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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 FcγRIIB was demonstrated in vitro using blocking antibody, and reconstitution experiments with wild-type and mutant FcγRIIB in NIH3T3IR 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 domain–containing inositol 5′-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 FcγRIIB 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.
In 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 β subunit (IRβ) 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 eNOS Ser1179 phosphorylation.8 Candidate cell surface receptors for CRP mediating this process include Fcγ receptors for IgG, which are classified as inhibitory receptors (FcγRIIB) and activating receptors (FcγRI and others). In immune response cells, immune complex binding to activating receptors causes the phosphorylation of the FcγRIIB cytoplasmic ITIM (immunoreceptor tyrosine-based inhibition motif) and the recruitment and activation of the Src homology 2 domain–containing phosphoinositide 5-phosphatases SHIP-1 and -2, which attenuate signaling downstream of PI3K by hydrolyzing 3-phosphoinositides.9–13 We have previously demonstrated that FcγRIIB is required for the antagonism of eNOS activation by CRP.8 However, the mechanism by which FcγRIIB mediates eNOS inhibition by CRP is unknown. In addition, whether SHIP is expressed in endothelial cells, whether SHIP regulates signaling to eNOS, and whether SHIP participates in CRP action in endothelium are unknown.
With a focus on these potential signaling molecules, the purpose of this study was to delineate the molecular mechanisms by which CRP alters insulin activation of eNOS. The experiments were performed using both cultured cells and a mouse model of rapid eNOS phosphorylation in vivo. We tested the hypothesis that CRP attenuates insulin signaling by binding to FcγRIIB and subsequent activation of SHIP-1 via the ITIM domain of the receptor.
Primary bovine aortic endothelial cells (BAECs) were obtained as previously described and used within 5 to 7 passages.14 NIH3T3IR fibroblast cells, which constitutively express insulin receptor (IR) for the optimal study of insulin signaling and are devoid of eNOS and FcγRIIB,15 were also used. Cells were transfected with cDNAs or small interfering (si)RNAs using LipofectAMINE 2000 (Invitrogen) and studied 48 hours later. cDNAs used in the experiments were human eNOS, hemagglutinin (HA)-tagged human FcγRIIB (WT-FcγRIIB), a mutant form of HA-tagged human FcγRIIB with alanine (A) substitutions for amino acids 308 to 312 (TYSLL) of the ITIM (FcγRIIB-ITIM5A), and constitutively active myristoylated human Akt (myrAkt). All constructs were confirmed by sequence analysis. In siRNA experiments, double-stranded RNA with sequence 5′-UGAGAUUCUUCACCAAGUUUU-3′ was designed to target the open reading frame of bovine SHIP-1 (GenBank accession no. 3635), and scrambled sequence served as control.
To assess changes in target protein phosphorylation in response to insulin in the absence or presence of CRP, cells were starved overnight in the absence of serum or phenol red in DMEM before exposure to insulin (500 nmol/L) for 3 minutes after 30 minutes of pretreatment with or without human recombinant CRP (Calbiochem). Based on preliminary dose-response studies (Figure I in the online data supplement, available at http://circres.ahajournals.org), experiments were performed with 25 μg/mL CRP. Whole cell lysates were obtained, and immunoblot analyses were performed. In selected experiments additional cells were treated with heat-inactivated CRP.8,16 Possible effects of the vehicle for CRP or of potential contamination of the recombinant protein with lipopolysaccharide have also been excluded by other strategies.8,16 The following antibodies were used: anti-actin antibody (Santa Cruz Biotechnology), anti–HA-probe antibody (Santa Cruz Biotechnology), anti-eNOS antibody (BD Biosciences Pharmingen), anti–phospho-Ser1179 eNOS antibody (Cell Signaling Technology), anti-Akt antibody (Cell Signaling Technology), anti–phospho-Ser473 Akt antibody (Cell Signaling Technology), anti-IRβ antibody (Santa Cruz Biotechnology), anti–phospho-Tyr1162/1163 IRβ (Santa Cruz Biotechnology), anti–IRS-1 antibody (Upstate Cell Signaling Solutions), anti–phospho-Tyr612 IRS-1 antibody (Upstate Cell Signaling Solutions), anti–SHIP-1 antibody (Santa Cruz Biotechnology), and anti–phospho-Tyr1020 SHIP1 antibody (Cell Signaling Technology). Findings by immunoblot analysis were confirmed in 3 or more independent experiments.
