ADAM-10 and -17 regulate endometriotic cell migration via concerted ligand and receptor shedding feedback on kinase signaling

A Disintegrin and Metalloproteinases (ADAMs) are the principal enzymes for shedding receptor tyrosine kinase (RTK) ectodomains and ligands from the cell surface. Multiple layers of activity regulation, feedback, and catalytic promiscuity impede our understanding of context-dependent ADAM “sheddase” function and our ability to predictably target that function in disease. This study uses combined measurement and computational modeling to examine how various growth factor environments influence sheddase activity and cell migration in the invasive disease of endometriosis. We find that ADAM-10 and -17 dynamically integrate numerous signaling pathways to direct cell motility. Data-driven modeling reveals that induced cell migration is a quantitative function of positive feedback through EGF ligand release and negative feedback through RTK shedding. Although sheddase inhibition prevents autocrine ligand shedding and resultant EGF receptor transactivation, it also leads to an accumulation of phosphorylated receptors (HER2, HER4, and MET) on the cell surface, which subsequently enhances Jnk/p38 signaling. Jnk/p38 inhibition reduces cell migration by blocking sheddase activity while additionally preventing the compensatory signaling from accumulated RTKs. In contrast, Mek inhibition reduces ADAM-10 and -17 activities but fails to inhibit compensatory signaling from accumulated RTKs, which actually enhances cell motility in some contexts. Thus, here we present a sheddase-based mechanism of rapidly acquired resistance to Mek inhibition through reduced RTK shedding that can be overcome with rationally directed combination inhibitor treatment. We investigate the clinical relevance of these findings using targeted proteomics of peritoneal fluid from endometriosis patients and find growth-factor–driven ADAM-10 activity and MET shedding are jointly dysregulated with disease.A Disintegrin and Metalloproteinases (ADAMs), especially ADAM-10 and -17, are the principal mediators of proteolytic ectodomain shedding on the cell surface (1). ADAMs and the closely related matrix metalloproteinases (MMPs) work together as “sheddases” to cleave hundreds of diverse transmembrane substrates including growth factor ligands, receptor tyrosine kinases (RTKs), adhesion molecules, and even proteases themselves from the cell surface. Unfortunately, little is known regarding how such a broad palette of proteolytic activity integrates to modulate behaviors such as cellular motility. Furthermore, extensive cross-talk and complexity among signaling networks, proteases, and their substrates make understanding sheddase regulation on a component-by-component basis challenging (2). Therapeutics have targeted sheddases and their substrates for the treatment of invasive diseases such as cancer, yet many of these inhibitors have failed in clinical trials (3). Therefore, a need exists for understanding how the balance of sheddase-mediated degradation integrates multiple layers of signaling networks to coordinately influence cell behavior in various disease contexts.Here we study how sheddase activity contributes to cell migration in the invasive disease of endometriosis, defined by the presence of endometrial-like tissue residing outside the uterus. Up to 10% of adult females and 40% of infertile women have the disease, which also exhibits comorbidity with several cancers (4, 5). Endometriosis currently has no cure: hormonal therapies merely manage the disease with significant side effects, and surgery provides only temporary relief for many, with recurrence rates as great as 40% within 5 y postoperation (6). Like cancer, endometriosis is associated with aberrant cell invasion into ectopic organ sites, and endometriotic tissues often exhibit dysregulated molecular pathways commonly perturbed in other invasive diseases. Mitogenic and inflammatory phospho-signaling [for example, phosphorylated extracellular-signal-related kinase 1/2 (p-Erk1/2), phosphorylated protein kinase B (p-Akt), and phosphorylated p38 mitogen-activated protein kinase (p-p38)], RTKs (including epidermal growth factor receptor, EGFR), and metalloproteinases have all been clinically associated with endometriosis (7, 8), and consequently represent attractive therapeutic strategies (9–11).Many challenges in developing targeted therapeutics stem from network-level complexities such as compensatory feedback, and recent work has demonstrated how critical such mechanisms are to achieving therapeutic success, especially in cancer (12, 13). Computational models of systems-level biochemical networks have shown promise as tools to understand how multiple enzymatic reactions integrate to impact overall biological behavior, often with the goal of aiding the design of personalized or combination therapies (14, 15). Considering its complex role in disease, sheddase regulation represents an ideal application of such network-level approaches. In this work, we apply the “cue–signal–response” (CSR) paradigm (14, 15) (Fig. 1A) to examine how disease-implicated growth-factor cues interact with experimentally monitored phospho-protein and protease networks (collectively referred to as signals), ultimately to influence cellular migration response. Computational modeling elucidates quantitative and predictive relationships among multiple layers of experimental data and offers testable hypotheses of context-dependent behavior and signaling feedback. We find ADAM-10 and -17 to be critical regulators of motility that are dynamically controlled through several signaling pathways, thereby affecting cell behavior through both positive feedback from EGF ligand release and negative feedback from Hepatocyte Growth Factor Receptor (HGFR; MET), Human Epidermal Growth Factor Receptor 2 (HER2), and HER4 RTK shedding. We find kinase inhibition generally reduces ADAM-10 and -17 activities, reduces subsequent RTK shedding, and consequently allows the accumulated RTKs to enhance downstream c-Jun N-terminal kinase (Jnk) and p38 signaling. Thus, here we demonstrate an ADAM-10 and -17–based mechanism of rapidly acquired resistance to kinase inhibition through reduced RTK shedding that can be overcome with combination therapy. Targeted proteomic analysis of clinical samples from endometriosis patients indeed confirms growth-factor–driven ADAM-10 activity and consequent MET shedding are dysregulated with disease. Overall, our results have wide implications for designing combination therapies and identifying context-dependent personalized therapeutic strategies for both kinase and protease inhibitors.Open in a separate windowFig. 1.CSR study design. (A) CSR overview: we stimulate endometriotic cells with a panel of growth factor cues; record multiple downstream signals comprising measurements of phospho-signaling, sheddase regulation, and sheddase substrate regulation; and use computational modeling to map these observations onto cell migration responses. (B) Overview of signals and responses included in the CSR dataset. All receptors shown were directly measured and/or stimulated. (C) Experimental timeline of CSR study. Dark colored lines denote measurement time points. At lower left, cell migration is depicted as single-cell tracks, where initial cell positions were centered for visualization.     

