Fig 2: Possible mechanisms by which NGF/TRKA increases the shedding of P75, promoting EOC progression. In EOC cells, TRKA is overexpressed (which increases cell proliferation and survival signaling) and P75 is downregulated (decreasing pro-apoptotic signaling). NGF/TRKA activates ADAM17 producing the shedding of P75 and generating the P75 membrane-bound carboxy-terminal fragment (P75-CTF) and P75-extracellular domain (P75-ECD). Hence, y-secretase performs the second shedding to P75, which generates a P75 soluble intracellular domain (P75-ICD) fragment. These results suggest a decrease in pro-apoptotic signaling and an enhanced TRKA signaling, which contribute to EOC progression.

Fig 3: Decreased ADAM17 activity in SpA SFMCs and PBMCs compared with RA SFMCs and PBMCs. (A) Relative ADAM17 mRNA expression measured by quantitative real-time PCR in synovial biopsies from SpA and RA patients (SpA, n = 21; RA, n = 17). (B) Semi-quantitative score (0–3) of the ADAM17 protein expression in the synovial lining layer and sublining, respectively, as assessed by immunohistochemistry (SpA, n = 20; RA, n = 20). (C) ADAM17 protein expression measured by immunohistochemistry in synovial biopsies from SpA and RA patients. Lower panels are magnified view of box. Scale bars, 100 µm (top), 10 µm (bottom). (D) Enzymatic activity of ADAM17 in SFMCs of SpA (n = 13) and RA (n = 9) patients. Normality was determined with a D’Agostino and Pearson omnibus test. The P value was determined by a Mann–Whitney test (A and D) or an unpaired t test (B). Values depicted are means ± SEM. Data are representative of two independent experiments (A, B, D, and E). RFU, relative fluorescence units.

Fig 4: Tumor-derived sNKG2DLs are shed by ADAM10 and ADAM17. (A) Detection of sMICA and sULBP-2 levels by ELISA in serum samples from 35 patients with NB and 10 healthy controls. (B) MICA/B and ULBP1-3 expression in the NB cell lines were assessed using flow cytometry. (C) After 2 days of culture, sMICA and sULBP1-3 production in the culture supernatant was assessed using ELISA. (D) Cell surface expression of ADAM10 and ADAM17 in NB SH-SY-5Y cells was analyzed by flow cytometry. (E-G) SH-SY-5Y cells were pretreated with the ADAM10/ADAM17 promotor PMA, ADAM10 inhibitor GI–X, or ADAM17 inhibitor TAPI-1 for 24 h. Cell surface MFI of MICA/B and ULBP2 was detected by flow cytometry. The levels of sMICA and sULBP2 in the culture supernatant were analyzed by ELISA. Data are presented as the mean ± SEM. P-values were determined using a Mann-Whitney U test in (A). ****P<0.001. s, soluble; ADAM, a disintegrin and metalloproteinase; NB, neuroblastoma; MFI, mean fluorescence intensity.

Fig 5: Structure and activity of hydroxamate drugs. (A) Structures of the four hydroxamates are shown on the right. The lipophilicity of each compound is indicated by the cLogP value (increased value indicates increased lipophilicity). The IC50 is the concentration of inhibitor (in nanomoles) necessary to decrease ADAM17 activity to 50% of its initial value when no inhibitor is added, as determined by the graph in (B). (B) Dose-response curves for NM-exposed corneas plus and minus different concentrations of inhibitors. Since the maximal ADAM17 activity with no inhibitor was 118 ng/mL, the IC50s were the concentrations of inhibitors yielding 59 ng/mL ADAM17 activity, read off the graph. There was no correlation between lipophilicity and IC50 (note that inhibitor concentrations are plotted on a log scale). The linear range of ADAM17 activity for all hydroxamates fell in the range of 0 to 3 nmol of drug, applied four times over the course of 22 hours post NM exposure. Data were analyzed by using one-way ANOVA followed by Duncan's multiple comparison tests. Dose-response studies (0.1, 0.3, 1, 3, 10, 30, and 100 nmol) of NM plus inhibitor samples were compared among data from the same inhibitor treatment. A value of P < 0.05 was considered statistically significant (*P < 0.05; color of * corresponds to the color of the line indicating each inhibitor as shown at the bottom).