Shedding of the Lymphocyte L-Selectin Adhesion Molecule Is Inhibited by a Hydroxamic Acid-based Protease Inhibitor

Abstract

Expression of the L-selectin adhesion molecule can be rapidly down-modulated by regulated proteolysis at a membrane-proximal site. The L-selectin secretase has remained undefined, and the secretase activity is resistant to a broad panel of common protease inhibitors. We have developed an L-selectin-alkaline phosphatase reporter, consisting of the ectodomain of human placental alkaline phosphatase fused to the membrane-proximal cleavage, transmembrane, and cytoplasmic domains of L-selectin, to aid in the screening for L-selectin secretase inhibitors. A hydroxamic acid-based metalloprotease inhibitor, KD-IX-73-4, inhibited release of the L-selectin-alkaline phosphatase reporter in a dose-dependent manner. The hydroxamic acid-based peptide was also found to inhibit wild type L-selectin down-regulation from the surfaces of phorbol myristate acetate-activated peripheral blood lymphocytes and phytohemagglutinin-stimulated lymphoblasts. Analysis of the proteolytic cleavage fragments of L-selectin confirmed that KD-IX-73-4 inhibited L-selectin proteolysis. Lymphocyte L-selectin was not down-regulated when co-cultured with formylmethionylleucylphenylalanine-stimulated neutrophils, suggesting that the putative secretase acts in cis with the membrane-bound L-selectin. These results suggest that the L-selectin secretase activity may involve a cell surface, zinc-dependent metalloprotease, although L-selectin shedding is not affected by EDTA and may be related to the recently described activity involved in processing of membrane-bound TNF-alpha.

Regulation of monocyte MMP-9 production by TNF-α and a tumour-derived soluble factor (MMPSF)

“…TGF-oc, M-CSF, FasL, etc. (Crowe et al, 1995;Kayagaki et al, 1995;Mullberg et al, 1995;Bennett et al, 1996;Couet et al, 1996;Drummond and Gearing, 1996;Feehan et al, 1996). Another parameter was obtained by gel filtration of CM revealing a peak of activity with a molecular mass of 89 kDa.…”

“…MMPIs were originally designed to reduce excessive MMP activity due to an imbalance of MMPs and their natural inhibitors (TIMPs). However, synthetic MMPIs also inhibit shedding of TNF-ox from its membrane spanning precursor (Gearing et al, 1994(Gearing et al, , 1995 and of other cytokines: TGF-oc, FasL, C) Cancer Research Campaign 1998 IL-6 receptor, stem cell factor, M-CSF, TNF-oc receptors, Lselectin and thyrotropin receptor ectodomain (Crowe et al, 1995;Kayagaki et al, 1995;Mullberg et al, 1995;Bennett et al, 1996;Couet et al, 1996;Drummond and Gearing, 1996;Feehan et al, 1996). Therefore, inhibition of release of cytokines that play a role in tumour development represents an alternative or additional mechanism of action of MMPIs.…”

Characterization of Growth Factor-induced Serine Phosphorylation of Tumor Necrosis Factor-α Converting Enzyme and of an Alternatively Translated Polypeptide

“…The MEK1 inhibitor U0126 and sequencing grade modified trypsin were purchased from Promega (San Luis Obispo, CA). TAPI-1, an inhibitor of metalloproteases, including TACE, was a gift from Dr. T. K. Kishimoto (Boehringer Ingelheim) (30,39). Phenanthroline, another metalloprotease/TACE inhibitor, was purchased from Sigma (47).…”

“…The detection of both the mature 100-kDa TACE and 20-kDa SPRACT under these conditions suggests that SPRACT is unlikely to be generated by autolysis during sample preparation. Previous studies (30,39) have shown that the metalloprotease inhibitor TAPI-1 inhibits the cleavage activity of TACE. Inclusion of TAPI-1 (10 M) and phenanthroline (10 mM) in the cell lysis buffer and throughout subsequent washes in immunoprecipitation did not decrease the level of SPRACT in such experiments (data not shown).…”

Effects of selective protein kinase C inhibitors on the proteolytic down-regulation of L-selectin from chemoattractant-activated neutrophils

“…Staurosporine, wortmannin, H89, KT5823, genistein, and K252b were purchased from Alexia (San Diego, CA); Gö-7874, Ro-31-8220, bisindolylmaleimide 1, rottlerin, and calphostin C were purchased from Calbiochem (San Diego, CA). KD-IX-73-4, a hydroxamic acid-based metalloprotease inhibitor, has previously been shown to block activation-induced L-selectin shedding [13,18]. All kinase inhibitors and KD-IX-73-4 were dissolved in dimethyl sulfoxide (DMSO) and diluted at least 1000-fold for all whole-cell assays.…”