epidermal-growth-factor and ilomastat

To investigate the potential regulation of matrix metalloproteinases (MMPs) by hepatocyte growth factor (HGF), and to identify individual MMPs essential for migration of human corneal epithelial cells.. Migration of human corneal epithelial cells (HCECs) was measured with a colony dispersion assay in response to concentrations of HGF (0-50 ng/mL). MMP activity in the conditioned media collected from the dispersion assay was assessed by zymography. The broad-spectrum MMP inhibitor ilomastat (1-100 microM) or an MMP-9-neutralizing antibody (1-10 microg/mL) were included in the dispersion assay to determine their effects on HCEC migration. Immunocytochemistry and in situ hybridization were used to localize MMP-1 in HCECs in the colony dispersion assay and in a human ex vivo corneal wound-healing model, respectively. ELISA for MMP-1 was performed on conditioned medium from migrating HCECs. Neutralizing antibodies to MMP-1 and -9 were added to an in vitro scratch-wound model to assess the effect on HCEC healing.. HCEC migration (P < 0.05) and MMP-2 and -9 released into the medium increased in response to HGF in a dose-dependent manner up to 20 ng/mL. Broad-spectrum MMP inhibition significantly reduced HCEC migration (P < 0.05). In contrast, neutralization of MMP-9 increased migration (P < 0.05). MMP-1 was found in association with HCECs at the migratory leading edge in both the dispersion and the ex vivo wound-healing experiments, and was found to be stimulated above basal levels by HGF. Neutralization of MMP-1 significantly decreased (P < 0.05), whereas neutralization of MMP-9 significantly increased (P < 0.05), scratch-wound closure.. This study provided novel data regarding HCEC migration in response to HGF and highlighted the importance of MMPs, particularly MMP-1 in migration and possibly reepithelialization in vivo. MMP-9 and/or -2 may be released by HCECs to remodel matrix behind the leading migratory front. Studies such as this are essential to assist in the safe and efficacious design of MMP inhibitors for therapeutic use in the eye.

Topics: Cell Movement; Cells, Cultured; Collagen Type I; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Epidermal Growth Factor; Epithelial Cells; Epithelium, Corneal; Hepatocyte Growth Factor; Humans; Hydroxamic Acids; Immunoenzyme Techniques; In Situ Hybridization; Indoles; Matrix Metalloproteinase 1; Matrix Metalloproteinase Inhibitors; Wound Healing

Colonic epithelial cell migration is required for normal differentiated cell function. This migratory phenotype is dependent upon wild-type adenomatous polyposis coli (Apc) expression. Non-tumorigenic murine colon epithelial cell lines with distinct Apc genotypes, i.e. young adult mouse colon (YAMC; Apc(+/+)) and immortomouse/Min colon epithelial (IMCE; Apc(Min/+) cells) were used to assess the association between the Apc genotype, cell motility and matrix metalloproteinase (MMP) activity. Cells were treated with epidermal growth factor (EGF; 1, 10 and 25 ng/ml), hepatocyte growth factor (HGF; 1, 10 and 25 ng/ml) and/or curcumin (0.1-100 microM). EGF (25 ng/ml) and HGF (25 ng/ml) induced a greater migratory response in YAMC compared with IMCE cells after 24 h (P < 0.05). Treatment with curcumin induced a greater or equivalent migratory response in IMCE than YAMC cells. When migrating cells were treated with Ilomastat (MMP inhibitor), migration was inhibited in both cell types. High concentrations of Ilomastat (25 and 50 microM) inhibited migration in both cell types, while low concentrations (10 microM) inhibited HGF-induced IMCE migration. Curcumin-induced migration was inhibited in both cell types at the highest concentration of Ilomastat (50 microM). Immuno-localization analysis of membrane type-1 (MT1)-MMP indicated that migration is associated with the redistribution of this protein from the endoplasmic reticulum to the plasma membrane. Addition of neutralizing polyclonal antibodies against MT1-MMP or a mixture of MT1, 2- and 3-MMPs demonstrated partial or complete inhibition of cell migration in both cell types, respectively. The data provide the first evidence that migration in non-tumorigenic murine colon epithelial cells is: (i) inducible by EGF and HGF in an Apc genotype-dependent manner, (ii) dependent on MT-MMP activity and (iii) inducible by curcumin in an Apc genotype-independent manner. The data suggest a potential mechanism by which curcumin may induce cells heterozygous for Apc to overcome defective cell migration, a phenotype associated with cell differentiation and apoptosis.

Topics: Adenomatous Polyposis Coli; Animals; Antineoplastic Agents; Cell Movement; Cells, Cultured; Colon; Curcumin; Epidermal Growth Factor; Gelatinases; Genes, APC; Genotype; Hepatocyte Growth Factor; Hydroxamic Acids; Indoles; Intestinal Mucosa; Matrix Metalloproteinase Inhibitors; Matrix Metalloproteinases; Mice; Protease Inhibitors