KLK5 Knockout HaCaT Cell Line

The KLK5 Knockout HaCaT Cell Line is a CRISPR/Cas9-edited human keratinocyte line in which the KLK5 gene has been disrupted, generating a stable loss-of-function model. Derived from the HaCaT immortalized keratinocyte background, this cell line provides a consistent platform for investigating KLK5-dependent processes. The knockout was achieved through CRISPR/Cas9-mediated gene editing, resulting in ablation of KLK5 protein expression while preserving the host cells’ differentiation capacity.

HaCaT cells are a spontaneously immortalized, non-tumorigenic human epidermal keratinocyte line established from adult skin. They retain the ability to differentiate and form stratified epidermal-like structures upon calcium stimulation, making them a physiologically relevant in vitro system for studying keratinocyte biology, skin barrier formation, cornification, and wound healing. This host background is widely used in dermatological research to model cutaneous homeostasis and disease.

KLK5 encodes a secreted serine protease central to epidermal desquamation and skin barrier maintenance. It cleaves corneodesmosomal adhesion proteins desmoglein 1 (DSG1), desmocollin 1 (DSC1), and corneodesmosin (CDSN) to facilitate controlled corneocyte shedding. KLK5 also activates protease-activated receptor 2 (PAR2), triggering inflammatory and pruritic signaling, and processes cathelicidin into the antimicrobial LL-37 peptide. Activity is inhibited by lymphoepithelial Kazal-type-related inhibitor (LEKTI; encoded by SPINK5) and influenced by extracellular calcium gradients. As part of the kallikrein cascade, KLK5 activates pro-KLK7 and pro-KLK14, amplifying stratum corneum proteolysis. Through these actions, KLK5 integrates desquamation, innate immunity, and PAR2 signaling to regulate barrier integrity and inflammation.

In HaCaT keratinocytes, KLK5 disruption provides a model for barrier dysfunction and inflammatory skin pathology. As a key effector downstream of SPINK5/LEKTI, KLK5 loss enables precise study of the LEKTI-KLK5 interplay relevant to Netherton syndrome, where SPINK5 mutations cause unopposed KLK5 activity. This knockout line is also valuable for atopic dermatitis, psoriasis, rosacea, and ichthyosis research, where dysregulated KLK5 and PAR2 signaling are implicated. Eliminating KLK5 allows researchers to uncouple its specific contributions from parallel proteolytic pathways and examine impacts on corneodesmosome stability, barrier integrity, and inflammatory mediator expression.

Key applications include skin barrier studies, desquamation research, inflammatory disease modeling, and drug screening for protease inhibitors. Representative assays are Western blot for KLK5, caseinolytic activity assays, immunofluorescence for DSG1/DSC1/CDSN, PAR2 cleavage and cathelicidin processing experiments, calcium-induced differentiation monitored by transepithelial electrical resistance, and scratch wound migration assays. This defined genetic model enables dissection of epidermal homeostasis and screening of modulators targeting the kallikrein?CPAR2?Ccathelicidin axis. For technical inquiries, please contact Ascent Research.