ADAM17 Activity Research Articles

Regulation of ADAM10 activity through microdomain-dependent intracellular calcium changes

A disintegrin and metalloproteinases (ADAMs) are transmembrane proteases that cleave other proteins close to the surface in a process called shedding. The prominent member ADAM10 has been linked to several pathologies such as Alzheimer’s disease, bacterial infection, cancer development and metastasis. Although the regulation of the ADAM10 activity by calcium influx and calmodulin inhibition has been reported, the spatiotemporal regulation of Ca2+-dependent ADAM10 activation and the required source of Ca2+ ions have not been thoroughly studied. In the present study, we observed the rapid Ca2+-dependent activation of ADAM10 in A549 lung carcinoma cells upon stimulation with ionomycin. The calmodulin-inhibitors trifluoperazine and ophiobolin A mediated delayed activation of ADAM10, which apparently did not depend on intracellular Ca2+ in the case of trifluoperazine. Furthermore, the surface translocation and release of ADAM10 in extracellular vesicles exhibited different kinetics and were only partially linked to catalytic activation. Finally, ADAM10 activation was observed after the entry of Ca2+ through certain channels, such as canonical members of transient receptor potential (TRP) channels. Therefore, the opening of particular channels for Ca2+ entry points and subsequent Ca2+ flux as well as the temporal aspects of the consequent increase in Ca2+ levels, must be considered for future therapeutic options involving the increasing or decreasing ADAM10 activity.

Cardiomyocyte-specific knockout of ADAM17 alleviates doxorubicin-induced cardiomyopathy via inhibiting TNFα–TRAF3–TAK1–MAPK axis

The pathogenesis of doxorubicin-induced cardiomyopathy remains unclear. This study was carried out to test our hypothesis that ADAM17 aggravates cardiomyocyte apoptosis induced by doxorubicin and inhibition of ADAM17 may ameliorate doxorubicin-induced cardiomyopathy. C57BL/6J mice were intraperitoneally injected with a cumulative dose of doxorubicin to induce cardiomyopathy. Cardiomyocyte-specific ADAM17-knockout (A17α-MHCKO) and ADAM17-overexpressing (AAV9-oeA17) mice were generated. In addition, RNA sequencing of the heart tissues in different mouse groups and in vitro experiments in neonatal rat cardiomyocytes (NRCMs) receiving different treatment were performed. Mouse tumor models were constructed in A17fl/fl and A17α-MHCKO mice. In addition, cardiomyocyte-specific TRAF3-knockdown and TRAF3-overexpressing mice were generated. ADAM17 expression and activity were markedly upregulated in doxorubicin-treated mouse hearts and NRCMs. A17α-MHCKO mice showed less cardiomyocyte apoptosis induced by doxorubicin than A17fl/fl mice, and cardiomyocyte ADAM17 deficiency did not affect the anti-tumor effect of doxorubicin. In contrast, AAV9-oeA17 mice exhibited markedly aggravated cardiomyocyte apoptosis relative to AAV9-oeNC mice after doxorubicin treatment. Mechanistically, doxorubicin enhanced the expression of transcription factor C/EBPβ, leading to increased expression and activity of ADAM17 in cardiomyocyte, which enhanced TNF-α shedding and upregulated the expression of TRAF3. Increased TRAF3 promoted TAK1 autophosphorylation, resulting in activated MAPKs pathway and cardiomyocyte apoptosis. ADAM17 acted as a positive regulator of cardiomyocyte apoptosis and cardiac remodeling and dysfunction induced by doxorubicin by upregulating TRAF3/TAK1/MAPKs signaling. Thus, targeting ADAM17/TRAF3/TAK1/MAPKs signaling holds a promising potential for treating doxorubicin-induced cardiotoxicity.

The metalloproteinase ADAM10 sheds angiotensin-converting enzyme (ACE) from the pulmonary endothelium as a soluble, functionally active convertase.

