MMP- and TIMP-secretion by human cutaneous keratinocytes and fibroblasts--impact of coculture and hydration.
Epithelial-mesenchymal interactions are important in wound healing and scarring, but are difficult to study in vitro. We have previously reported on an in vitro keratinocyte-fibroblast coculture system exploring these interactions and found that coculture modifies the levels of cytokines they secrete. The same coculture model was used to study changes in MMP- and TIMP-activity. We hypothesised that the previously shown decrease of collagen is partly due to increased MMPs. Adult human cutaneous keratinocytes and fibroblasts were cocultured under serum-free conditions. Keratinocytes were either kept at the air-liquid interface or hydrated. The conditioned medium was submitted to a multiplex sandwich enzyme-linked immunosorbent assay including gelatinases, collagenases, stromelysins, and tissue inhibitors of metalloproteinases. Collagen content was determined by western blot. Zymography depicted the gelatinases in conditioned media. For confirmation of the coculture results fibroblasts were treated with conditioned media from keratinocyte monocultures as well. MMP-1, MMP-9, and MMP-10 were mainly secreted by keratinocytes, whereas MMP-2, TIMP-1 and -2 by fibroblasts. MMP-13 was secreted by both cell types at comparable levels. Collagenases, gelatinases, MMP-3, and TIMPs increased significantly in cocultures compared to monocultures. Hydration of keratinocytes revealed a significant increase of MMP-3 and MMP-2, and a decrease of TIMP-2. Paracrine interactions between keratinocytes and fibroblasts modify strongly MMPs and TIMPs, whereas hydration of keratinocytes had a smaller impact in this context. The observed changes may be in part responsible for reduced collagen in coculture-conditioned media. The present coculture experiments reemphasise the role of epidermis in controlling scarring.
Tandara
Andrea A
AA
Division of Plastic and Reconstructive Surgery, Wound Healing Research Laboratory, Feinberg School of Medicine, Northwestern University, 675 North St. Clair, Suite 19-250, Chicago, IL 60611, USA.
Mustoe
Thomas A
TA
D015153:Blotting, Western; D002478:Cells, Cultured; D002921:Cicatrix / Q000503:physiopathology*; D018920:Coculture Techniques; D017077:Culture Media, Conditioned; D004797:Enzyme-Linked Immunosorbent Assay; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology*; D006801:Humans; D055103:Hypodermoclysis; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D020782:Matrix Metalloproteinases / Q000378:metabolism*; D012016:Reference Values; D012680:Sensitivity and Specificity; D012867:Skin / Q000166:cytology / Q000201:enzymology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism*
D017077:Culture Media, Conditioned; D019715:Tissue Inhibitor of Metalloproteinase-1; D019714:Tissue Inhibitor of Metalloproteinases; D019716:Tissue Inhibitor of Metalloproteinase-2; D020782:Matrix Metalloproteinases