eNOS activation in intact BAECs or NIH3T3IR cells in response to insulin (500 nmol/L) was assessed by measuring 14C-l-arginine conversion to 14C-l-citrulline using previously reported methods.17 In selected experiments, eNOS activity was evaluated in cells treated with CRP in the absence or presence of the FcγRIIB-specific blocking antibodies 2B6 or ch2B6-Agly (10 μg/mL, IgG1).18,19 IgG1 isotype control does not alter eNOS activity (data not shown). We also tested the impact of antibody IV 3mG1-Agly (10 μg/mL, IgG2b), which binds to FcγRIIA (MacroGenics Inc).18,19 Both 2B6 or ch2B6-Agly antibodies contain a mutation of the asparagine at position 297 in the Fc region to glutamine (N279Q) to remove the Fc glycosylation site. This strategy eliminates Fc receptor binding via the Fc region of the antibody.20,21 Stimulated activity is expressed as a percent of basal activity. All findings were confirmed in at least 3 independent experiments.
To determine whether CRP-induced changes in eNOS phosphorylation occur in vivo, we evaluated eNOS phosphorylation in mouse lung in response to insulin administration. The high density of endothelium, the specificity of eNOS expression to endothelium,22 and the ease of harvest make the murine lung an ideal model system for the study of rapid eNOS phosphorylation in vivo. The care and use of all study animals were approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center. Sixty minutes after IP injection with human recombinant CRP (250 μg) or an equal volume of vehicle, C57BL/6 male mice (12 to 16 weeks old) received an intravenous injection of bovine insulin (1 U/kg body weight) or an equal volume of saline, and 5 minutes later, the lungs were harvested and snap-frozen. Whole lung was homogenized in ice-cold lysis buffer (50 mmol/L Tris-HCl pH 7.6, 150 mmol/L NaCl, 1% [wt/vol] Triton X-100, 0.1% [wt/vol] SDS, 0.1% [wt/vol] Na-deoxycholate, 5 mmol/L Na-EDTA) containing protease inhibitors and phosphatase inhibitor cocktails (Sigma-Aldrich). The homogenate was sonicated (30 seconds, 50% pulse) on ice. After centrifugation at 3000g for 10 minutes at 4°C, the supernatant was used for immunoblotting. A limited number of additional studies were performed in aortas to determine whether the findings in the lung effectively mirror events in the systemic vasculature.
Differences in observations between multiple groups were evaluated by 1-way ANOVA after establishing equivalence of variances and normal distribution of data. Post hoc testing was performed by Student–Newman–Keuls. Values shown are means±SEM, with n=4 to 6. Significance was accepted at the 0.05 level of probability.
For comparison with parallel studies of proximal signaling events, we first evaluated the impact of CRP on insulin-induced eNOS phosphorylation at Ser1179 (Ser1177 for human eNOS) in BAECs. Consistent with our previous observations,8 phosphorylation of the enzyme was increased by insulin, and this was attenuated by CRP (Figure 1A). We next determined whether these processes are operative in vivo by evaluating eNOS phosphorylation in mouse lung in response to insulin administration. Sixty min after IP injection with vehicle or CRP that results in serum levels of 38±4 μg/mL, mice received IV insulin and lung eNOS phosphorylation was assessed 5 minutes later. In the representative immunoblots shown (Figure 1B), insulin caused an increase in eNOS phosphorylation, and the phosphorylation was attenuated following CRP treatment. Quantitative analysis demonstrated a 5.2-fold increase in eNOS phosphorylation by insulin, and full inhibition of this process by CRP (Figure 1C). Comparable findings were obtained in aorta (Online Figure II). Thus, CRP attenuates insulin-induced eNOS phosphorylation in vivo.
We next determined whether the CRP-related attenuation of eNOS phosphorylation in response to insulin is attributable to altered activation of the upstream kinase Akt. In BAECs, insulin caused an increase in Akt Ser473 phosphorylation, and this was blunted by CRP (Figure 2A). In vivo studies were then performed assessing Akt activation induced by insulin in the lung. Whereas insulin caused an increase in Akt phosphorylation in the lungs of vehicle-treated mice, there was a lack of Akt activation in the lungs of CRP-treated mice (Figure 2B). Summary data revealed a 2.4-fold increase in Akt phosphorylation by insulin and complete inhibition of this response by CRP (Figure 2C). Thus, the activation of the kinase immediately upstream of eNOS in the insulin signaling cascade is blunted by CRP in vivo.
