iRhom2 Expression Research Articles

Role of iRhom2 in Olfaction: Implications for Odorant Receptor Regulation and Activity-Dependent Adaptation.

The cell surface metalloprotease ADAM17 (a disintegrin and metalloprotease 17) and its binding partners iRhom2 and iRhom1 (inactive Rhomboid-like proteins 1 and 2) modulate cell-cell interactions by mediating the release of membrane proteins such as TNFα (Tumor necrosis factor α) and EGFR (Epidermal growth factor receptor) ligands from the cell surface. Most cell types express both iRhoms, though myeloid cells exclusively express iRhom2, and iRhom1 is the main iRhom in the mouse brain. Here, we report that iRhom2 is uniquely expressed in olfactory sensory neurons (OSNs), highly specialized cells expressing one olfactory receptor (OR) from a repertoire of more than a thousand OR genes in mice. iRhom2-/- mice had no evident morphological defects in the olfactory epithelium (OE), yet RNAseq analysis revealed differential expression of a small subset of ORs. Notably, while the majority of ORs remain unaffected in iRhom2-/- OE, OSNs expressing ORs that are enriched in iRhom2-/- OE showed fewer gene expression changes upon odor environmental changes than the majority of OSNs. Moreover, we discovered an inverse correlation between the expression of iRhom2 compared to OSN activity genes and that odor exposure negatively regulates iRhom2 expression. Given that ORs are specialized G-protein coupled receptors (GPCRs) and many GPCRs activate iRhom2/ADAM17, we investigated if ORs could activate iRhom2/ADAM17. Activation of an olfactory receptor that is ectopically expressed in keratinocytes (OR2AT4) by its agonist Sandalore leads to ERK1/2 phosphorylation, likely via an iRhom2/ADAM17-dependent pathway. Taken together, these findings point to a mechanism by which odor stimulation of OSNs activates iRhom2/ADAM17 catalytic activity, resulting in downstream transcriptional changes to the OR repertoire and activity genes, and driving a negative feedback loop to downregulate iRhom2 expression.

Increased Expression of Inactive Rhomboid Protein 2 in Circulating Monocytes after Acute Myocardial Infarction.

Increased TNF-α levels following acute myocardial infarction (AMI) contribute to impaired recovery of myocardial function. Interaction of inactive rhomboid protein 2 (iRhom2) with TNF-α converting enzyme (TACE) is required for TNF-α shedding from immune cells. We hypothesized that iRhom2 expression increases in circulating monocytes following AMI. Transcript levels of iRhom2, TACE and TNF-α were evaluated by quantitative real-time PCR in isolated monocytes of 50 AMI patients at admission (d1) and 3days (d3) after. We observed a significant increase in levels of iRhom2 mRNA expression in monocytes between d1-3, while TNF-α and TACE mRNA expression remained unchanged. At d3, iRhom2 mRNA expression positively correlated with levels of intermediate monocytes or serum TNF-α, and negatively with LV systolic function. iRhom2 may contribute to regulation of post-infarction inflammation and is associated with LV dysfunction following AMI. iRhom2 modulation should be evaluated as a potential therapeutic strategy to attenuate cardiac remodeling following AMI.

Deficiency in Inactive Rhomboid Protein2 (iRhom2) Alleviates Alcoholic Liver Fibrosis by Suppressing Inflammation and Oxidative Stress.