The renin-angiotensin-aldosterone system (RAAS) plays a critical role in the regulation of blood pressure and fluid balance, with angiotensin-converting enzyme (ACE) being a key transmembrane enzyme that converts angiotensin I to angiotensin II. Hence, ACE activity is an important drug target in cardiovascular pathologies such as hypertension. Our study demonstrates that human pulmonary microvascular endothelial cells (HPMECs) are an important source of proteolytically released ACE. The proteolytic release of transmembrane proteins, a process known as ectodomain shedding, is facilitated by membrane proteases called sheddases. By knockout and inhibition studies, we identified ADAM10 (A disintegrin and metalloprotease 10) as a primary sheddase responsible for ACE release in HEK293 cells. The function of ADAM10 as primary, constitutive sheddase of ACE was confirmed in HPMECs. Moreover, we demonstrated the physiological relevance of ADAM10 for ACE shedding in exvivo precision cut lung slices (PCLS) from human and mouse lungs. Notably, ADAM17 activity is not directly involved in ACE shedding but indirectly by regulating ACE mRNA and protein levels, leading to increased ADAM10-mediated ACE shedding. Importantly, soluble ACE generated by shedding is enzymatically active and can thereby participate in systemic RAAS functions. Taken together, our findings highlight the critical role of ADAM10 (directly) and ADAM17 (indirectly) in ACE shedding and RAAS modulation.

Lactate increases ADAM10 activity and reduces BACE1 activity in mouse brain.

The accumulation and aggregation of beta-amyloid (Aβ) peptides contributes to neuronal dysfunction and death. These Aβ peptides originate from a transmembrane protein known as amyloid precursor protein (APP), which can be processed via two competing pathways. Alpha-secretase (ADAM10) cleavage is thought to be neuroprotective while beta-secretase (BACE1) cleavage results in the production of Aβ. Aerobic exercise reduces BACE1 activity, but the mechanisms involved are unknown though several exercise-induced mediators such as lactate may be involved. The current study examined whether systemic lactate can alter APP processing and BACE1 and ADAM10 activity. Mice were randomly assigned to one of four groups (n=10 per group): (1) sedentary; (2) lactate-injection (1.0gkg-1 body mass); (3) exercise; and (4) exercise and oxamate (lactate dehydrogenase inhibitor; 750mgkg-1 body mass). Two hours following intervention, the hippocampus and prefrontal cortex (PFC) were collected. In the PFC lactate-injection and exercise resulted in higher ADAM10 activity compared to sedentary (exercise P=0.0215, lactate P=0.0038), in the hippocampus lactate-injection was higher compared to sedentary (lactate P=0.011), and this was absent in the presence of oxamate. Hippocampal BACE1 activity was lower in the lactate group compared to the exercise group (P=0.01). Oxamate resulted in higher BACE1 protein content compared to sedentary in the PFC (vs. sedentary P=0.048). These findings suggest that lactate is important for regulating ADAM10 activity and thereby shifts APP processing away from Aβ production. KEY POINTS: Exercise is known to alter the processing of amyloid precursor protein by reducing the activity of the rate-limiting enzyme BACE1 and increasing the activity of ADAM10. It is thought that exercise-induced factors are responsible for these enzymatic changes. This study examined if lactate accumulation plays a role in this process. Mice were assigned to one of four groups: sedentary, lactate, exercise and exercise + lactate. The findings demonstrate that lactate accumulation alters brain BACE1 and ADAM10 and shifts amyloid precursor protein processing away from beta-amyloid production.

Kaempferol reduces pyroptosis in acute lung injury by decreasing ADAM10 activity through the NLRP3/GSDMD pathway

Sepsis-associated acute lung injury (SA-ALI) is a significant problem in intensive care units worldwide, and no specific treatment is currently available. Therefore, it is crucial to identify new potential therapeutic targets for SA-ALI, in which inflammation and inflammation-activated pyroptosis play an important role. In recent years, a disintegrin and metalloprotease (ADAM)10 and ADAM17, which regulate inflammation and the immune system, have become essential targets. However, the efficacy of ADAM10/17 inhibition on pyroptosis in SA-ALI is unclear. Hence, we aimed to investigate the effects of GW280264X, a selective ADAM10/17 inhibitor, and kaempferol, a natural product frequently consumed by humans, on pyroptosis in lipopolysaccharide (LPS)-induced SA-ALI model in male and female mice. Enzyme-linked immunoassay (ELISA) was used to measure C-reactive protein (CRP), procalcitonin (PCT), interleukin (IL)-1β, IL-10, tumor necrosis factor-α (TNF-α), glucose-regulated protein-78 (GRP78) and vascular adhesion molecule-1 (VCAM-1) levels. Western blotting was used to measure levels of ADAM10, ADAM17, nod-like protein receptor-3 (NLRP3), gasdermin D (GSDMD/GSDMD-N), and caspase-1 (Cas-1). In addition, lung tissue was evaluated histopathologically. In the present study, reduced inflammation (CRP, PCT, IL-1β, VCAM-1), pyroptosis (NLRP3, GSDMD-N, Cas-1), endoplasmic reticulum stress (GRP78), and histopathological damage were found by decreasing ADAM10 levels using kaempferol. These results strongly suggest that kaempferol-mediated ADAM10 inhibition may be a promising therapeutic target against pyroptosis in SA-ALI, offering a potential breakthrough in the treatment of this condition.