To evaluate the causal relationship between the attenuated Akt activation and the blunted eNOS activation invoked by CRP, the impact of expression of constitutively active myrAkt on eNOS phosphorylation in response to insulin was determined in BAECs (Figure 2D). In contrast to the decrease in insulin-induced eNOS Ser1179 phosphorylation that occurred with CRP in sham-transfected cells, constitutively active Akt expression rescued the promotion of eNOS phosphorylation by insulin. Thus, the antagonism of Akt phosphorylation caused by CRP underlies the actions of the pentraxin on the capacity of insulin to activate eNOS.
To next determine whether CRP modifies the proximal signaling events that occur on insulin binding to IR in endothelial cells, stimulatory tyrosine phosphorylation of IR and IRS-1 was studied in BAECs. As shown in Online Figure III (A), CRP had no effect on Tyr1162/1163 phosphorylation of IRβ. However, CRP inhibited insulin-induced phosphorylation of IRS-1 at Tyr612, one of the critical tyrosine phosphorylation sites for binding and activation of PI 3K (Online Figure III, B).23
To first directly determine the involvement of FcγRIIB in CRP antagonism of insulin-mediated eNOS activation in endothelial cells, the effect of a blocking antibody specifically targeting FcγRIIB was evaluated in BAECs. It has been previously demonstrated that the antibody inhibits the actions of FcγRIIB in immune response cells.18,19 As shown in Figure 3, CRP inhibition of eNOS activation by insulin was completely reversed by 2 FcγRIIB blocking antibodies (2B6 or ch2B6-Agly), whereas the anti-FcγRIIA antibody IV.3mG1-Agly had no effect.
Complementary gain-of-function experiments were then performed in NIH3T3IR cells that do not express endogenous FcγRIIB. The cells were transfected with cDNA for eNOS and either sham vector or cDNA encoding FcγRIIB. In the sham-transfected cells, eNOS activation by insulin was not altered by CRP treatment (Figure 4A). In contrast, CRP inhibited insulin-induced eNOS activation in cells expressing FcγRIIB. We then determined whether the ITIM domain of FcγRIIB is required for CRP action. Cells expressing eNOS were cotransfected with cDNA for either wild-type FcγRIIB or a mutant form of FcγRIIB in which amino acids 308 to 312 (TYSLL) of the ITIM domain are substituted by alanine (FcγRIIB-ITIM5A). As shown in Figure 4B, CRP inhibited eNOS activation by insulin in cells expressing wild-type receptor, whereas it failed to do so in cells expressing the ITIM mutant. The requirement for the FcγRIIB ITIM in CRP antagonism of Akt phosphorylation by insulin was also evaluated (Figure 4C). In cells expressing wild-type FcγRIIB, Akt phosphorylation stimulated by insulin was blunted by CRP but not by heat-inactivated CRP. In contrast, in cells expressing the ITIM mutant, CRP did not inhibit Akt phosphorylation stimulated by insulin. These cumulative findings indicate that the ITIM domain of FcγRIIB mediates CRP antagonism of eNOS and the upstream changes in Akt activation that underlie the process.
Signaling by FcγRIIB in immune response cells entails ITIM domain-dependent activation of SHIP, which attenuates signaling downstream of PI3K.9–13 Having demonstrated that CRP-induced inhibition of signaling by insulin requires FcγRIIB and its ITIM, and knowing that insulin signaling is mediated by PI3K and Akt,24–26 we determined whether CRP antagonism of eNOS activation by insulin is mediated by SHIP. We first evaluated SHIP expression in endothelial cells and demonstrated SHIP-1 protein detection in BAECs by immunoblot analysis, with confirmation of its identity by knockdown using RNA interference (Figure 5A, inset). SHIP-2 was not detected (data not shown). We then determined the impact of SHIP-1 knockdown in BAECs on CRP antagonism of eNOS activation by insulin (Figure 5A). In control cells receiving scrambled double-stranded RNA, CRP inhibited eNOS activation by insulin. In contrast, SHIP-1 knockdown fully prevented the decrease in eNOS activation by insulin caused by CRP. To determine whether SHIP-1 mediates the alterations in phosphorylation by which CRP antagonizes eNOS, the impact of SHIP-1 knockdown on changes in eNOS Ser1179 phosphorylation was determined (Figure 5B). In control cells eNOS Ser1179 phosphorylation stimulated by insulin was attenuated by CRP, in agreement with the findings shown in Figure 1A. In contrast, SHIP-1 knockdown prevented the CRP-induced decrease in eNOS phosphorylation stimulated by insulin.