Chronic alcohol exposure can lead to liver pathology relating to inflammation and oxidative stress, which are two of the major factors in the incidence of liver fibrosis and even liver cancer. The underlying molecular mechanisms regarding hepatic lesions associated with alcohol are not fully understood. Considering that the recently identified iRhom2 is a key pathogenic mediator of inflammation, we performed in vitro and in vivo experiments to explore its regulatory role in alcohol-induced liver fibrosis. We found that iRhom2 knockout significantly inhibited alcohol-induced inflammatory responses in vitro, including elevated expressions of inflammatory cytokines (IL-1β, IL-6, IL-18, and TNF-α) and genes associated with inflammatory signaling pathways, such as TACE (tumor necrosis factor-alpha converting enzyme), TNFR1 (tumor necrosis factor receptor 1), and TNFR2, as well as the activation of NF-κB. The in vivo results confirmed that long-term alcohol exposure leads to hepatocyte damage and fibrous accumulation. In this pathological process, the expression of iRhom2 is promoted to activate the TACE/NF-κB signaling pathway, leading to inflammatory responses. Furthermore, the deletion of iRhom2 blocks the TACE/NF-κB signaling pathway and reduces liver damage and fibrosis caused by alcohol. Additionally, the activation of the JNK/Nrf2/HO-1 signaling pathway caused by alcohol exposure was also noted in vitro and in vivo. In the same way, knockout or deleting iRhom2 blocked the JNK/Nrf2/HO-1 signaling pathway to regulate the oxidative stress. Therefore, we contend that iRhom2 is a key regulator that promotes inflammatory responses and regulates oxidative stress in alcoholic liver fibrosis lesions. We posit that iRhom2 is potentially a new therapeutic target for alcoholic liver fibrosis.

Upregulated iRhom2 in the Hypothalamic Paraventricular Nucleus is Associated with TACE‐Mediated Shedding of Transforming Growth Factor‐α and Sympathetic Excitation in Heart Failure Rats

Tumor necrosis factor (TNF)‐α converting enzyme (TACE), also known as a disintegrin and metalloprotease (ADAM)17, is a key mediator of cell signaling by proteolytically cleaving extracellular domains of various cytokines and growth factors. TACE‐mediated shedding of transforming growth factor (TGF)‐α, has been shown to transactivate epidermal growth factor receptor (EGFR) to activate the mitogen‐activated protein kinase signaling pathway in the pathophysiological conditions. We previously reported that both TACE and TGF‐α are upregulated in the hypothalamic paraventricular nucleus (PVN, a critical cardiovascular and autonomic center) and contribute to the sympathetic excitation in heart failure (HF). However, the mechanisms by which TACE and TGF‐α are upregulated in the PVN in HF remain unclear. Recent evidence reveals that inactive Rhomboid proteins (iRhoms) including iRhom1 and iRhom2 are essential upstream regulators of TACE/ADAM17. The present study sought to determine the expression of iRhoms and its relationship with TACE‐mediated shedding of TGF‐α in the PVN in HF. Male rats underwent coronary artery ligation to induce HF or sham surgery (Sham). The left ventricular function and infarction size were assessed by echocardiography. Four weeks later, these animals were euthanized to collect cerebrospinal fluid (CSF), PVN tissues and blood for molecular and biochemical measurements. All values are expressed as the mean ± SD. Real‐time PCR showed abundant mRNA expression of both iRhom1 and iRhom2 in the PVN and brain cortex in Sham or HF rats. Compared with Sham rats, HF rats had significantly (*P<0.05) increased mRNA expression of iRhom2 (2.00 ± 0.57* vs 1.03 ± 0.28, HF vs Sham) but not iRhom1 (1.32 ± 0.41 vs 1.02 ± 0.21) in the PVN. Additionally, HF rats exhibited marked increases in mRNA expression of TACE (2.25 ± 1.15* vs 1.04 ± 0.36) in the PVN and the plasma level of norepinephrine (NE, a marker of sympathetic excitation, 12.39 ± 4.96* vs 5.82 ± 2.76 ng/mL), along with elevated levels of TGF‐α (23.54 ± 6.25* vs 10.08 ± 4.38 pg/mL) in CSF. The levels of epidermal growth factor (9.88 ± 3.59 vs 8.15 ± 4.24 pg/mL) in the CSF did not alter significantly in HF compared with Sham rats. Moreover, the linear regression analysis indicated that the mRNA expression of iRhom2 in the PVN was positively correlated with TACE level in the PVN (R2 = 0.62, P <0.01), TGF‐α level in the CSF (R2 = 0.64, P <0.01) and NE level in the plasma (R2 = 0.63, P <0.01). These results suggest that the upregulated expression of iRhom2 in the PVN may promote ectodomain shedding of TGF‐α by enhancing TACE expression to drive sympathetic activation in HF. Brain iRhom2 is a potential target for therapeutic intervention in HF.

