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.     

Antibodies to hepatitis E virus among chinese patients with acute hepatitis in Taiwan

The prevalence of antibodies to hepatitis E virus (anti-HEV) was investigated in patients with acute hepatitis, and correlated with the clinical features. Sera from 110 patients with acute hepatitis and 60 healthy controls were tested for anti-HEV, antibody to hepatitis C virus (anti-HCV), and hepatitis B surface antigen (HBsAg). There were significant differences in the prevalence of anti-HEV, anti-HCV, and HBsAg between patients and controls (21.8% vs. 0%, 16.3% vs. 1.6% and 58.1% vs. 18.0%, respectively). Anti-HEV was detected in 6 (25.0%) of 24 patients with anti-HCV, 6 (9.3%) of 64 patients with HBsAg, and another 6 (22.2%) of 27 patients with acute hepatitis non-A, non-B, non-C. Anti-HEV was found in 15 men and three women, whose ages ranged from 34 to 75 (median, 57) years old. The median age of patients with anti-HEV was older than that in patients without this antibody (57 vs. 38 years; P = 0.001). The prevalence of anti-HEV in patients with anti-HCV alone (35.2%) was higher than that (11.1%) in patients with HBsAg alone (P = 0.03). Compared to patients without anti-HEV, HEV-infected patients had a higher frequency of travel to a foreign country (P = 0.0001), had a lower HBsAg rate (P = 0.019), and had higher serum alkaline phosphatase levels (P = 0.04) and gamma-glutamyl transpeptidase levels (P = 0.01). In conclusion, HEV infection occurs in 22.2% of patients with acute hepatitis non-A, non-B, non-C. HEV superinfection may occur in patients with chronic hepatitis B or C virus infection. © 1994 Wiley-Liss, Inc.     

Effects of methylprednisolone on the neural conduction of the motor evoked potentials in spinal cord injured rats

Methylprednisolone(MP), a glucocorticoid steroid, has an anti-inflammatory action and seems to inhibit the formation of oxygen free radicals produced during lipid peroxidation in a spinal cord injury(SCI). However, the effects of MP on the functional recovery after a SCI is controversial. The present study was conducted to determine the effects of MP on the recovery of neural conduction following a SCI. A SCI was produced using the NYU spinal cord impactor. A behavioral test was conducted to measure neurological disorders, and motor evoked potentials (MEPs) were recorded. According to the behavioral test, using BBB locomotor scaling, MP-treated animals showed improved functional recoveries when compared to saline-treated animals. MEP latencies in the MP-treated group were shortened when compared to those in the control group. Peak amplitudes of MEPs were larger in the MP-treated group than those in the control group. The thresholds of MEPs tended to be lower in the MP-treated group than those in the control group. These results suggest that MP may improve functional recovery after a SCI.     

Approaches for Detection of Hepatitis B Virus Pre-S Gene Deletions and Pre-S Deleted Proteins and Their Application in Prediction of Higher Risk of Hepatocellular Carcinoma Development and Recurrence

Hepatocellular carcinoma (HCC) is among the most common and lethal human cancers worldwide and is closely associated with chronic hepatitis B virus (HBV) infection. Pre-S deleted proteins are naturally occurring mutant forms of HBV large surface proteins that are expressed by HBV surface genes harboring deletion mutations over the pre-S gene segments. It has been well demonstrated that HBV pre-S deleted proteins function as important oncoproteins, which promote malignant phenotypes of hepatocytes through the activation of multiple oncogenic signaling pathways and result in HCC formation. The oncogenic signaling pathways activated by pre-S deleted proteins have been verified as potential therapeutic targets for the prevention of HCC development. Moreover, the presence of pre-S gene deletions and the expression of pre-S deleted proteins in the blood and liver tissues of HBV-infected patients have been evaluated as valuable biomarkers for predicting a higher risk of HCC development and recurrence after curative surgical resection. Therefore, the precise detection of pre-S gene deletions and pre-S deleted proteins holds great promise as regards identifying the patients at higher risk of HCC development and recurrence, thus aiding in more timely and better treatments to improve their survival. This review summarizes the major approaches used for the detection of pre-S gene deletions and pre-S deleted proteins, including the approaches based on Sanger DNA sequencing, pre-S gene chips, next-generation sequencing and immunohistochemistry staining, and it highlights their important applications in the prediction of higher risks of HCC development and recurrence.     

