Single Mechanosensitive and Ca2+-Sensitive Channel Currents Recorded from Mouse and Human Embryonic Stem Cells

Cell-attached and inside-out patch clamp recording was used to compare the functional expression of membrane ion channels in mouse and human embryonic stem cells (ESCs). Both ESCs express mechanosensitive Ca²⁺ permeant cation channels (MscCa) and large conductance (200 pS) Ca²⁺-sensitive K⁺ (BK_Ca2+) channels but with markedly different patch densities. MscCa is expressed at higher density in mESCs compared with hESCs (70 % vs. 3 % of patches), whereas the BK_Ca2+ channel is more highly expressed in hESCs compared with mESCs (~50 % vs. 1 % of patches). ESCs of both species express a smaller conductance (25 pS) nonselective cation channel that is activated upon inside-out patch formation but is neither mechanosensitive nor strictly Ca²⁺-dependent. The finding that mouse and human ESCs express different channels that sense membrane tension and intracellular [Ca²⁺] may contribute to their different patterns of growth and differentiation in response to mechanical and chemical cues.     

MiR-218 regulated cardiomyocyte differentiation and migration in mouse embryonic stem cells by targeting PDGFRα

MicroRNAs (miRNAs) have been identified as key players in cardiogenesis and heart pathophysiological processes. However, many miRNAs are still not recognized for their roles in cardiomyocytes differentiation. In this study, we evaluated the effects of microRNA-218 (miR-218) in cardiomyocyte differentiation of the mouse embryonic stem cells (ESCs) in vitro. The percentage of the beating embryoid bodies (EBs) in miR-218 mimic-treated cells was reduced to 32% compared with miR-218 mimic negative control (56%) on day 5 + 3. The amplitude of the intracellular Ca²⁺ transients in the cardiomyocytes derived from ESCs was reduced upon miR-218 overexpression, followed by the decreased calcium-related proteins and cell junction proteins expressions. Besides, miR-218 expression in ESCs was related to the directional spreading ability of EBs during differentiation. The increased expression of miR-218 could promote the migration of ESCs in vitro, while the decreased expression of miR-218 could inhibit the migration by the transwell experiment. Meanwhile, miR-218 could regulate cell migration–related proteins Cdc42 and Rac1. Platelet-derived growth factor receptor α (PDGFRα) was further confirmed to be a direct target of miR-218 both physically and functionally by dual-luciferase reporter assay. Our data further described that overexpression of PDGFRα rescued the miR-218-mediated inhibition of cardiomyocyte differentiation and restored the miR-218-mediated promotion of cell migration. In conclusion, miR-218 was demonstrated to exert an inhibitory function and promoted cell migration via targeting PDGFRα during cardiomyocyte differentiation from ESCs. The current study revealed the role of miR-218 and may provide an important hint for cardiomyocyte differentiation of ESCs and induced pluripotent stem cells.     

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.     

Involvement of calcium-sensing receptor in cardiac hypertrophy-induced by angiotensinII through calcineurin pathway in cultured neonatal rat cardiomyocytes

Cardiac hypertrophy is a common pathological change accompanying cardiovascular disease. Recently, some evidence indicated that calcium-sensing receptor (CaSR) expressed in the cardiovascular tissue. However, the functional involvement of CaSR in cardiac hypertrophy remains unclear. Previous studies have shown that CaSR caused accumulation of inositol phosphate to increase the release of intracellular calcium. Moreover, Ca²⁺-dependent phosphatase calcineurin (CaN) played a vital role in the development of cardiac hypertrophy. Therefore, we investigated the expression of CaSR in cardiac hypertrophy-induced by angiotensin II (AngII) and the effects of CaSR activated by GdCl₃ on the related signaling transduction pathways. The results showed that AngII induced cardiac hypertrophy and up-regulated the expression of CaSR, meanwhile increased the intracellular calcium concentration ([Ca²⁺]_i) and activated CaN hypertrophic signaling pathway. Compared with AngII alone, the above changes were further obvious when adding GdCl₃. But the effects of GdCl₃ on the cardiac hypertrophy were attenuated by CsA, a specific inhibitor of CaN. In conclusion, these results suggest that CaSR is involved in cardiac hypertrophy-induced by AngII through CaN pathway in cultured neonatal rat cardiomyocytes.     

