Differentiation of Human Adipose Derived Stem Cells into Smooth Muscle Cells Is Modulated by CaMKIIγ

Abstract

The multifunctional Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is known to participate in maintenance and switches of smooth muscle cell (SMC) phenotypes. However, which isoform of CaMKII is involved in differentiation of adult mesenchymal stem cells into contractile SMCs remains unclear. In the present study, we detected γ isoform of CaMKII in differentiation of human adipose derived stem cells (hASCs) into SMCs that resulted from treatment with TGF-β1 and BMP4 in combination for 7 days. The results showed that CaMKIIγ increased gradually during differentiation of hASCs as determined by real-time PCR and western blot analysis. The siRNA-mediated knockdown of CaMKIIγ decreased the protein levels and transcriptional levels of smooth muscle contractile markers (a-SMA, SM22a, calponin, and SM-MHC), while CaMKIIγ overexpression increases the transcriptional and protein levels of smooth muscle contractile markers. These results suggested that γ isoform of CaMKII plays a significant role in smooth muscle differentiation of hASCs.

Figure 1

Human ASCs were induced to differentiate into SMCs by treatment with TGF-β1 and BMP4 for 7 days. (a) Quantitative real-time PCR experiments were carried out to measure the expression level of smooth muscle specific markers. (b, c) Time course of the expression of SMC differentiation markers detected by qRT-PCR and western blot analysis, respectively. (d) Immunofluorescent staining of smooth muscle specific markers (red), respectively. Nuclear were counterstained with DAPI (blue). Scale bars: 100 μm. Data represent means ± SE, n = 3 (^∗ P < 0.05 versus controls).

Figure 2

Expression of CaMKII isoforms in hASCs subjected to combined treatment with TGF-β1 and BMP4 for 7 days. mRNA (a) and protein (b) expressions of CaMKII isoforms after smooth muscle differentiation of hASCs. (c) qRT-PCR analysis of CaMKIIγ expression at 1, 3, 5, and 7 days, respectively. (d) Expression of CaMKIIγ detected by western blot at 1, 3, 5, and 7 days of stimulation. ^∗ P < 0.05 when (a) versus none, (b) versus day 0, and (d) versus day 0.

Figure 3

Cell transfection efficiency. CaMKII mRNA (a) and protein (b) expression levels in hASCs transfected with CaMKIIγ and control. (c) qRT-PCR analysis of CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ in hASCs transfected with siRNA targeting CaMKIIγ (si-CaMKIIγ) and nontargeting siRNA (si-control) for 48 hours, respectively. (d) Detection of CaMKII isoforms by western blot in hASCs transfected with siRNA targeting CaMKIIγ (si-CaMKIIγ) and nontargeting siRNA (si-control). ^∗ P < 0.05 when (a) versus ad-GFP and (c) si-control.

Figure 4

Transfection of CaMKIIγ upregulated expression of smooth muscle contractile markers. (a) qRT-PCR analysis of smooth muscle specific markers. (b) Western blot analysis of smooth muscle contractile proteins in CaMKIIγ and control group, respectively. Data represent means ± SE, n = 3 (^∗ P < 0.05 versus si-control).

Figure 5

Expression of smooth muscle contractile markers was downregulated in induced hASCs transfected with siRNA against CaMKIIγ. (a) Expression of smooth muscle contractile markers in si-CaMKIIγ and si-control group determined by qRT-PCR. Data represent means ± SE, n = 3 (^∗ P < 0.05 versus si-control). (b) Western blot analysis of smooth muscle contractile proteins in si-CaMKIIγ and si-control group, respectively. Data represent means ± SE, n = 3 (^∗ P < 0.05 versus si-control). (c) Immunofluorescent staining of a-SMA, SM22a, and SM-MHC (red) in si-CaMKII and si-control group, respectively. Nuclear were stained with DAPI (blue). Scale bars: 100 μm.