Increase of laminin 5 synthesis in human keratinocytes by acute wound fluid, inflammatory cytokines and growth factors, and lysophospholipids

Abstract

Laminin 5 is known to induce the adhesion, spreading and migration of human keratinocytes. In skin wound healing, laminin 5 deposition beneath migrating keratinocytes occurs early and is followed by the formation of hemidesmosomes and then basement membrane. To identify factors that regulate the synthesis and secretion of laminin 5 by human keratinocytes during acute wound healing. Laminin 5 synthesis by human keratinocytes was determined by a specific sandwich enzyme-linked immunosorbent assay. To determine the total amount of laminin 5 synthesized, laminin 5 deposited on culture dishes and inside cells was solubilized by detergent solution and determined separately from conditioned medium, and the total laminin 5 synthesis was calculated. A quantitative polymerase chain reaction method was used to measure the expression levels of laminin 5 genes, LAMA3, LAMB3 and LAMC2, which correspond to the alpha3, beta3 and gamma2 chains of laminin 5. We also examined the effects of lysophospholipids, proinflammatory cytokines and growth factors, which are components in acute wound fluids, on laminin 5 synthesis in keratinocytes. Human acute wound fluid at days 1, 2 and 3 stimulated laminin 5 synthesis in cultured human keratinocytes in a concentration-dependent manner, although findings are restricted to one case. Human serum also increased laminin 5 production by human keratinocytes as strongly as the wound fluid did, suggesting that the major active components in acute wound fluid may be derived from those in human serum. Lysophospholipids such as lysophosphatidic acid (LPA), lysophosphatidylcholines (LPCs) and sphingosine-1-phosphate (S1P) increased laminin 5 synthesis in a concentration-dependent manner. Among growth factors, epidermal growth factor, insulin-like growth factor-1, interferon-gamma and keratinocyte growth factor increased laminin 5 production in keratinocytes, while platelet-derived growth factor, hepatocyte growth factor and basic fibroblast growth factor were ineffective. Although interleukin-1alpha had no effect, transforming growth factor (TGF)-alpha, tumour necrosis factor (TNF)-alpha and TGF-beta1 also stimulated laminin 5 synthesis, and TGF-alpha and TGF-beta1 showed a synergistic effect. Neutralizing antibodies to TGF-alpha and TGF-beta1 markedly inhibited the enhanced laminin 5 synthesis by human serum, suggesting that TGF-alpha and TGF-beta1 are important components to increase laminin 5 in human serum. In line with the increase of laminin 5 synthesis, the expression levels of all three laminin 5 genes were also augmented by TGF-alpha and TGF-beta1. Laminin 5 synthesis in human keratinocytes was augmented by inflammatory cytokines and growth factors such as TGF-alpha, TGF-beta1 and TNF-alpha, and lysophospholipids such as S1P, LPA and LPCs, which are supposed to be present in acute wound fluid. The increased laminin 5 protein in the wound area presumably enhances wound repair by stimulating adhesion and migration of keratinocytes on the wound bed and by facilitating basement membrane formation at the dermal-epidermal junction.