Galectin-1 attenuates cardiomyocyte hypertrophy through splice-variant specific modulation of CaV1.2 calcium channel

Pressure overload-induced cardiac hypertrophy occurs in response to chronic blood pressure increase, and dysfunction of Ca_V1.2 calcium channel involves in cardiac hypertrophic processes by perturbing intracellular calcium concentration ([Ca²⁺]_i) and calcium-dependent signaling. As a carbohydrate-binding protein, galectin-1 (Gal-1) is found to bind with Ca_V1.2 channel, which regulates vascular Ca_V1.2 channel functions and blood pressure. However, the potential roles of Gal-1 in cardiac Ca_V1.2 channel (Ca_V1.2_CM) and cardiomyocyte hypertrophy remain elusive. By whole-cell patch clamp, we find Gal-1 decreases the I_Ca,L with or without isoproterenol (ISO) application by reducing the channel membrane expression in neonatal rat ventricular myocytes (NRVMs). Moreover, Gal-1 could inhibit the current densities of Ca_V1.2_CM by an alternative exon 9*-dependent manner in heterologously expressed HEK293 cells. Of significance, overexpression of Gal-1 diminishes ISO or KCl-induced [Ca²⁺]_i elevation and attenuates ISO-induced hypertrophy in NRVMs. Mechanistically, Gal-1 decreases the ISO or Bay K8644-induced phosphorylation of intracellular calcium-dependent signaling proteins δCaMKII and HDAC4, and inhibits ISO-triggered translocation of HDAC4 in NRVMs. Pathologically, we observe that the expressions of Gal-1 and Ca_V1.2_E9* channels are synchronously increased in rat hypertrophic cardiomyocytes and hearts. Taken together, our study indicates that Gal-1 reduces the channel membrane expression to inhibit the currents of Ca_V1.2_CM in a splice-variant specific manner, which diminishes [Ca²⁺]_i elevation, and attenuates cardiomyocyte hypertrophy by inhibiting the phosphorylation of δCaMKII and HDAC4. Furthermore, our work suggests that dysregulated Gal-1 and Ca_V1.2 alternative exon 9* might be attributed to the pathological processes of cardiac hypertrophy, and provides a potential anti-hypertrophic target in the heart.     

DHRS4L2非编码RNA调控CPNE6基因表达

DHRS4/NRDR基因编码一种属于SDR家族的酶,在维甲酸合成、类固醇代谢和苯甲基代谢中发挥生物合成催化作用.DHRS4基因定位于14q11-2,有两个相似的拷贝基因,分别为DHRS4L2和DHRS4L1.我们前期发现了DHRS4L2基因一个上游转录起始位点,命名为DHRS4L2-Ea.在本研究中,我们用RT-PCR和双脱氧测序法发现一个新的从DHRS4L2-Ea转录的选择性剪接亚型DHRS4L2-900a(KC237374).同时RT-PCR结果显示在SK-N-SH细胞DHRS4L2-Ea选择性剪接亚型中DHRS4L2 iso(AY616183)表达最多,为主要亚型.在SK-N-SH细胞过表达DHRS4L2-800a(AY920361)使DHRS4L2-Ea 基因下游CPNE6 mRNA表达下调.在HeLa细胞过表达DHRS4L2 800a(AY920361)或DHRS4L2-900a(KC237374) 进一步表明DHRS4L2 Ea抑制CPNE6表达的作用.定量PCR结果显示si-RNA抑制DHRS4L2-Ea表达使CPNE6 mRNA表达上调.亚硫酸盐测序结果显示在SK-N-SH转染DHRS4L2-800a(AY920361)的样本中CPNE6基因DNA CpG甲基化增加.综上所述,本研究揭示DHRS4L2表达的非编码RNA抑制其下游基因CPNE6的表达.     

The Alternative Splicing of Cytoplasmic Polyadenylation Element Binding Protein 2 Drives Anoikis Resistance and the Metastasis of Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) represents an anomalous subset of breast cancer with a greatly reduced (30%) 5-year survival rate. The enhanced mortality and morbidity of TNBC arises from the high metastatic rate, which requires the acquisition of AnR, a process whereby anchorage-dependent cells become resistant to cell death induced by detachment. In this study TNBC cell lines were selected for AnR, and these cell lines demonstrated dramatic enhancement in the formation of lung metastases as compared with parental cells. Genetic analysis of the AnR subclones versus parental cells via next generation sequencing and analysis of global alternative RNA splicing identified that the mRNA splicing of cytoplasmic polyadenylation element binding 2 (CPEB2), a translational regulator, was altered in AnR TNBC cells. Specifically, increased inclusion of exon 4 into the mature mRNA to produce the CPEB2B isoform was observed in AnR cell lines. Molecular manipulations of CPEB2 splice variants demonstrated a key role for this RNA splicing event in the resistance of cells to anoikis. Specifically, down-regulation of the CPEB2B isoform using siRNA re-sensitized the AnR cell lines to detachment-induced cell death. The ectopic expression of CPEB2B in parental TNBC cell lines induced AnR and dramatically increased metastatic potential. Importantly, alterations in the alternative splicing of CPEB2 were also observed in human TNBC and additional subtypes of human breast cancer tumors linked to a high metastatic rate. Our findings demonstrate that the regulation of CPEB2 mRNA splicing is a key mechanism in AnR and a driving force in TNBC metastasis.     

cDNA cloning and expression of the mouse Na/H antiporter (NHE-1) and a potential splice variant

A cDNA for the Mus musculus Na/H exchanger-isoform 1 (NHE-1) was identified in a BALB/c myoblast library by its hybridization to rat NHE-1 sequences. Analysis of the clone showed it to display extensive homology with NHE-1 clones from other mammalian species; however, the region of interspecific homology was abruptly interrupted in the midst of the open reading frame by 166 bp of unrelated sequence. This extra sequence is likely to be an unspliced intron 9. Aside from the retained intron 9, the NHE-1 cDNA clone is otherwise fully processed, with all of the other ten introns removed and containing a poly(A) tract. From PCR results this variant represents a significant but minor population of NHE-1 RNAs. The variant message does associate with polysomes thereby suggesting it to be translated into protein. The location of the retained intron in the carboxy terminus of the protein is such that its translation would produce a protein predicted to be still capable of effecting Na and H translocation but whose regulatory features would be markedly altered.Amino acid sequence comparison of the mouse NHE-1 (derived from the fully processed message) with that of other mammalian species demonstrated two exceptionally divergent regions; the C-terminal cytoplasmic tail (residues 750-790), containing a region of 6-8 contiguous acidic amino acids variably composed of aspartate and glutamate residues, and the N-terminal extracellular domain that includes an N-linked glycosylation site (residues 60-80).