Upregulations of Gata4 and oxytocin receptor are important in cardiomyocyte differentiation processes of P19CL6 cells

Oxytocin induces P19 cells to differentiate into cardiomyocytes possibly through the oxytocin/oxytocin receptor system. We added oxytocin to the growth medium of P19CL6, a subline of P19, but they did not differentiate into cardiomyocytes as indicated by RT-PCR and Western blotting results. During the cardiac commitment time of P19CL6 cells, the mRNA expression levels of the oxytocin receptor were upregulated by the addition of oxytocin as well as DMSO, but an upregulation of Gata4 expression levels was only observed for the cells induced by DMSO. The in silico analysis of the upstream sequence of the oxytocin receptor predicted putative binding sites for Gata4 and Nkx2.5. These results suggest that upregulations of the oxytocin receptor and Gata4 are important for cardiomyocyte differentiation processes.     

稳定转染MiR-499对P19CL6细胞向心肌细胞分化的影响

小鼠miR-499基因包含在心肌重链肌球蛋白Myh7b基因的第19内含子中,并且在心肌细胞中特异表达,然而其在心肌细胞中表达的生物学功能和意义尚不清楚.利用可体外分化为心肌细胞的P19CL6细胞建立稳定表达miR-499的细胞株对研究miR-499的生物学功能具有重要意义.根据小鼠miR-499基因序列,设计PCR引物...     

全反式维甲酸体外诱导P19细胞向心肌方向分化

目的探讨不同浓度全反式维甲酸(all-trans retinoic acid,atRA)诱导P19细胞向心肌分化的效力。方法细胞分成P19细胞组,2nm/L atRA诱导组,5nm/L atRA诱导组,8nm/L atRA诱导组。各组细胞经过诱导、聚集培养、聚集体贴壁培养10天后,RT-PCR检测GATA-4,α-肌球蛋白重链(α-myosin heavychain,α-MHC)的mRNA表达,免疫荧光双标检测α-sarcomeric actin和cTnT蛋白共表达,Western blot检测cTnT的蛋白表达。结果 atRA可诱导聚集P19细胞表达GA-TA-4、a-MHC mRNA;α-sarcomeric actin和cTnT的表达和共表达增加;5nm/L atRA组,8nm/L atRA组GATA-4、a-MHCmRNA的表达量显著高于P19细胞组;5nm/L atRA组,8nm/L atRA组两种蛋白的表达和共表达量显著高于P19细胞组,以5nm/L atRA组最高,显著高于其它组。结论 atRA可诱导聚集P19细胞向心肌分化,其中5nm/L atRA组效果最好。     

Proteomics analysis of mature seed of four peanut cultivars using two-dimensional gel electrophoresis reveals distinct differential expression of storage, anti-nutritional, and allergenic proteins

Profiles of total seed proteins isolated from mature seeds of four peanut cultivars, New Mexico Valencia C (NM Valencia C), Tamspan 90, Georgia Green, and NC-7, were studied using two-dimensional gel electrophoresis coupled with nano-electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC–MS/MS). Two-dimensional gels stained with silver nitrate revealed a total of 457, 516, 556, and 530 protein spots in NM Valencia C, Tamspan 90, Georgia Green, and NC-7, respectively. Twenty abundant protein spots showing differences in relative abundance among these cultivars were analyzed by nESI-LC–MS/MS, resulting in identification of 14 non-redundant proteins. The majority of these proteins belonged to the globulin fraction consisting of arachin (glycinin and Arah3/4) and conarachin seed storage proteins as well as other allergen proteins. The expression of some of these identified protein spots was cultivar-specific. For example, allergen Arah3/Arah4 and conarachin protein spots were only detected in Tamspan 90 and NC-7, whereas the Gly1 protein spot was detected only in NM Valencia C and NC-7. Moreover, a galactose-binding lectin protein spot with anti-nutritive properties was only present in Tamspan 90. Other proteins showing differences in relative abundance among the four cultivars included 13-lipoxygenase, fructose-biphosphate aldolase, and glyceraldehyde 3-phosphate dehydrogenase. Together, these results suggest that identified proteins might serve as potential markers for cultivar differentiation and may be associated with underlying sensory and nutritional traits of peanut cultivars.     