Inflammatory activation of surface molecule shedding by upregulation of the pseudoprotease iRhom2 in colon epithelial cells

The metalloproteinase ADAM17 contributes to inflammatory and proliferative responses by shedding of cell-surface molecules. By this ADAM17 is implicated in inflammation, regeneration, and permeability regulation of epithelial cells in the colon. ADAM17 maturation and surface expression requires the adapter proteins iRhom1 or iRhom2. Here we report that expression of iRhom2 but not iRhom1 is upregulated in intestinal tissue of mice with acute colitis. Our analysis of public databases indicates elevated iRhom2 expression in mucosal tissue and epithelial cells from patients with inflammatory bowel disease (IBD). Consistently, expression of iRhom2 but not iRhom1 is upregulated in colon or intestinal epithelial cell lines after co-stimulation with tumor necrosis factor (TNF) and interferon gamma (IFNgamma). This upregulation can be reduced by inhibition of Janus kinases or transcription factors NF-kappaB or AP-1. Upregulation of iRhom2 can be mimicked by iRhom2 overexpression and is associated with enhanced maturation and surface expression of ADAM17 which then results in increased cleavage of transforming growth factor (TGF) alpha and junctional adhesion molecule (JAM)-A. Finally, the induction of these responses is suppressed by inhibition of iRhom2 transcription. Thus, inflammatory induction of iRhom2 may contribute to upregulated ADAM17-dependent mediator and adhesion molecule release in IBD. The development of iRhom2-dependent inhibitors may allow selective targeting of inflammatory ADAM17 activities.

Increased expression of inactive rhomboid protein 2 in circulating monocytes after acute myocardial infarction

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG; Sachbeihilfe HE6092/2) DGK (Deutsche Gesellschaft für Kardiologie) Otto-Hess-Promotionsstipendium Purpose Tumor necrosis factor-alpha (TNF-α) levels in the blood increase in the course of an acute myocardial infarction (AMI) and TNF-α is involved in the impaired recovery of myocardial function following AMI. The interaction of inactive rhomboid protein 2 (iRhom2) with the TNF-α converting enzyme (TACE) is required for the proper shedding of TNF-α from the cell surface of immune cells. We hypothesized, that iRhom2 expression increases in circulating monocytes of patients following AMI. Methods Circulating monocytes were MACS-sorted from peripheral blood of 50 patients with AMI (NSTEMI, n = 16; STEMI n = 34) at the day of admission (day 1) and 3 days after admission. 50 patients with chronic coronary syndrome (CCS) were recruited as control. mRNA was isolated from sorted monocytes and expression levels of iRhom2, TACE and TNF-α were evaluated by real-time RT-PCR. TNF-α protein levels were assessed in the serum. Levels of circulating classical, intermediate and non-classical monocyte subsets were determined by flow cytometry. Results There was a significant increase of iRhom2 mRNA expression levels in monocytes (by 14%, p = 0.012), levels of circulating intermediate monocytes (p < 0.001), and TNF-α serum levels (p < 0.001) at day 3 following AMI compared to day 1 reaching levels similar to those observed in CCS patients. In contrast, TNF-α and TACE mRNA expression in circulating monocytes did not differ between day 1 and 3 following AMI. iRhom2 mRNA expression in monocytes on day 3 following AMI correlated with TACE mRNA expression in monocytes (r = 0.72, p < 0.001), TNF-α serum levels (r = 0.33, p = 0.019), and levels of circulating intermediate monocytes (r = 0.37, p = 0.009). Conclusions Following AMI, iRhom2 expression in circulating monocytes increases in parallel to serum levels of TNF-α and levels of circulating intermediate monocytes. These findings suggest that iRhom2 contributes to inflammation following AMI and thus may serve as a potential therapeutic target.

