The LG4 module of the laminin ␣3 chain (␣3 LG4), a component of epithelial-specific laminin-5, has cell attachment activity and binds syndecan (Utani, A., Nomizu, M., Matsuura, H., Kato, K., Kobayashi, T., Takeda, U., Aota, S., Nielsen, P. K., and Shinkai, H. LG4 is mediated through the IL-1␤ autocrine loop in keratinocytes but the mechanism of the induction in fibroblasts is different. Our study suggests that the laminin ␣3 LG4 module may play an important role in tissue remodeling by inducing MMP-1 expression during wound healing.Laminin is a heterotrimeric glycoprotein specific to the basement membrane and has many biological functions, including cell adhesion, migration, cell proliferation, differentiation, angiogenesis, and tumor invasion (for reviews, see Refs. 1 and 2). At least 15 laminin isoforms (laminin-1 to -15) have been identified with 11 genetically distinct chains: 5 ␣ chains, 3 ␤ chains, and 3 ␥ chains. Three chains assemble into a cross-shaped heterotrimer (␣␤␥) through coiled-coil interaction at the long arm of the cross (3, 4). Laminin-5 is specific to epithelial cells and a component of the anchoring filament. Laminin-5 forms a complex with the hemidesmosome apparatus by interacting with laminin-6 and -7, collagen VII (5, 6), and fibulin-1 and -2 (7, 8). Laminin-5 consists of the ␣3, ␤3, and ␥2 chains. The ␣3 chain contains a large globular module (G module) 1 at the C terminus, which consists of a tandem repeat of five homologous LG modules (LG1-LG5), each module containing about 200 amino acid residues autonomous folding unit (9). The LG subdomains of laminin ␣ chains have been shown to bind heparin, ␣ 3 ␤ 1 , ␣ 6 ␤ 4 integrins, ␣-dystroglycan, and syndecan (Ref. 10; for review, see Ref. 11) and are implicated as active regions for various biological functions.Syndecans, cell surface heparan sulfate proteoglycans, have been shown to bind the G module of laminin ␣ chains and are involved in laminin-mediated biological functions. We previously demonstrated that keratinocytes and fibroblasts bound LG4 of the ␣3 G module via syndecans (12). Neurite outgrowth of PC12 cells was induced by ␣3 LG4 via syndecans (13). We also showed that HT1080 cells bound to the C-terminal G module of the laminin ␣4 chain through syndecans (14). The interaction of laminin ␣1 LG4 and syndecan family or heparan sulfate proteoglycans was essential for embryonic basement membrane assembly (15).Although laminin ␣3 LG4 -5 is processed in the keratinocyte culture medium (16,17), the unprocessed laminin ␣3 chain was found in a cell layer of the provisional edge of the cell sheet in cultured keratinocytes. In vivo, the unprocessed ␣3 chain has been identified especially in the newly synthesized epidermal basement membrane in wounds but disappears from the mature basement membrane (Refs. 18 and 19; for review, see Ref. 20).Matrix metalloproteinase-1 (MMP-1) is expressed in the basal keratinocytes at the leading edge of re-epithelization (Refs. 21 and 22; for review, see Ref. 23). At the wound edge, MMP-1 degrades collage...

