The Enzymatic Activity of Lysyl Oxidas-like-2 (LOXL2) Is Not Required for LOXL2-induced Inhibition of Keratinocyte Differentiation

Jennie Lugassy,Shelly Zaffryar-Eilot,Sharon Soueid,Amit Mordoviz,Victoria Smith,Ofra Kessler,Gera Neufeld

doi:10.1074/jbc.m111.261016

Abstract

Lysyl oxidase-like-2 (LOXL2) induces tumor progression and fibrosis. It also inhibits the differentiation of keratinocytes promoting development of squamous cell carcinomas. Stimulation of HaCaT skin keratinocytes with exogenous LOXL2 or overexpression of LOXL2 in these cells inhibits their differentiation as manifested by inhibition of calcium or vitamin D-induced involucrin expression. The inhibition was abrogated by the LOXL2 function-blocking monoclonal antibody AB0023 as well as by an anti-LOXL2 polyclonal antibody. Surprisingly, a point-mutated form of LOXL2 (LOXL2(Y689F)) lacking enzymatic activity, as well as a LOXL2 deletion mutant lacking the entire catalytic domain, also inhibited calcium or vitamin D-induced up-regulation of involucrin expression, suggesting that the enzymatic activity of LOXL2 is not required for this activity. This conclusion was supported by experiments that showed that β-aminoproprionitrile, an irreversible competitive inhibitor of the enzymatic activity of all lysyl oxidases, is unable to abolish the LOXL2-induced inhibition of HaCaT cell differentiation. The activity of LOXL2(Y689F) required the presence of the fourth scavenger receptor-cysteine-rich (SRCR) domain of LOXL2, which is also the binding target of AB0023. Epitope-tagged LOXL2(Y689F) was internalized at 37 °C by HaCaT cells. The internalization was inhibited by AB0023 and by competition with unlabeled LOXL2, suggesting that these cells may express a LOXL2 receptor. Our results suggest that agents that inhibit the enzymatic activity of LOXL2 may not suffice to inhibit completely the effects of LOXL2 on complex processes that involve altered states of cellular differentiation.

Highlights

It undergoes differentiation when exposed to calcium or to additional inducers of keratinocyte differentiation such as vitamin D, which is manifested by the up-regulation of the expression of keratinocyte differentiation markers such as involucrin, keratin-10, or filaggrin [30]
We have cloned a 3-kb fragment containing part of the Lysyl oxidase-like-2 (LOXL2) promoter (Fig. 1C). This DNA fragment was able to down-regulate the expression of a luciferase reporter gene fused downstream to it when HaCaT cells transfected with this construct were exposed to high calcium concentrations or when they were stimulated with vitamin D, suggesting that the decreased LOXL2 mRNA concentrations found in differentiating HaCaT cells were due to inhibition of LOXL2 transcription (Fig. 1D)
It was reported that oxidation of the transcription factor snail by intracellular LOXL2 is part of the mechanism by which LOXL2 inhibits the differentiation of keratinocytes and promotes the progression of dermal malignancies such as squamous cell carcinoma of the skin [13, 16]

Summary

Background

Lysyl oxidase-like-2 (LOXL2) induces tumor progression and fibrosis It inhibits the differentiation of keratinocytes promoting development of squamous cell carcinomas. A point-mutated form of LOXL2 (LOXL2Y689F) lacking enzymatic activity, as well as a LOXL2 deletion mutant lacking the entire catalytic domain, inhibited calcium or vitamin D-induced up-regulation of involucrin expression, suggesting that the enzymatic activity of LOXL2 is not required for this activity This conclusion was supported by experiments that showed that ␤-aminoproprionitrile, an irreversible competitive inhibitor of the enzymatic activity of all lysyl oxidases, is unable to abolish the LOXL2-induced inhibition of HaCaT cell differentiation. Silencing LOX expression, in contrast to the silencing of LOXL2, impairs keratinocyte differentiation [17] Enzymes such as heparanase have been recently found to induce biological responses by mechanisms that do not require enzyme activity [18]. We provide preliminary evidence suggesting that keratinocytes express a functional LOXL2 receptor on their cell surface

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION