Identification Of NOD2 As A Novel Target Of RNA-Binding Protein HuR:Evidence From Nadph Oxidase-Mediated HuR Signaling In Diabetic Nephropathy

BackgroundDiabetic nephropathy (DN) is one of the major micro-vascular complications of diabetes mellitus (DM). Mesangial cells are the key point that leads to glomerulus sclerosis and DN progression by proliferating and synthetizing excessive extra-cellular matrix. Dysfunction of mesangial cells is mainly due to abnormal expression of many proteins. Recent studies have revealed the importance of mRNA stability to the regulation of protein expression. HuR is the most well-known RNA-binding protein that can regulate mRNA stability. It can bind to the AU-rich element (ARE) in 3’UTR of many target mRNAs. Studies have shown the existence of HuR in mesangial cells and its abnormal expression and/or activity are highly related to the changes of mesangial morphology or function induced by multiple inflammatory factors. But we still do not know what role HuR plays in dysfunctional mesangial cells induced by high blood glucose.Activation of innate immune system and subsequently releasing of inflammatory mediators play important parts in the development of DN. The innate immune system can recognize pathogen-associated molecular pattern (PAMP) and danger-associated molecular pattern (DAMP) by pathogen recognition receptor (PRR). NOD-like receptor (NLR), an important sub-family of PRR, can activate immune cells or renal intrinsic cells in multiple kidney diseases. By helping releasing inflammatory factors, NOD2 can lead to tissue damage. A recent study from our laboratory has demonstrated the contribution of NOD2, one important member of NLR, to the pathogenesis of DN. We found that NOD2 was up-regulated in kidney biopsy samples from DN patients, and Nod2 gene knockout ameliorated diabetic renal injury in mice. But the mechanism of the up-regulation of NOD2 in high blood glucose remains unknown.By gene sequence alignment, we found the existence of AREs in 3’UTR of NOD2 mRNA that might be recognized by HuR. One of the AREs is highly conserved among different species which strongly suggests that NOD2 might be the possible target of HuR. One of the central questions of our study is therefore to elucidate whether or not HuR acts as a key posttranscriptional regulator of NOD2 expression.Reactive oxygen species (ROS) is a kind of free radical that is widely distributed in all kinds of cells and tissues. NADPH oxidases, mainly NOX4, are thought to be responsible for most of the ROS in kidney. Reports said that ROS could regulate the shuttling of HuR between nucleus and cytoplasm. However, the mechanisms of NOX4 acting on HuR and its relationship with chronic inflammation in DN remains unknown. As a result, it is of significant importance to elucidate the NOX4 mediated oxidation-reduction state changes in DN and the subsequent changes of innate immune system and inflammatory factors. This will greatly improve our understanding of the pathogenic mechanisms of DN.Objectives1. Determine the expression pattern and function of HuR in DN. Reveal the therapeutic effect of gene interference targeted to HuR in DN.2. Elucidate whether HuR can modulate NOD2 mRNA stability and the related mechanism.3. Discuss the action of NOX4-mediated oxidative stress on HuR/NOD2 regulation.Methods1 Role of RNA-binding protein HuR in DN and related mechanisms1.1 HuR expression in human renal tissues and its relationship with urinary protein of DN patientsThe renal section samples, including normal control, DN samples and diabetes mellitus non-nephropathy (DM-NN) samples were collected from Shandong Provincial Hospital Affiliated to Shandong University and the Department of Pathology, School of Medicine, Shandong University. Immunohistochemical (IHC) staining was used to observe expression of HuR. Urinary protein excretion amounts of DN patients in 24h were collected in order to find the relationship with HuR expression by statistical analysis.1.2 Impact of cortex HuR interference on DN in rat modelsMale Sprague-Dawley rats were randomized into groups:control, DN, scramble control, shRNA control, DN+scramble, DN+shRNA. Each group had 10 rats. Rats were uninephrectomized before tail-vein injection of STZ to induce DN. At the same time, Lentivirus vector