Reciprocal regulation of LOX and LOXL2 expression during cell adhesion and terminal differentiation in epidermal keratinocytes

BackgroundClassical lysyl oxidase (LO) is involved in the stabilization and repair of extracellular matrix by the oxidization of lysine residues in collagen and elastin. Five genetically distinct species of LOs (LOX and LOXL1–4) have been identified, but their functions remain unclear.ObjectivesTo investigate the regulation of LOs during differentiation and cell adhesion of cultured keratinocytes.MethodsLO mRNA and polypeptides levels in the suspension and adhesive cultures were measured using real-time PCR, immunoblot and immunofluorescence assays. Localization of LOX, LOXL1 and LOXL2 gene transcripts in normal epidermis was assessed by in situ hybridization.ResultsIn the epidermis and cultured keratinocytes, LOX, LOXL1 and LOXL2 mRNA levels were predominant, and LOXL3 and LOXL4 were minor components. When cultured keratinocytes were induced to terminal differentiation, LOX expression increased 12-fold and LOXL2 expression decreased 0.1-fold, while LOXL1 mRNA level was essentially unchanged. When growth-arrested cells were allowed to adhere to extracellular matrices, LOXL2 mRNA level was stimulated 7-fold by fibronectin or type IV collagen substrate, whereas LOX mRNA level was decreased 0.1-fold by all substrates tested. Reciprocal regulation of LOX and LOXL2 expression during differentiation and adhesion was confirmed by immunoblot analysis and double immunofluorescent observation of cultured cells using anti-LOX and anti-LOXL2 antibodies. In situ hybridization analysis of normal epidermis revealed LOXL2 mRNA mainly in the lower epidermis, LOX mRNA in the upper layer of epidermis and LOXL1 throughout the epidermis.ConclusionLOX expression in cultured keratinocytes is related to keratinization whereas LOXL2 expression is related to cell–matrix interaction.