Role of ADAM-9 Disintegrin-Cysteine-rich Domains in Human Keratinocyte Migration

Abstract

ADAM-9 belongs to a family of transmembrane, disintegrincontaining metalloproteinases involved in protein ectodomain shedding and cell-cell and cell-matrix interactions. The aim of this study was to analyze the expression of ADAM-9 in skin and to assess the role of this proteolytic/adhesive protein in skin physiology. In normal skin, ADAM-9 expression was detected in both the epidermis and dermis and in vitro in keratinocytes and fibroblasts. Here we report that ADAM-9 functions as a cell adhesion molecule via its disintegrin-cysteine-rich domain. Using solid phase binding assays and antibody inhibition experiments, we demonstrated that the recombinant disintegrin-cysteine-rich domain of ADAM-9 specifically interacts with the ␤1 integrin subunit on keratinocytes. This was corroborated by co-immunoprecipitation. In addition, engagement of integrin receptors by the disintegrin-cysteine-rich domain resulted in ERK phosphorylation and increased MMP-9 synthesis. Treatment with the ERK inhibitor PD98059 inhibited MMP-9 induction. Furthermore, the presence of the soluble disintegrin-cysteine-rich domain did not interfere with cell migration on different substrates. However, keratinocytes adhering to the immobilized disintegrin-cysteine-rich domain showed increased motility, which was partially due to the induction of MMP-9 secretion. In summary, our results indicate that the ADAM-9 adhesive domain plays a role in regulating the motility of cells by interaction with ␤1 integrins and modulates MMP synthesis.Degradation of the extracellular matrix is a prerequisite for tissue repair but also for cell migration and for release of bound factors and bioactive peptides. Different proteases have been implicated in these processes, such as the matrix metalloproteinase (MMP), 2 serine, cysteine, and aspartic protease families. In recent years, the family of proteases (a disintegrin and metalloproteinase (ADAM)) has drawn attention because the manifold proteolytic and adhesive activities of the different ADAM family members were attributed a pivotal role in physiological and pathological situations.The ADAM family includes ϳ30 members of proteins containing disintegrin-and metalloprotease-like domains. Most of the family members share a common well conserved domain structure, including a prodomain, metalloprotease, disintegrinlike, cysteine-rich, EGF-like, and a short cytoplasmic domain (reviewed in Refs. 1 and 2).Structurally, the ADAMs are most closely related to the P-III snake venom metalloproteases. However, in contrast to snake venom metalloproteases, most ADAMs possess EGF-like, transmembrane, and cytoplasmic domains. Half of the ADAM proteins are predicted to be active metalloproteinases, although the identification of specific substrates is still lacking for most of them. Various cell surface proteins are shed by ADAMs, such as IL-6 receptor, FAS-ligand, transforming growth factor-␣, tumor necrosis factor-␣, heparin-binding EGF, and L-selectin. The release of soluble forms of these proteins might lead to autocrine and di...

Characterization of a Cysteine-Rich Protein Specifically Expressed in the Silk Gland of CaddisflyStenopsyche marmorata(Trichoptera; Stenopsychidae)

“…Further evidence of the adhesive function of cysteine has been found in many cell adhesion proteins, since they can promote cell adhesion through their cysteine-rich domains. [19][20][21][22][23] Cysteine is assumed to play a role in maintaining the topology of charged amino acids on the molecular surface by the formation of intramolecular disulfidebonds.…”

The Disintegrin-like and Cysteine-rich domains of ADAM-9 Mediate Interactions between Melanoma Cells and Fibroblasts

“…The cells were resuspended in HEPES buffer (1 mM Hepes, 0.5% BSA, 1 mM of each CaCl 2 and MnCl 2 ). Adhesion assays were performed as described before (10). Briefly, 96-well tissue culture plates were coated with recombinant DC-9 and His 6 peptide (0.6 M) at 4°C overnight.…”

“…We have recently shown that ADAM-9 is expressed in both human and mouse epidermis. In keratinocytes, the adhesive activity of ADAM-9 leads to modulation of MMP-9 expression and cell migration (10). Further, proteolysis via ADAM-9 in keratinocytes has been shown to be important for the constitutive shedding of collagen XVII (11).…”

Platelet integrin α6β1 controls lung metastasis through direct binding to cancer cell–derived ADAM9

“…Although studies have often focused on the proteolytic activity of members of this family, there is increasing evidence that they play a role in cell adhesion through direct interaction with integrins. ADAM9 is a widely expressed non-Arg-Gly-Asp-containing molecule, which has been shown to bind to αvβ5 on myeloma cells, α3β1 on keratinocytes, and α6β1 on fibroblasts (28,29,(31)(32)(33)(34). These studies raise the possibility that ADAM9 could mediate cell-platelet interactions to regulate dissemination of cancer cells.…”

“…Recombinant DC-9 was produced as previously described (33). Briefly, HEK293-EBNA cells were transfected by JetPei (Polyplus) with pCEP-pu BM40-cHis-Dis-Cys plasmid giving rise to a fusion protein with a His6 tag placed in frame with disintegrin-cysteine-rich coding regions of ADAM9.…”