Participation of PLK1 and FOXM1 in the hyperplastic proliferation of pulmonary artery smooth muscle cells in pulmonary arterial hypertension.

Jamie L Wilson,Lizhen Wang,Zeyu Zhang,Peter Polgar,Nicholas S Hill,Vinicio A De Jesus Perez

doi:10.1371/journal.pone.0221728

Abstract

Vascular smooth muscle cells from the pulmonary arteries (HPASMC) of subjects with pulmonary arterial hypertension (PAH) exhibit hyperplastic growth. The PAH HPASMC display an increased sensitivity to fetal bovine serum (FBS) and undergo growth at a very low, 0.2%, FBS concentration. On the other hand, normal HPASMC (obtained from non-PAH donors) do not proliferate at low FBS (0.2%). A previous genomic study suggested that the nuclear factor, FOXM1 and the polo like kinase 1 (PLK1) are involved in promoting this hyperplastic growth of the PAH HPASMC. Here we find that limiting the action of FOXM1 or PLK1 not only restricts the hyperplastic proliferation of the PAH HPASMC but also modulates the FBS stimulated growth of normal HPASMC. The PAH HPASMC exhibit significantly elevated PLK1 and FOXM1 expression and decreased p27 (quiescence protein) levels compared to normal HPASMC. Regulation of the expression of FOXM1 and PLK1 is accompanied by the regulation of downstream expression of cell cycle components, Aurora B, cyclin B1 and cyclin D1. Expression of these cell cycle components is reversed by the knockdown of FOXM1 or PLK1 expression/activity. Furthermore, the knockdown of PLK1 expression lowers the protein level of FOXM1. On the other hand, inhibiting the action of FOXO1, a growth inhibitor, further increases the expression of FOXM1 in PAH HPASMC. Although PLK1 and FOXM1 clearly participate in PAH HPASMC hyperplasia, at this time it is not clear whether their increased activity is the primary driver of the hyperplastic behavior of the PAH HPASMC or merely a component of the pathway(s) leading to this response.

Highlights

In a previous communications we showed that smooth muscle cells from the pulmonary artery of pulmonary arterial hypertension (PAH) subjects display abnormal behavior which manifests as hyperplastic growth [1] and dysregulated migration [2]
PAH human pulmonary artery smooth muscle cells (HPASMC) proliferate under normally quiescent cell culture condition (0.2% fetal bovine serum (FBS) concentration) [1]
To confirm the growth results obtained with use of the MTT assay, we measured cell proliferation under the same conditions using BrdU incorporation and obtained results which mirrored those obtained with the MTT assay (Fig 1E). Since these experiments measured proliferation during asynchronous cell growth, we looked at the effect of FOXM1 and polo-like kinase 1 (PLK1) inhibitors on BrdU incorporation when HPASMC were synchronized by withholding FBS in the culture medium followed by release into the cell cycle with serum addition

Summary

Introduction

In a previous communications we showed that smooth muscle cells from the pulmonary artery of PAH subjects display abnormal behavior which manifests as hyperplastic growth [1] and dysregulated migration [2]. The hyperplastic growth phenomenon has been demonstrated by others [3,4,5]. Regardless, these human pulmonary artery smooth muscle cells (HPASMC) isolated and cultured from subjects both with or without PAH retain their in vivo phenotype as illustrated by their expression of both alpha smooth muscle actin and H-caldesmon [1] and constriction in response to endothelin 1 [6]. The oncogene FOXM1 and cell cycle regulator, polo-like kinase 1 (PLK1), were of particular note as possibly contributing to this hyperplastic growth as they were upregulated in PAH HPASMC [7]

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Results

Conclusion