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Receptor for Advanced Glycation End Products Mediates NADPH Oxidase Signaling in Human Skeletal Muscle
thesisposted on 01.08.2019 by Brian Keith Blackburn
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Type 2 Diabetes Mellitus (T2DM) is a major public health burden, emerging as the 7th leading cause of death in the United States over the past decade (1). The natural history of T2DM, also described as the glucose tolerance continuum (Figure 1), is a multifactorial condition that results in chronic hyperglycemia superimposed with insulin resistance and glucose intolerance (2,3). While no tissue is immune to the damaging effects of chronic hyperglycemia, its impact on skeletal muscle may be most critical for whole body metabolism as skeletal muscle accounts for approximately 40% of total body mass and more than 80% of insulin-mediated glucose uptake (4). Skeletal muscle responds to chronic hyperglycemia with increased production of reactive oxygen species (ROS) and activation of pro-inflammatory signaling pathways (5,6), thus potentiating a pro-oxidant and pro-inflammatory environment. The precise mechanisms that underlie these metabolic abnormalities are still poorly defined (7). Elucidation of the molecular events implicit in hyperglycemia induced ROS production are important for the treatment and prevention of T2DM and the subsequent development of complications such as retinopathy, neuropathy and nephropathy. Of the many pathological events associated with T2DM, the most deleterious may be the increased expression of the receptor of advanced glycation end products (RAGE). RAGE is a multi-ligand receptor that activates nuclear factor-kappa beta (NF-κB) and subsequent production of inflammatory cytokines. The mechanisms by which RAGE induces NF-κB have been strongly associated with ROS production (8,9,10). Further, RAGE induced NF-κB signaling also initiates the self-transcription of RAGE, thus producing a futile cycle that generates greater RAGE protein expression at the cell membrane. These molecular events propagate RAGE signaling, promote ROS production and exacerbate inflammation (11,12). Thus, RAGE expressing cells are susceptible to multiple cellular insult, which can lead to the development of future complications. Although the exact mechanisms by which these events occur are unclear, RAGE induced ROS generation may be dependent on NADPH Oxidase II (NOX II), a membrane bound enzyme complex containing a NOX II-gp91phox subunit that produces superoxide (O2.-) by one electron reduction of molecular oxygen (13,14). Under homeostatic conditions, the NOX II enzyme complex assembles, and disassembles to produce episodic bursts of O2.-. This process is tightly regulated by the phosphorylation of p47phox, the principle NOX II subunit that initiates enzyme complex assembly through the colocalization of additional cytosolic factors, including p67phox, p40phox and Rac 1/2 (Figure 2), (15,16,17). NOX II induced O2.- is a transient event, and disassociation of p47phox from the NADPH complex terminates NOX II action. Under pathological conditions, such as T2DM, p47phox phosphorylation is upregulated, provoking heightened NADPH oxidase assembly and increased O2.- production (18,19). One potential kinase that may be modulating increased p47phox phosphorylation is non-tyrosine kinase (Src). Src is recruited by ligand bound transmembrane receptors that are absent of intrinsic tyrosine kinase activity (20). When phosphorylated at tyrosine 419, Src undergoes a conformational change that exposes the SH1 domain and kinase site (21,22). Once active, Src signals cell stress responses by phosphorylating downstream targets associated with oxidative stress and inflammation (23). Interestingly, RAGE activation has been shown to initiate Src kinase phosphorylation and subsequent NADPH oxidase complex assembly (24,25). RAGE is absent of intrinsic tyrosine kinase activity and once ligand bound, the RAGE cytoplasmic domain associates with the FH1 domain of mammalian diaphanous-1 (mDia-1), which has been shown to transmit RAGE signaling to downstream targets (26). Active mDia-1 has been reported to bind with the SH3 domain of Src kinase, converting Src from inactive to an active state. (27,28). In various tissue types, Src has been shown to phosphorylate and initiate the translocation of both p47phox and p67phox for NADPH oxidase complex assembly. Likewise, increased Src activity has also been shown to be central in the development of insulin resistance and diabetic nephropathy (29,30). Thus, it appears likely that a RAGE/NOX II signaling axis may be linked by mDia-1 dependent Src kinase activation (Figure 2). RAGE signal transduction and concomitant NOX II O2.- production disrupts the balance between ROS generation and scavenging. Thus, maintaining the integrity of antioxidant defense is critical for optimal cellular function and prevention of a pro-oxidative environment. Several decades of dietary and nutraceutical research efforts suggest that antioxidant supplementation may be advantageous in preventing oxidative stress and the development of many chronic diseases (31,32). However, results from randomized control trials have been unconvincing in the setting of preventative therapy (33,34). Thus, an alternative strategy may be to enhance the expression of endogenous defenses, or to reveal new mechanisms of intrinsic antioxidant defense. Intriguingly, a study conducted by Noubade et al. (2014), demonstrated that NOX II enzyme activity is regulated by a novel, leucine-rich protein, dubbed negative regulator of reactive oxidative species (NRROS, also known as LRRC33). NRROS directly competes with the p22phox subunit of NADPH oxidase for NOX II co-localization. NRROS-bound NOX II is then flagged for degradation by intracellular proteases, thereby promoting a favorable balance between ROS production and removal. Paradoxically, NRROS protein is predisposed to degradation through pro-inflammatory signaling responses, suggesting that ROS and subsequent inflammation reduce NRROS defense (35). Further, NRROS limits toll like receptor (TLR) inflammation, and tags TLRs for degradation in a similar manner as NOX II (36). TLRs and RAGE share analogous mechanistic properties, common ligands and signaling pathways and accumulating evidence points towards their co-operative interaction in the host immune response (37). Thus, NRROS may be a fundamental scavenger of transmembrane proteins that augment the oxidative and inflammatory milieu. It appears evident that a RAGE/NOX II axis fundamentally augments ROS production, and the loss of counter-regulatory NRROS may play an important role in the development of diabetic complications. While a RAGE/NOX II axis has been proposed and studied in vascular and kidney tissues, its role in skeletal muscle has not been elucidated. Likewise, tissue specific expression and temporal regulation of NRROS has not previously been characterized. Skeletal muscle is a vast, highly metabolic and plastic organ, making it an innovative platform to investigate diabetes (38). Further, skeletal muscle expresses RAGE several fold greater than vascular and neuronal tissues. To investigate these mechanisms in human skeletal muscle, we aim to determine the regulators of RAGE signaling on NADPH oxidase activity and the potential protective role of NRROS. As a critical first step in answering these questions, the applicant has generated preliminary data to confirm that both RAGE and NOX II (Figure 3D) proteins are expressed in human skeletal muscle tissue. Importantly, older obese subjects with T2DM (age 55±3 yrs; BMI 34±2 kg/m2, n=2) displayed greater (p<0.05) protein expression of both RAGE and NOX II compared to young lean healthy controls (age 27±3 yrs; BMI 23±2 kg/m2, n=2) (Figure 3A/B), Further, NRROS was found to be expressed in both human skeletal muscle tissue (age 23±2 yrs; BMI 22±1 kg/m2, n=2) and primary human skeletal muscle cell lines (Figure 3C/D).