Targeting iRhom2/ADAM17 attenuates COVID-19-induced cytokine release from cultured lung epithelial cells

The COVID-19 pandemic, caused by SARS-CoV-2, continues to pose a significant global health challenge, with acute respiratory distress syndrome (ARDS) being a major cause of mortality. Excessive cytokine release (cytokine storm) has been causally related to COVID-19-associated ARDS. While TNF-α inhibitors have shown potential in reducing inflammation, their broad effects on TNF-α signaling, including both pro- and anti-inflammatory pathways, present significant challenges and side effects in clinical use. Therefore, more precise therapeutic targets are urgently needed. ADAM17 is a key enzyme driving cytokine release, but its broad presence complicates direct inhibition. Targeting iRhom2, a regulator specific to immune cells that controls ADAM17's activity, offers a more focused and effective approach to reducing cytokine release. In this study, we hypothesized that targeted inhibition of ADAM-17/iRhom2 attenuates COVID-19-induced cytokine release in cultured lung epithelial cells. Human primary bronchial/tracheal epithelial cells challenged with COVID-19 pseudo-viral particles resulted in elevated cytokine release, which was attenuated following siRNA-mediated silencing of ADAM17 and iRhom2. Targeting ADAM-17/iRhom2 pathway may thus represent a strategy to overcome the COVID-19-associated ARDS.

Neuroprotection by ADAM10 inhibition requires TrkB signaling in the Huntington’s disease hippocampus

Synaptic dysfunction is an early pathogenic event leading to cognitive decline in Huntington’s disease (HD). We previously reported that the active ADAM10 level is increased in the HD cortex and striatum, causing excessive proteolysis of the synaptic cell adhesion protein N-Cadherin. Conversely, ADAM10 inhibition is neuroprotective and prevents cognitive decline in HD mice. Although the breakdown of cortico-striatal connection has been historically linked to cognitive deterioration in HD, dendritic spine loss and long-term potentiation (LTP) defects identified in the HD hippocampus are also thought to contribute to the cognitive symptoms of the disease. The aim of this study is to investigate the contribution of ADAM10 to spine pathology and LTP defects of the HD hippocampus. We provide evidence that active ADAM10 is increased in the hippocampus of two mouse models of HD, leading to extensive proteolysis of N-Cadherin, which has a widely recognized role in spine morphology and synaptic plasticity. Importantly, the conditional heterozygous deletion of ADAM10 in the forebrain of HD mice resulted in the recovery of spine loss and ultrastructural synaptic defects in CA1 pyramidal neurons. Meanwhile, normalization of the active ADAM10 level increased the pool of synaptic BDNF protein and activated ERK neuroprotective signaling in the HD hippocampus. We also show that the ADAM10 inhibitor GI254023X restored LTP defects and increased the density of mushroom spines enriched with GluA1-AMPA receptors in HD hippocampal neurons. Notably, we report that administration of the TrkB antagonist ANA12 to HD hippocampal neurons reduced the beneficial effect of GI254023X, indicating that the BDNF receptor TrkB contributes to mediate the neuroprotective activity exerted by ADAM10 inhibition in HD. Collectively, these findings indicate that ADAM10 inhibition coupled with TrkB signaling represents an efficacious strategy to prevent hippocampal synaptic plasticity defects and cognitive dysfunction in HD.

Examination of Akt and GSK3β in BDNF-mediated reductions in BACE1 activity in neuronal cells.

Brain-derived neurotrophic factor (BDNF) content and signaling has been identified as one potential regulator of amyloid precursor protein (APP) processing. Recently published work has demonstrated that BDNF reduces BACE1 activity while also elevating the inhibition of GSK3β in the prefrontal cortex of male C57BL/6J mice. These results provide evidence that BDNF alters APP processing by reducing BACE1 activity, which may act through GSK3β inhibition. The purpose of this study was to further explore the role of GSK3β in BDNF-induced regulation on BACE1 activity. We utilized a cell culture and an invitro activity assay model to pharmacologically target BDNF and GSK3β signaling to confirm its involvement in the BDNF response. Treatment of differentiated SH-SY5Y neuronal cells with 75 ng/mL BDNF resulted in elevated pTrkB content, pAkt content, pGSK3β content, and reduced BACE1 activity. An invitro BACE1 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF reduced BACE1 activity; however, in the presence of TrkB or Akt inhibition, this effect was abolished. An invitro ADAM10 activity assay utilizing mouse prefrontal cortex (n = 6/group) supplemented with BDNF, BDNF + ANA12 (Trkb antagonist), or BDNF + wortmannin (Akt inhibitor) demonstrated that BDNF did not alter ADAM10 activity. However, inhibiting BDNF signaling reduced ADAM10 activity. Collectively these studies suggest that GSK3β inhibition may be necessary for BDNF-induced reductions in BACE1 activity. These findings will allow for the optimization of future therapeutic strategies by selectively targeting TrkB activation and GSK3β inhibition.

