Modulation of endothelin-A receptor, Galpha subunit, and RGS2 expression during H9c2 cardiomyoblast differentiation

In cardiac myocytes, growth responses depend on activation of G protein-coupled receptors interacting with Gq/11 protein subfamily members. Endothelin receptors of the ETA subtype belong to this receptor group inducing hypertrophic responses. To understand the role of ETA receptors and signal transduction proteins in modulating cell growth, we analyzed the pharmacological profile of this receptor, its level of expression together with those of Galpha subunits and the RGS2 protein in cardiomyoblasts differentiating into the cardiac phenotype. H9c2 rat cardiomyoblasts were grown in the presence of 10% fetal bovine serum (FBS) or 1% FBS plus all-trans-retinoic acid to induce the cardiac phenotype. The pharmacological properties of ETA receptors were investigated by competition-binding experiments, whereas the protein expression profile was analyzed by immunoblot and immunocytochemistry. The pharmacological profile of ETA receptors changed during differentiation of cardiomyoblasts into cardiomyocytes, and the amount of expressed receptor appeared to increase. Immunocytochemistry also showed a marked increase of receptor expression on cell membranes of differentiated cardiomyocytes. Among the other signaling proteins examined, both Galphaq/11 and RGS2 expression decreased in cells with the cardiac phenotype. Our results demonstrate that the expression of key proteins (ETA receptor, Galphaq/11, and RGS2) involved in signal transduction of hypertrophic stimuli is modulated during cell differentiation and correlates with the cardiac phenotype.     

Depletion of the actin bundling protein SM22/transgelin increases actin dynamics and enhances the tumourigenic phenotypes of cells

Background  

SM22 has long been studied as an actin-associated protein. Interestingly, levels of SM22 are often reduced in tumour cell lines, while they are increased during senescence possibly indicating a role for SM22 in cell fate decisions via its interaction with actin. In this study we aimed to determine whether reducing levels of SM22 could actively contribute to a tumourigenic phenotype.     

Expression cloning identifies transgelin (SM22) as a novel repressor of 92-kDa type IV collagenase (MMP-9) expression

The 92-kDa gelatinase (MMP-9) expression is prerequisite for tissue remodeling in physiology and cancer. However, there are few known regulators of MMP-9 expression. Using an expression cloning strategy, we identified transgelin (SM22), a 22-25-kDa actin-binding protein localized to the cell membrane and cytoplasm, as a novel regulator of MMP-9 expression. Overexpression of a SM22 cDNA in HT1080 cells decreased MMP-9 mRNA/protein levels and diminished in vitro invasion of the latter rescued with exogenous MMP-9. Conversely, small interfering RNA-mediated knockdown of SM22 elevated MMP-9 synthesis, and uterus from SM22-null mice showed strong MMP-9 immunoreactivity compared with wild type animals. The ability of SM22 to repress MMP-9 expression required an intact amino terminus calponin homology domain. MMP-9 expression is driven by ERK signaling and SM22 targeted this pathway as evidenced by (a) the transience in MAPK activation and (b) blunted stimulation of the MMP-9 promoter by a constitutively active MEK expression vector. Progressive deletion analysis located the SM22 responsive region of the MMP-9 promoter to the proximal 90-bp region harboring an AP-1 motif subsequently implicated by site-directed mutagenesis. Furthermore, nuclear extract from the SM22 transfectants showed diminished c-Fos binding to this motif and SM22 expression reduced the activity of an AP-1-driven reporter by 75%. Thus, SM22 adds to a short list of repressors of MMP-9 expression, achieving this by reducing AP-1-dependent trans-activation of the gene by way of compromised ERK activation. Diminished transgelin expression in several cancers may thus partly account for the elevated MMP-9 expression evident in these tumors.     

Down-regulation of phospholipase D during differentiation of mouse F9 teratocarcinoma cells.

Phospholipase D has been recognized as playing an important role in signal transduction in many types of cells. We investigated the expression of phospholipase D during the differentiation of F9 embryonal teratocarcinoma cells. The ADP ribosylation factor-dependent phospholipase D activity, as measured by an in vitro assay, and H2O2-induced phospholipase D activity and phospholipase D protein content in whole cells were decreased during the differentiation of F9 cells induced by a combination of dibutyryl cyclic AMP and all-trans retinoic acid. In contrast, these changes were not observed when cells were induced by retinoic acid. These results suggest that down-regulation of phospholipase D protein is associated with differentiation of F9 cells to a parietal endoderm lineage.     

