosin II-B is required for normal development of the mouse heart.

We used targeted gene disruption in mice to ablate nonmuscle myosin heavy chain B (NMHC-B), one of the two isoforms of nonmuscle myosin II present in all vertebrate cells. Approximately 65% of the NMHC-B-/- embryos died prior to birth, and those that were born suffered from congestive heart failure and died during the first day. No abnormalities were detected in NMHC-B+/- mice. The absence of NMHC-B resulted in a significant increase in the transverse diameters of the cardiac myocytes from 7.8 +/- 1.8 micron (right ventricle) and 7.8 +/- 1.3 micron (left ventricle) in NMHC-B+/+ and B+/- mice to 14.7 +/- 1.1 micron and 13.8 +/- 2.3 micron, respectively, in NMHC-B-/- mice (in both cases, P < 0.001). The increase in size of the cardiac myocytes was seen as early as embryonic day 12.5 (4.5 +/- 0.2 micron for NMHC-B+/+ and B+/- vs. 7. 2 +/- 0.6 micron for NMHC-B-/- mice (P < 0.01)). Six of seven NMHC-B-/- newborn mice analyzed by serial sectioning also showed structural cardiac defects, including a ventricular septal defect, an aortic root that either straddled the defect or originated from the right ventricle, and muscular obstruction to right ventricular outflow. Some of the hearts of NMHC-B-/- mice showed evidence for up-regulation of NMHC-A protein. These studies suggest that nonmuscle myosin II-B is required for normal cardiac myocyte development and that its absence results in structural defects resembling, in part, two common human congenital heart diseases, tetralogy of Fallot and double outlet right ventricle.

Expression of nonmuscle myosin heavy chain-B isoform in the vessel wall of porcine coronary arteries after balloon angioplasty.

Nonmuscle myosin heavy chain-B isoform (NMMHC-B) is expressed by proliferating vascular smooth muscle cells (SMCs), and its expression in primary lesions has been proposed to be predictive of restenosis after atherectomy. The present study was designed to study the time-course expression of NMMHC-B after angioplasty of porcine coronary arteries by in situ hybridization and immunohistochemistry. Domestic juvenile swine underwent percutaneous transluminal coronary angioplasty (PTCA) of the left anterior descending and circumflex coronary arteries with standard clinical angioplasty catheters. To identify proliferating cells, 5'-bromo-2'-deoxyuridine (BrdU) was administered and detected by immunohistochemistry on serial sections. Vessels were examined at 3, 7, and 14 days after balloon angioplasty, and uninjured coronary vessels were used as controls. Normal arteries showed hybridization to 35S-labeled NMMHC-B riboprobes localized mainly in the medial layer. NMMHC-B expression in the adventitia was markedly increased 3 days after balloon angioplasty. Seven and 14 days after injury, NMMHC-B mRNA-containing cells were localized in the adventitia and neointima at the arterial injury site. Cell proliferation, as indicated by BrdU staining, colocalized with NMMHC-B mRNA expression 3 and 7 days after angioplasty. These data indicate that cells proliferating in the adventitia and neointima express NMMHC-B; however, its expression is not limited to the proliferative state, since NMMHC-B mRNA was also found in quiescent SMCs of normal coronary arteries and in nonproliferating adventitial and neointimal cells 14 days after angioplasty.

Cloning of the cDNA encoding human nonmuscle myosin heavy chain-B and analysis of human tissues with isoform-specific antibodies.

Previously, we reported the sequence of cDNA clones encoding amino acids 63 through 723 of the human nonmuscle myosin heavy chain-B isoform. In this paper, we present the derived sequence of the remaining 1303 amino acids along with 5' and 3' untranslated sequences. We made use of the differences between the derived nonmuscle myosin heavy chain-A and -B amino acid sequences to raise isoform-specific antibodies. Immunoblot analysis reveals a differential expression of both myosin heavy chain isoforms in a variety of human adult and foetal tissues and cells. When extracts of human adult aorta were subjected to gel electrophoresis, two distinct Coomassie Blue-stained bands and a fused band were seen migrating at approximately 200 kDa. These bands can be detected with four different specific antibodies recognizing the two different smooth muscle myosin heavy chain isoforms (204 kDa and 200 kDa) and the two different nonmuscle myosin heavy chain isoforms (A and B). Using immunohistochemistry, we confirmed the presence of the four different isoforms in adult and foetal aortas.

Evidence for inserted sequences in the head region of nonmuscle myosin specific to the nervous system. Cloning of the cDNA encoding the myosin heavy chain-B isoform of vertebrate nonmuscle myosin.

The complete amino acid sequence of a vertebrate nonmuscle myosin heavy chain-B isoform (MHC-B, 1976 amino acids, 229 kDa) has been deduced by using cDNA clones from chicken brain libraries. The chicken nonmuscle MHC-B shows overall similarity in primary structure to other MHCs in the areas contributing to the ATP-binding site and actin-binding site. Similar to other nonsarcomeric MHC IIs, there is a short uncoiled tail sequence at the carboxyl terminus of the molecule. It is in the uncoiled tail sequence that the greatest number of differences in amino acids sequence between MHC-A and B were found, which allowed generation of isoform-specific antibodies. These antibodies were used to determine the relative content of MHC-A and MHC-B in various tissues. During the cloning of the cDNA encoding chicken brain MHC-B, we found a 63-nucleotide insertion encoding 21 amino acids located in the head region of the MHC near to the actin-binding site and a 30 nucleotide insertion encoding 10 amino acids near to the ATP-binding site. Analysis using S-1 nuclease showed that both inserts are expressed in a tissue-dependent manner; mRNA containing the inserts is present in tissues of the nervous system, but is absent from other non-muscle cells, which contain the noninserted isoform of MHC-B. Similar inserts were found in corresponding positions in human cerebellar mRNA. Antibodies raised against a peptide synthesized based on the 21 amino acid insert found in chickens recognize a MHC isoform in the same tissues that are enriched for the mRNA. These insertions appear to be a mechanism for generating additional MHC-B isoforms specific to the nervous system.