Activation of the NLRP3 Inflammasome Is Associated with Valosin-Containing Protein Myopathy

Aberrant activation of the NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome, triggers a pathogenic inflammatory response in many inherited neurodegenerative disorders. Inflammation has recently been associated with valosin-containing protein (VCP)-associated diseases, caused by missense mutations in the VCP gene. This prompted us to investigate whether NLRP3 inflammasome plays a role in VCP-associated diseases, which classically affects the muscles, bones, and brain. In this report, we demonstrate (i) an elevated activation of the NLRP3 inflammasome in VCP myoblasts, derived from induced pluripotent stem cells (iPSCs) of VCP patients, which was significantly decreased following in vitro treatment with the MCC950, a potent and specific inhibitor of NLRP3 inflammasome; (ii) a significant increase in the expression of NLRP3, caspase 1, IL-1β, and IL-18 in the quadriceps muscles of VCP^R155H/+ heterozygote mice, an experimental mouse model that has many clinical features of human VCP-associated myopathy; (iii) a significant increase of number of IL-1β⁽⁺⁾F4/80⁽⁺⁾Ly6C⁽⁺⁾ inflammatory macrophages that infiltrate the muscles of VCP^R155H/+ mice; (iv) NLRP3 inflammasome activation and accumulation IL-1β⁽⁺⁾F4/80⁽⁺⁾Ly6C⁽⁺⁾ macrophages positively correlated with high expression of TDP-43 and p62/SQSTM1 markers of VCP pathology in damaged muscle; and (v) treatment of VCP^R155H/+ mice with MCC950 inhibitor suppressed activation of NLRP3 inflammasome, reduced the F4/80⁽⁺⁾Ly6C⁽⁺⁾IL-1β⁽⁺⁾ macrophage infiltrates in the muscle, and significantly ameliorated muscle strength. Together, these results suggest that (i) NLRP3 inflammasome and local IL-1β⁽⁺⁾F4/80⁽⁺⁾Ly6C⁽⁺⁾ inflammatory macrophages contribute to pathogenesis of VCP-associated myopathy and (ii) identified MCC950 specific inhibitor of the NLRP3 inflammasome with promising therapeutic potential for the treatment of VCP-associated myopathy.     

Correlation of oxidative status with BMI and lung function in COPD

OBJECTIVES: The imbalance in oxidative status together with nutrition depletion and low body weight play a vital role in the pathogenesis and severity of chronic obstructive pulmonary disease (COPD). The study was undertaken to ascertain if a relationship existed between oxidative status and BMI in COPD. In addition, association of oxidative status and BMI with lung function of the disease was also examined. MATERIALS AND METHODS: In 202 COPD patients and 136 healthy controls plasma lipid peroxidation (LPO), reduced glutathione (GSH), glutathione peroxidase (GPx), catalase (CAT) activities, BMI and FEV(1)% predicted were looked for interactions. RESULTS: The patients had increased LPO (p=0.006) and decreased antioxidants (GSH, p=0.005; GPx, p=0.035 and CAT, p=0.008, respectively). Of note are the correlations of oxidative stress markers with BMI and FEV(1)% predicted in the patients. LPO inversely and GSH, GPx, and CAT positively correlated with both BMI (p=0.007, p<0.001, p=0.045 and p=0.009, respectively), and FEV(1)% of predicted (LPO, p=0.001; GSH, p<0.001; GPx, p=0.043 and CAT, p<0.001) in the patients. Further, a positive correlation existed between BMI and FEV(1)% predicted (p=0.016) in COPD. CONCLUSION: The intimate relationship of oxidative status with BMI and lung function, and the direct correlation between BMI and FEV(1) may potentiate severity of the disease.