Gangliosides suppression of murine lymphoproliferation and interleukin 1 production

Studies were carried out to determine whether inhibition of gangliosides on lymphoproliferation was related to interleukin (IL)-1. The results showed that gangliosides, GM1 and GT1b were able to inhibit the proliferation of spleen lymphocytes from C57BL/6J mice in dose-dependent fashion, whereas asialo-GM1 was not inhibitory. However, gangliosides, GM1 and asialo-GM1 did not suppress the production of IL-1 in Salmonella typhosa lipopolysaccharide (LPS)-induced peritoneal adherent cells. Various types of LPS including S. enteritidis, S. minnesota and Escherichia coli 055:B5 were used to stimulate the production of IL-1 in adherent cell cultures. The IL-1 production was not affected by gangliosides, GD1a and GD1b. Although GT1b suppressed IL-1 production of human monocytes to 82% of control level it did not, however, affect the IL-1 production of murine adherent cells. Thus, the inhibitory mechanism of gangliosides on murine immune cells remains unclear, and warrants further study.     

Comparative cytotoxicity of five current dentin bonding agents: Role of cell cycle deregulation

To compare the cytotoxicity of three nano-dentin bonding agents (nano-DBAs) and two non-nano-DBAs using Chinese hamster ovary (CHO-K1) cells. We found that nano fillers were not the major contributing factor in DBA cytotoxicity, as analyzed by colony forming assay and 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Exposure of CHO-K1 cells to all three tested total-etching DBAs led to G₀/G₁ cell cycle arrest, whereas exposure to higher concentrations of two tested nano-DBAs induced G₂/M arrest. All five DBAs further induced apoptosis at the highest concentration, as analyzed by propidium iodide staining flow cytometry. The toxicity of all DBAs (1:4000 v/v or higher) is related to increased reactive oxygen species (ROS) production, as analyzed by single cell DCF fluorescence flow cytometry. These results indicate that clinical application of DBAs may be potentially toxic to dental pulp tissues. Cytotoxicity of DBAs is associated with ROS production, cell cycle deregulation and apoptosis. Presence of methacrylate monomers such as PENTA and UDMA is possibly the major cytotoxic factor for DBAs. Further studies on other toxicological endpoints of nano-DBAs are necessary to highlight their safe use.     

Alterations in vitamin D status and anti-microbial peptide levels in patients in the intensive care unit with sepsis

Background  

Vitamin D insufficiency is common in hospitalized patients. Recent evidence suggests that vitamin D may enhance the innate immune response by induction of cathelicidin (LL-37), an endogenous antimicrobial peptide produced by macrophages and neutrophils. Thus, the relationship between vitamin D status and LL-37 production may be of importance for host immunity, but little data is available on this subject, especially in the setting of human sepsis syndrome and other critical illness.     

Production and perception of mandarin tone in adults with cerebral palsy

The primary objective of the present study was to document tone production and intelligibility deficits in Mandarin‐speaking persons with cerebral palsy (CP). Spastic, athetoid, and mixed types of CP were studied, along with a control group, to investigate the possibility of tone production and intelligibility deficits that were differentially affected by type of CP. Speakers produced a 78‐word list that included contrasts for all four Mandarin tones. Intelligibility data were obtained for these words from 26 native speakers of Mandarin, and F0 contour analyses were implemented in CSpeech (Milenkovic) for a subset of the words. Mean tone intelligibility for speakers with CP was 73%, significantly different from the 91% value for control speakers. F0 contour analysis showed that speakers with CP maintained some contrast among the F0 contours for the four tones, but with less distinction than contrasts observed for control speakers. Discussion focuses on the role of tone in a model of the dysarthria associated with CP.