Potential Mechanisms Underlying COVID-19-Mediated Central and Peripheral Demyelination: Roles of the RAAS and ADAM-17.

Demyelination is among the most conspicuous neurological sequelae of SARS-CoV-2 infection (COVID-19) in both the central (CNS) and peripheral (PNS) nervous systems. Several hypotheses have been proposed to explain the mechanisms underlying demyelination in COVID-19. However, none have considered the SARS-CoV-2's effects on the renin-angiotensin-aldosterone system (RAAS). Therefore, our objective in this review is to evaluate how RAAS imbalance, caused by direct and indirect effects of SARS-CoV-2 infection, could contribute to myelin loss in the PNS and CNS. In the PNS, we propose that demyelination transpires from two significant changes induced by SARS-CoV-2 infection, which include upregulation of ADAM-17 and induction of lymphopenia. Whereas, in the CNS, demyelination could result from RAAS imbalance triggering two alterations: (1) a decrease in angiotensin type II receptor (AT2R) activity, responsible for restraining defense cells' action on myelin; (2) upregulation of ADAM-17 activity, leading to impaired maturation of oligodendrocytes and myelin formation. Thus, we hypothesize that increased ADAM-17 activity and decreased AT2R activity play roles in SARS-CoV-2 infection-mediated demyelination in the CNS.

ADAM10 as a major activator of reactive oxygen species production and klotho shedding in podocytes under diabetic conditions

Early stages of diabetes are characterized by elevations of insulin and glucose concentrations. Both factors stimulate reactive oxygen species (ROS) production, leading to impairments in podocyte function and disruption of the glomerular filtration barrier. Podocytes were recently shown to be an important source of αKlotho (αKL) expression. Low blood Klotho concentrations are also associated with an increase in albuminuria, especially in patients with diabetes. We investigated whether ADAM10, which is known to cleave αKL, is activated in glomeruli and podocytes under diabetic conditions and the potential mechanisms by which ADAM10 mediates ROS production and disturbances of the glomerular filtration barrier.In cultured human podocytes, high glucose increased ADAM10 expression, shedding, and activity, NADPH oxidase activity, ROS production, and albumin permeability. These effects of glucose were inhibited when cells were pretreated with an ADAM10 inhibitor or transfected with short-hairpin ADAM10 (shADAM10) or after the addition soluble Klotho. We also observed increases in ADAM10 activity, NOX4 expression, NADPH oxidase activity, and ROS production in αKL-depleted podocytes. This was accompanied by an increase in albumin permeability in shKL-expressing podocytes. The protein expression and activity of ADAM10 also increased in isolated glomeruli and urine samples from diabetic rats. Altogether, these results reveal a new mechanism by which hyperglycemia in diabetes increases albumin permeability through ADAM10 activation and an increase in oxidative stress via NOX4 enzyme activation. Moreover, αKlotho downregulates ADAM10 activity and supports redox balance, consequently protecting the slit diaphragm of podocyteσ under hyperglycemic conditions.

Combination of ADAM17 knockdown with eplerenone is more effective than single therapy in ameliorating diabetic cardiomyopathy.