Fisetin protects against high fat diet-induced nephropathy by inhibiting inflammation and oxidative stress via the blockage of iRhom2/NF-κB signaling

Promoted inflammation enhances the development of nephropathy in obesity. Fisetin (3,3′,4′,7-tetrahydroxyflavone, FIS) is a naturally occurring dietary flavonoid, and exhibits anti-inflammatory and anti-oxidative properties. Inactive rhomboid protein 2 (iRhom2), an inactive member of the rhomboid intramembrane proteinase family, is an essential inflammation-associated regulator. Here, we attempted to investigate the protective mechanisms of FIS against high fat diet (HFD)-induced nephropathy, with particular focus on iRhom2. We found that HFD induced systematic and renal pro-inflammatory cytokine production. Furthermore, iRhom2 expression was markedly elevated in kidney of HFD-fed mice, and in PAL-incubated macrophages, accompanied with high phosphorylation of NF-κB. Significant oxidative stress was observed in kidney of HFD-fed mice through suppressing Nrf-2/HO-1 signaling. Moreover, activation of iRhom2/NF-κB signaling and oxidative stress by PAL was detected in macrophages, which were effectively reversed by FIS. Importantly, we showed that iRhom2 knockdown significantly abrogated the ability of FIS to restrain inflammation and oxidative stress induced by PAL in macrophages, indicating that iRhom2 might be a potential therapeutic target for FIS during nephropathy treatment. Together, these results revealed that FIS could mitigate HFD-induced renal injury by regulating iRhom2/NF-κB and Nrf-2/HO-1 signaling pathways.

4-Hydroxyisoleucine relieves inflammation through iRhom2-dependent pathway in co-cultured macrophages and adipocytes with LPS stimulation

Background4-Hydroxyisoleucine (4-HIL) is an active ingredient extracted from Trigonella foenum-graecum L., a Chinese traditional herbal medicine, which exerts the efficacy of anti-obesity and anti-diabetes. We previously reported that 4-HIL potentiates anti-inflammatory and anti-insulin resistance effects through down-regulation of TNF-α and TNF-α converting enzyme (TACE) in 3 T3-L1 adipocytes and HepG2 cells. In the present study, we further investigate the effects and mechanisms of 4-HIL on obesity-induced inflammation in RAW264.7 macrophages and 3 T3-L1 adipocytes co-culture system.MethodsRAW264.7 macrophages and 3 T3-L1 adipocytes were co-cultured to mimic the microenvironment of adipose tissue. siRNA-iRhom2 transfection was performed to knockdown iRhom2 expression in RAW264.2 macrophages. The mRNA and protein expression of iRhom2 and TACE were measured by real-time quantitative PCR (RT-qPCR) and western blotting. The production of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), IL-6 and IL-10 were evaluated by ELISA. The ratio of M2/M1 was detected by flow cytometry.Results4-HIL significantly repressed the mRNA and protein levels of iRhom2 and TACE in RAW264.7 macrophages after LPS stimulated. Meanwhile, the levels of pro-inflammatory cytokines, including TNF-α, MCP-1, and IL-6, were substantially suppressed by 4-HIL in the co-culture system. Moreover, the level of anti-inflammatory cytokine IL-10 was increased significantly by 4-HIL in the co-culture system after LPS stimulation. Additionally, the ratio of M2/M1 was also increased by 4-HIL in the co-culture system after LPS stimulation. Finally, these effects of 4-HIL were largely enhanced by siRNA-iRhom2 transfection.ConclusionTaken together, our results indicated that obesity-induced inflammation was potently relieved by 4-HIL, most likely through the iRhom2-dependent pathway.

Reactive oxygen species-responsive dexamethasone-loaded nanoparticles for targeted treatment of rheumatoid arthritis via suppressing the iRhom2/TNF-α/BAFF signaling pathway

