4B, repeating the experiment using selleck chem Dasatinib the ADAM17/TACE-specific Innozyme TACE activity assay confirmed that the increase was due, at least in part, to KK-dependent activation of ADAM17. The KK effect was detectable within 5 min of exposure and persisted for at least 15 min. FIGURE 4. Plasma kallikrein causes PAR-dependent ADAM17 activation in vascular smooth muscle cells. A, serum-deprived R-VSMCs in 96-well plates were treated with varying concentrations of plasma KK for 4 h in the presence of the ADAM10/17-specific fluorogenic peptide … Because ADAM17 is regulated by multiple stimuli, including many G protein-coupled receptors (26, 27), we next tested whether there was a link between KK-dependent PAR activation and its activation of ADAM17 in R-VSMC.
For these experiments, R-VSMCs were preincubated in the presence of PAR1 or PAR2 antagonist peptides prior to exposing the cells to KK. As these peptides are related to the internal PAR1/2 ligand sequence, we first had to exclude the possibility that they would also behave as pseudosubstrate inhibitors of plasma KK. As shown in Fig. 4C, neither peptide had any effect upon KK activity when assayed in a cell-free system. Similarly, neither peptide interfered with the ability of recombinant ADAM17/TACE to cleave its fluorogenic substrate, KPLGL-Dpa-AR-NH2, in a cell-free assay (data not shown). However, as shown in Fig. 4D, the PAR1 and PAR2 antagonist peptides blocked KK-induced ADAM activation in R-VSMCs, suggesting that KK cleavage of PARs occurs upstream of ADAM activation.
The two inhibitors in combination were somewhat more effective that either alone; however, the difference was not statistically significant. Plasma Kallikrein Stimulates ADAM-dependent Amphiregulin Release and EGF Receptor Transactivation in Primary Aortic Vascular Smooth Muscle ADAMs are known to play a key role in the regulated shedding of at least six of the known EGF family ligands, transforming growth factor-��, EGF, HB-EGF, betacellulin, epiregulin, and AR (46). Of these, HB-EGF and AR have been implicated in the control of VSMC proliferation and development of vascular disease (28,�C30, 47,�C49). To determine whether the KK-induced increase in ADAM activity increased VSMC shedding of EGF family growth factors, we looked for the presence of AR and HB-EGF in the culture medium after KK exposure.
Because species-specific antibodies Drug_discovery recognizing rat AR and HB-EGF are not available, we employed primary H-VSMC for these experiments. As shown in Fig. 5A, KK exposure increased in AR shedding compared with vehicle-treated control cells. As shown in Fig. 5B, KK-stimulated AR release was ADAM-dependent, because it was sensitive to the broad spectrum MMP inhibitor GM6001. Although we were unable to detect HB-EGF shedding in response to plasma KK (data not shown), we did observe increased shedding of another ADAM17/TACE substrate, TNF-��, consistent with the results of our direct assay of ADAM17/TACE activity.
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