Control of TGF-beta signaling and endothelial-to-mesenchymal transition by protein tyrosine phosphatases

Background and Aim: Cellular differentiation and dedifferentiation events, including endothelial-to-mesenchymal transition (EndMT), give rise to fibroblast-like cells in the vasculature and are driven by transforming growth factor-beta (TGFbeta). Phosphorylation of the transcriptional repressor SNAI1 was shown to reduce EndMT by increasing its nuclear export. Protein tyrosine phosphatase-1B (PTP1B) negatively regulates growth factor signaling by receptor tyrosine kinase dephosphorylation, and previous work has implicated vascular PTP1B deficiency in the pathophysiology of vascular fibrosis. The aim of this study is to determine the role of PTP1B in the control of SNAI1 transcriptional activity by phosphorylation and how this affects EndMT and vascular fibrosis.

Methods: Human aortic endothelial cells (HAoECs) from a young (28 years-of-age) and an old (73 years-of-age) male donor were purchased from PromoCell. PTP1B downregulation in HAoECs was achieved by PTP1B small interfering RNA (siRNA) for 48 hours, followed by stimulation with recombinant human TGFbeta1 for short (30 min, 120 min and 24 hours) and long (2, 3 and 4 days) time periods. EndMT was assessed using qPCR, western blot and immunofluorescence microscopy analysis. Primary endothelial cells were isolated from mice with tamoxifen-inducible, Tie.ERT2-Cre mediated deletion of PTP1B (End.PTP1B-KO). 

Results: Western blot analysis of HAoECs after 48 hours of siRNA transfection showed successful downregulation of PTP1B protein levels (>80% in both young and old donors), and absence of PTP1B was confirmed using immunofluorescence microscopy. PTP1B siRNA-treated “young” HAoECs exhibited lower protein levels of the endothelial tight junction protein VE-cadherin, similar to TGFbeta1-stimulated control siRNA-treated cells. A similar, but more prolonged response was observed in “old” HAoECs. Moreover, and importantly, PTP1B downregulation enhanced the cellular response to TGFbeta1 stimulation, as suggested by findings of elevated SMAD3 phosphorylation and SNAI1 expression. Protein levels of total and phosphorylated (at the inhibitory serine-9 site) of glycogen synthase kinase-3 beta (GSK3beta), a serine-threonine kinase involved in cellular energy metabolism regulation and potential upstream regulator of SNAI1, were markedly increased in endothelial cells lacking PTP1B. Immunofluorescence time course analyses revealed that, in the absence of PTP1B and in the presence of TGFbeta1 stimulation, phosphorylated SNAI1 (at serine 11) and GSK3beta were primarily observed in the cell nucleus compared to TGFbeta1 stimulated HAoECs transfected with control siRNA. Cellular fractionation analysis showed higher total and phosphorylated GSK3beta levels in the nuclear fraction of HAoECs upon TGFbeta1 stimulation. A significant increase in the mRNA expression of mesenchymal markers, such as SMA and COL1A, and transcription factors involved in the regulation of endothelial-to-mesenchymal transition, such as SNAI1, TWIST, and ZEB1 was observed upon siRNA-mediated PTP1B downregulation and TGFbeta1 stimulation. Elevated mRNA transcripts levels of SMA and SNAI1 in response to TGFbeta1 were confirmed in primary endothelial cells isolated from End.PTP1B knockout mice.

Conclusion: The results of this ongoing project suggest that absence of PTP1B enhances the response of endothelial cells to TGFbeta1 and EndMT by altering the phosphorylation-dependent nuclear translocation of GSK3beta and SNAI1 regulation.