Rheumatoid arthritis (RA) is an immune-mediated inflammatory disease that results in synovitis, cartilage destruction, and even loss of joint function. The frequent and long-term administration of anti-rheumatic drugs often leads to obvious adverse effects and patient non-compliance. Therefore, to specifically deliver dexamethasone (Dex) to inflamed joints and reduce the administration frequency of Dex, we developed Dex-loaded reactive oxygen species (ROS)-responsive nanoparticles (Dex/Oxi-αCD NPs) and folic acid (FA) modified Dex/Oxi-αCD NPs (Dex/FA-Oxi-αCD NPs) and validated their anti-inflammatory effect in vitro and in vivo. In vitro study demonstrated that these NPs can be effectively internalized by activated macrophages and the released Dex from NPs significantly downregulated the expression of iRhom2, TNF-α, and BAFF in activated Raw264.7. In vivo experiments revealed that Dex/Oxi-αCD NPs, especially Dex/FA-Oxi-αCD NPs significantly accumulated at inflamed joints in collagen-induced arthritis (CIA) mice and alleviated the joint swelling and cartilage destruction. Importantly, the expression of iRhom2, TNF-α, and BAFF in the joint was inhibited by intravenous injection of Dex/Oxi-αCD NPs and Dex/FA-Oxi-αCD NPs. Collectively, our data revealed that Dex-loaded ROS-responsive NPs can target inflamed joints and attenuate arthritis, and the ‘iRhom2-TNF-α-BAFF’ pathway plays an important role in the treatment of RA with the NPs, suggesting that this pathway may be a novel target for RA therapy.

Association of iRhom1 and iRhom2 expression with prognosis in patients with cervical cancer and possible signaling pathways.

Several proteins in the iRhom family function as oncogenic regulators in certain cancers. However, the function of these proteins in cervical cancer (CC) is unknown. The relationship of iRhom1 and iRhom2 expression with the clinicopathological features and prognosis of patients with CC was investigated, and their possible molecular mechanisms were examined using in vitro experiments. The expression of iRhom1 and iRhom2 in CC samples of 83 patients was determined by immunohistochemistry (IHC), and the associations of their expression with the clinicopathological features of patients were determined. The relationship of iRhom1, iRhom2, and Ki-67 expression with survival rates was determined using Kaplan-Meier analysis and Cox regression analyses. HeLa cells were analyzed using MTT assays, cell cycle analysis, and apoptosis assays. The results revealed that CC tissues had higher levels of iRhom1 and iRhom2 than adjacent normal tissues. Increased expression of iRhom1, iRhom2, and K-i67 was significantly associated with tumor stage, size, and parametrium invasion. High expression of iRhom1, iRhom2 and Ki-67 was correlated with poor outcomes. Cancer stage and iRhom2 expression were independent prognostic indicators of CC. Knockdown of iRhom1 and iRhom2 in HeLa cells inhibited cell proliferation, promoted the G1 phase and relieved S-phase arrest, and induced apoptosis. Genomic microarray analysis indicated that iRhom2 knockdown altered several pathways with roles in oncogenesis, including the expression of five genes in the Wnt/β-catenin pathway. Western blotting in HeLa cells revealed that iRhom1 knockdown significantly suppressed the expression of β-catenin, Myc, p-EGFR and TGFBR2, and increased the expression of FAS; iRhom2 knockdown significantly suppressed the expression of β-catenin, GSK3β, p-EGFR and Myc. These results were consistent with the genomic microarray data. Collectively, the results indicated that iRhom1 and iRhom2 may function as oncogenes in CC and are potential therapeutic targets.

Regulation of iRhom-2/Tumor Necrosis Factor-α Converting Enzyme Pathway and Oxidative Stress Protects the Renal Injury with Anemonin in Streptozotocin-Induced Diabetic Nephropathy Neonatal Rat Model

Diabetic nephropathy (DN) is a chronic complication of diabetes, and thus the present investigation evaluates the nephroprotective effect of anemonin against streptozotocin (STZ)-induced DN rats. Diabetes was induced by intraperitoneal administration of STZ (50 mg/kg) on day 2 and 3 postnatal, and rats were kept as such for the duration of 12 weeks. Thereafter, rats were treated with anemonin 75 and 150 mg/kg per oral for the period 4 week which means between the period of 12–16 weeks. Effect of anemonin was estimated by determining the blood glucose, markers of nephropathy, and mediators of inflammation in the serum and activity of tumor necrosis factor-α (TNF-α)converting enzyme (TACE) in the kidney tissue of DN rats. Moreover, reverse transcriptase polymerase chain reaction and western blot assay were determined in the kidney tissue homogenate of DN rats. Histopathology study was done by Periodic acid-Schiff’s and masson staining for the pathological changes and apoptosis of podocytes in the kidney tissue of DN rats. Moreover, production of reactive oxygen species (ROS) was estimated in the kidney tissue by 2’,7’-dichlorofluorescein staining. Data of study reveal that anemonin significantly reduces (p < 0.01) the blood glucose and markers of renal injury in the serum and urine of DN rats. There was a reduction in the level of cytokines in the serum, and production of ROS and activity of TACE were reduced in the kidney tissue of the anemonin-treated group than in the DN group. Expression of iRhom-2, TACE, TNF-α, and inducible nitric oxide synthase protein and histopathology of kidney tissue were attenuated in the anemonin-treated group in DN rats. In conclusion, data of study reveal that treatment with anemonin ameliorates progression of renal injury by regulating TACE/iRhom-2 signaling pathway.