bearing scramble or shRNA-HuR was delivered into corresponding group’s renal parenchymal one day after STZ injection. All rats were sustained for 12 weeks. Fast blood glucose and 24h urinary protein excretion were monitored to evaluate whether the DM/DN models were successful. IHC was used to observe the expression of HuR in DN renal cortex. Use PAS staining to observe morphological changes and ELISA to examine the inflammatory factors after HuR interference.1.3 Impact of high glucose on HuR expression and shuttling in RMCRat glomerulus mesangial cells (RMC) were cultured in vitro and stimulated by gradient glucose concentrations or gradient time courses. Cytoplasm, nucleus and total protein were extracted. Western blot was used to measure HuR expression in different cellular components. Immunofluorescence was carried out to observe HuR location and distribution in RMC. Cycloheximide (CHX, 10μg/mL) was admitted before high glucose to stop total protein synthesis. Then different cellular components were extracted and Western blot was used to examine HuR shuttling.2 Regulation of NOD2 mRNA stability by HuR2.1 NOD2 expression in human renal tissues and its relationship with HuRIHC staining was used to observe NOD2 expression in different human renal tissues. Statistical methods were employed to carry out the correspondence analysis of HuR and NOD2 expression.2.2 Impact of HuR interference on NOD2 expression in DN rat renal cortexIHC and Western blot were used to detect HuR and NOD2 expression in renal cortex of DN rats. Western blot and immunofluorescence staining were used to observe NOD2 changes after interfering HuR in renal cortex.2.3 NOD2 regulation by HuR in RMCWestern blot and RT-PCR were used to detect protein and mRNA changes of NOD2 in high glucose stimulated RMC. Use Lentivirus vector bearing shRNA-HuR to interfere HuR expression. Western blot was used to detect the interference efficiency and NOD2 changes after infection.2.4 Mechanisms of NOD2 regulation by HuRActinomycin D (Act D) was used to stop RNA synthesis. Afterwards, we tested NOD2 mRNA remnant at different time points by RT-PCR to determine NOD2 mRNA eliminating rates with or without HuR interference. We searched for the latent HuR binding sequences in 3’UTR of NOD2 mRNA, and tested the binding efficiencies of different ARE sequences of NOD2 with HuR using RNA-EMSA to identify the exact binding site of HuR in NOD2 mRNA.To confirm the function of 3’UTR to NOD2 mRNA stability, we integrated the sequence into Dual-Luciferase reporter plasmid. RMC was transfected with the recombined plasmid. By analyzing the luciferase expression intensity, we could know the 3’UTR function in normal/HG conditions.3 HuR-NOD2 pathway regulation by NADPH oxidase3.1 Oxidative stress in renal cortex of DN ratsDetect NOXs expression in rat renal cortex using Western blot. DHE probe was used to test super oxide anion.Amplex red was used to test hydrogen peroxide.3.2 Oxidative stress of high glucose stimulated RMCWestern blot was used to detect NOX4 expression in high glucose stimulated RMC. DHE probe was used to test super oxide anion. Amplex red was used to test hydrogen peroxide.3.3 Role of NOX4 in HuR regulated NOD2 expressionUse siRNA to interfere NOX4 expression and the interference efficiency was determined by Western blot. The different components of cells were extracted after HG stimulation. HuR and NOD2 protein levels were determined by Western blot. Immunofluorescence was used to observe HuR distribution changes. Act D was admitted to stop RNA synthesis and mRNA eliminating rate was determined by RT-PCR as described previously. Renal cell line NRK-52E was cultured in vitro in order to determine HuR and NOX4 expression pattern by Western blot after high glucose stimulation.Results1 Role of RNA-binding protein HuR in DN and related mechanisms1.1 HuR expression in human renal tissues and its relationship with urinary protein of DN patientsIHC staining showed the up-regulation of HuR in glomerulus of DN patients. HuR expression was positively correlated with patients’ urinary protein level.1.2 Impact of cortex HuR interference on DN in rat modelsIHC showed the up-regulation of HuR in renal cortex of DN rats which was consistent with that of patients’ samples.Physiological data of HuR interfered DN rats was