The tetraspanin CD151-ARSA mutant inhibits angiogenesis via the YRSL sequence

Abstract

Previous studies have shown that the tetraspanin CD151 is essential for pathological or physiological angiogenesis. However, the cellular signaling mechanism and the role of the CD151 YRSL sorting motif in in vitro vasculogenesis remains unknown. In this study, the results showed that both CD151 and CD151-ARSA gene delivery were capable of increasing the expression of CD151 at the protein level in human umbilical vein endothelial cells (HUVECs). Moreover, there was no significant difference in CD151 protein expression between the CD151 group and the CD151-ARSA group. Overexpression of CD151 promoted HUVEC cell proliferation, migration and capillary network formation in vitro. However, in the CD151-ARSA group, the abilities of cell proliferation, migration and capillary network formation were all decreased, compared with the CD151 group. Furthermore, the activation of PI3K, Akt and ERK signaling pathways was attenuated in the CD151-ARSA mutant group compared with the CD151 group. This study suggests that the YRSL motif of CD151 plays a key role in CD151-induced angiogenesis. Our observations provide insights into a new mechanism of CD151 regulating angiogenesis via vesicle trafficking.

Figure 1

Expression of CD151 protein after transfection in HUVECs. (A) The gene sequences of CD151 and CD151-ARSA. The CD151 mutation (CD151-ARSA) changed the motif of YRSL245-248 to ARSA245-248. (B) HUVECs transfected with rAAV-GFP observed using an inverted fluorescence microscope 7 days after transfection (the same field). (C) Western blot analysis. (D) Quantitative analysis of CD151 protein expression. β-actin was used as an internal loading control. The mean density of CD151 in control group was defined as 100%. Each experiment was performed at least in triplicate. ^*p<0.05 vs. control and GFP group. HUVECs, human umbilical vein endothelial cells; rAAV, recombinant adeno-associated virus; GFP, green fluorescent protein.

Figure 2

Effects of CD151 and CD151-ARSA transfection on HUVECs. Cell Counting Kit-8 (CCK-8) assays were performed at 24 and 48 h after gene delivery. The CD151 group showed promoted proliferation ability. In the CD151-ARSA mutation group, the proliferation of HUVECs was decreased significantly at 24 or 48 h after delivery. Three independent experiments were carried out, and each experiment was in triplicate. ^*p<0.05 vs. control and GFP group. ^#p<0.05 vs. CD151 group. HUVECs, human umbilical vein endothelial cells; GFP, green fluorescent protein.

Figure 3

Effects of CD151 and CD151-ARSA transfection on the migration of HUVECs. (A) Cell migration was assessed by a cell wound-healing assay and observed at 0, 12, 24 and 48 h after rAAV transfection. (B) Quantitative analysis of HUVEC migration. Each experiment was performed at least in triplicate. ^*p<0.05 vs. control and GFP group. ^#p<0.05 vs. CD151 group. HUVECs, human umbilical vein endothelial cells; GFP, green fluorescent protein.

Figure 4

Effects of CD151 and CD151-ARSA transfection on the capillary network formation of HUVECs. (A) Representative photomicrographs observed at 12, 24 and 48 h after rAAV transfection on Matrigel showed that HUVECs assembled into capillary network structures. (B) Quantitative analysis of capillary network formation. ^*p<0.05 vs. control and GFP group. ^#p<0.05 vs. CD151 group. HUVECs, human umbilical vein endothelial cells; GFP, green fluorescent protein.

Figure 5

Effects of CD151 and CD151-ARSA transfection on PI3K/Akt and ERK signaling pathways. Western blot analysis for PI3K, phosphorylated Akt, total Akt, phosphorylated ERK and total ERK. Inhibitors of MAPK (apigenin) and PI3K (LY294002) were applied to HUVECs following transfection with CD151 or CD151-ARSA. (A-C) The protein levels of PI3K, Akt, phospho-Akt and quantitative analysis. (D and E) Levels of ERK1, phospho-ERK1 and quantitative analysis. Inhibitor of PI3K (LY294002, 15 μM); inhibitor of MAPK (apigenin, 25 μM). HD group (HD-Fugene 6 transfection reagent) was used as a control group. Each experiment was performed at least in triplicate. ^*p<0.05 vs. control, GFP and HD groups. ^#p<0.05 vs. CD151 group. ^&p<0.05 vs. CD151-ARSA group (no signaling pathway inhibitor group). HUVECs, human umbilical vein endothelial cells; GFP, green fluorescent protein.