Impaired AMPK activity contributes to the inflammatory phenotype and the reduced phagocytosis capacity of VASP-deficient macrophages

Abstract

Macrophage polarization plays an important role in tissue regeneration. Numerous factors and signaling molecules affect polarization processes. Here we investigated the consequences of the genetic deletion of vasodilator-stimulated phosphoprotein (VASP), which increases macrophage M1 polarization through augmented signal transducer and activator of transcription 1 (STAT1) signaling, and AMP-activated protein kinase (AMPK), which attenuates inflammation by inhibiting STAT1 expression and signaling. While a basal activity of AMPK (phosphorylation on Thr172) was detected in macrophages from wild-type mice, AMPK phosphorylation was significantly reduced in VASP-deficient M1 macrophages in vitro and the expression of the pro-inflammatory cytokines TNFα and IL-1β was increased in these cells. Consistent with the role of AMPK in macrophage phagocytosis, VASP-/- macrophage phagocytosis was also significantly impaired. Interestingly, impaired phagocytosis could be rescued by exogenous activation of AMPK. Mechanistically, we found that VASP binds directly to protein phosphatase 1 regulatory subunit 6 (PP1-R6) and we hypothesize that VASP-binding to PP1-R6/PP1 limits the PP1-dependent de-phosphorylation of AMPK in wild-type cells. Conversely, AMPK dephosphorylation by the PP1-R6/PP1 complex is enhanced in the absence of VASP. In summary, we have identified a link between VASP and AMP-activated protein kinase (AMPK) activity, which may contribute to the pro-inflammatory phenotype of VASP-deficient macrophages.

Cardiovascular Functions of Ena/VASP Proteins: Past, Present and Beyond

“…In line with the role of Ena/VASP proteins in Listeria movement, travelled distance and speed of rocketing phagosomes in VASP-deficient Dictyostelium cells were markedly impaired [ 179 ]. Similar to the results in VASP −/− macrophages [ 63 ], loss of VASP in Dictyostelium resulted in substantial defects in phagocytosis and macropinocytosis. Given that VEGF-induced internalization and signaling of VEGFR2 is largely mediated by macropinocytosis [ 235 ], these findings also provide a rational for the impaired VEGFR2 internalization in EVL-deficient endothelial cells [ 120 ].…”

“…This includes the focal adhesion proteins zyxin, vinculin and LPP [ 47 , 50 , 51 , 52 , 53 ], the lamellipodia/leading edge proteins lamellipodin [ 54 ], Rap1-GTP-interacting adaptor molecule (RIAM) [ 55 ], protocadherin FAT1 [ 56 ], and WASP [ 57 ], as well as the stress fiber associated protein palladin [ 58 ]. Further ActA-like motifs are found in the two transmembrane guidance cue molecules, namely roundabout [ 59 ] and semaphoring 6A-1 [ 60 ], the filamin-c binding protein Xin [ 61 ], the fyn-binding and SLP-76 associated protein (fyb/SLAP) [ 62 ], which is important during T-cell activation, as well as the regulatory subunit 6 of protein phosphatase 1 (PP1-R6) [ 63 ]. The Ena/VASP EVH1 domain has also been shown to directly interact with Abi1, a component of the WAVE regulatory complex [ 64 ].…”

“…Given that AMPK is an important regulator of macrophage polarization and AMPK activation inhibits STAT1 expression [ 171 , 172 , 173 , 174 ], the role of AMPK in determining the phenotype of VASP-deficient macrophages was investigated. While a basal activity of AMPK (phosphorylation on Thr172) was detected in macrophages from wild-type mice, AMPK phosphorylation was significantly reduced in VASP-deficient M1 macrophages in vitro and the expression of the pro-inflammatory cytokines TNFα and IL-1β was increased in these cells [ 63 ]. A link between VASP and AMPK phosphorylation in macrophages has not been reported to date.…”

“…VASP −/− macrophages displayed significantly reduced phagocytic capacity compared to wild-type controls. Interestingly, activation of AMPK with berberine increased phagocytosis in VASP-deficient cells to a level that was statistically indistinguishable from wild-type macrophages [ 63 ]. Thus, impaired AMPK activity in VASP −/− macrophages may, at least in part, explain the decreased phagocytosis capacity.…”

“…Phosphorylation of AMPK on Thr-172 is dynamically regulated by several kinases and phosphatases [ 180 ]. Mechanistically, VASP bound directly to protein phosphatase 1 (PP1) regulatory subunit 6 (PP1-R6) [ 63 ], which recruits PP1 to dephosphorylate and thereby inactivate AMPK [ 181 ]. Notably, PP1-R6 contains a conserved peptide motif ( Figure 7 B) that closely resembles the high affinity EVH1 binding motif of ActA [ 47 ].…”