The known mechanism of activation of SHIP-1 in hematopoietic cells entails phosphorylation of Tyr1020.27,28 We therefore assessed SHIP-1 Tyr1020 phosphorylation in BAECs as an indicator of SHIP-1 activation by CRP in endothelial cells (Figure 5C). CRP caused an increase in SHIP-1 phosphorylation that was evident at 15 minutes and sustained through 60 minutes. In vivo studies were further performed assessing SHIP-1 phosphorylation induced by CRP in the lung. Compared with lungs from vehicle-treated mice, CRP caused an increase in the phosphorylation of the phosphatase (Figure 5D). Summary data showed a 3.1-fold increase in SHIP-1 phosphorylation by CRP (Figure 5E). These cumulative results indicate that SHIP-1 is expressed in endothelial cells and that it is activated by CRP to cause the attenuation of eNOS stimulation by insulin.
Insulin plays an important role in cardiovascular health via its direct actions on endothelium to stimulate eNOS and activate the production of the atheroprotective signaling molecule NO.1 Insulin resistance is an independent risk factor for cardiovascular disease, and in patients with type 2 diabetes, elevated CRP levels are associated with endothelial dysfunction.29,30 We previously demonstrated direct mechanistic linkage between CRP and impaired insulin action in cultured endothelial cells, showing that the pentraxin attenuates insulin-induced eNOS Ser1179 phosphorylation and enzyme activation, leading to a diminished capacity of insulin to prevent monocyte adhesion to endothelial cells.8 In the present work, we have elucidated the molecular basis by which this occurs, demonstrating that it is mediated by FcγRIIB and its ITIM, by activation of the phosphatase SHIP-1, and by the resulting blunted activation of Akt which resides immediately upstream of eNOS (Figure 6).
The mechanisms by which insulin signaling leads to eNOS activation via Ser1179 phosphorylation have been interrogated to great depth in cultured endothelial cells.1,26 To evaluate insulin signaling to eNOS in vivo, we devised a model to investigate rapid eNOS phosphorylation in mouse lung. This strategy takes advantage of the high density of endothelium, the specificity of eNOS expression to the endothelium,22 and the ease of rapidly harvesting murine lung. Using this model, we demonstrated that insulin causes rapid eNOS Ser1179 phosphorylation in vivo and then showed that this is blunted by CRP administration. Comparable findings were made in aorta. Thus, CRP antagonism of insulin activation of eNOS, which we previously demonstrated in cultured endothelial cells, occurs in vivo.
The impairment of insulin-induced Akt activation by CRP was also demonstrated both in cultured endothelial cells and in vivo in the lung. Just as importantly, a causal linkage between attenuated Akt activation and blunted eNOS activation was demonstrated by rescue of normal insulin-induced eNOS phosphorylation in CRP-treated endothelial cells expressing constitutively active myrAkt. The findings for CRP and Akt phosphorylation in the lung reveal that the function of the kinase immediately upstream of eNOS, which also regulates multiple other key processes in endothelium, is adversely modified in vivo by a factor that is strongly associated with endothelial dysfunction and cardiovascular disease risk in humans.5–7,29,30
The central role of FcγRIIB in CRP-induced impairment of insulin signaling to eNOS was demonstrated by multiple strategies. Blocking monoclonal antibody to FcγRIIB caused complete prevention of CRP antagonism of insulin signaling in cultured endothelial cells. In NIH3T3IR cells a gain-of-function strategy provided another paradigm in which the role of FcγRIIB was demonstrated. We further found that the ITIM of FγRIIB is required for CRP antagonism of eNOS and the upstream impairment in Akt activation that underlies it. These observations indicate that the receptor not only mediates CRP binding to the endothelium as has been observed in human aortic endothelial cells31 but that it also induces the signaling events that modify kinase activity and downstream eNOS activity.
