Stress Response of Human Cell Lines to Ultraviolet B

0 downloads 0 Views 197KB Size Report
Introduction. Increased expression of a specific set of genes as a consequence of ex- tracellular stress is typically observed in all organisms. Heat shock proteins.
Stress Response of Human Cell Lines to Ultraviolet B Irradiation J. Peng1,3, T.J. Piva2,4, G.L. Jones*1, G.M. Boyle2, P.G. Parsons2 and K. Watson1 School of Biological, Biomedical and Molecular Sciences, Human Biology, University of New England, Armidale, Australia; 2Melanoma Genomics Group, Queensland Institute of Medical Research, Brisbane, Australia; 3Buck Institute for Aging Research, Novato, CA, U.S.A., 4Faculty of Life Sciences, Royal Melbourne Institute of Technology University, Bundoora, Victoria, Australia 1

Introduction Increased expression of a specific set of genes as a consequence of extracellular stress is typically observed in all organisms. Heat shock proteins (hsps) are a set of evolutionarily conserved proteins, some constitutively expressed and others induced in response to physiological and environmental stresses (1). However, changes in expression of many stress-inducible genes often occur under conditions that are ultimately lethal to the cell. UVB has been reported to initiate a variety of hsp expression in mouse keratinocytes (2), as well as the induction of hsp72 in cultured human fibroblasts (3) and keratinocytes (4). This stress response may be extremely important in the protection of human skin from UV-induced injury. The ability of UVB to induce the expression of hsps in human skin cell is important because these proteins are critical for the survival of cells exposed to a variety of environmental stresses. In particular, a suboptimal stress response in skin cells may predispose skin to melanoma development and photoageing. Nevertheless, heat shock protein function offers a potential therapeutic target for modulation of UV-irradiation skin carcinogenesis and ageing. Reactive oxygen species (ROS) have been implicated in UVB induced damage to skin. The endogenous antioxidant capacity of skin, including nonenzymatic low molecular weight antioxidants and enzymes, may be a major determinant in its response to UVB oxidative mediated damage. Antioxidants may also modulate the expression of genes whose products are involved in carcinogenesis, ageing and inflammation (5). NF-κB may be a central target for oxidants in the UV response but signaling pathways in©2003 by MEDIMOND S.r.l.

D627C0002

79

80 Free Radicals and Oxidative Stress: Chemistry, Biochemistry and Pathophysiological Implications

volved in the antioxidant protection against UVB irradiation are not well understood. Large-scale gene expression analysis with cDNA arrays, provide the opportunity to observe the broad effects of UVB-irradiation and antioxidants on signaling pathways. The results presented here address the hypothesis that antioxidants may ameliorate UV-induced skin damage through the concerted modulation of gene expression.

Results and Discussion UVB induced stress response in human skin cells - Western blot analyses were performed to identify UVB-inducible stress proteins using the human

UVB (J/m2)

0

100

200

500

Post-UVB 4 h

Control

MM96L

HaCaT

NFF

melanocytes

melanocytes

Control

Post-UVB 4 h

HaCaT

Post-UVB 2 h

MM96L

C

HO-1 Post-UVB 8 h

Control

A

Post-UVB 4 h

hsp70

B

MM96L HaCaT NFF melanocytes FIG. 1. Effect of UVB on stress protein and NF-NB subunit p65 syntheses. A. Cells were exposed to different levels of UVB and incubated in media at 37qC for 8 h as described in the Materials and Methods section. Equal amounts of protein (20 Pg) were electrophoresed by SDS-PAGE and Western blot analysis performed with specific antibody for hsp70. B. Cells were exposed to UVB (200 J/m2) and incubated in media at 37qC for 4 or 8 h. Western blot analysis was performed with specific antibodies for hsp70 and HO-1 (hsp32). C. Cells were exposed to 500 J/m2 UVB and incubated in media at 37qC for 2 or 4 h. Western blot analysis of nuclear extracts was performed with specific antibody for p65.