The renin-angiotensin-aldosterone system (RAAS) members, especially Ang II and aldosterone, play key roles in the pathogenesis of diabetic cardiomyopathy (DCM). Angiotensin-converting enzyme inhibitors or angiotensin-receptor blockers combined with aldosterone receptor antagonists (mineralocorticoid receptor antagonists) have substantially improved clinical outcomes in patients with DCM. However, the use of the combination has been limited due to its high risk of inducing hyperkalemia. Type 1 diabetes was induced in 8-week-old male C57BL/6J mice by intraperitoneal injection of streptozotocin at a dose of 55mg/kg for 5 consecutive days. Adeno-associated virus 9-mediated short-hairpin RNA (shRNA) was used to knock down the expression of ADAM17 in mice hearts. Eplerenone was administered via gavage at 200mg/kg daily for 4weeks. Primary cardiac fibroblasts were exposed to high glucose (HG) in vitro for 24h to examine the cardiac fibroblasts to myofibroblasts transformation (CMT). Cardiac collagen deposition and CMT increased in diabetic mice, leading to cardiac fibrosis and dysfunction. In addition, ADAM17 expression and activity increased in the hearts of diabetic mice. ADAM17 inhibition and eplerenone treatment both improved diabetes-induced cardiac fibrosis, cardiac hypertrophy and cardiac dysfunction, ADAM17 deficiency combined with eplerenone further reduced the effects of cardiac fibrosis, cardiac hypertrophy and cardiac dysfunction compared with single therapy in vivo. High-glucose stimulation promotes CMT in vitro and leads to increased ADAM17 expression and activity. ADAM17 knockdown and eplerenone pretreatment can reduce the CMT of fibroblasts that is induced by high glucose levels by inhibiting TGFβ1/Smad3 activation; the combination of the two can further reduce CMT compared with single therapy in vitro. Our findings indicated that ADAM17 knockout could improve diabetes-induced cardiac dysfunction and remodeling through the inhibition of RAAS overactivation when combined with eplerenone treatment, which reduced TGF-β1/Smad3 pathway activation-mediated CMT. The combined intervention of ADAM17 deficiency and eplerenone therapy provided additional cardiac protection compared with a single therapy alone without disturbing potassium level. Therefore, the combination of ADAM17 inhibition and eplerenone is a potential therapeutic strategy for human DCM.

Sex Dependent Exacerbation of Salt Sensitive Hypertension and Cardiac Dysfunction Is Associated With Upregulation of miRNA Targeting ADAM17 and ACE2 in the Brain

Background: The DOCA-salt model has been widely used to study salt-sensitive hypertension and we previously reported that it is associated with ADAM17-mediated ACE2 shedding in the central nervous system. However, previously reported data are mainly from male mice, and it is unclear whether female mice exhibit the same phenotypes. Objective: We aimed to compare sex specific development of salt sensitive hypertension and cardiac dysfunction and investigate miRNA regulation associated with these changes. Methods: C57 mice (male and females, n = 8 -15) underwent uninephrectomy and telemetry implantation (DSI Inc.) for blood pressure (BP) conscious recording. After baseline recording, a DOCA pellet (50 mg, 21 days, Innovative Research of America) was implanted subcutaneously and drinking water was replaced by 1% NaCl solution to induce salt sensitive hypertension. Cerebral Spinal fluid (CSF) was collected at baseline and after 3 weeks of DOCA-salt treatment for exosome miRNA sequencing (Creative Biolab). In another set of mice, cardiac function was assessed by echocardiography (Visualsonics Vevo F2) and invasive left ventricular hemodynamics (Transonic). Mortality from each group was analyzed as well. Results: Baseline BP was not different between male and female mice. Following DOCA-salt treatment for 3 weeks, BP was increased in both sexes, with higher systolic BP observed in males compared to females (active phase: 173.0 ± 19.6 vs. 154.2 ± 19.1 mmHg, p<0.05). Although ejection fraction was not changed in either male or female mice, mitral peak early diastolic filling velocity and tissue doppler showed that E/E’ was increased in both sexes compared to baselines (Male: 33.4 ± 4 vs. 26.6 ± 1.9; Female: 30.3 ± 5.0 vs. 26.4 ± 4), with a greater increase in males (25.3% vs. 14.4%). In addition, hemodynamics revealed increased left ventricular end-diastolic pressure (11.1 ± 1.9 vs. 4.5 ± 0.5 mmHg, p<0.01) and relaxation time constant Tau (6.7 ± 1.0 vs. 4.9 ± 0.5 msec, p<0.01) in DOCA-salt treated males compared to non-treated controls, with no changes shown in female mice. These data suggest worsened cardiac diastolic function in males compared to females following DOCA-salt treatment. Moreover, a 20% mortality was observed in males while no death occured in females, confirming a more severe phenotype in males. Furthermore, miRNAseq analysis of CSF exosomes showed several upregulated miRNA in males but not in females following DOCA-salt treatment. Notably, miR206 associated with increased ADAM17 activity was increased by 8-fold in males but only 3-fold n females. Conclusion: DOCA-salt treatment promotes exacerbated hypertension, impaired cardiac function and mortality in a sex dependent manner, associated with upregulation of key miRNA targeting the brain renin-angiotensin system. The changes of ADAM17 and ACE2 targeted exosome miRNAs in the central nervous system might contribute to the sex difference in the cardiovascular dysfunction phenotypes in this model. This work was supported in part by a research grant from the National Institutes of Health (HL163588). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

GW501516-Mediated Targeting of Tetraspanin 15 Regulates ADAM10-Dependent N-Cadherin Cleavage in Invasive Bladder Cancer Cells.