Having implicated FcγRIIB and it ITIM, we investigated the potential participation of SHIP, which is recruited to the ITIM of the receptor in antibody complex-activated immune response cells.9–13 SHIP-1 protein expression was demonstrated in cultured endothelial cells, its knockdown prevented CRP antagonism of insulin-induced eNOS phosphorylation and enzyme activation, and SHIP-1–activating phosphorylation occurred in response to CRP. It has been previously shown that SHIP-1 mRNA is upregulated in human umbilical vein endothelial cells in response to vascular endothelial growth factor A, and SHIP-1 protein abundance increases in the endothelium of the ovary during angiogenesis.32 However, the present work is the first to demonstrate a function for SHIP-1 in endothelium, and it identifies the phosphatase as an important modulator of endothelial cell Akt, which is critically involved in the regulation of vascular homeostasis and angiogenesis in numerous paradigms.33 Although the other isoform of SHIP, SHIP-2, is widely expressed in various tissues,9–11 SHIP-2 was not detected in BAECs. Because SHIP-1 knockdown completely prevented CRP action, we conclude that the contribution of SHIP-2, if any, would be minimal.
Our findings regarding FcγRIIB and SHIP-1 contrast with those of Xu et al, who reported that blocking antibody to the activating Fc receptor FcγRI but not blocking antibody to FcγRIIB partially reversed the effect of CRP on insulin signaling in endothelium.34 However, because we previously demonstrated a role for FcγRIIB in CRP antagonism of eNOS in vivo,8 because our blocking Ab to FcγRIIB is specific to that isoform and optimally selected as a blocking Ab,20,21 and because SHIP-1 activation is logically coupled to FcγRIIB, we conclude that FcγRIIB and SHIP-1 are critically involved in these processes.
In the interrogation of upstream signaling events, we observed that CRP has no effect on insulin-induced IR tyrosine phosphorylation but that it inhibits IRS-1 Tyr612 phosphorylation. We previously demonstrated that IRS-1 Tyr612 is one of the critical tyrosine phosphorylation sites for binding and activation of PI3K.23 Xu et al previously reported that CRP antagonism of insulin signaling depends on the phosphorylation of JNK and the phosphorylation of IRS-1 at Ser307 by the Rho-A kinase/SYK pathway.34 Because an increase in the phosphorylation of the inhibitory Ser307 of IRS-1 decreases the phosphorylation of Tyr612,35,36 our results for Tyr612 phosphorylation are generally consistent with their observations. More importantly, although it occurs, we believe that modified IRS-1 phosphorylation is not the primary mechanism by which insulin signaling to eNOS is altered by CRP, because there was complete rescue of insulin signaling to eNOS with SHIP-1 knockdown.
It is controversial whether the actions of CRP on the cardiovascular system are mediated by native, pentameric CRP, or monomeric (m)CRP. mCRP has been detected in normal human blood vessels and in inflamed tissues,37–39 and it has been reported that mCRP promotes a proinflammatory phenotype in cultured endothelial cells. However, in earlier studies mCRP actions on endothelial cells were modified by anti-CD16 (FcγRIII) blocking antibody and not by anti-FcγRII antibody,40 and there is a recent report that mCRP actually mediates responses in human endothelial cells via plasma membrane insertion rather than binding to surface FcγRs.41 Most importantly, it has been demonstrated that native CRP impairs endothelial function in vivo, whereas mCRP does not.42 When these observations are considered along with our previous demonstration that CRP antagonism of eNOS in vivo requires FcγRIIB,8 we conclude that although mCRP may have biological impact, it is most likely not the primary form of the protein mediating actions of relevance to vascular health in vivo.
The endothelial actions of insulin that are an important component of the interplay between cardiovascular and metabolic homeostasis have been appreciated for more than a decade.43 More recently, we and others have demonstrated that CRP, a well-recognized risk factor for both endothelial dysfunction and insulin resistance,29,30 directly attenuates the endothelial actions of insulin. Having now identified the requirements for and the basis for FcγRIIB and SHIP-1 participation in this process, the mechanistic linkages between CRP and insulin and eNOS have been further elucidated. We postulate that endothelial FcγRIIB and SHIP-1 are potential new drug targets worthy of pursuit in our efforts to optimize the cardiovascular and metabolic actions of insulin in the setting of numerous chronic inflammatory conditions.
We are indebted to Christopher Longoria and Amanda Cross for technical assistance.
Sources of Funding
This work was supported by NIH grants HL75473 (to P.W.S.). Additional support was provided by the Crystal Charity Ball Center for Pediatric Critical Care Research and the Lowe Foundation (to P.W.S.).
E.B. is employed by MacroGenics Inc, whose (potential) product was studied in the present work. Additionally, E.B. has filed patent applications related to the work that is described in the present study.