Differential modulation of stress-inflammation responses by plant polyphenols in cultured normal human keratinocytes and immortalized HaCaT cells Saveria Pastore a , Daniela Lulli a , Alla I. Potapovich a,b , Paolo Fidanza a , Vladimir A. Kostyuk a,b , Elena Dellambra a , Chiara De Luca a , Riccardo Maurelli a , Liudmila G. Korkina a, * a Lab. Tissue Engineering and Skin Pathophysiology, Dermatology Institute (Istituto Dermopatico dell’Immacolata, IDI IRCCS), Rome 00167, Italy b Biochemistry Department, Byelorussian State University, Minsk 220050, Byelorussia 1. Introduction Acute inflammatory response in the skin is a local protective reaction to various stresses including injury, microbial invasion, solar irradiation, and environmental pollutants [1]. On the other hand, chronic or excessive environmental and endogenous stresses or altered skin structure/functions may lead to persistent inflammation, which is considered a causative reason for skin cancers and premature ageing [2,3]. Keratinocytes appear to be not only primary sensors of stressful conditions but also major players of the extremely complex immune response in the skin conducting an orchestrated recruitment and functions of other immune cells involved in a stress-inflammation skin reaction [4]. Therefore cultured keratinocytes have become a prototype model for screening of anti-inflammatory, photo-protective, and cancer preventive substances for topical application [5–8]. There is a steadily growing interest in the skin protection by plant polyphenols (PPs) although the mechanisms by which these natural compounds exert their beneficial effects are not fully understood [9–12]. The skin benefits of PPs have been largely attributed to their classical antioxidant activity. However, in the last 5–10 years, evidence from numerous in vitro skin cell studies suggests that PPs can influence cellular functions by multiple other mechanisms, such as direct interaction with several receptors, modulation of intracellular signal transduction and transcription of a number of genes as well as post-translational modulation of enzymatic activities [2,13–15]. The hypothesis has been recently formulated and first publications have appeared that PPs-related modulation may also depend on their interaction with epigenomic processes [9,16,17]. It should be noted that the majority of the molecular pathways targeted by PPs are redox-dependent although free radical scavenging and metal chelating properties Journal of Dermatological Science 63 (2011) 104–114 A R T I C L E I N F O Article history: Received 2 February 2011 Received in revised form 23 March 2011 Accepted 19 April 2011 Keywords: HaCaT cells Lipopolysaccharide Normal human epidermal keratinocytes Plant polyphenols TNF-alpha UVA A B S T R A C T Background: Environmental and endogenous stresses to skin are considered causative reasons for skin cancers, premature ageing, and chronic inflammation. Screening of substances with preventive and/or curative properties is currently based on mechanistic studies of their effects towards stress-induced responses in skin cell cultures. Objective: We compared effects of plant polyphenols (PPs) on the constitutive, UVA-, LPS-, or TNF-alpha- induced inflammatory responses in cultured normal human epidermal keratinocytes (NHEK) and immortalized HaCaT cells. Methods: Representatives of three classes of PPs, flavonoids, stilbenoids, and phenylpropanoids were studied. Their effects on mRNA were determined by qRT-PCR; protein expression was assayed by Western blot and bioplexed ELISA; phosphorylation of Akt1, ERK1/2, EGFR, and NFkappaB was quantified by intracellular ELISA or Western blot. Results: PPs or their combination with UVA or LPS induced strong up-regulation of stress responses in HaCaT but not in NHEK. In addition, compared to NHEK, HaCaT responded to TNF-alpha with higher synthesis of MCP-1, IP-10 and IL-8, concomitant with stronger NFkappaB activation. PPs down-regulated the chemokine release from both cell types, although with distinct effects on NFkappaB, Akt1, ERK, and EGFR activation. Conclusion: Results of pharmacological screenings obtained by using HaCaT should be cautiously considered while extending them to primary keratinocytes from human epidermis. ß 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. * Corresponding author at: Laboratory of Tissue Engineering and Skin Patho- physiology, Istituto Dermopatico dell’Immacolata (IDI IRCCS), Via Monti di Creta 104, Rome 00167, Italy. Tel.: +39 06 66464258; fax: +39 06 66464253. E-mail address: l.korkina@idi.it (L.G. Korkina). Contents lists available at ScienceDirect Journal of Dermatological Science jou r nal h o mep ag e: w ww .elsevier .co m /jds 0923-1811/$36.00 ß 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2011.04.011