Bladder cancer aggressiveness is correlated with abnormal N-cadherin transmembrane glycoprotein expression. This protein is cleaved by the metalloprotease ADAM10 and the γ-secretase complex releasing a pro-angiogenic N-terminal fragment (NTF) and a proliferation-activating soluble C-terminal fragment (CTF2). Tetraspanin 15 (Tspan15) is identified as an ADAM10-interacting protein to induce selective N-cadherin cleavage. We first demonstrated, in invasive T24 bladder cancer cells, that N-cadherin was cleaved by ADAM10 generating NTF in the extracellular environment and leaving a membrane-anchored CTF1 fragment and that Tspan15 is required for ADAM10 to induce the selective N-cadherin cleavage. Targeting N-cadherin function in cancer is relevant to preventing tumor progression and metastases. For antitumor molecules to inhibit N-cadherin function, they should be complete and not cleaved. We first showed that the GW501516, an agonist of the nuclear receptor PPARβ/δ, decreased Tspan15 and prevented N-cadherin cleavage thus decreasing NTF. Interestingly, the drug did not modify ADAM10 expression, which was important because it could limit side effects since ADAM10 cleaves numerous substrates. By targeting Tspan15 to block ADAM10 activity on N-cadherin, GW501516 could prevent NTF pro-tumoral effects and be a promising molecule to treat bladder cancer. More interestingly, it could optimize the effects of the N-cadherin antagonists those such as ADH-1 that target the N-cadherin ectodomain.

The possible role of platelet APP processing in the pathophysiology of Alzheimer’s Disease

IntroductionAlzheimer’s disease (AD) stands as the most prevalent form of dementia. Alzheimer’s Disease is acknowledged to have a complex origin, a gradual neurodegenerative progression, and a wide-ranging clinical profile marked primarily by progressive memory loss, cognitive decline, and various functional impairments that significantly diminish the quality of life: Key characteristics of AD encompass the presence of amyloid plaques, which are characterized by the pathological accumulation of insoluble β-amyloid (Aβ) aggregates within the brain tissue and blood vessel walls.Several reports have indicated the existence of cerebral abnormalities within platelets in individuals afflicted by AD.ObjectivesThe objective of this investigation was to review studies investigating the metabolism of APP in platelets among individuals with AD to identify potential dependable peripheral indicators leading to novel approaches to its management and treatment.MethodsA systematic review according to the PRISMA guidelines was carried out, by accessing the PubMed database up to June 2023. The authors screened the titles and the abstracts of all the potentially relevant papers on the basis of a strict list of exclusion and inclusion criteria.ResultsA total of thirty-two studies were included. The evidence points towards the observation that AD individuals exhibit various modifications in platelet APP processing when compared to matched healthy controls, that are frequently associated with the severity of cognitive impairment and functional independence. The majority of the evidence supports changes in platelet ADAM-10 activity, β-secretase activity, APP ratio, a state of heightened platelet activation or hyper-responsiveness, and a potential release of platelet APP via vesicular mechanisms, which may ultimately contribute to Aβ production.ConclusionsPlatelets offer a promising peripheral model for detecting and evaluating molecular changes associated with AD, as they hold the potential to provide vital insights into the development of an effective diagnostic tool and open doors to innovative therapeutic approaches.Disclosure of InterestNone Declared

Abstract 6990: RHBDF1 is a potential modulator of EGFR activation and oncogenic biomarker in triple-negative breast cancer