Overexpression of calreticulin modulates protein kinase B/Akt signaling to promote apoptosis during cardiac differentiation of cardiomyoblast H9c2 cells

Calreticulin is a Ca(2+)-binding molecular chaperone of the lumen of the endoplasmic reticulum. Calreticulin has been shown to be essential for cardiac and neural development in mice, but the mechanism by which it functions in cell differentiation is not fully understood. To examine the role of calreticulin in cardiac differentiation, the calreticulin gene was introduced into rat cardiomyoblast H9c2 cells, and the effect of calreticulin overexpression on cardiac differentiation was examined. Upon culture in a differentiation medium containing fetal calf serum (1%) and retinoic acid (10 nm), cells transfected with the calreticulin gene were highly susceptible to apoptosis compared with controls. In the gene-transfected cells, protein kinase B/Akt signaling was significantly suppressed during differentiation. Furthermore, protein phosphatase 2A, a Ser/Thr protein phosphatase, was significantly up-regulated, implying suppression of Akt signaling due to dephosphorylation of Akt by the up-regulated protein phosphatase 2A via regulation of Ca(2+) homeostasis. Thus, overexpression of calreticulin promotes differentiation-dependent apoptosis in H9c2 cells by suppressing the Akt signaling pathway. These findings indicate a novel mechanism by which cytoplasmic Akt signaling is modulated to cause apoptosis by a resident protein of the endoplasmic reticulum, calreticulin.     

SM22alpha promoter targets gene expression to vascular smooth muscle cells in vitro and in vivo

BACKGROUND: Gene transfer into vascular smooth muscle cells (vsmcs) holds promise for studying the pathogenesis of arterial disorders. However, a potential limitation of vectors with heterologous promoters is organ toxicity resulting from unrestricted transgene expression. Vascular smooth muscle cell-specific gene expression could increase the safety of vectors for vascular diseases. MATERIALS AND METHODS: To develop vectors that target gene expression to vsmcs, we constructed vectors encoding human placental alkaline phosphatase (hpAP) and chloramphenicol transferase (CAT) driven by a 441-bp region of the murine SM22alpha promoter (AdSM22alpha-hpAP). RESULTS: Transfection of AdSM22alpha-hpAP into vascular and nonvascular cells resulted in the expression of alkaline phosphatase (AP) in primary arterial and venous smcs, but not in primary endothelial cells or National Institutes of Health (NIH) 3T3 cells. Expression of AP was observed on 32.5 +/- 1.4% of primary pig vsmcs-infected AdSM22alpha-hpAP at a multiplicity of infection (MOI) of 500; whereas, infection with AdCMV-hpAP resulted in 100 +/- 0.0% expression at a MOI of 250. In vitro, expression from the heterologous cytomegalovirus (CMV) promoter was approximately 10(3)-fold higher in vsmcs, compared with the SM22alpha promoter. Following introduction of AdSM22alpha-hpAP vectors into balloon-injured pig arteries, AP recombinant protein was detected in neointimal (2.23 +/- 1.14%) and medial (0.56 +/- 0.21%) smcs, but not in endothelial or adventitial cells. In contrast, AdCMV-hpAP vectors led to AP expression in intimal endothelial and smcs cells (39.14 +/- 10.09%) and medial smcs (2.84 +/- 1.05%). AP expression was not observed in endothelial or vsmcs following transfection with the control vector, adenoviral vector lacking E1 (AddeltaE1). CONCLUSIONS: The SM22alpha promoter programs recombinant gene expression exclusively to vascular smcs in vitro and in vivo. Although expression levels are lower than with heterologous promoters, these vectors may provide a safe and effective tool for gene therapy of vascular diseases.     

Effects of resveratrol on H2O2‐induced apoptosis and expression of SIRTs in H9c2 cells

Resveratrol, a polyphenol found in fruits, has been demonstrated to activate Sir2. Though many studies have demonstrated that resveratrol can activate SIRT1, whether it has effect on other sirtuins (SIRT2–7) are unknown. The present study shows that exposure of H9c2 cells to 50 µM H₂O₂ for 6 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry (FCM), but pretreatment of resveratrol (20 µM) eliminated H₂O₂‐induced apoptosis. Resveratrol also prevented H₂O₂‐induced caspase‐3 activation. Exposure of cells to resveratrol caused rapid activation of SIRT1,3,4,7. Sirtuin inhibitor, nicotinamide (20 mM) attenuated resveratrol's inhibitory effect on cell apoptosis and caspase‐3 activity. These results suggest that resveratrol protects cardiomyocytes from H₂O₂‐induced apoptosis by activating SIRT1,3,4,7. J. Cell. Biochem. 107: 741–747, 2009. © 2009 Wiley‐Liss, Inc.     

