ERK1/2 pathway regulates coxsackie and adenovirus receptor expression in mouse cardiac stem cells

Abstract

Cardiac stem cells (CSCs) are the most promising and effective candidates for the therapy of cardiac regenerative diseases; however, they have marked limitations. For instance, the implantation of CSCs is hampered by factors such as their sustainability and long-term durability. Gene modification appears to be the most effective method of optimizing CSCs and gene therapy trials have demonstrated that efficient gene transfer is key to achieving therapeutic efficacy. However, the transduction ability of adenovirus (Ad) is limited. Previous studies have reported that low expression of coxsackie and adenovirus receptor (CAR) in target cells decreases the transduction efficiency. A promising method for improving Ad-mediated gene transfer is to increase CAR expression in target cells. The present study investigated the effect of the Raf-mitogen-associated protein kinase (MAPK) kinase (MEK)-extracellular signal-associated protein kinase (ERK) signaling pathway on the expression of CAR on CSCs, as this pathway decreases cell-cell adhesion via cell surface molecules. The results demonstrated that interference with the Raf-MEK-ERK signaling pathway by knockdown of ERK1/2 upregulated the expression of CAR. The entry of the Ad into the cells was increased following inhibition of ERK1/2. Moreover, following knockdown of CAR, the entry of Ad into cells was decreased. However, knockdown of c-Jun N-terminal kinase and p38 as other components of the MAPK pathway did not affect CAR expression. Therefore, CAR expression in CSCs may be mediated via the Raf-MEK-ERK signaling pathway. Upregulation of CAR by knockdown of ERK1/2 may significantly improve Ad-mediated genetic modification of CSCs in the treatment of cardiovascular diseases.

Keywords: cardiac stem cells; coxsackie and adenovirus receptor; extracellular signal-regulated kinase 1/2; gene therapy.

Figure 1.

Characteristics of CAR expression and inhibition of mitogen-activated protein kinase on murine CSCs. Immunofluorescence analysis of CAR expression on CSCs and ISK cells. The nuclei were counter-stained with 4′,6-diamidino-2-phenylindole (original magnification, ×400). ISK, ishikawa cells; CSC, cardiac stem cells; CAR, coxsackie and adenovirus receptor.

Figure 2.

Cellular protein was collected after individual transfection with non-targeting, JNK, ERK1/2 or p38 siRNAs for 48 h, followed by detection of ERK1/2, JNK and p38 protein expression by western blot analysis. *P<0.05 vs. NC. ISK, ishikawa cells; CSC, cardiac stem cell; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; NC, negative control.

Figure 3.

Effects of mitogen-activated protein kinase silencing on CAR expression in mCSCs. mCSCs were individually treated with siRNA-ERK1/2, -JNK, -P38 or -NC (control) for 48 h. (A) Western blot analysis of CAR protein expression. Representative immunoblots and densitometrically quantified CAR protein levels. (B) Effect of ERK1/2 silencing on the subcellular CAR protein distribution observed by immunofluorescence microscopy. DNA was visualized by 4′,6-diamidino-2-phenylindole staining. (C) CAR mRNA expression determined by reverse-transcription quantitative polymerase chain reaction with normalization to GAPDH. The bar graphs represent the mean values of triplicate measurements ± standard error of the mean. *P<0.05 vs. NC. mCSC, murine cardiac stem cell; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; CAR, coxsackie and adenovirus receptor; NC, negative control.

Figure 4.

Changes in viral uptake following ERK1/2 silencing. The infectivity of a GFP-expressing, non-replicating adenovirus at a multiplicity of infection of 10 was evaluated after 48 h treatment with siRNA-ERK1/2 or siRNA-NC (control group) (A) using fluorescence-assisted cell sorting analysis. Values are expressed as the mean of triplicate measurements ± standard error. *P<0.05 vs. NC. (B) The infectivity was also assessed using fluorescence microscopy (scale bar, 200 µm). Under the same conditions, the left panel shows transmitted light images and the right panels show green fluorescence. JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; NC, negative control; GFP, green fluorescence protein. CAR, coxsackie and adenovirus receptor; mCSC, murine cardiac stem cell.

Figure 5.

Changes in viral uptake and CAR expression after silencing of ERK1/2. mCSCs were treated with siRNA-CAR alone or siRNA-CAR with siRNA-ERK1/2 for 48 h. siRNA-NC-treated mCSCs were used as a control group. (A) CAR protein expression was detected by immunoblot analysis and densitometrically quantified. The bar graphs represent the mean values of triplicate measurements ± standard error of the mean. *P<0.05 vs. NC. (B) The infectivity of a GFP-expressing, non-replicating adenovirus at a multiplicity of infection of 10 was evaluated by fluorescence microscopy (scale bar, 100 µm). Under the same conditions, the left panel shows transmitted light images and the right panels show fluorescence. ERK, extracellular signal-regulated kinase; siRNA, small interfering RNA; NC, negative control; GFP, green fluorescence protein; CAR, coxsackie and adenovirus receptor; mCSC, murine cardiac stem cell.