A member of tetraspanin CD151 is a scaffold protein of laminin-binding integrins and it plays an important role in stable interaction between cells and basement membrane. Although the upregulation of CD151 in tumor cells is thought to accelerate tumor invasion and metastasis, detailed pathological investigation on CD151 and its association with integrins has not been well documented, yet. In the present study, we showed that the expression levels of CD151 and its associated integrin subunits in epidermal carcinoma cell HSC5 were higher than those in immortalized epidermal cell HaCaT. By the stimulation of epidermal growth factor, CD151 was dissociated from cell surface and dispersed in the cytoplasm, and a3b1 integrin was concomitantly internalized. To understand the significance of CD151 in tumor cell dynamics, CD151 in HSC5 was knocked down (HSC5 CD151À ), and the expression of integrin subunits and matrix metalloproteinases (MMPs) were investigated. In HSC5 CD151À, striking morphological alteration on Matrigel and laminin, and cytoskeletal rearrangements were demonstrated. a3b1 integrin was internalized in part, and a6b4 integrin was re-distributed from basal site to cell periphery. Quantitative RT-PCR, Western blot and zymography revealed that the expression levels of MMP2, MMP7 and MMP9 were markedly downregulated in HSC5 CD151À. Immunoprecipitation assay demonstrated that MMP7 was co-immunoprecipitated with CD151. In double stainings, MMP7 was colocalized with CD151 at the leading edge of lamellipodia under migratory status. These results elucidated the importance of CD151 as one of the key molecules for integrin-dependent carcinoma-stroma interaction. It is indicated that CD151 might contribute not only to cell stabilization by associating with adhesion complexes but also to cell migration by inducing integrins re-localization and MMPs production.

Syndecans function as receptors for extracellular matrix (ECM) with integrins in cell spreading. However, the molecular mechanism of their specific involvement in cell migration or in wound healing has not been elucidated yet. Here, we report that a synthetic peptide, PEP75, which contains the syndecan-binding sequence of the laminin alpha 3LG4 module, induces keratinocyte migration in in vitro and in vivo. Soluble PEP75 induced the clustering of syndecan-4 and conformation-modified integrin beta1 colocalized with syndecan-4 in soluble PEP75-induced clusters. Treatment of cells in solution with PEP75 resulted in the exposure of the P4G11 antibody epitope of integrin beta1 in immunostaining as well as in flow cytometry and augmented integrin beta1-dependent cell adhesion to ECM. Pulldown assays demonstrated that PEP75 bound to syndecan-4, but not to integrin beta1. A siRNA study revealed a role for syndecan-4 in PEP75-induced up-regulation of P4G11 antibody binding and migration of HaCaT cells. We conclude that binding of soluble PEP75 to syndecan-4 induces the coupling of integrin beta1, which is associated with integrin beta1-conformational changes and activation, and leads to keratinocyte migration. To activate integrin function through syndecans could be a novel therapeutic approach for chronic wound.

Laminin alpha3 chain, a functionally key subunit of laminin-5, contains a large globular module (G module) which consists of a tandem repeat of five homologous LG modules (LG1-5). We previously demonstrated that the LG4 module of laminin alpha3 chain (alpha3 LG4) induces a matrix metalloproteinase-1 (MMP-1) expression through the interaction with syndecans leading to MAPK activation/IL-1beta expression signaling loop (Utani et al., J. Biol. Chem. 278, 34483-34490, 2003). Here, we show that a recombinant alpha3 LG4 and synthetic peptides containing syndecan binding motif induced a cell motility and a MMP-9 expression in ketarinocytes. The synthetic peptide (A3G756)-induced cell migration and MMP-9 upregulation were inhibited by each application of a heparin and an IL-1 receptor antagonist (IL-1RA), suggesting the involvement of syndecans and IL-1beta autocrine. Furthermore, the A3G756-induced cell motility was inhibited by an MMP-9 inhibitor and a neutralizing antibody of MMP-9, indicating induced cell motility was dependent on an MMP-9 activity. Taken these together, laminin-5 alpha3 LG4 module may play an important role in re-epithelialization at tissue remodeling.

ABSTRACT. Lactoferrin has several biological activities, including antitumor activities in some human and animal tumor cells. Clinical trials have been carried out in human medicine based on these effects. However, the antitumor effects of lactoferrin in veterinary medicine remain unknown. In this in vitro study, we demonstrated that co-incubation of canine mammary gland tumor cells (CIPp and CHMp) and bovine lactoferrin induced growth arrest of tumor cells. This growth arrest was associated with induction of G1 arrest. Furthermore, this effect was stronger in tumor cells than in normal cells. These findings demonstrate that bovine lactoferrin has anti-tumor activity in canine mammary tumors and has the potential for use in tumor-bearing dogs. KEY WORDS: bovine lactoferrin, canine mammary gland tumor cell, G1 arrest.