Endoplasmic reticulum stress-induced iRhom2 up-regulation promotes macrophage-regulated cardiac inflammation and lipid deposition in high fat diet (HFD)-challenged mice: Intervention of fisetin and metformin

Endoplasmic reticulum stress (ERS) has been implicated in obesity-associated cardiac remodeling and dysfunction. Inactive rhomboid protein 2 (iRhom2), also known as Rhbdf2, is an inactive member of the rhomboid intramembrane proteinase family, playing an essential role in regulating inflammation. Nevertheless, the role of ERS-meditated iRhom2 pathway in metabolic stress-induced cardiomyopathy remains unknown. In the study, we showed that 4-PBA, as an essential ERS inhibitor, significantly alleviated high fat diet (HFD)-induced metabolic disorder and cardiac dysfunction in mice. Additionally, lipid deposition in heart tissues was prevented by 4-PBA in HFD-challenged mice. Moreover, 4-PBA blunted the expression of iRhom2, TACE, TNFR2 and phosphorylated NF-κB to prevent HFD-induced expression of inflammatory factors. Further, 4-PBA restrained HFD-triggered oxidative stress by promoting Nrf-2 signaling. Importantly, 4-PBA markedly suppressed cardiac ERS in HFD mice. The anti-inflammation, anti-ERS and anti-oxidant effects of 4-PBA were verified in palmitate (PAL)-incubated macrophages and cardiomyocytes. In addition, promoting ERS could obviously enhance iRhom2 signaling in vitro. Intriguingly, our data demonstrated that PAL-induced iRhom2 up-regulation apparently promoted macrophage to generate inflammatory factors that could promote cardiomyocyte inflammation and lipid accumulation. Finally, interventions by adding fisetin or metformin significantly abrogated metabolic stress-induced cardiomyopathy through the mechanisms mentioned above. In conclusion, this study provided a novel mechanism for metabolic stress-induced cardiomyopathy pathogenesis. Therapeutic strategy to restrain ROS/ERS/iRhom2 signaling pathway could be developed to prevent myocardial inflammation and lipid deposition, consequently alleviating obesity-induced cardiomyopathy.

PPARγ maintains the metabolic heterogeneity and homeostasis of renal tubules.

BackgroundThe renal tubules, which have distant metabolic features and functions in different segments, reabsorb >99% of approximately 180 l of water and 25,000 mmol of Na + daily. Defective metabolism in renal tubules is involved in the pathobiology of kidney diseases. However, the mechanisms underlying the metabolic regulation in renal tubules remain to be defined.MethodsWe quantitatively compared the proteomes of the isolated proximal tubules (PT) and distal tubules (DT) from C57BL/6 mouse using tandem mass tag (TMT) labeling-based quantitative mass spectrometry. Bioinformatics analysis of the differentially expressed proteins revealed the significant differences between PT and DT in metabolism pathway. We also performed in vitro and in vivo assays to investigate the molecular mechanism underlying the distant metabolic features in PT and DT.FindingsWe demonstrate that the renal proximal tubule (PT) has high expression of lipid metabolism enzymes, which is transcriptionally upregulated by abundantly expressed PPARα/γ. In contrast, the renal distal tubule (DT) has elevated glycolytic enzyme expression, which is mediated by highly expressed c-Myc. Importantly, PPARγ transcriptionally enhances the protease iRhom2 expression in PT, which suppresses EGF expression and secretion and subsequent EGFR-dependent glycolytic gene expression and glycolysis. PPARγ inhibition reduces iRhom2 expression and increases EGF and GLUT1 expression in PT in mice, resulting in renal tubule hypertrophy, tubulointerstitial fibrosis and damaged kidney functions, which are rescued by 2-deoxy-d-glucose treatment.InterpretationThese findings delineate instrumental mechanisms underlying the active lipid metabolism and suppressed glycolysis in PT and active glycolysis in DT and reveal critical roles for PPARs and c-Myc in maintaining renal metabolic homeostasis. FUND: This work was supported by the National Natural Science Foundation of China (grants 81572076 and 81873932; to Q.Z.), the Applied Development Program of the Science and Technology Committee of Chongqing (cstc2014yykfB10003; Q.Z.), the Program of Populace Creativities Workshops of the Science and Technology Committee of Chongqing (Q.Z.), the special demonstration programs for innovation and application of techniques (cstc2018jscx-mszdX0022) from the Science and Technology Committee of Chongqing (Q.Z.).

