Abstract
Background
Classical 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.
Objectives
To investigate the regulation of LOs during differentiation and cell adhesion of cultured
keratinocytes.
Methods
LO 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.
Results
In 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.
Conclusion
LOX expression in cultured keratinocytes is related to keratinization whereas LOXL2
expression is related to cell–matrix interaction.
Abbreviations:
EDTA (ethylenediaminetetraacetic acid), NEM (N-ethylmaleimide), PMSF (phenylmethylsulfonyl fluoride), SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis), TCA (trichloroacetic acid)Keywords
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Article info
Publication history
Published online: July 12, 2010
Accepted:
March 19,
2009
Received in revised form:
March 6,
2009
Received:
January 16,
2009
Identification
Copyright
© 2009 Japanese Society for Investigative Dermatology. Published by Elsevier Inc. All rights reserved.