N

N

Ioannina, Greece, June 26-29, 2003

81

cell lines MM96L HaCaT, NFF and melanocytes exposed to UVB (100 500 J/m2). By contrast with sham-irradiated cells, an increase in hsp70 synthesis was observed in UVB-irradiated cells, corresponding to the major hsp found in mammalian cells (Figure 1A). As shown in Figure 1B, the synthesis of hsp70 was induced in these cells following exposure to 200 J/m2 UVB and recovery at 37°C for 4 and 8 h. On the other hand, under the same UVB exposure conditions, changes in the constitutive expression of haem oxygenase-1 (HO-1, hsp32) in MM96L and HaCaT cells were minimal. Interestingly, Western blot analysis revealed that UVB (500 J/m2) induced p65 (Rel A) accumulation in the nucleus at 2 h and 4 h post-irradiation in different human cell lines (Figure 1C). Heat shock induced cell survival following UVB irradiation - The effect of prior heat shock on subsequent UVB irradiation was investigated in HaCaT, NFF and melanocytes. Cells were heat shocked at 42°C for 1 h, allowed to recover at 37°C for 3 h, and then exposed to 500 J/m2 UVB. A mild heat shock prior to UVB significantly increased survival and tolerance to UVB irradiation damage in all three cell lines. Furthermore, significant up-regulation of hsp70 and hsp90 was observed, following heat shock of the human cell lines (Figure 2A). By contrast with UVB alone, treatment of cells with a heat shock prior to UVB irradiation decreased NF-κB nuclear localization following exposure to irradiation (Figure 2B), particularly in NFF cells. Effect of NAC on UVB irradiation – In order to examine whether the cellular redox status can modulate the effects of UVB-irradiation, the influence of the antioxidant NAC (N-acetylcysteine) on NF-κB nuclear localization, cell viability and cell cycle status was investigated. A partial N A

hsp90 C

HS

hsp70 C

B

HS

HaCaT

HaCaT

NFF

NFF

melanocytes

melanocytes

+ HS  HS C UV C UV

FIG. 2. Effect of UVB and heat shock on stress protein and NF-NB subunit p65 syntheses. A. C, control cells incubated at 37qC throughout; HS, heat shocked cells incubated at 42qC for 1 h and then allowed to recover at 37qC for 3 h. Equal amounts of protein (20 Pg) were electrophoresed by SDS-PAGE and Western blot analysis performed with specific antibodies for hsp90 and hsp70. B. Cells were incubated at either 37qC or 42qC for 1 h and then allowed to recover at 37qC for 3 h. Cells were washed twice with PBS, left in PBS and exposed to 500 J/m2 UVB. Following UVB or sham exposure, cells were incubated in media at 37qC for 2 h. Equal amounts of nuclear protein extracts (20 Pg) were electrophoresed by SDS-PAGE and Western blot analysis performed with specific antibody for p65.

N

82 Free Radicals and Oxidative Stress: Chemistry, Biochemistry and Pathophysiological Implications

inhibition of UVB-induced NF-kB translocation was observed with NAC (Figure 3A). The cellular oxidative stress induced by the free radical initiator, AAPH, was shown to greatly enhance UVB-irradiation cytotoxicity in MM96L and HaCaT cells (Figure 3B). As shown in Figure 3B, pre-incubation of HaCaT cells with NAC (10 mM) offered protection (e.g. 50-60% survival at 200 J/m2, compared with 10 - 30% survival in the presence of NAC) against subsequent AAPH oxidative stress. UVB-induced changes in gene expression in HaCaT cells - In order to further characterize the molecular events associated with UVB exposure on HaCaT cells, both Atlas Human and Atlas Cancer cDNA array filters were screened with radiolabelled cDNA derived from poly(A)+ RNA from control and UVB (200 J/m2) post-irradiation 4 h samples. In the case of up-regulated genes, the minimum normalized count and the minimum ratio in treated samples were set greater than 20,000 and 2-fold (UVB-treated to control), respectively. In the case of down-regulated genes, the minimum normalized count in the control sample was set greater than 20,000 and the minimum ratio in treated samples was set less than 0.5-fold. Based on these limits, the majority (80%) of genes that were screenedN (2200) were down-regulated  NAC C