硫化氢通过调控SIRT1抑制阿霉素诱导的H9c2细胞损伤

目的探讨硫化氢（H₂S）对阿霉素（DOX）诱导的H9c2细胞损伤的影响及其作用机制。 方法H₂S对DOX心肌毒性保护作用的实验分组为：对照组（Control组）,5?μmol/?L DOX处理组（A组）,5?μmol/L DOX和400?μmol/L NaHS共同处理组（B组）,400?μmol/L NaHS单独处理组（C组）,5?μmol/L DOX、400?μmol/L NaHS和15?μmol/L Sirtinol共同处理组（D组）,15?μmol/L Sirtinol单独处理组（E组）。SIRT1是否参与H₂S抗DOX心肌毒性作用机制的实验分组为：对照组（Control组）,5?μmol/L DOX处理组（F组）,5?μmol/L DOX和400?μmol/L NaHS共同处理组（G组）,5?μmol/L DOX、400?μmol/L NaHS和15?μmol/L Sirtinol共同处理组（H组）,15?μmol/L Sirtinol单独处理组（I组）。使用MTT法检测细胞活力;Elisa法检测细胞MDA以及SOD水平;DCFH-?DA荧光探针法检测ROS水平;采用Western Blot法检测SIRT1蛋白表达。使用单因素方差分析法进行统计学分析。 结果NaHS预处理可抑制DOX导致的H9c2细胞活力下降：Control组,A组、B组、C组细胞活力分别为100﹪、（54.58±1.58）﹪、（85.05±4.31）﹪、（100.22±4.46）﹪ （F = 134.9,P < 0.001）。NaHS预处理可减弱DOX引起的H9c2细胞ROS、MDA水平的增加以及SOD水平的降低：Control组的ROS、MDA和SOD水平分别是100﹪、（34.18±1.56） μmol/g、（53.69±1.44） U/?mg;A组的ROS、MDA和SOD水平分别是（174.90±12.65）﹪、（72.65±2.66） μmol/g、（31.80±2.05） U/?mg;B组的ROS、MDA和SOD水平分别是（126.08±6.25）﹪、（44.59±1.92） μmol/g、（48.06±1.56） U/mg;C组的ROS、MDA和SOD水平分别是（91.86±1.66）﹪、（32.93±1.56）?μmol/?g、（55.93±1.58）?U/?mg （F?= 83.26,P < 0.001;F = 271.4,P < 0.001;F = 127.0,P < 0.001）。F组（6、12、24?h）H9c2细胞SIRT1蛋白表达水平分别是（0.45±0.03）、（0.27±0.02）、（0.25±0.03）,较Control组（1.00±0.00）降低（F = 611.1,P < 0.001）。本研究还发现,NaHS预处理H9c2细胞能阻止DOX引起的SIRT1蛋白表达下调：Control组、F组、G组、H组的SIRT1蛋白表达水平分别是（1.00±0.00）、（0.31±0.03）、（0.60±0.04）、（1.09±0.09）（F = 123.4,P?2S对DOX诱导的H9c2细胞活力降低的抑制作用：Control组,F组、G组、H组、I组细胞活力分别为100﹪、（54.58±1.58）﹪、（85.37±3.62）﹪、（71.11±2.11）﹪、（97.53±1.45）﹪ （F = 238.2,P < 0.001）。Sirtinol预处理可明显逆转H₂S对DOX导致的H9c2细胞ROS和MDA含量增加及SOD水平降低的抑制作用：Control组的ROS、MDA和SOD水平分别是100﹪、（35.84±2.22）μmol/?g、（53.03±3.16） U/mg;F组的ROS、MDA和SOD水平分别是（184.6±11.33）﹪、（74.78±5.30）μmol/g、（29.26±0.85）U/mg;G组的ROS、MDA和SOD水平分别是（126.5±7.57）﹪、（41.95±3.43）μmol/g、（52.61±2.26）U/mg;H组的ROS、MDA和SOD水平分别是（174.7±5.50）﹪、（67.69±1.52） μmol/g、（35.33±1.95） U/mg,I组的ROS、MDA和SOD水平分别是（98.03±2.86）﹪、（37.66±2.49）μmol/g、51.14 U/mg（F = 112.0,P < 0.001;F = 93.73,P < 0.001;F = 84.92,P < 0.001）。 结论H₂S通过调控SIRT1抑制DOX诱导的H9c2细胞损伤。