IRhom2 promotes atherosclerosis through macrophage inflammation and induction of oxidative stress

Atherosclerosis is a complex chronic inflammatory disease that is characterized by the formation of lipid-rich plaques on the inner walls of the arteries. Inactive rhomboid protein 2 (iRhom2) was recently determined as a necessary regulator for the shedding of tumor necrosis factor-alpha (TNF-α) in immune cells. In the present study, we aimed to explore the effects of iRhom2 on the inflammatory response and oxidative stress induced by low-density lipoprotein (ox-LDL) in RAW264.7 and THP-1-derived macrophages. The expression levels of iRhom2 were also investigated in apolipoprotein E knockout (ApoE−/-) mice fed a high-fat diet (HFD). iRhom2 was significantly induced by ox-LDL in macrophages, as confirmed by Western blotting and RT-qPCR analysis. Furthermore, iRhom2 knockdown showed significant suppressive effects on the activation of ox-LDL-induced RAW264.7 and THP-1-derived macrophages through reducing TACE and TNFR2 expressions, and the inactivation of the IκBα/NF-κB signaling pathway. A reduction in reactive oxygen species (ROS) generation, malondialdehyde (MDA) levels, and nitric oxide (NOX) activity and an increase in glutathione peroxidase (GSH-Px) activity were determined in the absence of iRhom2 expression. In addition, the NF-E2 related factor-2 (Nrf-2)/heme oxygenase-1 (HO-1) pathway was also upregulated in ox-LDL-treated macrophages subjected to iRhom2 inhibition. Moreover, suppression of iRhom2 expression inactivated PI3K/AKT pathway activation, contributing to ROS reduction in ox-LDL-stimulated macrophages. iRhom2 was also significantly expressed in ApoE−/- mice fed HFD. Finally, we observed increased serum levels of TNF-α, TNFR1, and TNFR2 in patients with coronary artery atherosclerosis as compared to healthy volunteers. In conclusion, our findings suggested that iRhom2 played a key role in the pathogenesis of atherosclerosis, and that iRhom2 might be a potential therapeutic target against atherosclerosis.

Abstract 493: Neutrophil and Macrophage Cell Surface Colony-Stimulating Factor-1 is Shed by a Disintegrin and Metalloprotease 17 and Contributes to Stimulating Macrophage Proliferation in Inflammation

