A study of keratinocyte differentiation and adhesion in vitro

In this study, I used the serum-free MCDB 153 culture system to investigate calcium-induced keratinocyte differentiation. Treating normal human keratinocytes with high extracellular calcium concentrations (1mM) increased the proportion of cells expressing differentiation-specific proteins. I showed that this was not caused by calcium-induced cell-cycle arrest, nor was it a consequence of stratification. However, the expression of differentiation-specific proteins was preceded by the formation of cadherin-mediated cell-cell adhesions. The likely importance of the cadherin-mediated adhesions in initiation the differentiation program was confirmed in two ways. Firstly, clustering cell-surface E-cadherin in low extracellular calcium using monoclonal antibodies increased the proportion of keratinocytes expressing differentiation-specific proteins. Secondly, suppressing the formation of cadherin-mediated cell-cell adhesions using synthetic peptide analogous to the cadherin recognition domain attenuated the calcium-induced expression of differentiation-specific proteins. These data are consistent with a role for the cadherin-mediated cell-cell adhesions in initiating the keratinocyte differentiation program in response to calcium in vitro. A second aspect of this project involved an investigation of the role played by the Src-family of protein tyrosine kinases at calcium-induced cadherin-mediated adherens junctions. The ubiquitously expressed members of this family, c-Src, Fyn and c-Yes were localised to the cadherin-mediated adhesions formed in response to high extracellular calcium. Treating adherent keratinocytes maintained in low extracellular calcium with a specific Src kinase inhibitor, PD162531, induced the assembly of cadherin-mediated cell-cell adhesions leading to the formation of contiguous groups of cells similar to those seen in response to high extracellular calcium. The data presented are consistent with a role for the Src kinases in regulating adherens junction turnover but do not exclude a role also in modulating aspects of differentiation