ML281 Does Not Function as an STK33 Inhibitor but Modulates Proliferation and Differentiation of Keratinocytes Derived from Hypertrophic Scars

Post-burn hypertrophic scar (HTS) formation is influenced by the dynamic balance between keratinocyte proliferation and differentiation, a process critical for maintaining skin homeostasis. Serine/threonine kinase 33 (STK33) has emerged as a potential therapeutic target in oncology. However, its role in HTS formation remains unclear, and its effects on keratinocytes are poorly understood. We isolated human HTS-derived keratinocytes (HTSKs) from post-burn HTS tissues and treated them with ML281, originally developed as an STK33 inhibitor. We examined markers associated with keratinocyte phenotypes and functions, including proliferation (proliferating cell nuclear antigen, c-Myc, keratins 5, 14, 6, 16, and 17), epithelial–mesenchymal transition (EMT; snail1, slug, twist1, e-cadherin, n-cadherin, and vimentin), differentiation (keratins 1 and 10, involucrin, loricrin, Notch1, p21, and p27), and apoptosis (cytochrome c, cleaved caspase3, Bid, Bad, Bax, and Bcl-2). mRNA and protein expression levels were assessed using reverse transcription–quantitative PCR, Western blotting, and immunocytochemistry. In HTSKs, ML281 increased STK33 enzymatic activity and STK33 mRNA and protein expression, rather than inhibiting STK33 activity. ML281 treatment reduced the expression of proliferation-associated markers, promoted an EMT-associated phenotype, modulated the expression of differentiation-associated markers, and induced apoptosis-associated changes. Collectively, these findings suggest that ML281 alters post-burn HTSK phenotypes associated with proliferation, EMT, differentiation, and apoptosis.