High extracellular calcium attenuates adipogenesis in 3T3-L1 preadipocytes

We studied the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_e) on adipocyte differentiation. Preadipocytes exposed to continuous [Ca²⁺]_e higher than 2.5 mmol/l accumulated little or no cytoplasmic lipid compared to controls in 1.8 mmol/l [Ca²⁺]_e. Differentiation was monitored by Oil Red O staining of cytoplasmic lipid and triglyceride assay of accumulated lipid, by RT-PCR analysis of adipogenic markers, and by the activity of glycerol-3-phosphate dehydrogenase (GPDH). Elevated [Ca²⁺]_e inhibited expression of peroxisome proliferator-activated receptor γ, CCAAT/enhancer binding protein α, and steroid regulatory binding element protein. High [Ca²⁺]_e significantly inhibited differentiation marker expression including adipocyte fatty acid binding protein, and GPDH. The decrease in Pref-1 expression that accompanied differentiation also was prevented by high [Ca²⁺]_e. Treatment of 3T3-L1 cells with high [Ca²⁺]_e did not significantly affect cell number or viability and did not trigger apoptosis. Levels of intracellular Ca⁺² remained unchanged in various [Ca²⁺]_e. Treatment of 3T3-L1 with pertussis toxin (PTX) partially restored lipid accumulation and increased differentiation markers in cells treated with 5 mmol/l [Ca²⁺]_e. ‘Classical’ parathyroid cell Ca²⁺ sensing receptors (CaSR) were not detected either by RT-PCR or by Western blotting. These results suggest that continuos exposure to high [Ca²⁺]_e inhibits preadipocyte differentiation and that this may involve a G-protein-coupled mechanism mediated by a novel Ca²⁺ sensor or receptor.     

Functional Characteristics of ES Cell–derived Cardiac Precursor Cells Identified by Tissue-specific Expression of the Green Fluorescent Protein

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions.In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac α-actin promoter. First, fluorescent areas in ES cell–derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca²⁺ homeostasis plays a key role in cardiac function, we investigated how Ca²⁺ channels and Ca²⁺ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca²⁺ imaging experiments proved the functional expression of the L-type Ca²⁺ current (I_Ca) starting from day 7 of EB development. On day 7, using 10 mM Ca²⁺ as charge carrier, I_Ca was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of I_Ca proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, I_Ca was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP₃-sensitive intracellular Ca²⁺ stores and Ca²⁺-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca²⁺ stores, detected by caffeine-induced Ca²⁺ release, appeared in most GFP-positive cells 1–2 d after I_Ca. Coexpression of both I_Ca and ryanodine-sensitive Ca²⁺ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs.Thus, the functional expression of voltage-dependent L-type Ca²⁺ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP₃ receptors and sarcoplasmic Ca²⁺-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.     

Cellular responses to TGFβ and TGFβ receptor expression in human colonic epithelial cells require CaSR expression and function

CaSR and TGFβ are robust promoters of differentiation in the colonic epithelium. Loss of cellular responses to TGFβ or loss of CaSR expression is tightly linked to malignant progression. Human colonic epithelial CBS cells, originally developed from a differentiated human colon tumor, retain CaSR expression and function, TGFβ responsiveness and TGFβ receptor expression. Thus, these cells offer a unique opportunity in determining the functional linkage (if any) between CaSR and TGFβ. Knocking down CaSR expression abrogated TGFβ-mediated cellular responses and attenuated the expression of TGFβ receptors. Ca²⁺ or vitamin D treatment induced CaSR expression with a concurrent up-regulation of TGFβ receptor expression. Ca²⁺ or vitamin D, however, did not induce CaSR in CaSR knocked down cells and without CaSR; there was no up-regulation of TGFβ receptor. It is concluded that TGFβ receptor expression and TGFβ mediated responses requires CaSR expression and function.     

