Receptor for advanced glycation end product (RAGE)-dependent signaling has been implicated in ischemia/reperfusion injury in the heart, lung, liver, and brain. Because macrophages contribute to vascular perturbation and tissue injury in hypoxic settings, we tested the hypothesis that RAGE regulates early growth response-1 (Egr-1) expression in hypoxia-exposed macrophages. Molecular analysis, including silencing of RAGE, or blockade of RAGE with sRAGE (the extracellular ligandbinding domain of RAGE), anti-RAGE IgG, or anti-AGE IgG in THP-1 cells, and genetic deletion of RAGE in peritoneal macrophages, revealed that hypoxia-induced up-regulation of Egr-1 is mediated by RAGE signaling. In addition, the observation of increased cellular release of RAGE ligand AGEs in hypoxic THP-1 cells suggests that recruitment of RAGE in hypoxia is stimulated by rapid production of RAGE ligands in this setting. Finally, we show that mDia-1, previously shown to interact with the RAGE cytoplasmic domain, is essential for hypoxia-stimulated regulation of Egr-1, at least in part through protein kinase C II, ERK1/2, and c-Jun NH 2 -terminal kinase signaling triggered by RAGE ligands. Our findings highlight a novel mechanism by which an extracellular signal initiated by RAGE ligand AGEs regulates Egr-1 in a manner requiring mDia-1.The occlusion of blood vessels or insufficient blood flow to diseased tissues occurs with the onset and progression of many pathological states (1-6). Macrophages accumulate in large numbers in such ischemic/hypoxic areas and respond to oxygen signaling mechanisms involving a number of transcription factors (7-9). One such transcription factor, early growth response-1 (Egr-1), 2 an inducible zinc finger transcription factor, is rapidly up-regulated in macrophages in coordinating inflammatory and procoagulant response to hypoxia (8, 9).The generation of advanced glycation end products (AGEs) has been implicated in ischemia/reperfusion injury in the heart (10 -14). AGE-modified proteins are able to activate macrophages and stimulate secretion of cytokines and inflammatory factors (15-17). A major mechanism by which AGEs exert their cellular effects is by ligation of the multiligand receptor for AGE (RAGE) (18). In addition, our previous findings demonstrated that AGE-RAGE-dependent membrane translocation of protein kinase C (PKC) II and consequent activation of JNK signaling in the heart and in endothelial cells subjected to hypoxia directly impact on regulation of Egr-1 (19). However, ligands of RAGE are not simply tethered to this receptor.Studies in vivo and in vitro revealed that cytoplamic domain of RAGE is essential for RAGE ligand-triggered signal transduction because deletion of the cytoplasmic domain of RAGE blocks ligands from inducing signaling and modulating gene expression (20). In addition, the cytoplasmic domain of RAGE interacts with a member of the formin homology domain proteins, diaphanous or mDia-1, which has been identified as a binding partner of the RAGE cytoplasmic domain (21). Previous...
Objective Diabetic subjects are at higher risk of ischemic peripheral vascular disease (PVD). We tested the hypothesis that advanced glycation end products (AGEs) and their receptor (RAGE) block neoangiogenesis and blood flow recovery after hind limb ischemia induced by femoral artery ligation (FAL) through modulation of immune/inflammatory mechanisms. Approach and Results Wild type (WT) mice rendered diabetic with streptozotocin and subjected to unilateral FAL displayed increased accumulation and expression of AGEs and RAGE in ischemic muscle. In diabetic WT mice, FAL attenuated neoangiogenesis and impaired blood flow recovery, in parallel with reduced macrophage content in ischemic muscle and suppression of early inflammatory gene expression, including chemokine (C-C motif) ligand 2 (Ccl2) and early growth response gene 1 (Egr1) versus non-diabetic mice. Deletion of Ager or transgenic expression of Glo1 (reduces AGEs) restored adaptive inflammation, neoangiogenesis and blood flow recovery in diabetic mice. In diabetes, deletion of Ager increased circulating Ly6Chi monocytes and augmented macrophage infiltration into ischemic muscle tissue after FAL. In vitro, macrophages grown in high glucose display inflammation that is skewed to expression of tissue damage versus tissue repair gene expression. Further, macrophages grown in high versus low glucose demonstrate blunted macrophage-endothelial cell interactions. In both settings, these adverse effects of high glucose were reversed by Ager deletion in macrophages. Conclusions These findings indicate that RAGE attenuates adaptive inflammation in hind limb ischemia; underscore microenvironment-specific functions for RAGE in inflammation in tissue repair versus damage; and illustrate that AGE/RAGE antagonism may fill a critical gap in diabetic PVD.