hhlim促进DMSO诱导的P19细胞向心肌分化

为了确定hhlim是否参与胚胎期的心肌分化和发育过程,用可表达hhlim蛋白和hhlim反义RNA的真核表达质粒转染P19胚胎干细胞,经G418筛选得到稳定表达hhlim和hhlim反义RNA的P19细胞克隆后,观察hhlim对P19细胞向心肌分化和发育的影响.结果显示,Nkx2.5和GATA-4在未被外源性hhlim基因转染的P19细胞中不表达.DMSO刺激细胞2天后,GATA-4开始表达,3天后Nkx2.5的表达活性显著升高.hhlim的过表达不但有利于P19细胞的存活和生长,而且还可以使Nkx2.5和GATA-4的表达比对照细胞提前1天.反义hhlim细胞株被DMSO诱导5天后,细胞仍呈集落化生长.同时,Nkx2.5和GATA-4开始表达的时间明显延滞.结果表明,hhlim能促进P19细胞向心肌细胞分化,其作用是通过促进转录因子GATA-4和Nkx2.5的表达而实现的.     

Cellular differentiation regulates expression of Cl- transport and cystic fibrosis transmembrane conductance regulator mRNA in human intestinal cells.

The gene defective in cystic fibrosis has recently been shown to code for a membrane protein designated the "cystic fibrosis transmembrane conductance regulator" (CFTR) protein. While it has been shown that detectable levels of the mRNA for the normal CFTR protein are present in epithelial cells from different tissues, factors which regulate CFTR expression have not been identified. A clonal cell line originating from a human colon adenocarcinoma (HT29-18) differentiates to multiple epithelial cell types when deprived of glucose in the culture medium. In these studies, mRNA isolated from these cells was examined by hybridization to a 1.45-kilobase cDNA probe which encodes transmembrane portions of the CFTR protein between exons 13 and 19. Cellular differentiation of HT29-18 causes a 9-18-fold increase in CFTR mRNA abundance versus the mRNA for the structural proteins actin and tubulin. Cellular differentiation also causes a 5-fold increase in second messenger-regulated Cl- transport which is sensitive to a Cl- channel blocker (diphenylamine 2-carboxylate). Subclones of HT29-18 which are committed to differentiate to either a mucin-secreting (HT29-18-N2) or an "enterocyte-like" (HT29-18-C1) phenotype have also been examined. In both subclones, elevated levels of CFTR mRNA are observed when compared with undifferentiated HT29-18 cells. However, during cellular differentiation, the regulation of CFTR mRNA abundance and membrane enzyme expression by the subclones is different from HT29-18. The results show that elevated CFTR mRNA occurs in multiple differentiated intestinal epithelial cell types, despite a phenotype-specific regulation of membrane protein expression. This suggests that CFTR expression plays a role in the differentiated functions of multiple epithelial phenotypes and that both cellular differentiation and cellular phenotypes are factors which regulate CFTR expression.     

Search for novel stress-responsive protein components using a yeast mutant lacking two cytosolic Hsp70 genes, SSA1 and SSA2

Heat shock proteins (Hsp) 70 are a ubiquitous family of molecular chaperones involved in many cellular processes. A yeast strain, ssa1/2, with two functionally redundant cytosolic Hsp70s (SSA1 and SSA2) deleted shows thermotolerance comparable to mildly heat-shocked wild type yeast, as well as increased protein synthesis and ubiquitin-proteasome protein degradation. Since mRNA abundance does not always correlate well with protein expression levels it is essential to study proteins directly. We used a gel-based approach to identify stress-responsive proteins in the ssa1/2 mutant and identified 43 differentially expressed spots. These were trypsin-digested and analyzed by nano electrospray ionization liquid chromatography tandem mass spectrometry (nESI-LC-MS/MS). A total of 22 non-redundant proteins were identified, 11 of which were confirmed by N-terminal sequencing. Nine proteins, most of which were up-regulated (2-fold or more) in the ssa1/2 mutant, proved to be stress-inducible proteins such as molecular chaperones and anti-oxidant proteins, or proteins related to carbohydrate metabolism. Interestingly, a translational factor Hyp2p up-regulated in the mutant was also found to be highly phosphorylated. These results indicate that the cytosolic Hsp70s, Ssa1p and Ssa2p, regulate an abundance of proteins mainly involved in stress responses and protein synthesis.     

