Extracellular matrix proteins induce changes in intracellular calcium and cyclic AMP signalling systems in cultured human keratinocytes.

The purpose of this study was to investigate whether extracellular matrix proteins which influence human keratinocyte behaviour are capable of altering intracellular signalling systems in these cells. The effects of extracellular matrix proteins on two major signal transduction pathways, intracellular calcium and cyclic adenosine monophosphate (cyclic AMP), were investigated. The extracellular matrix proteins examined were the basement membrane preparation matrigel, collagens type I and IV, vitronectin and its active tripeptide component Arg-Gly-Asp (RGD). Acute additions of matrigel, vitronectin and RGD caused rapid transient increases in intracellular calcium and, together with collagen type I, also caused sustained elevations in basal calcium when cells were grown on these substrates. Cyclic AMP production was unaffected by acute exposure to these extracellular matrix proteins. Culture of cells on matrigel, collagen type I or IV, however, significantly reduced basal cyclic AMP accumulation and increased the response of the cells to the receptor-independent agonist forskolin. It is concluded that in vitro some extracellular matrix proteins can initiate both acute and sustained changes in intracellular signalling in human keratinocytes.

Influence of extracellular matrix proteins on human keratinocyte attachment, proliferation and transfer to a dermal wound model.

The aim of this study was to investigate whether prior culture of cells on ECM proteins might positively influence the performance of keratinocytes when cells are transferred to a dermal in vitro wound bed model. Keratinocytes were cultured using a method for producing cultured epithelial autografts for severely burned patients (essentially using Green's medium, a mitogen-rich medium containing fetal calf serum, cholera toxin, EGF, insulin, transferrin and triiodothyronine). Cells were cultured either on irradiated 3T3 fibroblasts (as in the standard Rheinwald and Green technique) or, alternatively, on collagen I, collagen IV, matrigel, RGD, vitronectin or fibronectin. Under these conditions matrigel, collagen I and IV enhanced initial attachment, RGD, vitronectin, fibronectin and irradiated 3T3 fibroblasts did not. Proliferation of cells was positively influenced by matrigel, collagen I and IV and irradiated 3T3 fibroblasts; of these, however, only matrigel and 3T3 fibroblasts had sustained significant effects on keratinocyte proliferation over 4 days. Cells on fibronectin showed significantly reduced proliferation. An acellular non-viable dermis was then used to mimic the homograft allodermis onto which cultured epithelial autograft sheets are grafted clinically and cells cultured on the various ECM proteins for 96 h were transferred to this in vitro wound model. None of the substrates enhanced keratinocyte performance on this model. It was concluded that under these conditions some ECM proteins can significantly affect keratinocyte attachment and, to a lesser extent, proliferation but that the culture of keratinocytes on these ECM proteins does not appear to confer any lasting benefit to the attachment of these keratinocytes to an in vitro wound-bed model.

A calmodulin-like protein as an extracellular mitogen for the keratinocyte.

This study investigated the importance of extracellular calmodulin to the proliferation of the keratinocyte. Normal keratinocytes in culture produced a calmodulin-like protein in their culture media, the level of which increased abruptly and transiently during their growth. This protein was calmodulin-like, in that it specifically bound to a calmodulin affinity column, exhibited calmodulin-like immunoreactivity in both an ELISA and on immunoblots when immunostained with a monoclonal antibody against calmodulin, had an apparent M(r) between 18,000 and 20,000, and stimulated activity in a calmodulin-dependent phosphodiesterase enzyme assay. Addition of exogenous pure calmodulin was of no further mitogenic benefit to the keratinocytes, and slightly reduced proliferation under the culture conditions used. However, addition of either a neutralizing antibody to calmodulin, or W7-agarose, to the culture media of proliferating cells markedly inhibited their proliferation. Accordingly, a calmodulin-like protein was found to satisfy all but one of the criteria for its action as an autocrine growth factor for the keratinocyte. We propose that the lack of mitogenic response to calmodulin in vitro is due to the cell meeting its own requirement for extracellular calmodulin.