The oncogenic epidermal growth factor receptor (EGFR) is commonly overexpressed in solid cancers. The tyrosine kinase activity of EGFR has been a major therapeutic target for cancer; however, the efficacy of EGFR tyrosine kinase inhibitors to treat cancers has been challenged by innate and acquired resistance at the clinic. Accumulating evidence suggests that EGFR possesses kinase-independent pro-survival functions, and that cancer cells are more vulnerable to reduction of EGFR protein than to inhibition of its kinase activity. The molecular mechanism underlying loss-of-EGFR-induced cell death remains largely unknown. In this study, we show that, unlike inhibiting EGFR kinase activity that is known to induce pro-survival non-selective autophagy, downregulating EGFR protein, either by siRNA, or by a synthetic EGFR-downregulating peptide (Herdegradin), kills prostate and ovarian cancer cells via selective mitophagy by activating the mTORC2/Akt axis. Furthermore, Herdegradin induced mitophagy and inhibited the growth of orthotopic ovarian cancers in mice. This study identifies anti-mitophagy as a kinase-independent function of EGFR, reveals a novel function of mTORC2/Akt axis in promoting mitophagy in cancer cells, and offers a novel approach for pharmacological downregulation of EGFR protein as a potential treatment for EGFR-positive cancers.
Type I interferons such as interferon-beta (IFN-β) play essential roles in the host innate immune response to herpes simplex virus type I (HSV-1) infection. The transcription of type I interferon genes is controlled by nuclear factor-κB (NF-κB) and interferon regulatory factor (IRF) family members including IRF3. NF-κB activation depends on the phosphorylation of inhibitor of κB (IκB), which triggers its ubiqitination and degradation. It has been reported that neddylation inhibition by a pharmacological agent MLN4924 potently suppresses lipopolysaccharide (LPS)-induced proinflammatory cytokine production with the accumulation of phosphorylated IκBα. However, the role of neddylation in type I interferon expression remains unknown. Here, we report that neddylation inhibition with MLN4924 or upon UBA3 deficiency led to accumulation of phosphorylated IκBα, impaired IκBα degradation, and impaired NF-κB nuclear translocation in the early phase of HSV-1 infection even though phosphorylation and nuclear translocation of IRF3 were not affected. The blockade of NF-κB nuclear translocation by neddylation inhibition becomes less efficient at the later time points of HSV-1 infection. Consequently, HSV-1-induced early phase IFN-β production significantly decreased upon MLN4924 treatment and UBA3 deficiency. NF-κB inhibitor JSH-23 mimicked the effects of neddylation inhibition in the early phase of HSV-1 infection. Moreover, the effects of neddylation inhibition on HSV-1-induced early phase IFN-β production diminished in the presence of NF-κB inhibitor JSH-23. Thus, neddylation contributes to HSV-1-induced early phase IFN-β production through, at least partially, promoting NF-κB activation.
Background: Interferon regulatory factor 8 (IRF8), which is induced by peripheral nerve injury (PNI), plays a key role in activating spinal microglia to release inflammatory cytokines in a p38- dependent way, thereafter results in formation of central sensitization. Pulsed radiofrequency (PRF) on dorsal root ganglion (DRG) alleviates neuropathic pain and inhibits the microglial activation in chronic constriction injury (CCI) rats. However, the consequences of PRF on spinal IRF8 of CCI rats remains unknown. Objectives: We explore if PRF on DRG of rats with CCI could restrain IRF8, microglia, and p38 hyperactivity in the spinal cord to alleviate neuropathic pain. Study Design: A randomized, controlled animal study. Setting: Department of Pain Management, Fujian Provincial Hospital, Fujian Key Laboratory of Geriatrics, Provincial Clinic College of Fujian Medical University. Methods: The changes in pain behaviors and the expressions of IRF8, Iba1 and p-p38 in the spinal cord of CCI rats which were administrated with antisense/ mismatch oligodeoxynucleotide of IRF8 were studied. Rats in CCI+AS ODN group, CCI+MM ODN group or CCI+NS group were intrathecally treated with antisense oligodeoxynucleotide of IRF8, mismatch oligodeoxynucleotide of IRF8 or same volume 0.9% NaCl once daily respectively, beginning from the day after nerve transection 12 hours and lasting for 7 days. The effects of PRF on L4-5 DRG of rats with CCI were investigated. PRF was applied adjacent to the L4-5 DRG at an intensity of 45 V for 6 minutes after CCI, whereas the control rats were treated without radiofrequency current. The withdrawal thresholds were studied and the spinal levels of IRF8, ionized calcium-binding adapter molecule 1 (Iba1, microglia characteristic marker) and p-p38 were calculated by ELISA, western blot, RT-PCR, and immunofluorescence. Results: Intrathecal administration of antisense oligodeoxynucleotide of IRF8 led to the reversal of CCI-induced allodynia, lower activation of spinal microglia and p-p38. Withdrawal thresholds were partially recovered after a single PRF treatment for 14 days. CCI-induced IRF8 upregulation, microglia hyperactivity, and p38 phosphorylation in the spinal cord were reduced due to PRF treatment. However, PRF did not alter pain behaviors and pain signals in normal rats. Limitations: In our study, one time point was selected just to assess the levels of microglia, and p-p38. The changes of IRF8, microglia, p-p38 in the ipsilateral DRG were not investigated. A more detailed study on how PRF on the DRG could further relieve NP is needed. Conclusions: Restraining IRF8, microglia and p38 hyperactivity in the spinal cord of CCI rats involved in the contribution to the long-lasting analgesia of PRF. Keywords: Neuropathic pain, pulsed radiofrequency, dorsal root ganglion,
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