Protein kinase Cδ and c‐Abl kinase are required for transforming growth factor β induction of endothelial–mesenchymal transition in vitro

Abstract

Objective. The origin of the mesenchymal cells responsible for the intimal fibrosis in systemic sclerosis (SSc) has not been fully identified. The present study was undertaken to investigate whether subendothelial mesenchymal cells may emerge through transdifferentiation of endothelial cells (ECs) into myofibroblasts via endothelial-mesenchymal transition (EndoMT) in vitro and to explore the signaling pathways involved in this process.Methods. Primary mouse pulmonary ECs isolated by immunomagnetic methods with sequential anti-CD34 and anti-CD102 antibody selection were cultured in monolayers. Cell morphology and diacetylated lowdensity lipoprotein uptake assays confirmed their EC characteristics. The induction of EndoMT was assessed by determination of ␣-smooth muscle actin (␣-SMA), type I collagen, and VE-cadherin expression, and the expression of the transcriptional repressor Snail-1 was analyzed. The signaling pathways involved were examined using small-molecule kinase inhibitors and RNA interference.Results. Transforming growth factor ␤1 (TGF␤1) induced ␣-SMA and type I collagen expression and inhibited VE-cadherin. These effects were mediated by a marked increase in Snail-1 expression and were abolished by treatment with either the c-Abl tyrosine kinase inhibitor imatinib mesylate or the protein kinase C␦ (PKC␦) inhibitor rottlerin. The inhibitory effects of imatinib mesylate and rottlerin were mediated by inhibition of phosphorylation of glycogen synthase kinase 3␤ at residue Ser 9 . These observations were confirmed in experiments using small interfering RNA specific for c-Abl and PKC␦.Conclusion. These results indicate that c-Abl and PKC␦ are crucial for TGF␤-induced EndoMT and that imatinib mesylate and rottlerin or similar kinase inhibitor molecules may be effective therapeutic agents for SSc and other fibroproliferative vasculopathies in which EndoMT plays a pathogenetic role.

Transforming Growth Factor-β1 Inhibits Trophoblast Cell Invasion by Inducing Snail-mediated Down-regulation of Vascular Endothelial-cadherin Protein

“…Treatment with vascular endothelial growth factor (VEGF) up-regulates VE-cadherin expression by activating the ERK and p38 MAPK signaling pathways in human umbilical vein endothelial cells and HTR-8/SVneo cells (45). TGF-␤1 down-regulates VE-cadherin in human multilineage progenitor cells and mouse pulmonary endothelial cells (46,47). However, in microvascular endothelial cells from the bovine corpus luteum, TGF-␤1 treatment alters the localization of VE-cadherin in cellular junctions without affecting its total expression levels (48).…”

The origin of the myofibroblast in fibroproliferative vasculopathy: Does the endothelial cell steer the pathophysiology of systemic sclerosis?

“…Finally, they provide evidence that TGF␤ induces the process of EndoMT via activation of the c-Abl and PKC␦ kinases, since highly specific inhibition of their kinase activity with imatinib mesylate and rottlerin, respectively, or by knockdown of their transcripts with siRNA abrogated TGF␤-induced ␣-SMA and Snail-1 expression at both the messenger RNA and protein levels (23). In particular, imatinib mesylate and rottlerin appear to abrogate TGF␤-induced EndoMT through the inhibition of c-Abl-and PKC␦-mediated glycogen synthase kinase 3␤ (GSK3␤) phosphorylation at residue Ser 9 , respectively, with subsequent phosphorylation and proteasomal degradation of Snail-1.…”

“…In fact, transfection of Snail-1 siRNA caused a significant inhibition of TGF␤-induced ␣-SMA expression in pulmonary ECs (23).…”

“…In a report in this issue of Arthritis & Rheumatism, Li and Jimenez provide evidence that the tyrosine kinase c-Abl and protein kinase C␦ (PKC␦) are crucial for TGF␤ induction of EndoMT in vitro (23). Indeed, they show that imatinib mesylate and rottlerin or similar small-molecule kinase inhibitors may be effective therapeutic agents for the fibroproliferative vasculopathy of SSc and other fibrotic disorders in which EndoMT plays a pathogenetic role.…”

“…In particular, imatinib mesylate and rottlerin appear to abrogate TGF␤-induced EndoMT through the inhibition of c-Abl-and PKC␦-mediated glycogen synthase kinase 3␤ (GSK3␤) phosphorylation at residue Ser 9 , respectively, with subsequent phosphorylation and proteasomal degradation of Snail-1. In fact, GSK3␤ phosphorylation at residue Ser 9 is known to block GSK3␤ activity, resulting in stabilization, transport, and accumulation of Snail-1 in the nucleus, where it may exert its transcriptional regulatory activity with consequent induction of mesenchymal cell markers (e.g., ␣-SMA and type I collagen) and repression of EC-specific markers (e.g., VE-cadherin) (19,20,23). The net result is the transition to a myofibroblastic phenotype, as summarized in Figure 7 of the report by Li and Jimenez (23).…”