Environmental pro-oxidants induce altered envelope protein profiles in human keratinocytes

Abstract

Cornified envelopes (CEs) of human epidermis ordinarily consist of transglutaminase-mediated cross-linked proteins and are essential for skin barrier function. However, in addition to enzyme-mediated isopeptide bonding, protein cross-linking could also arise from oxidative damage. Our group recently demonstrated abnormal incorporation of cellular proteins into CEs by pro-oxidants in woodsmoke. In this study, we focused on 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), mesquite liquid smoke (MLS), and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), to further understand the mechanisms through which environmental pro-oxidants induce CE formation and alter the CE proteome. CEs induced by the ionophore X537A were used for comparison. Similar to X537A, DMNQ- and MLS-induced CE formation was associated with membrane permeabilization. However, since DMNQ is non-adduct forming, its CEs were similar in protein profile to those from X537A. By contrast, MLS, rich in reactive carbonyls that can form protein adducts, caused a dramatic change in the CE proteome. TCDD-CEs were found to contain many CE precursors, such as small proline-rich proteins and late cornified envelope proteins, encoded by the epidermal differentiation complex. Since expression of these proteins is mediated by the aryl hydrocarbon receptor (AhR), and its well-known target protein, CYP1A1, was exclusively present in the TCDD group, we suggest that TCDD alters the CE proteome through persistent AhR activation. This study demonstrates the potential of environmental pro-oxidants to alter the epidermal CE proteome and indicates that the cellular redox state has an important role in CE formation.

Human Keratinocyte Responses to Woodsmoke Chemicals

“…These findings resemble the relatively high keratin content for envelope formation induced by liquid smoke extract (50%) but much lower (2%) for cells permeabilized with the reactive oxygen generating chemical DMNQ or the ionophore X537A. 20 3.4. Gene Expression.…”

“…To show amine incorporation into protein independent of cellular transglutaminase, cultures were treated with 20 mM iodoacetamide for 1 h before harvest to inactivate this activity. 20 2.6. Sample Processing for Proteomic Profiling.…”

“…Noteworthy responses upon treatment include induction of cytochrome P4501A1, markers of oxidative stress and inflammatory mediators, and, at a higher concentration, cross-linked envelope formation, a lethal event. 20 Although health effects correlate closely with the PM2.5 level, recent studies have demonstrated different levels of toxicity among different chemical classes as well as PM from different sources, suggesting potentially distinctive effects from PM with different chemical compositions. Profoundly different effects of pollutant chemicals on keratinocyte envelope formation have recently been observed.…”

“…23 Our previous work indicates that the exposure of keratinocytes to the woodsmoke extract perturbs protein incorporation into envelopes and in this way could affect dermatological disorders. 20 Since woodsmoke extract is a complex chemical mixture, the present work has focused on studying commercially available chemicals (phenols, catechols, and carbonyls) known to be or similar to those in aqueous smoke extract. 19 The hypothesis being explored is that individual chemical moieties in the extract are responsible for the specific responses observed.…”

“…Noteworthy responses upon treatment include induction of cytochrome P4501A1, markers of oxidative stress and inflammatory mediators, and, at a higher concentration, cross-linked envelope formation, a lethal event. 20 …”