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. 2016 Dec 16;60(4):2711.
doi: 10.4081/ejh.2016.2711.

Aortic dissection is associated with reduced polycystin-1 expression, an abnormality that leads to increased ERK phosphorylation in vascular smooth muscle cells

J Feng  1 , S Ge, L Zhang, H Che, C Liang
Affiliations

Aortic dissection is associated with reduced polycystin-1 expression, an abnormality that leads to increased ERK phosphorylation in vascular smooth muscle cells

J Feng et al. Eur J Histochem. .

Abstract

The vascular smooth muscle cell (VSMC) phenotypic switch is a key pathophysiological change in various cardiovascular diseases, such as aortic dissection (AD), with a high morbidity. Polycystin-1 (PC1) is significantly downregulated in the VSMCs of AD patients. PC1 is an integral membrane glycoprotein and kinase that regulates different biological processes, including cell proliferation, apoptosis, and cell polarity. However, the role of PC1 in intracellular signaling pathways remains poorly understood. In this study, PC1 downregulation in VSMCs promoted the expression of SM22α, ACTA2 and calponin 1 (CNN1) proteins. Furthermore, PC1 downregulation in VSMCs upregulated phospho-MEK, phospho-ERK and myc, but did not change phospho-JNK and phospho-p38. These findings suggest that the MEK/ERK/myc signaling pathway is involved in PC1-mediated human VSMC phenotypic switch. Opposite results were observed when an ERK inhibitor was used in VSMCs downregulated by PC1. When the C-terminal domain of PC1 (PC1 C-tail) was overexpressed in VSMCs, the expression levels of phosphor-ERK, myc, SM22α, ACTA2 and CNN1 proteins were downregulated. The group with the overexpressed mutant protein (S4166A) in the PC1 C-tail showed similar results to the group with the downregulated PC1 in VSMCs. These results suggest that the Ser at the 4166 site in PC1 is crucial in the PC1 mediated MEK/ERK/myc signaling pathway, which might be the key pathophysiological cause of AD.

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Conflict of interest statement

Conflict of interest: the authors declare no conflict of interest.

Figures

Figure 1.
Figure 1.
PC1 was downregulated in the aorta of AD. A) HE staining and Masson’s trichrome staining of aortas from AD patients and normal individuals (ctrl); red arrows indicate aortic fractures. B) PC1 immunohistochemistry of aortas from AD patients and normal individuals (ctrl). Scale bars: 100 μm.
Figure 2.
Figure 2.
PC1 mRNA and protein levels in AD patients and normal persons. A) Expression of PC1 mRNA in the control and AD; AD patient has a significantly lower expression of PC1 than the control. B) Western blot of four cases of AD and control. C) Normalization of the PC1 protein of the AD and control groups; AD patient has a significantly lower expression of PC1 than the control. Each experimental condition was performed in triplicate and applied in three independent experiments. Values are expressed as mean ± SD; *P<0.01.
Figure 3.
Figure 3.
PC1 knockdown promoted VSMC proliferation. A) VSMC was infected by LV-PC1-kd for 72 h, and Q-PCR was used to detect the efficiency. B) Western blot was used to detect the efficiency of LV-PC1-kd. C) Cell proliferation was detected through CCK8 kit and valuated by an absorbance at 450 nm; two groups of VSMCs were detected: one served as control, and the other was infected with lentivirus containing PC1-shRNA that can knockdown PC1. D) Cell cycle of two groups of VSMCs detected through flow cytometry in a representative experiment. E) Cell ratios in the G0/G1, S, and G2/M phases of the two VSMC groups. F) Transwell assay of PC1-kd VSMCs in a representative experiment. The VSMCs migrated through transwell chamber were colored purple by 0.05% crystal violet; scale bars: 50 μm. G) The number of migration cells in the transwell assay of each VSMC group. Each experimental condition was performed in triplicate and applied in three independent experiments. Values are expressed as mean ± SD; *P<0.01.
Figure 4.
Figure 4.
PC1 knockdown promoted the VSMC phenotypic switch. A) mRNA expression levels of SM22α, ACTA2, and CNN1 were detected through RT-PCR; two groups of VSMCs were detected: one served as control, and the other was infected with lentivirus containing PC1-shRNA that can knockdown PC1. B) Protein levels of SM22α, ACTA2, and CNN1 detected through Western blot. C) Relative levels of SM22α, ACTA2, and CNN1 were normalized to tubulin through grayscale scanning. Each experimental condition was performed in triplicate and applied in three independent experiments. Values are expressed as mean ± SD; *P<0.01.
Figure 5.
Figure 5.
Effect of PC1 on MAPK pathways. A) Phosphorylated p38, ERK, JNK, PI3K and MEK were detected through Western blot; two VSMCs groups were detected: one served as the control, and the other was infected with lentivirus containing PC1-shRNA that can knockdown PC1. B) Relative level of phosphorylation was normalized to total specific protein, such as phosphor-ERK/ERK, through gray scale scanning. C) Protein expression levels of myc and cylinD1 were detected through Western blot. D) Relative mRNA levels of myc and cyclinD1 in the two VSMC groups were detected through RT-PCR. Each experimental condition was performed in triplicate and applied in three independent experiments. Values are expressed as mean ± SD; *P<0.01.
Figure 6.
Figure 6.
ERK inhibitor SCH772984 can inhibit the effects of PC1 knockdown on VSMCs. A) Cell proliferation was detected through a CCK8 kit and evaluated by absorbance at 450 nm; three groups were detected: VSMC control, without lentivirus and SCH772984; LV-PC1-kd + DMSO, VSMCs with lentivirus with PC1-shRNA and DMSO as solvent control; LV-PC1-kd + Comp., VSMCs containing lentivirus with PC1-shRNA and ERK inhibitor SCH772984. B) Cell cycles of the three VSMC groups were detected through flow cytometry; the result is from a representative experiment. C) Cell ratios of the G0/G1, S, G2/M phases in the three VSMC groups. D) Protein expression levels of ACTA2, SM22α and cyclinD1 were detected through Western blot. Each experimental condition was performed in triplicate and applied in three independent experiments. Values are expressed as mean ± SD; *P<0.01.
Figure 7.
Figure 7.
Function of PC1 C-tail and the mutant PC1 C-tail formed at S4166A. A) Diagrams of PC1, PC1 C-tail and S4166A mutant site; blue strip represents the extracellular region, the green strip represents the helical structure, and red strip represents the cytoplasmic region. B) Protein levels of phospho-ERK, ERK, ACTA2, SM22α, CNN1, cyclinD1, and myc in four VSMC groups were detected: Ctrl, VSMCs without any transfection; LV-PC1-kd, VSMCs infected with lentivirus with PC1-shRNA that can knockdown PC1; PC1, VSMCs infected by lentivirus with PC1 C-tail coding sequence that can promote PC1 C-tail expression; and PC1(S4166A), VSMCs infected by lentivirus with mutant PC1 C-tail at the S4166A coding sequence that can express the mutant form of the PC1 C-tail. C) Diagram of signaling pathway in VSMC phenotypic switch induced by PC1.
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