Calcium sensing receptor in developing human airway smooth muscle

Airway smooth muscle (ASM) regulation of airway structure and contractility is critical in fetal/neonatal physiology in health and disease. Fetal lungs experience higher Ca²⁺ environment that may impact extracellular Ca²⁺ ([Ca²⁺]_o) sensing receptor (CaSR). Well-known in the parathyroid gland, CaSR is also expressed in late embryonic lung mesenchyme. Using cells from 18-22 week human fetal lungs, we tested the hypothesis that CaSR regulates intracellular Ca²⁺ ([Ca²⁺]_i) in fetal ASM (fASM). Compared with adult ASM, CaSR expression was higher in fASM, while fluorescence Ca²⁺ imaging showed that [Ca²⁺]_i was more sensitive to altered [Ca²⁺]_o. The fASM [Ca²⁺]_i responses to histamine were also more sensitive to [Ca²⁺]_o (0–2 mM) compared with an adult, enhanced by calcimimetic R568 but blunted by calcilytic NPS2143. [Ca²⁺]_i was enhanced by endogenous CaSR agonist spermine (again higher sensitivity compared with adult). Inhibition of phospholipase C (U73122; siRNA) or inositol 1,4,5-triphosphate receptor (Xestospongin C) blunted [Ca²⁺]_o sensitivity and R568 effects. NPS2143 potentiated U73122 effects. Store-operated Ca²⁺ entry was potentiated by R568. Traction force microscopy showed responsiveness of fASM cellular contractility to [Ca²⁺]_o and NPS2143. Separately, fASM proliferation showed sensitivity to [Ca²⁺]_o and NPS2143. These results demonstrate functional CaSR in developing ASM that modulates airway contractility and proliferation.     

Identification of novel DNA methylation inhibitors via a two-component reporter gene system

Background

The extracellular calcium-sensing receptor (CaSR) belongs to family C of the G protein coupled receptors. Whether the CaSR is expressed in the pulmonary artery (PA) is unknown.

Methods

The expression and distribution of CaSR were detected by RT-PCR, Western blotting and immunofluorescence. PA tension was detected by the pulmonary arterial ring technique, and the intracellular calcium concentration ([Ca²⁺]_i) was detected by a laser-scanning confocal microscope.

Results

The expressions of CaSR mRNA and protein were found in both rat pulmonary artery smooth muscle cells (PASMCs) and PAs. Increased levels of [Ca²⁺]_o (extracellular calcium concentration) or Gd³⁺ (an agonist of CaSR) induced an increase of [Ca²⁺]_i and PAs constriction in a concentration-dependent manner_. In addition, the above-mentioned effects of Ca²⁺ and Gd³⁺ were inhibited by U73122 (specific inhibitor of PLC), 2-APB (specific antagonist of IP₃ receptor), and thapsigargin (blocker of sarcoplasmic reticulum calcium ATPase).

Conclusions

CaSR is expressed in rat PASMCs, and is involved in regulation of PA tension by increasing [Ca²⁺]_i through G-PLC-IP₃ pathway.     

Vascular endothelial growth factor promotes cardiomyocyte differentiation of embryonic stem cells

Embryonic stem cells (ESCs) overexpressing the vascular endothelial growth factor (VEGF) improve cardiac function in mouse models of myocardial ischemia and infarction by mechanisms that are poorly understood. Here we studied the effects of VEGF on cardiomyocyte differentiation of mouse ESCs in vitro. We used flow cytometry to determine the expression of alpha-myosin heavy chain (alpha-MHC), cardiac troponin I (cTn-I), and Nkx2.5 in differentiated ESCs. VEGF (20 ng/ml) significantly enhanced alpha-MHC, cTn-I, and Nkx2.5 expression in differentiated ESCs. Western blot analysis confirmed these findings. We found that VEGF receptor FMS-like tyrosine kinase-1 (Flt-1) and fetal liver kinase-1 (Flk-1) expression increased during ESC differentiation. Antibodies against Flk-1 totally blocked and against Flt-1 partially blocked VEGF-induced NKx2.5-positive-stained cells. The ERK inhibitor PD-098059 abolished VEGF-induced cardiomyocyte differentiation of ESCs. Our results suggest that VEGF promotes cardiomyocyte differentiation predominantly by ERK-mediated Flk-1 activation and, to a lesser extent, by Flt-1 activation. These findings may be of significance for stem cell and growth factor therapies to regenerate failing cardiomyocytes.     

Extracellular calcium stimulates osteogenic differentiation of human adipose-derived stem cells by enhancing bone morphogenetic protein-2 expression