Regulation of a group of abundant mRNA sequences during Friend cell differentiation

The changes occurring in the pattern of genes expressed at the polysomal level during induction of Friend cell differentiation with 1.5% dimethylsulfoxide (DMSO) have been examined in two ways. First, homologous and heterologous hybridization experiments between cDNA and polysomal poly(A)⁺ mRNA from differentiated and undifferentiated cells show that about 8000 mRNAs are expressed at both stages of differentiation, the major change being the accumulation of α+β-globin mRNA after DMSO treatment. The vast majority of the mRNA sequences do not change qualitatively, remaining homologous between the undifferentiated and differentiated state. However, in addition to the accumulation of α+β-globin mRNA there is a decrease, after DMSO treatment, in the concentration of abundant and semiabundant sequences found in undifferentiated cells. From control studies with Friend cell variants and fractionated cDNA probes enriched in these sequences, it is shown that the decrease in the abundance of these mRNAs is related to the process of differentiation and not an artefact of DMSO treatment. Comparison of the polysomal poly(A)⁺ mRNAs in differentiated cells to those in pluripotential embryonal carcinoma (EC) cells shows that the vast majority of the sequences are homologous and hence not erythropoiesis specific. Second, comparison of these mRNA populations by in vitro translation and analysis of the protein products on two-dimensional gels also shows that among the more abundant proteins very few qualitatively new proteins appear after differentiation and that the majority are the same as those translated in EC mRNA. There are several proteins prominent in undifferentiated cells which diminish after DMSO treatment, in agreement with the findings from the cDNA studies.     

Insulin induces proliferation and cardiac differentiation of P19CL6 cells in a dose‐dependent manner

Insulin is a peptide hormone produced by beta cells of the pancreas. The roles of insulin in energy metabolism have been well studied, with most of the attention focused on glucose utilization, but the roles of insulin in cell proliferation and differentiation remain unclear. In this study, we observed for the first time that 10 nmol/L insulin treatment induces cell proliferation and cardiac differentiation of P19CL6 cells, whereas 50 and 100 nmol/L insulin treatment induces P19CL6 cell apoptosis and blocks cardiac differentiation of P19CL6 cells. By using real‐time polymerase chain reaction (PCR) and Western blotting analysis, we found that the mRNA levels of cyclin D1 and α myosin heavy chain (α‐MHC) are induced upon 10 nmol/L insulin stimulation and inhibited upon 50/100 nmol/L insulin treatment, whereas the mRNA levels of BCL‐2‐antagonist of cell death (BAD) exists a reverse trend. The similar results were observed in P19CL6 cells expressing GATA‐6 or peroxisome proliferator‐activated receptor α (PPARα). Our results identified the downstream targets of insulin, cyclin D1, BAD, α‐MHC, and GATA‐4, elucidate a novel molecular mechanism of insulin in promoting cell proliferation and differentiation.     

2D-difference gel electrophoretic proteomic analysis of a cell culture model of alveolar rhabdomyosarcoma

To evaluate the consequences of expression of the protein encoded by PAX3-FOXO1 (P3F) in the pediatric malignancy alveolar rhabdomyosarcoma (A-RMS), we developed and evaluated a genetically defined in vitro model of A-RMS tumorigenesis. The expression of P3F in cooperation with simian virus 40 (SV40) Large-T (LT) antigen in murine C3H10T1/2 fibroblasts led to robust malignant transformation. Using 2-dimensional-difference gel electrophoresis (2D-DIGE), we compared proteomes from lysates from cells that express P3F + LT versus from cells that express LT alone. Analysis of 2D gel spot patterns by DeCyder image analysis software indicated 93 spots that were different in abundance. Peptide mass fingerprint analysis of the 93 spots by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis identified 37 nonredundant proteins. 2D-DIGE analysis of cell culture media conditioned by cells transduced by P3F + LT versus by LT alone found 29 spots in the P3F + LT cells leading to the identification of 11 nonredundant proteins. A substantial number of proteins with potential roles in tumorigenesis and myogenesis were detected, most of which have not been identified in previous wide-scale expression studies of RMS experimental models or tumors. We validated the 2D gel image analysis findings by Western blot analysis and immunohistochemistry (IHC). Thus, the 2D-DIGE proteomics methodology described here provided an important discovery approach to the study of RMS biology and complements the findings of previous mRNA expression studies.