title: The enzymatic activity of lysyl oxidas-like-2 (LOXL2) is not required for LOXL2-induced inhibition of keratinocyte differentiation.
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.
authors.affiliation: Cancer and Vascular Biology Research Center, Bruce Rappaport Faculty of Medicine, Technion, Israel Institute of Technology, Haifa 31096, Israel.
authors.forename: Jennie
authors.initials: J
authors.lastname: Lugassy
authors.forename: Shelly
authors.initials: S
authors.lastname: Zaffryar-Eilot
authors.forename: Sharon
authors.initials: S
authors.lastname: Soueid
authors.forename: Amit
authors.initials: A
authors.lastname: Mordoviz
authors.forename: Victoria
authors.initials: V
authors.lastname: Smith
authors.forename: Ofra
authors.initials: O
authors.lastname: Kessler
authors.forename: Gera
authors.initials: G
authors.lastname: Neufeld
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000629:Aminopropionitrile / Q000494:pharmacology; D019138:Antibodies, Blocking / Q000494:pharmacology; D058846:Antibodies, Monoclonal, Murine-Derived / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D057809:HEK293 Cells; D006801:Humans; D015603:Keratinocytes / Q000166:cytology / Q000201:enzymology*; D017354:Point Mutation; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D017434:Protein Structure, Tertiary; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D014807:Vitamin D / Q000494:pharmacology; D014815:Vitamins / Q000494:pharmacology
chemical_list: D019138:Antibodies, Blocking; D058846:Antibodies, Monoclonal, Murine-Derived; D011498:Protein Precursors; D014815:Vitamins; D014807:Vitamin D; D000629:Aminopropionitrile; C032253:involucrin; D000594:Amino Acid Oxidoreductases; C119091:LOXL2 protein, human; D002118:Calcium