G Protein–Coupled Receptor Kinase-5 Attenuates Atherosclerosis by Regulating Receptor Tyrosine Kinases and 7-Transmembrane Receptors

Abstract

Objective G protein-coupled receptor kinase-5 (GRK5) is a widely expressed Ser/Thr kinase that regulates several atherogenic receptors, and may activate or inhibit NFκB. This study sought to determine whether and by what mechanisms GRK5 affects atherosclerosis. Methods and Results Grk5−/−/Apoe−/− mice developed 50% greater aortic atherosclerosis than Apoe−/− mice, and demonstrated greater proliferation of macrophages and smooth muscle cells (SMCs) in atherosclerotic lesions. In Apoe−/− mice, carotid interposition grafts from Grk5−/− mice demonstrated greater up-regulation of cell adhesion molecules than grafts from WT mice, and subsequently more atherosclerosis. By comparing Grk5−/− with WT cells, we found that GRK5 desensitized two key atherogenic receptor tyrosine kinases: the PDGF receptor-β (PDGFRβ) in SMCs, by augmenting ubiquitination/degradation; the colony stimulating factor-1 receptor (CSF-1R) in macrophages, by reducing CSF-1-induced tyrosyl phosphorylation. GRK5 activity in monocytes also reduced migration promoted by the 7-transmembrane receptor for MCP-1 (CCR2). Whereas GRK5 diminished NFκB-dependent gene expression in SMCs and endothelial cells, it had no effect on NFκB activity in macrophages. Conclusions GRK5 attenuates atherosclerosis through multiple cell type-specific mechanisms, including reduction of SMC and endothelial cell NFκB activity, and desensitization of receptor-specific signaling through the monocyte CCR2, macrophage CSF-1R, and the SMC PDGFRβ.

Ubiquitin-specific Protease 20 Regulates the Reciprocal Functions of β-Arrestin2 in Toll-like Receptor 4-promoted Nuclear Factor κB (NFκB) Activation

“…Cell Lines-Smooth muscle cells (SMCs) were isolated by enzymatic digestion of aortas stripped of adventitia and endothelial cells, according to published protocols (14,31,32). They were split 1:4 for each passage and not used after passage 7 (freshly isolated cells ϭ passage 1).…”

“…9). Carotid Endothelial Denudation-Carotid endothelial denudation was performed on mice anesthetized with pentobarbital (50 mg/kg) using a 0.36-mm-diameter coronary guide wire (Cordis), as we described previously (14,31). Four weeks after endothelial denudation, injured common carotids were harvested from anesthetized mice after 20 min of perfusion-fixation (80 mm Hg) with 10% formalin in PBS.…”

“…Histology-For carotid artery morphometry, paraffin-embedded specimens were sliced at 5 m and stained with a modified Masson's trichrome and Verhoeff's elastic tissue stain as we described previously (14,31). Computerized planimetry with ImageJ TM was performed as described previously (14, 31) by observers blinded to sample identity.…”

“…Immunofluorescence staining was performed on aortas embedded in OCT compound or paraffin-embedded carotids after samples were sliced at 5 m (14, 31). Tissue sections were probed with rabbit IgGs specified above, followed by anti-rabbit IgG conjugated to Alexa-488, Alexa-548, or Alexa-594, as described previously (14,31), or with Cy3-conjugated 1A4 anti-SMC ␣-actin (SigmaAldrich), as described previously (14,31). Nuclei were counterstained with Hoechst 33342 (10 g/ml) during the secondary antibody incubation.…”

Role of G protein-coupled receptor kinases in cell migration

“…GRK5 attenuates atherosclerosis by desensitizing CCR2 in monocytes and inhibiting their migration [34]. In epithelial cells, a functional screening identified GRK6 as a critical mediator in integrin-mediated cell adhesion and migration of tumor cells [35], and GRK6 deficiency reportedly promotes CXCR2 receptor-mediated tumor progression and metastasis in a lung carcinoma model [36].…”