Original contribution
Impairment of proteasome function upon UVA- and UVB-irradiation of human keratinocytes

https://doi.org/10.1016/S0891-5849(02)00816-XGet rights and content

Abstract

The major environmental influence for epidermal cells is sun exposure and the harmful effect of UV radiation on skin is related to the generation of reactive oxygen species that are altering cellular components including proteins. It is now well established that the proteasome is responsible for the degradation of oxidized proteins. Therefore, the effects of UV-irradiation on proteasome have been investigated in human keratinocyte cultures. Human keratinocytes were irradiated with 10 J/cm2 of UVA and 0.05 J/cm2 of UVB and proteasome peptidase activities were measured in cell lysates using fluorogenic peptides. All three peptidase activities were decreased as early as 1 h and up to 24 h after irradiation of the cells. Increased levels of oxidized and ubiquitinated proteins as well as proteins modified by the lipid peroxidation product 4-hydroxy-2-nonenal were also observed in irradiated cells. However, immunopurified 20S proteasome exhibited no difference in both peptidase specific activities and 2D gel pattern of subunits in irradiated cells, ruling out the possibility that the 20S proteasome could be a target for the UV-induced damage. Finally, extracts from irradiated keratinocytes were able to inhibit degradation by the proteasome, demonstrating the presence of endogeneous inhibitors, including 4-hydroxy-2-nonenal modified proteins, generated upon UV-irradiation.

Introduction

Exposure of human skin to UV-irradiation over a period of years results in chronic photodamage, also called photoaging [1]. Photoaging induces morphological changes such as wrinkling and sagging due to general alteration of all the epidermal and dermal components of the skin including the cutaneous cells [2]. UVA radiation (320–400 nm) constitutes more than 90% of the terrestrial UV solar energy and penetrates more efficiently than UVB (280–320 nm) in the proliferative basal layers of the epidermis [3]. Although it is well accepted that low levels of reactive oxygen species (ROS) are continuously produced in vivo and are involved in physiological processes, there is accumulating evidence for the damaging effects of higher concentrations of ROS (hydroxyl radical, superoxide anion, and singlet oxygen) generated in vivo after UVA- and UVB-irradiation of the skin [4], [5], [6]. Generation of ROS following irradiation with UVA [7] and UVB [8] requires the absorption of photons by endogenous photosensitizer molecules [9]. The UVA-induced skin damage is mainly related to cell photosensitization involving endogenous sensitizers such as NAD(P)H flavines, or porphyrins [10], [11], [12]. The photosensitized reactions produce ROS such as singlet oxygen and oxygen radicals, which react with DNA, resulting in transient and permanent genetic damage, and with lipids and proteins, resulting in the activation of cytoplasmic signal transduction pathways that are related to growth, differentiation, and replicative senescence [5], [9], [13], [14]. The peroxidation of lipids and oxidation of glutathione have been shown to be the immediate consequences of UVA-irradiation of human skin [15], [16], [17]. UVB-dependent generation of hydroxyl radicals and lipids peroxides has been demonstrated in murine skin and in keratinocytes [8], [18]. Therefore, UV radiation can damage proteins by direct oxidation or by covalent binding of lipid peroxidation breakdown products that result in loss in protein function and/or enzymatic activity [19], [20], [21], [22].

The proteasome is the major proteolytic system that has been implicated in removal of abnormal and oxidatively damaged proteins [23], [24]. The 20S proteasome is made of 28 subunits arranged as four stacked rings. The two outer rings are formed by α subunits and the two inner rings are formed by β subunits that carry the proteolytic activities. The core 20S proteasome can combine with the 19S regulator complex to form a 26S proteolytic complex. This complex is responsible for the ATP- and ubiquitin-dependent proteolysis and the degradation of key molecules in signal transduction, cell cycle control, and immune response [25]. Recent studies of our laboratory on the effect of aging in human keratinocytes and epidermis have evidenced a decreased proteasome activity and content with age [26] and age-related alterations of proteasome subunits [27]. These results may explain, at least in part, the accumulation of highly oxidized and aggregated proteins in the cytosol of cells during skin aging [26]. There is evidence that aging and photoaging have, at least in part, overlapping biochemical mechanisms which make it interesting to study the effects of UV-irradiation on proteasome function [28], [29].

The fate of proteasome subjected to oxidative stress in vitro and in vivo has been studied by many groups, using different oxidant treatments [30], [31], [32], [33], [34], [35], [36], [37], [38]. We have recently shown that 20S proteasome is inactivated during ischemia-reperfusion injury [39]. The current study was undertaken to characterize the effects of UV-irradiation of human keratinocytes on proteasome peptidase activities in order to assess if alterations in proteasome function may contribute to photoaging. Our results demonstrate that UV-irradiation of keratinocytes induces a significant decline in proteasome function and is correlated with an increase in oxidatively modified and ubiquitinated proteins. Among oxidatively modified proteins, we have shown that some cytosolic proteins undergo modification by the lipid peroxidation product 4-hydroxy-2-nonenal (HNE). HNE is believed to be largely responsible for the cytopathological effects observed during oxidative stress in vivo [21]. It can react with different proteins and impair their function [40], [41], [42], [43]. Moreover, HNE-modified proteins have been previously reported to be resistant to degradation by the proteasome and to act as potent inhibitors of the proteasome in vitro [44], [45]. No inactivation of the 20S core enzyme nor dissociation of the 26S proteasome were observed upon UV-irradiation of keratinocytes, while extracts from UV-irradiated cells were inhibiting FITC-casein degradation by the proteasome. In addition, removal of HNE-modified proteins was found to lower the inhibitory activity in the extracts. Therefore, these HNE-modified proteins as well as other highly oxidized proteins that are generated upon UV-irradiation of keratinocytes appear as good candidates to account for proteasome inhibition.

Section snippets

Immunochemical reagents

Polyclonal anti-HNE-antibodies specific to 1:1amino acid-HNE Michael adducts [46] were provided by Dr L. I. Szweda (CWRU, Cleveland, OH, USA). Polyclonal anti-ubiquitin antibodies specific to ubiquitin were obtained from Dako (Dako S.A., Trappes, France). Antibodies specific to proteasome subunits S7, MSS1 (19S ATPase subunits); S14 (19S non-ATPase subunit), Y (MCP 421); Z (MCP 168), and all α subunits (MCP 231) were obtained from Affiniti Research Products Limited (Mamhead, Exeter, UK).

Human keratinocyte primary culture

All

Proteasome activities and content in UV-irradiated keratinocytes

Normal human keratinocytes from primary cultures were submitted to different doses of UVA and UVB and harvested 24 h after the end of the irradiation. An irradiation dose of 10 J/cm2 UVA and 0.05 J/cm2 UVB was chosen since it is a sublethal dose that both keeps cellular viability as high as 90% (see Table 1) and inhibits proteasome activity that was monitored by assaying chymotrypsin-like, peptidylglutamyl-peptide hydrolase, and trypsin-like peptidase activities in cell lysates, using

Discussion

In this study we have shown that proteasome activity is compromised by UV-irradiation in human keratinocytes. Indeed, irradiation of the cells by UVA (10 J/cm2) and UVB (0.05 J/cm2) results in declines in proteasome chymotrypsin-like, peptidylglutamyl-peptide hydrolase, and trypsin-like activities in cytosolic extracts. The declines were observed as early as 1 h after irradiation and lasted for 24 h (Fig. 1). No recovery of proteasome peptidase activities was observed even 96 h after

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