Increase of laminin 5 synthesis in human keratinocytes by acute wound fluid, inflammatory cytokines and growth factors, and lysophospholipids
Increase of laminin 5 synthesis in human keratinocytes by acute wound fluid, inflammatory cytokines and growth factors, and lysophospholipids
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DOI：
10.1111/j.1365-2133.2004.06175.x
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发表时间：
2004-11-01
影响因子：
10.3
通讯作者：
Nishiyama, T
中科院分区：
文献类型：
--
作者：
Amano, S;Akutsu, N;Nishiyama, T
背景层粘连蛋白5可诱导角质形成细胞的黏附、扩散和迁移。在皮肤创面愈合过程中，层粘连蛋白5在迁移中的角质形成细胞下层粘连蛋白5沉积较早，随后形成半桥粒，继而形成基底膜。目的确定在皮肤创伤愈合过程中影响角质形成细胞合成和分泌层粘连蛋白5的因素。为了测定层粘连蛋白5的合成总量，将沉积在培养皿和细胞内的层粘连蛋白5用洗涤剂溶液溶解，并与条件培养液分开测定，计算层粘连蛋白5的合成总量。用定量聚合酶链式反应方法检测与层粘连蛋白5的α3、β3和Gamma2链对应的层粘连蛋白5基因LAMA3、LAMB3和LAMC2的表达水平。我们还检测了急性创面液中的溶血磷脂、促炎细胞因子和生长因子对角质形成细胞层粘连蛋白5合成的影响。人血清对人角质形成细胞产生层粘连蛋白5的作用与伤口液一样强，提示急性伤口液中的主要活性成分可能来源于人血清中的活性成分。溶血磷脂酸(LPA)、溶血磷脂酰胆碱(LPCS)和1-磷酸鞘氨醇(S1P)等溶血磷脂以浓度依赖的方式促进层粘连蛋白5的合成。在生长因子中，表皮生长因子、胰岛素样生长因子-1、干扰素-γ和角质形成细胞生长因子促进角质形成细胞层粘连蛋白5的产生，而血小板衍生生长因子、肝细胞生长因子和碱性成纤维细胞生长因子作用不明显。尽管白介素1α无此作用，但转化生长因子α、肿瘤坏死因子α和转化生长因子β1也可促进层粘连蛋白5的合成，且两者具有协同作用。抗转化生长因子-α和转化生长因子-β1的中和抗体显著抑制人血清层粘连蛋白5的合成，提示转化生长因子-α和转化生长因子-β1是增加人血清层粘连蛋白5的重要成分。随着层粘连蛋白5合成的增加，3种层粘连蛋白5基因的表达水平也被转化生长因子α、转化生长因子β1、肿瘤坏死因子α和溶血磷脂S1P、LPA、LPCS等促进层粘连蛋白5的表达。创面中增加的层粘连蛋白5蛋白可能通过刺激角质形成细胞在创面床上的黏附和迁移，以及通过促进真皮-表皮交界处基底膜的形成来促进创面修复。
Background 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.Objectives To identify factors that regulate the synthesis and secretion of laminin 5 by human keratinocytes during acute wound healing.Methods 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.Results 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.Conclusions 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.