Human tissue kallikrein 14 induces the expression of IL-6, IL-8, and CXCL1 in skin fibroblasts through protease-activated receptor 1 signaling

Human tissue kallikrein 14 (KLK14) is protease with trypsin/chymotrypsin specificity that is abundant in the skin. It is involved in skin desquamation and wound healing by cleaving cell–cell adhesion molecules and extracellular matrix components. In the process of wound healing, a paracrine communication between the epithelium, human skin fibroblasts (HSFs), and immune cells is essential for proper regulation. Previous reports highlighted stimulation of interleukin-6 (IL-6), interleukin-8 (IL-8), and growth-regulated alpha protein (CXCL1) production by keratinocyte-conditioned medium in fibroblast cells and implicated these cytokines in cancer. Here, we hypothesize that KLK14 may be a paracrine mediator released by keratinocytes that activates fibroblasts via proteinase-activated receptor (PAR) pathway, affecting the HSF secretome. Semiquantitative real-time PCR and ELISA demonstrated that proteolytically active KLK14 induced the expression of IL-6, IL-8, and CXCL1 by 15-, 847-, and 50-fold, respectively, and resulted in the release of the proteins in ng/ml quantities from stimulated HSFs to the culture medium. Through the implementation of the PAR-1 antagonist RWJ 56110, we demonstrated that the KLK14-mediated release of IL-6 and IL-8 is dependent on PAR-1 activation. Contrarily, PAR-1 activation was shown to function as a limiting factor in the KLK14-mediated CXCL1-releasing pathway. Furthermore, human recombinant IL-6, IL-8, and CXCL1 enhanced closure of a cell-free gap in an HaCaT cell monolayer, mimicking wound healing of keratinocytes in the skin. KLK14-stimulated HSF conditioned media also induced wound healing in the HaCaT model in an IL-6-dependent manner, as a cytokine-neutralizing antibody significantly decreased this activity. Thus, KLK14 in the skin may participate in paracrine signaling between fibroblasts and keratinocytes, in that keratinocyte-secreted KLK14 initiates the release of IL-6, IL-8, and CXCL1 from fibroblasts, which in turn act on proximal keratinocytes to trigger their migration for wound closure. Our findings add to the understanding of the role of KLK14 in the related processes of wound healing and tumor development.