Bone morphogenetic protein-2 (BMP-2) promotes the differentiation of non-osteogenic mesenchymal cells to osteogenic cells. In this study, we isolated human adipose-derived stem cells (hASCs) and investigated the effects of recombinant human BMP-2 (rhBMP-2) and extracellular Ca²⁺ concentration ([Ca²⁺]_out) on the osteogenic differentiation of hASCs. rhBMP-2 promoted calcium deposition in hASCs and stimulated the mRNA expressions of six proteins known to be involved in the osteogenic differentiation of hASCs: Runx2, osterix, alkaline phosphatase, osteonectin, bone sialoprotein and osteocalcin. Elevation of [Ca²⁺]_out enhanced the level of alkaline phosphatase enzyme, increased the mRNA expressions of Runx2 and osteocalcin and induced the expressions of BMP-2 mRNA and protein in hASCs. Elevation of [Ca²⁺]_out transiently increased the intracellular Ca²⁺ concentration ([Ca²⁺]_in) due to activation of the calcium-sensing receptor (CaSR). The Ca²⁺-induced expressions of BMP-2 mRNA and protein were inhibited by the calmodulin antagonist, W-7. Furthermore, elevation of [Ca²⁺]_out decreased the cytoplasmic level of phosphorylated nuclear factor of activated T-cell-2 (NFAT-2) and increased the nuclear level of dephosphorylated NFAT2. Taken together, these results suggest that rhBMP-2 promotes the osteogenic differentiation of hASCs. Furthermore, an increase in [Ca²⁺]_out enhances the expression of BMP-2 via activation of the CaSR, elevation of [Ca²⁺]_in and stimulation of Ca²⁺/calmodulin-dependent NFAT-signaling pathways.     

Platelet-derived growth factor-BB induces pulmonary venous smooth muscle cells proliferation by upregulating calcium sensing receptor under hypoxic conditions

Pulmonary hypertension (PH) is characterized by pulmonary vascular remodeling, which exists in both pulmonary arteries and pulmonary veins. Pulmonary vascular remodeling stems from excessive proliferation of pulmonary vascular myocytes. Platelet-derived growth factor-BB (PDGF-BB) is a vital vascular regulator whose level increases in PH human lungs. Although the mechanisms by which pulmonary arterial smooth muscle cells respond to PDGF-BB have been studied extensively, the effects of PDGF-BB on pulmonary venous smooth muscle cells (PVSMCs) remain unknown. We herein examined the involvement of calcium sensing receptor (CaSR) in PDGF-BB-induced PVSMCs proliferation under hypoxic conditions. In PVSMCs isolated from rat intrapulmonary veins, PDGF-BB increased the cell number and DNA synthesis under normoxic and hypoxic conditions, which was accompanied by upregulated CaSR expression. The influences of PDGF-BB on proliferation and CaSR expression in hypoxic PVSMCs were greater than that in normoxic PVSMCs. In hypoxic PVSMCs superfused with Ca²⁺-free solution, restoration of extracellular Ca²⁺ induced an increase of [Ca²⁺]_i, which was significantly smaller than that in PDGF-BB-treated hypoxic PVSMCs. The positive CaSR modulator spermine enhanced, whereas the negative CaSR modulator NPS2143 attenuated, the extracellular Ca²⁺-induced [Ca²⁺]_i increase in PDGF-BB-treated hypoxic PVSMCs. Furthermore, the spermine enhanced, whereas the NPS2143 inhibited, PDGF-BB-induced proliferation in hypoxic PVSMCs. Silencing CaSR with siRNA attenuated the extracellular Ca²⁺-induced [Ca²⁺]_i increase in PDGF-BB-treated hypoxic PVSMCs and inhibited PDGF-BB-induced proliferation in hypoxic PVSMCs. In conclusion, these results demonstrated that CaSR mediating PDGF-BB-induced excessive PVSMCs proliferation is an important mechanism involved in the initiation and progression of PVSMCs proliferation under hypoxic conditions.     

Molecular Mechanisms of Calcium-sensing Receptor-mediated Calcium Signaling in the Modulation of Epithelial Ion Transport and Bicarbonate Secretion

