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The well-known insulin-like growth factor 1 (IGF1)/IGF-1 receptor (IGF-1R) signaling pathway is overexpressed in many tumors, and is thus an attractive target for cancer treatment. However, results have often been disappointing due to crosstalk with other signals. Here, we report that IGF-1R signaling stimulates the growth of hepatocellular carcinoma (HCC) cells through the translocation of IGF-1R into the ER to enhance sarco-endoplasmic reticulum calcium ATPase 2 (SERCA2) activity. In response to ligand binding, IGF-1Rβ is translocated into the ER by β-arrestin2 (β-arr2). Mass spectrometry analysis identified SERCA2 as a target of ER IGF-1Rβ. SERCA2 activity is heavily dependent on the increase in ER IGF-1Rβ levels. ER IGF-1Rβ phosphorylates SERCA2 on Tyr990 to enhance its activity. Mutation of SERCA2-Tyr990 disrupted the interaction of ER IGF-1Rβ with SERCA2, and therefore ER IGF-1Rβ failed to promote SERCA2 activity. The enhancement of SERCA2 activity triggered Ca2+ER perturbation, leading to an increase in autophagy. Thapsigargin blocked the interaction between SERCA2 and ER IGF-1Rβ and therefore SERCA2 activity, resulting in inhibition of HCC growth. In conclusion, the translocation of IGF-1R into the ER triggers Ca2+ER perturbation by enhancing SERCA2 activity through phosphorylating Tyr990 in HCC.
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Aim To investigate the inotropic effect of PF-04957325, a phosphodiesterase type 8 inhibitor, in normal rats and its underlying molecular mechanism. Methods The techniques of in vivo rat left ventricular pressure-volume loop (P-V loop) and isolated perfusion rat heart were used to analyze the hemodynamics and positive inotropic effect of rat hearts. The Ca transient induced by field stimulation was used to analyze the hemodynamics of sarcoplasmic reticulum (SR) Ca + uptaking. Western blot was used to analyze the phosphorylation levels of SR phospolamban (PLB) and ryanodine receptor type 2 (RyR2). Results The P-V loop experiment indicated that PF-04957325 (0.5 mg · kg
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Heart failure from various cardiovascular diseases is a serious threat to human health. Systolic and diastolic dysfunction are the basic characteristics of heart failure. SERCA2a, a key enzyme for calcium transport, regulates intracellular free calcium ion concentration, affecting the myocardial diastolic process. This article mainly summarized the structure and function of SERCA2a gene, the expression and regulation of SERCA2a in heart failure, and the current situation of drug therapy, gene therapy and clinical research targeting SERCA2a gene.
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BACKGROUND AND OBJECTIVES: Insulin secretion entirely depends on Ca²⁺ influx and sequestration into endoplasmic reticulum (ER) of β-cells, performed by Sarco-ER Ca²⁺-ATPase 2b (SERCA2b). In diabetes, SERCA2b is decreased in the β-cells leading to impaired intracellular Ca²⁺ homeostasis and insulin secretion. Adipose mesenchymal stem cells (AMSCs) play a potential role in transplantation in animal models. The present study aimed at investigating and comparing the therapeutic effect of non-transfected AMSCs and SERCA2b gene transfected AMSCs on the pancreas of induced diabetes type 1 in rat. METHODS AND RESULTS: 58 adult male albino rats were divided into: Donor group: 22 rats, 2 for isolation, propagation and characterization of AMSCs and SERCA2b transfected AMSCs, in addition 20 for isolated islet calcium level assessment. Group I (Control Group): 6 rats, Group II (Diabetic Group): 10 rats, 50 mg streptozotocin (STZ) were injected intraperitoneal (IP), Group III (AMSCs Group): 10 rats, 1×10⁶ AMSCs were injected intravenous and Group IV (SERCA2b transfected AMSCs Group): 10 rats, 1×10⁶SERCA2b transfected AMSCs were injected as in group III. Groups I, II, III and IV were sacrified 3 weeks following confirmation of diabetes. Serological, histological, morphometric studies and quantitative polymerase chain reaction (qPCR) were performed. Nuclear, cytoplasmic degenerative and extensive fibrotic changes were detected in the islets of group II that regressed in groups III and IV. Isolated islet calcium, blood glucose, plasma insulin and qPCR were confirmative. CONCLUSIONS: AMSCs and SERCA2b gene transfected AMSCs therapy proved definite therapeutic effect, more obvious in response to SERCA2b gene transfected AMSCs.
