C-Reactive Protein Inhibits Insulin Activation of Endothelial Nitric Oxide Synthase via the Immunoreceptor Tyrosine-Based Inhibition Motif of FcγRIIB and SHIP-1

Abstract

Abstract-Insulin promotes the cardiovascular protective functions of the endothelium including NO production by endothelial NO synthase (eNOS), which it stimulates via Akt kinase which phosphorylates eNOS Ser1179. C-reactive protein (CRP) is an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. We previously showed that CRP inhibits eNOS activation by insulin by blunting Ser1179 phosphorylation. We now elucidate the underlying molecular mechanisms. We first show in mice that CRP inhibits insulin-induced eNOS phosphorylation, indicating that these processes are operative in vivo. In endothelial cells we find that CRP attenuates insulin-induced Akt phosphorylation, and CRP antagonism of eNOS is negated by expression of constitutively active Akt; the inhibitory effect of CRP on Akt is also observed in vivo. A requirement for the IgG receptor Fc␥RIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant Fc␥RIIB in NIH3T3 IR cells revealed that these processes require the ITIM (immunoreceptor tyrosine-based inhibition motif) of the receptor. Furthermore, we find that endothelium express SHIP-1 (Src homology 2 domaincontaining inositol 5Ј-phosphatase 1), that CRP induces SHIP-1 stimulatory phosphorylation in endothelium in culture and in vivo, and that SHIP-1 knockdown by small interfering RNA prevents CRP antagonism of insulin-induced eNOS activation. Thus, CRP inhibits eNOS stimulation by insulin via Fc␥RIIB and its ITIM, SHIP-1 activation, and resulting blunted activation of Akt. These findings provide mechanistic linkage among CRP, impaired insulin signaling in endothelium, and greater cardiovascular disease risk in type 2 diabetes. Key Words: C-reactive protein Ⅲ insulin Ⅲ eNOS Ⅲ Fc␥RIIB Ⅲ SHIP1 I n addition to its critical role in metabolism, insulin promotes the cardiovascular protective functions of the endothelium. These include the activation of NO production by endothelial NO synthase (eNOS). 1 The resulting NO regulates vasodilation, angiogenesis, thrombosis, hemostasis, and vascular smooth muscle cell growth and migration, 2 and it also attenuates monocyte adhesion, which is among the initiating steps in the development of atherosclerosis. 3,4 Linking cardiovascular and metabolic homeostasis, insulin activation of eNOS also promotes blood flow that augments glucose disposal in insulin target tissues, particularly skeletal muscle. Insulin stimulates eNOS enzymatic activity by insulin receptor (IR)-dependent activation of phosphatidylinositol 3-kinase (PI3K) and Akt kinase, leading to eNOS Ser1179 phosphorylation. Upstream insulin signaling entails the phosphorylation of the IR ␤ subunit (IR␤) and IR substrate-1 (IRS-1). 1 C-reactive protein (CRP) is a member of the pentraxin family and an acute-phase reactant that is positively correlated with cardiovascular disease risk in patients with type 2 diabetes. 5-7 Recently, we showed that CRP inhibits eNOS activation by insulin by blunting eNO...

Hyposialylated IgG activates endothelial IgG receptor FcγRIIB to promote obesity-induced insulin resistance

“…Whereas Con-IgG had no effect, HFD-IgG inhibited insulin-induced eNOS activation ( Figure 5A). To then determine whether FcγRIIB is required, BAECs were transfected with control RNAi or RNAi targeting FcγRIIB to downregulate receptor expression (Figure 5B), and IgG actions were compared with those of CRP, a positive control FcγRIIB ligand known to inhibit eNOS (28). In cells with a normal complement of endogenous FcγRIIB, CRP inhibited insulin-stimulated eNOS activation as expected, and HFD-IgG had a similar effect ( Figure 5C).…”

“…Next, to identify the actions of IgG that adversely affect glucose homeostatic mechanisms via FcγRIIB in endothelium, we took advantage of the knowledge that in cultured endothelial cells, FcγRIIB stimulation antagonizes insulin activation of endothelial NOS (eNOS) (28). We compared eNOS activation by insulin in cultured bovine aortic endothelial cells (BAECs) exposed to IgG isolated from control mice (Con-IgG) versus IgG isolated from mice who had received a HFD (HFD-IgG).…”

“…Primary BAECs were obtained as previously described (28), primary HAECs were purchased from Lonza, and cells were used within 5 to 7 passages. To evaluate the requirement for FcγRIIB in actions of IgG on BAECs, cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific).…”

Chronic CRP-Exposure Inhibits VEGF-Induced Endothelial Cell Migration

“…CRP has also been reported to induce the expression of proinflammatory proteins in endothelial cells 21,40,44) and to downregulate endothelial NO synthase (eNOS) protein levels 22) . The potential of CRP to directly interact with endothelial cell signalling is further supported by a recent study from Tanigaki and collegues, who described the inhibition of insulin-induced eNOS activation by CRP via the immunoreceptor tyrosinebased inhibitin motif of FcgammaRIIB in endothelial cells 45) . These results point towards the specific interaction of CRP with endothelial signal transduction through activation of the immunoreceptor FcgammaRIIB.…”

Association of C‐reactive protein with high disease activity in systemic sclerosis: Results from the Canadian Scleroderma Research Group

“…CRP antagonizes endothelial cell nitric oxide synthase mediated by the coupling of Fc␥ receptor I (Fc␥RI) to Fc␥RIIB (52)(53)(54). Patients with elevated CRP levels had more cardiopulmonary manifestations and these are among the most common SSc-related deaths (55).…”