Lysyl Oxidase-Like and Lysyl Oxidase Are Present in the Dermis and Epidermis of a Skin Equivalent and in Human Skin and Are Associated to Elastic Fibers

Abstract

Elastic fiber formation involves the secretion of tropoelastin which is converted to insoluble elastin by cross-linking, initiated by the oxidative deamination of lysine residues by lysyl oxidase. Five lysyl oxidase genes have been discovered. This study deals with the expression of two isoforms, LOX and LOX-like (LOXL), in human foreskin and in a human skin-equivalent (SE) model that allows the formation of elastic fibers. In this model, keratinocytes are added to a dermal equivalent made of fibroblasts grown on a chitosan-cross-linked collagen-GAG matrix. LOX and LOXL were detected by immunohistochemistry in the dermis and the epidermis of both normal skin and in a SE. This expression was confirmed by in situ hybridization on the SE. LOX and LOXL expression patterns were confirmed in human skin. The ultrastructural localization of LOXL was indicative of its association with elastin-positive materials within the SE and human skin, though interaction with collagen could not be discarded. LOX was found on collagen fibers and could be associated with elastin-positive materials in the SE and human skin. LOXL and LOX were detected in keratinocytes where LOX was mainly expressed by differentiating keratinocytes, in contrast to LOXL that can be found in both proliferating and differentiating fibroblasts. These data favor a role for LOXL in elastic fiber formation, together with LOX, and within the epidermis where both enzymes should play a role in post-translational modification of yet unknown substrates.

The Pro-regions of Lysyl Oxidase and Lysyl Oxidase-like 1 Are Required for Deposition onto Elastic Fibers

“…Heart, lung, and skin, for example, have overlapping localization of LOX and LOXL. Furthermore, both enzymes co-localize to the same elastic fibers of human skin using immunoelectron microscopy (13). These findings suggest that even if LOX and LOXL have the same catalytic activity and are expressed in the same tissues, they appear to not have completely redundant roles in elastic fibers formation and homeostasis.…”

“…Antibodies-A polyclonal antibody against human recombinant LOX (anti-LOX 228 -279 ) was used as described (13) for LOX immunolocalization and Western blot analysis. For LOXL Western blot analysis, a polyclonal antibody against human recombinant LOXL (anti-LOXL 355-416 ) was used as described (13).…”

“…For LOXL Western blot analysis, a polyclonal antibody against human recombinant LOXL (anti-LOXL 355-416 ) was used as described (13). A new polyclonal antibody against recombinant mouse LOXL (anti-LOXL 204 -393 ) was used for immunolocalization of LOXL.…”

“…Transient Transfection Constructs and Mutagenesis-The transfection constructs used for this study 5 were derived from a previously characterized full-length murine cDNA of LOX and from a full-length human cDNA of LOXL (13,15). Chimeric V5 fusion proteins were inserted in-frame with the C-terminal V5/His tag within the pcDNA4-V5/His vector (Invitrogen).…”

Intracellular localization of the matrix enzyme lysyl oxidase in polarized epithelial cells

“…Although epithelia face the extracellular matrix with their basal surfaces, there is poor knowledge about a potential role of LOX in epithelial cell types. A few reports however did provide evidence for LOX expression in some epithelial tissues based on immunohistochemistry data and one study detected LOX mRNA expression in cultured rabbit pigment epithelium cells (Omori et al, 2002, Hayashi et al, 2004, Noblesse et al, 2004Fogelgren et al, 2005). Additionally, recent findings indicate that LOX is a critical factor when breast epithelial tumor cells acquire a migratory and invasive phenotype in vitro (Kirschmann et al, 1999;Kirschmann et al, 2002;Payne et al, 2005).…”