Abstract Recently, our proteomics study on breast cancer (BC) reported a possible association of RHBDF1 with triple-negative breast cancer (TNBC). The overall survival of TNBC patients on METABRIC data set implicated that the RHBDF1 high-expression group has a shorter life expectancy than the RHBDF1 low-expression group. Although many TNBC patients have high expression levels of RHBDF1 and poor prognosis, the mechanism of RHBDF1 in the pathogenesis of TNBC remained unclear. Rhomboid 5 homolog 1 (RHBDF1) is a catalytically inactive pseudoprotease on the Golgi and endoplasmic reticulum membrane. Despite its inactive status of peptidase, it regulates the activity of ADAM17 protease that cleaves the extracellular portion of EGFR ligands such as TGF alpha and EGF. Eventually, RHBDF1 and ADAM17 complex activate the EGFR signaling pathway, thereby increasing cell proliferation, migration, and tumorigenesis. First of all, we recruited the clinical data of the overall survival (OS) and disease-free survival (DFS) in the hospital of Seoul National University (SNUH cohort, n= 203) and compared IHC score 0 or 1 with survival rate. To investigate the underlying biological mechanism of RHBDF1 in TNBC, the effect of knockdown or overexpression of RHBDF1 was observed in TNBC cell lines (MDA-MB-468, HCC1143 and BT20) using siRNA (small interfering RNA) and lentiviral systems. Cell proliferation assay was performed to clarify whether RHBDF1 affects cell growth. Then, we estimated the ability of migration, invasion, and sphere formation to confirm the characteristics of cancer cells including cancer stemness and metastasis. SNUH cohort with TNBC patients showed that the RHBDF1 high-expression group has a poorer disease-free survival (DFS) than the low-expression group (p<0.05). In vitro assay, RHBDF1 knockdown inhibited proliferation in MDA-MB-468 and HCC1143. RHBDF1 downregulation also decreased the ability of migration and invasion. We found that RHBDF1 activates specific cell growth signaling pathways and increases cell mobility in TNBC. The sphere formation assay was performed to measure cancer stemness characteristics. As a result, RHBDF1 knockdown TNBC cell lines couldn’t form spheres relative to RHBDF1 overexpressed TNBC cell lines. In conclusion, we demonstrated that RHBDF1, a potential triple-negative breast cancer biomarker, is highly amplified in TNBC patients and TNBC cell lines. Its functions of accelerating cell proliferation, metastasis and cancer stemness were unveiled. But we need to study ultimately how RHBDF1 regulates ADAM17 activity at Golgi and endoplasmic reticulum membrane and which transcription factor activates RHBDF1 expression. Citation Format: Hayeon Kim, Soo Young Park, Da Sol Kim, Cheng Hyun Lee, Min Ji Song, Han Suk Ryu. RHBDF1 is a potential modulator of EGFR activation and oncogenic biomarker in triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6990.

Pathological mutations reveal the key role of the cytosolic iRhom2 N-terminus for phosphorylation-independent 14-3-3 interaction and ADAM17 binding, stability, and activity

The protease ADAM17 plays an important role in inflammation and cancer and is regulated by iRhom2. Mutations in the cytosolic N-terminus of human iRhom2 cause tylosis with oesophageal cancer (TOC). In mice, partial deletion of the N-terminus results in a curly hair phenotype (cub). These pathological consequences are consistent with our findings that iRhom2 is highly expressed in keratinocytes and in oesophageal cancer. Cub and TOC are associated with hyperactivation of ADAM17-dependent EGFR signalling. However, the underlying molecular mechanisms are not understood. We have identified a non-canonical, phosphorylation-independent 14-3-3 interaction site that encompasses all known TOC mutations. Disruption of this site dysregulates ADAM17 activity. The larger cub deletion also includes the TOC site and thus also dysregulated ADAM17 activity. The cub deletion, but not the TOC mutation, also causes severe reductions in stimulated shedding, binding, and stability of ADAM17, demonstrating the presence of additional regulatory sites in the N-terminus of iRhom2. Overall, this study contrasts the TOC and cub mutations, illustrates their different molecular consequences, and reveals important key functions of the iRhom2 N-terminus in regulating ADAM17.

Apoptosis-mediated ADAM10 activation removes a mucin barrier promoting T cell efferocytosis

Efferocytic clearance of apoptotic cells in general, and T cells in particular, is required for tissue and immune homeostasis. Transmembrane mucins are extended glycoproteins highly expressed in the cell glycocalyx that function as a barrier to phagocytosis. Whether and how mucins may be regulated during cell death to facilitate efferocytic corpse clearance is not well understood. Here we show that normal and transformed human T cells express a subset of mucins which are rapidly and selectively removed from the cell surface during apoptosis. This process is mediated by the ADAM10 sheddase, the activity of which is associated with XKR8-catalyzed flipping of phosphatidylserine to the outer leaflet of the plasma membrane. Mucin clearance enhances uptake of apoptotic T cells by macrophages, confirming mucins as an enzymatically-modulatable barrier to efferocytosis. Together these findings demonstrate a glycocalyx regulatory pathway with implications for therapeutic intervention in the clearance of normal and transformed apoptotic T cells.

Neurotensin induces sustainable activation of the ErbB-ERK1/2 pathway, which is required for developmental competence of oocytes in mice.