Subject(s)
Adult , Animals , Humans , Male , Rats , Blood Glucose , Calcium , Cytoplasm , Endoplasmic Reticulum , Homeostasis , Insulin , Mesenchymal Stem Cells , Models, Animal , Pancreas , Plasma , Polymerase Chain Reaction , Stem Cells , Streptozocin , Tissue DonorsABSTRACT
Objective To investigate the effect and mechanism of recombinant adenovirus associated vi-rus type 1 (rAAV1) mediated SERCA2a gene transfer on cardiac function in canine with heart failure (HF). Methods 20 healthy male beagle dogs were randomly divided into control group,HF group,HF+EGFP group and HF+SERCA2a group,5 dogs in each group. The canine cardiac function,apoptosis and MMP-9 expression in cardiac myocytes were detected by TUNEL and immunohistochemistry methods. Results The left ventricular dia-stolic diameter (LVDD),left ventricular systolic diameter (LVSD) and left ventricular posterior wall dimension (LVPWD)in the HF and HF+EGFP groups,were significantly higher than those of the control and HF+SERCA2a groups,and the ejection fraction(EF)in the former two groups was significantly lower than that of the control group and HF+SERCA2a group(P<0.05). The left ventricular systolic pressure(LVSP)and left ventricular maximal rate of pressure rise(+dp/dtmax)in the HF and HF+EGFP groups were both significantly lower than those of the control group and HF+SERCA2a group,while left ventricular end diastolic pressure(LVEDP)and left ventricular maximal rate of pressure decline(-dp/dtmax)significantly higher than those of the control and HF+SERCA2a groups(P<0.05). The apoptosis index of HF+SERCA2a group was significantly lower than that of group HF and group HF+EGFP(P<0.05). The MMP-9 light density of HF+SERCA2a group was significantly higher than that of the control group(P < 0.05). Conclusion RAAV1 mediated CA2a SER gene transfer can effectively improve the cardiac function of HF dog,probably by inhibiting the apoptosis of cardiac muscle cells and down-regulating the expression of MMP-9.
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Objective To investigate the effect and mechanism of recombinant adenovirus associated vi-rus type 1 (rAAV1) mediated SERCA2a gene transfer on cardiac function in canine with heart failure (HF). Methods 20 healthy male beagle dogs were randomly divided into control group,HF group,HF+EGFP group and HF+SERCA2a group,5 dogs in each group. The canine cardiac function,apoptosis and MMP-9 expression in cardiac myocytes were detected by TUNEL and immunohistochemistry methods. Results The left ventricular dia-stolic diameter (LVDD),left ventricular systolic diameter (LVSD) and left ventricular posterior wall dimension (LVPWD)in the HF and HF+EGFP groups,were significantly higher than those of the control and HF+SERCA2a groups,and the ejection fraction(EF)in the former two groups was significantly lower than that of the control group and HF+SERCA2a group(P<0.05). The left ventricular systolic pressure(LVSP)and left ventricular maximal rate of pressure rise(+dp/dtmax)in the HF and HF+EGFP groups were both significantly lower than those of the control group and HF+SERCA2a group,while left ventricular end diastolic pressure(LVEDP)and left ventricular maximal rate of pressure decline(-dp/dtmax)significantly higher than those of the control and HF+SERCA2a groups(P<0.05). The apoptosis index of HF+SERCA2a group was significantly lower than that of group HF and group HF+EGFP(P<0.05). The MMP-9 light density of HF+SERCA2a group was significantly higher than that of the control group(P < 0.05). Conclusion RAAV1 mediated CA2a SER gene transfer can effectively improve the cardiac function of HF dog,probably by inhibiting the apoptosis of cardiac muscle cells and down-regulating the expression of MMP-9.
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Ca2+ pumps are important players in smooth muscle contraction. Nevertheless, little information is available about these pumps in the vas deferens. We have determined which subtype of sarco(endo)plasmic reticulum Ca2+-ATPase isoform (SERCA) is expressed in rat vas deferens (RVD) and its modulation by calmodulin (CaM)-dependent mechanisms. The thapsigargin-sensitive Ca2+-ATPase from a membrane fraction containing the highest SERCA levels in the RVD homogenate has the same molecular mass (∼115 kDa) as that of SERCA2 from the rat cerebellum. It has a very high affinity for Ca2+ (Ca0.5 = 780 nM) and a low sensitivity to vanadate (IC50 = 41 µM). These facts indicate that SERCA2 is present in the RVD. Immunoblotting for CaM and Ca2+/calmodulin-dependent protein kinase II (CaMKII) showed the expression of these two regulatory proteins. Ca2+ and CaM increased serine-phosphorylated residues of the 115-kDa protein, indicating the involvement of CaMKII in the regulatory phosphorylation of SERCA2. Phosphorylation is accompanied by an 8-fold increase of thapsigargin-sensitive Ca2+ accumulation in the lumen of vesicles derived from these membranes. These data establish that SERCA2 in the RVD is modulated by Ca2+ and CaM, possibly via CaMKII, in a process that results in stimulation of Ca2+ pumping activity.