much better than their counterparts. Pathological damage was also relieved. Inflammatory factors were reduced too.1.3 Impact of high glucose on HuR expression and shuttling in RMCHuR was up-regulated in total protein and accumulated in cytoplasm in a concentration/time gradient dependent pattern. Immunofluorescence showed translocation of HuR from nucleus to cytoplasm. Accumulation of HuR in cytoplasm could still be observed by Western blot after cycloheximide was admitted to stop total protein synthesis.2 Regulation of NOD2 mRNA stability by HuR2.1 NOD2 expression in human renal tissues and its relationship with HuRIHC staining showed the up-regulation of NOD2 expression in glomerulus of DN patients. Statistical analysis revealed a positive relationship between HuR and NOD2 expression.2.2 Impact of HuR interference on NOD2 expression in DN rat renal cortexIHC and Western blot showed the up-regulation of both HuR and NOD2 expression in renal cortex of DN rats, which was consistent with that in human samples. NOD2 expression was obviously attenuated by HuR interference in renal cortex of DN rats.2.3 NOD2 regulation by HuR in RMCNOD2 mRNA and protein were time dependently up-regulated by high glucose stimulation. But interference of HuR could obviously attenuate NOD2 expression.2.4 Mechanisms of NOD2 regulation by HuRActinomycin D (Act D) was used to stop RNA synthesis. NOD2 mRNA degradation tested by RT-PCR was much slower when stimulated by high glucose. But HuR interference could attenuate this phenomenon.There were four AREs in rat NOD2 3’UTR. Sequence alignment revealed that ARE4 was highly conserved among different species. RNA-EMSA revealed that only NOD2-ARE4 probe could form complex with kidney extracts. In vitro experiments found that cytoplasm of RMC treated by HG can form the complex in a concentration dependent pattern. Super shift could be seen when anti-HuR antibody was introduced into the reaction system, which proved the involvement of HuR in the formation of complex. The interaction of HuR and NOD2-ARE4 was confirmed using the recombined HuR protein as a positive control. Dual-Luciferase reporter assay revealed that NOD2 3’UTR-containing plasmid had a profoundly down-regulated enzyme activity than control. But high glucose stimulation could inhibit the reduction. Together these results proved that HuR could bind to NOD2 ARE4 and stabilize its mRNA.3 HuR-NOD2 pathway regulation by NADPH oxidase3.1 Oxidative stress in renal cortex of DN ratsNOX4 was profoundly up-regulated in renal cortex of DN rats. O2·- and H2O2 were also increased.3.2 Oxidative stress of high glucose stimulated RMCHigh glucose could stimulate NOX4 expression in RMC and increase O2·- and H2O2 levels.3.3 Role of NOX4 in HuR regulated NOD2 expressionsiRNA-NOX4 could attenuate HuR expression and cytoplasmic accumulation in RMC stimulated by high glucose. Immunofluorescence also showed weakened high glucose induced HuR shuttling when NOX4 was silenced. In addition, high glucose induced NOD2 up-regulation and mRNA stabilization were also attenuated along with NOX4 silence. Increased NOX4 and HuR expression were observed in NRK-52E when stimulated by high glucose, indicating that the oxidative stress modulated HuR might be the common mechanism in renal parenchymal cells.Conclusions1. HG could activate NADPH oxidase, mainly NOX4, in mesangial cell. Increased reactive oxygen species lead to HuR up-regulation and accumulation in cytoplasm.2. HuR bound to 3’UTR (mainly ARE4) of NOD2 mRNA in cytoplasm. As a result, NOD2 mRNA was stabilized and protein was increased.3. Innate immune reaction mediated by NOD2 was activated which lead to increased inflammatory factors in renal cortex, proliferated mesangial cells, accumulated matrix and aggravated kidney damage.4. Silencing of HuR could obviously down-regulate NOD2 expression and ameliorate renal damage of DN rats, suggesting gene therapy targeted to HuR might have a latent treatment effect to diabetic nephropathy.InnovationThis is the first time to prove that high glucose could increase HuR accumulation by NOX4 in RMC cytoplasm, and as a result, NOD2 mRNA was stabilized.In vivo experiment of DN rats suggested the gene interference targeted to HuR could relieve the diabetic renal damage, which provided us a new way for DN treatment.