LH induces the expression of EGF-like factors and their shedding enzyme (ADAM17) in granulosa cells (GCs), which is essential for ovulation via activation of the ErbB-ERK1/2 pathway in cumulus cells (CCs). Neurotensin (NTS) is reported as a novel regulator of ovulation, whereas the NTS-induced maturation mechanism in oocytes remains unclear. In this study, we focused on the role of NTS in the expression of EGF-like factors and ErbBs, and ADAM17 activity, during oocyte maturation and ovulation in mice. The expression and localization in GC and CC were examined. Next, hCG and NTS receptor 1 antagonist (SR) were injected into eCG-primed mice, and the effects of SR on ERK1/2 phosphorylation were investigated. Finally, we explored the effects of SR on the expression of EGF-like factors and ErbBs, and ADAM17 activity in GC and CC. NTS was significantly upregulated in GC and CC following hCG injection. SR injection suppressed oocyte maturation and ERK1/2 phosphorylation. SR also downregulated part of the expression of EGF-like factors and their receptors, and ADAM17 activity. NTS induces oocyte maturation through the sustainable activation of the ERK1/2 signaling pathway by upregulating part of the EGF-like factor-induced pathway during oocyte maturation in mice.

Hepatocellular RECK as a Critical Regulator of Metabolic Dysfunction-associated Steatohepatitis Development

Reversion-inducing cysteine-rich protein with Kazal motifs (RECK) is an extracellular matrix regulator with anti-fibrotic effects. However, its expression and role in metabolic dysfunction-associated steatohepatitis (MASH) and hepatic fibrosis are poorly understood. We generated a novel transgenic mouse model with RECK overexpression specifically in hepatocytes to investigate its role in Western diet (WD)-induced liver disease. Proteomic analysis and invitro studies were performed to mechanistically link RECK to hepatic inflammation and fibrosis. Our results show that RECK expression is significantly decreased in liver biopsies from human patients diagnosed with MASH and correlated negatively with severity of metabolic dysfunction-associated steatotic liver disease (MASLD) and fibrosis. Similarly, RECK expression is downregulated in WD-induced MASH in wild-type mice. Hepatocyte-specific RECK overexpression significantly reduced hepatic pathology in WD-induced liver injury. Proteomic analysis highlighted changes in extracellular matrix and cell-signaling proteins. Invitro mechanistic studies linked RECK induction to reduced ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) and ADAM17 activity, amphiregulin release, epidermal growth factor receptor activation, and stellate cell activation. Our invivo and mechanistic invitro studies reveal that RECK is a novel upstream regulator of inflammation and fibrosis in the diseased liver, its induction is hepatoprotective, and thus highlights its potential as a novel therapeutic in MASH.

Modulation of gut microbiota with probiotics as a strategy to counteract endogenous and exogenous neurotoxicity.

The existing data demonstrate that probiotic supplementation affords protective effects against neurotoxicity of exogenous (e.g., metals, ethanol, propionic acid, aflatoxin B1, organic pollutants) and endogenous (e.g., LPS, glucose, Aβ, phospho-tau, α-synuclein) agents. Although the protective mechanisms of probiotic treatments differ between various neurotoxic agents, several key mechanisms at both the intestinal and brain levels seem inherent to all of them. Specifically, probiotic-induced improvement in gut microbiota diversity and taxonomic characteristics results in modulation of gut-derived metabolite production with increased secretion of SFCA. Moreover, modulation of gut microbiota results in inhibition of intestinal absorption of neurotoxic agents and their deposition in brain. Probiotics also maintain gut wall integrity and inhibit intestinal inflammation, thus reducing systemic levels of LPS. Centrally, probiotics ameliorate neurotoxin-induced neuroinflammation by decreasing LPS-induced TLR4/MyD88/NF-κB signaling and prevention of microglia activation. Neuroprotective mechanisms of probiotics also include inhibition of apoptosis and oxidative stress, at least partially by up-regulation of SIRT1 signaling. Moreover, probiotics reduce inhibitory effect of neurotoxic agents on BDNF expression, on neurogenesis, and on synaptic function. They can also reverse altered neurotransmitter metabolism and exert an antiamyloidogenic effect. The latter may be due to up-regulation of ADAM10 activity and down-regulation of presenilin 1 expression. Therefore, in view of the multiple mechanisms invoked for the neuroprotective effect of probiotics, as well as their high tolerance and safety, the use of probiotics should be considered as a therapeutic strategy for ameliorating adverse brain effects of various endogenous and exogenous agents.