Subject(s)
Animals , Male , Rats , Calcium-Binding Proteins/metabolism , Calcium-Transporting ATPases/metabolism , Calmodulin/metabolism , Protein Serine-Threonine Kinases/metabolism , Vas Deferens/metabolism , Muscle Contraction , Phosphorylation , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolismABSTRACT
Ventricular hypertrophy is one of the major myocardial responses to pressure overload (PO). Most studies on early myocardial response focus on the days or even weeks after induction of hypertrophic stimuli. Since mechanotransduction pathways are immediately activated in hearts undergoing increased work load, it is reasonable to infer that the myocardial gene program may be regulated in the first few hours. In the present study, we monitored the expression of some genes previously described in the context of myocardial hypertrophic growth by using the Northern blot technique, to estimate the mRNA content of selected genes in rat myocardium for the periods 1, 3, 6, 12 and 48 h after PO stimuli. Results revealed an immediate switch in the expression of genes encoding alpha and beta isoforms of myosin heavy chain, and up-regulation of the cardiac isoform of alpha actin. We also detected transitory gene regulation as the increase in mitochondrial cytochrome c oxidase 1 gene expression, parallel to down-regulation of genes encoding sarco(endo)plasmic reticulum Ca+2 ATPase and sodium-calcium exchanger. Taken together, these results indicate that initial myocardial responses to increased work load include alterations in the contractile properties of sarcomeres and transitory adjustment of mitochondrial bioenergetics and calcium availability.
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We have shown that myocardial dysfunction induced by food restriction is related to calcium handling. Although cardiac function is depressed in food-restricted animals, there is limited information about the molecular mechanisms that lead to this abnormality. The present study evaluated the effects of food restriction on calcium cycling, focusing on sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban (PLB), and ryanodine channel (RYR2) mRNA expressions in rat myocardium. Male Wistar-Kyoto rats, 60 days old, were submitted to ad libitum feeding (control rats) or 50 percent diet restriction for 90 days. The levels of left ventricle SERCA2, PLB, and RYR2 were measured using semi-quantitative RT-PCR. Body and ventricular weights were reduced in 50 percent food-restricted animals. RYR2 mRNA was significantly decreased in the left ventricle of the food-restricted group (control = 5.92 ± 0.48 vs food-restricted group = 4.84 ± 0.33, P < 0.01). The levels of SERCA2 and PLB mRNA were similar between groups (control = 8.38 ± 0.44 vs food-restricted group = 7.96 ± 0.45, and control = 1.52 ± 0.06 vs food-restricted group = 1.53 ± 0.10, respectively). Down-regulation of RYR2 mRNA expressions suggests that chronic food restriction promotes abnormalities in sarcoplasmic reticulum Ca2+ release.
Subject(s)
Animals , Male , Rats , Calcium-Binding Proteins/metabolism , Down-Regulation/physiology , Food Deprivation/physiology , Ryanodine Receptor Calcium Release Channel/metabolism , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Calcium-Binding Proteins/genetics , Down-Regulation/genetics , Rats, Inbred WKY , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/genetics , Ryanodine Receptor Calcium Release Channel/genetics , Sarcoplasmic Reticulum Calcium-Transporting ATPases/geneticsABSTRACT
Objective To observe the abnormity of heart function in rats with pressure overload-induced left ventricular hypertrophy and the changes of NCX,SERCA2a expression in myocardial tissues. Methods Cardiac hypertrophy was induced by clipping the abdominal aorta in rats. The cardiac hypertrophy was evaluated by Left ventricular weight index(LVWI,left ventricular weight/body weight). NCX, SERCA2a mRNA and protein expressions in left ventricular tissues were determined by half-quantitative RT-PCR and Western blot normalized to abundance of GAPDH mRNA and protein,respectively. Results LVSP and LVEDP were obviously enhanced(P
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Objective To explore the protective effect of vitamin E on the rabbit bladder after partial outlet obstruction artificially setup. Methods A total of 28 New Zealand white male rabbits were divided into 4 groups (group A in 6,group B in 6,group C in 8 and group D in 8).Group A,B and C were fed a regular diet,and group D were placed on a diet enriched with 600 mg vitamin E.After 4 weeks partial outlet obstruction was created in groups C and D,while group B underwent sham operation. After 4 weeks of obstruction each rabbit was sedated and cystometry was repeated.After cystometry the bladder was weighed.The gene expression of sarcoplasmic endoplasmic reticulum,calcium,magnesium,adenosine triphosphatase (SERCA2) in bladder was detected by using RT-PCR assay,while the protein level of SERCA2 was measured by Western blot analysis. Results All parameters measured were approximately identical in nomal rabbits(Group A) and shum operation rabbits(group B).Thus,these 2 groups were combined as the control group(Group A and B).Partial outlet obstruction resulted in bladder weight increased significantly in obstructed groups given vehicle group C(13.07?1.71)g and those vitamin E group D(11.80?2.01)g,4-fold higher than in the control group (2.81?0.30)g(P