Coculture Modulates Laminin Synthesis and mRNA Levels in Epidermal Keratinocytes and Dermal Fibroblasts

Abstract

To investigate the role of cell-cell interactions between keratinocytes and fibroblasts at the dermal-epidermal junction in the regulation of extracellular matrix protein expression, we have developed a cell coculture model that allows both cell types to internet on a reciprocal basis and yet be isolated as pure populations for quantitative analysis. Using porous membrane inserts as a substrate for keratinocytes grown in coculture over fibroblast monolayers, we report that coculture stimulates cell growth and total protein synthesis in both cell types when compared to monocultured controls. Both keratinocytes and fibroblasts synthesize laminin B1 and B2 chains with an additional subunit comigrating with laminin M chain detected in keratinocytes but only faintly visible in fibroblasts. Laminin A chain synthesis could not be detected. Although laminin subunit composition did not change in either cell type, fractional laminin synthesis increases by 41.0 ± 2.3% in cocultured keratinocytes and decreases by 73.8 ± 8.1% in cocultured fibroblasts when compared to monocultured controls. In cocultured keratinocytes, steady-state mRNA levels for laminin B1, B2, and M chains increased by 69.4, 63.5, and 136.8%, respectively, when compared to monocultured controls. However, in cocultured fibroblasts, laminin B1 and B2 chain mRNA decreased by 74.2 and 72.7%, respectively. Laminin M chain mRNA could not be detected in fibroblasts. These results suggest that synthesis and expression of laminin is regulated through reciprocal cell-cell interactions in fibroblasts and keratinocytes grown in coculture.