Epithelial ion transport is mainly under the control of intracellular cAMP and Ca²⁺ signaling. Although the molecular mechanisms of cAMP-induced epithelial ion secretion are well defined, those induced by Ca²⁺ signaling remain poorly understood. Because calcium-sensing receptor (CaSR) activation results in an increase in cytosolic Ca²⁺ ([Ca²⁺]_cyt) but a decrease in cAMP levels, it is a suitable receptor for elucidating the mechanisms of [Ca²⁺]_cyt-mediated epithelial ion transport and duodenal bicarbonate secretion (DBS). CaSR proteins have been detected in mouse duodenal mucosae and human intestinal epithelial cells. Spermine and Gd³⁺, two CaSR activators, markedly stimulated DBS without altering duodenal short circuit currents in wild-type mice but did not affect DBS and duodenal short circuit currents in cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice. Clotrimazole, a selective blocker of intermediate conductance Ca²⁺-activated K⁺ channels but not chromanol 293B, a selective blocker of cAMP-activated K⁺ channels (KCNQ1), significantly inhibited CaSR activator-induced DBS, which was similar in wild-type and KCNQ1 knockout mice. HCO₃⁻ fluxes across epithelial cells were activated by a CFTR activator, but blocked by a CFTR inhibitor. CaSR activators induced HCO₃⁻ fluxes, which were inhibited by a receptor-operated channel (ROC) blocker. Moreover, CaSR activators dose-dependently raised cellular [Ca²⁺]_cyt, which was abolished in Ca²⁺-free solutions and inhibited markedly by selective CaSR antagonist calhex 231, and ROC blocker in both animal and human intestinal epithelial cells. Taken together, CaSR activation triggers Ca²⁺-dependent DBS, likely through the ROC, intermediate conductance Ca²⁺-activated K⁺ channels, and CFTR channels. This study not only reveals that [Ca²⁺]_cyt signaling is critical to modulate DBS but also provides novel insights into the molecular mechanisms of CaSR-mediated Ca²⁺-induced DBS.     

Calmodulin Regulates Ca2+-sensing Receptor-mediated Ca2+ Signaling and Its Cell Surface Expression

The Ca²⁺-sensing receptor (CaSR) is a member of family C of the GPCRs responsible for sensing extracellular Ca²⁺ ([Ca²⁺]_o) levels, maintaining extracellular Ca²⁺ homeostasis, and transducing Ca²⁺ signaling from the extracellular milieu to the intracellular environment. In the present study, we have demonstrated a Ca²⁺-dependent, stoichiometric interaction between CaM and a CaM-binding domain (CaMBD) located within the C terminus of CaSR (residues 871–898). Our studies suggest a wrapping around 1–14-like mode of interaction that involves global conformational changes in both lobes of CaM with concomitant formation of a helical structure in the CaMBD. More importantly, the Ca²⁺-dependent association between CaM and the C terminus of CaSR is critical for maintaining proper responsiveness of intracellular Ca²⁺ responses to changes in extracellular Ca²⁺ and regulating cell surface expression of the receptor.     

Agmatine modulates calcium handling in cardiomyocytes of hibernating ground squirrels through calcium-sensing receptor signaling

True hibernators are remarkable group of mammals whose hearts are resistant to such stressors as deep hypothermia, ischemia, arrhythmia. Capability of cardiac cells from hibernating species to effectively rule Ca²⁺ homeostasis during torpor is poorly studied. Better understanding of these mechanisms could allow to introduce new strategies for improvement the cardiac performance and may be useful for cardiovascular medicine. Here for the first time we have shown that the regulation of Ca²⁺ handling and thereby cardiomyocyte contractility by endogenous neurotransmitter agmatine occurs through the modulation of calcium-sensing receptor (CaSR). In isolated cardiocytes of hibernating ground squirrels generating stationary Ca²⁺ transients in the absence of actual myocellular excitation, low doses of this polyamine (up to 500 μM) induce the G_βγ-dependent activation of PI₃-kinase with subsequent stimulation of Akt-kinase and nitric oxide (NO) production by endothelial NO-synthase (eNOS). NO production abolishes Ca²⁺ oscillations in virtue of the enhancement of Ca²⁺ reuptake by sarco(endo)plasmic Ca²⁺ ATPase (SERCA). Simultaneously, the activation of phospholipase A₂ (PLA₂) and arachidonic-acid dependent Ca²⁺ entry occur providing replenishment of Ca²⁺ store. High concentrations of agmatine (> 2 mM) induce other CaSR-mediated pathways involving phospholipase C (PLC) pathway, the formation of inositoltriphosphate (IP₃) and diacylglicerol (DAG) followed by induction of their targets: IP₃ receptors and protein kinase C isoforms (PKC), respectively. Furthermore, it is also responsible for the stimulation of PLA₂ and elevation of intracellular calcium caused by arachidonic acid-regulated Ca²⁺-permeable (ARC) channels. Additionally, there is a potent store-operated Ca²⁺ entry (SOC) in cardiomyocyte. Negative (NPS 2143) and positive (R 568) allosteric modulators of CaSR recapitulate effects of low and high agmatine doses on Ca²⁺ handling and NO synthesis. These facts and the alteration of agmatine influence in response to an increase of extracellular Ca²⁺, which is the direct agonist of CaSR, may confirm the participation of CaSR in regulation of Ca²⁺ handling and excitability of cardiomyocytes by agmatine.