FBW7 regulates endothelial functions by targeting KLF2 for ubiquitination and degradation

Abstract

F-box and WD repeat domain-containing 7 (FBW7), the substrate-binding subunit of E3 ubiquitin ligase SCF FBW7 (a complex of SKP1, cullin-1 and FBW7), plays important roles in various physiological and pathological processes. Although FBW7 is required for vascular development, its function in the endothelium remains to be investigated. In this study, we show that FBW7 is an important regulator of endothelial functions, including angiogenesis, leukocyte adhesion and the endothelial barrier integrity. Using RNA interference, we found that the depletion of FBW7 markedly impairs angiogenesis in vitro and in vivo. We identified the zinc finger transcription factor Krüppel-like factor 2 (KLF2) as a physiological target of FBW7 in endothelial cells. Knockdown of FBW7 expression resulted in the accumulation of endogenous KLF2 protein in endothelial cells. FBW7-mediated KLF2 destruction was shown to depend on the phosphorylation of KLF2 via glycogen synthase kinase-3 (GSK3) at two conserved phosphodegrons. Mutating these phosphodegron motifs abolished the FBW7-mediated degradation and ubiquitination of KLF2. The siRNAmediated knockdown of FBW7 showed that KLF2 is an essential target of FBW7 in the regulation of endothelial functions. Moreover, FBW7-mediated KLF2 degradation was shown to be critical for angiogenesis in teratomas and in zebrafish development. Taken together, our study suggests a role for FBW7 in the processes of endothelial cell migration, angiogenesis, inflammation and barrier integrity, and provides novel insights into the regulation of KLF2 stability in vivo.

KLF2 is a rate-limiting transcription factor that can be targeted to enhance regulatory T-cell production

“…All of these results are consistent with KLF2 stability being a key rate-determining step during Treg induction, and for that reason we repeated transfection/induction experiments using a mutant form of KLF2 that is no longer recognized by the E3 ubiquitin ligase complex, SCF FBW7 (35). SCF FBW7 is a multisubunit RING-finger ligase that targets proteins for 26S proteasomal destruction, and in this context KLF2 has recently been identified as a substrate in endothelial cells (35). Despite the fact that several E3 ubiquitin ligases have a documented role in promoting KLF2 degradation (34-36), we found that CD4 + CD25 + FoxP3 + frequencies were enhanced when CD4 + CD25 − T cells were transfected with the mutant form of KLF2 before ex vivo Treg induction (Fig.…”

“…5G). All of these results are consistent with KLF2 stability being a key rate-determining step during Treg induction, and for that reason we repeated transfection/induction experiments using a mutant form of KLF2 that is no longer recognized by the E3 ubiquitin ligase complex, SCF FBW7 (35). SCF FBW7 is a multisubunit RING-finger ligase that targets proteins for 26S proteasomal destruction, and in this context KLF2 has recently been identified as a substrate in endothelial cells (35).…”

Krüppel-like factors in mammalian stem cells and development

“…Scale bar: 0.2 amino acid changes per site. (Frontelo et al, 2007;Nuez et al, 1995;Ouyang et al, 1998;Perkins et al, 1995;Siatecka et al, 2007;Zhang et al, 2001b) (Anderson et al, 1995;Basu et al, 2005;Carlson et al, 2006;Kuo et al, 1997a;Kuo et al, 1997b;Lee et al, 2006;Nakagawa et al, 2008;Wang et al, 2013;Wani et al, 1999a,b;Wu et al, 2005;Xie et al, 2011;Zhang et al, 2004 (Chaib et al, 2001;Chen et al, 2002;Chen et al, 2005a;Conkright et al, 1999;Du et al, 2007;Ge et al, 2015;He et al, 2009;Liu et al, 2010;McConnell et al, 2009;McConnell et al, 2007;Nakagawa et al, 2008;Nandan et al, 2010;Nandan et al, 2008;Oishi et al, 2008;Oishi et al, 2005;Qin et al, 2015;Shindo et al, 2002;Shinoda et al, 2008;Takeda et al, 2010;Tong et al, 2006;Wan et al, 2008;...…”

Peripheral tolerance can be modified by altering KLF2-regulated Treg migration

“…4E), we speculated that enhancing KLF2 expression within the Treg compartment could be used to promote peripheral tolerance. To test this premise, we established an acute model of graft-versus-host disease (aGVHD) using irradiated major histocompatibility complex (MHC) haplotype H2 d animals that received allogeneic T cells (MHC haplotype H2 b ) in concert with CD62L low Tregs; alternatively, CD62L low Tregs were transduced with a nondegradative form of KLF2 (16,28) before cotransfer. Consistent with previous reports (29,30), CD62L low Tregs were unable to prevent aGVHD (Fig.…”