Recent studies highlight a role for macrophage proliferation post monocyte recruitment in inflammatory conditions, such as atherosclerosis. However, the mechanisms regulating macrophage proliferation are not well understood. Using an acute peritonitis model, we identified a 40 ± 8% reduction (mean ± SEM; n=5; p&lt;0.01) in macrophages in the S phase of the cell cycle 40 hrs post thioglycollate injection in mice lacking the transmembrane protease ADAM17 in hematopoietic cells. ADAM17 is a member of a disintegrin and metalloprotease family that cleaves many cell surface proteins involved in inflammation. The macrophage proliferation defect in ADAM17 null chimeras was associated with a 60 ± 10% (n=5; p&lt;0.001) decrease in soluble macrophage colony-stimulating factor (CSF)-1 in the peritoneum, and was rescued by intraperitoneal injection of CSF-1. We demonstrate that neutrophils, the first innate immune cell that migrates into inflammatory sites, and macrophages are both major sources of cell surface (cs)CSF-1 in this acute inflammation model, adding to the current knowledge that stromal, epithelial and endothelial cells are sources of CSF-1. We show that ADAM17 is critical in mediating csCSF-1 release in the inflamed peritoneum, and that this release following neutrophil extravasation is associated with elevated expression of iRhom2, a member of the rhomboid-like superfamily, which promotes ADAM17 maturation and trafficking to the cell surface. Accordingly, deletion of hematopoietic iRhom2 is sufficient to prevent csCSF-1 release from neutrophils and macrophages, thus recapitulating the defect in macrophage proliferation seen in ADAM17 null cells. Our data indicate that csCSF-1 release and macrophage proliferation is self-limiting in states of acute inflammation due to the transient nature of leukocyte recruitment and temporally restricted csCSF-1 expression and release. In chronic inflammation such as atherosclerosis, we show that macrophage proliferation is decreased by 30 ± 7% (n=6; p&lt;0.01) in atherosclerotic lesions of ADAM17 hematopoietic null LDL receptor-deficient mice fed a high fat diet for 16 weeks. Together, these results demonstrate a novel mechanism governing macrophage proliferation in both acute and chronic inflammation.

P63 is a key regulator of iRHOM2 signalling in the keratinocyte stress response

Hyperproliferative keratinocytes induced by trauma, hyperkeratosis and/or inflammation display molecular signatures similar to those of palmoplantar epidermis. Inherited gain-of-function mutations in RHBDF2 (encoding iRHOM2) are associated with a hyperproliferative palmoplantar keratoderma and squamous oesophageal cancer syndrome (termed TOC). In contrast, genetic ablation of rhbdf2 in mice leads to a thinning of the mammalian footpad, and reduces keratinocyte hyperproliferation and migration. Here, we report that iRHOM2 is a novel target gene of p63 and that both p63 and iRHOM2 differentially regulate cellular stress-associated signalling pathways in normal and hyperproliferative keratinocytes. We demonstrate that p63–iRHOM2 regulates cell survival and response to oxidative stress via modulation of SURVIVIN and Cytoglobin, respectively. Furthermore, the antioxidant compound Sulforaphane downregulates p63–iRHOM2 expression, leading to reduced proliferation, inflammation, survival and ROS production. These findings elucidate a novel p63-associated pathway that identifies iRHOM2 modulation as a potential therapeutic target to treat hyperproliferative skin disease and neoplasia.

Endogenous Transmembrane TNF-Alpha Protects Against Premature Senescence in Endothelial Colony Forming Cells.

Transmembrane tumor necrosis factor-α (tmTNF-α) is the prime ligand for TNF receptor 2, which has been shown to mediate angiogenic and blood vessel repair activities in mice. We have previously reported that the angiogenic potential of highly proliferative endothelial colony-forming cells (ECFCs) can be explained by the absence of senescent cells, which in mature endothelial cells occupy >30% of the population, and that exposure to a chronic inflammatory environment induced premature, telomere-independent senescence in ECFCs. The goal of this study was to determine the role of tmTNF-α in the proliferation of ECFCs. Here, we show that tmTNF-α expression on ECFCs selects for higher proliferative potential and when removed from the cell surface promotes ECFC senescence. Moreover, the induction of premature senescence by chronic inflammatory conditions is blocked by inhibition of tmTNF-α cleavage. Indeed, the mechanism of chronic inflammation-induced premature senescence involves an abrogation of tmTNF/TNF receptor 2 signaling. This process is mediated by activation of the tmTNF cleavage metalloprotease TNF-α-converting enzyme via p38 MAP kinase activation and its concurrent export to the cell surface by means of increased iRhom2 expression. Thus, we conclude that tmTNF-α on the surface of highly proliferative ECFCs plays an important role in the regulation of their proliferative capacity.

AB0154 Pathogenic Role of Irhom2 in Seropositive Rheumatoid Arthritis

BackgroundTumor necrosis factor (TNF)-α is a major pathologic cytokine in rheumatoid arthritis (RA) and the current target of RA treatment has been on blocking the TNF-α. iRHOM2 is a regulator...