UV

+ NAC C UV

A

B

HaCaT

C

MM96L

100

100

N

75

Viability (%)

Viability (%)

80

50

25

60 40 20 0

0 0

100

200

UVB (J/m2)

300

0

100

200

UVB (J/m2)

FIG. 3. Effect of NAC on UVB-induced NF-NB translocation and cell viabilities in HaCaT cells. A. Cells were incubated with or without 10 mM NAC for 1 h, then exposed to 500 J/m2 UVB. Following UVB or sham exposure, cells were incubated in medium with or without 10 mM NAC for 4 h. Equal amounts of nuclear protein extracts (20 Pg) were electrophoresed by SDS-PAGE and Western blot analysis performed with specific antibody for p65. B. Cell viabilities following the treatments in A were determined by the Sulforhodamine B calorimetric assay (13). Points are mean r SD, n = 5. MM96L cells, control (), 20 mM AAPH (i), 40 mM AAPH (x). HaCaT cells, control (2), 10 mM NAC (x), 40 mM AAPH (¡), 40 mM AAPH + 10 mM NAC (i).

Ioannina, Greece, June 26-29, 2003

83

following exposure to UVB Of the genes that increased in expression as a result of UVB irradiation, 30% were mediators of the stress response, including the heat shock proteins hsp27 and hsp70, glutathione peroxidase (GPX1, GPX2), thioredoxin peroxidase 1 (TDPX1), anti-apoptosis protein (RP105 and IEX-1) and glutaredoxin.By contrast, and consistent with Western blot analysis, there was only a 1.8-fold increase in mRNA level of HO-1 following UVB exposure. A general cell defensive response was also consistent with reduction in the expression of genes involved in cell proliferation and cell cycle progression. These included down-regulation in genes associated with cyclindependent kinase activity, such as cyclin-dependent kinase 4 inhibitor (CDKN2A), cyclins (CCND1 and CCNB1), cyclin-dependent kinase regulatory subunit (CKS1 and CKS2) and M-phase inducer phosphatase 2 (CDC25B). A generalised decrease in proliferation enzymes was suggested by the decline in the expression of genes involved in DNA replication (TOPI, TOP 2A, RPA70 and DNA-PK), translation (RNA polymerase II elongation factor p15 subunit) and protein synthesis (60S ribosomal protein L5 and L6, EIFá). The direction of changes in other functional categories, such as signal transduction and transcription, support a UVB irradiation-related trend consistent with cell oxidant stress defence. The effect of antioxidants on the gene expression profile of UVB irradiated cells, was investigated using cultures of HaCaT cells that were incubated with 10 mM NAC at 37°C for 1 h prior to exposure to 200 J/m2 UVB. Following UVB-exposure, cells were allowed to recover in fresh culture media for 4 h. NAC preincubation was shown to attenuate the UVB irradiationrelated changes in gene expression profiles, in particular the stress-inducible genes which were up-regulated by UVB. Of the genes that displayed consistent irradiation-associated alterations, 74% were suppressed by NAC. This result supports the general trend of UV stressed cells displaying the hallmarks of disruption in oxidant homeostasis.

Acknowledgments This work was partially supported by internal research grants from the University of New England (UNE) and the National Health and Medical Research Council of Australia. JP is the recipient of a Postgraduate Research Scholarship (UNERS).

References 1. Morimoto, R.I., Tissieres, A and Georgopoulos, C. (1994) The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, New York. 2. Maytin, E. V. (1992) Differential effects of heat shock and UVB light upon stress protein expression in epidermal keratinocytes. J Biol Chem 267, 2318923196

84 Free Radicals and Oxidative Stress: Chemistry, Biochemistry and Pathophysiological Implications 3. Cao, Y., Ohwatari, N., Matsumoto, T., Kosaka, M., Ohtsuru, A. and Yamashita, S. (1999) TGF-beta1 mediates 70-KDa heat shock protein induction due to ultraviolet irradiation in human skin fibroblasts. Pflugers Arch 438, 239244 4. Maytin, E. V., Wimberly, J. M. and Kane, K. S. (1994) Heat shock modulates UVB-induced cell death in human epidermal keratinocytes: evidence for a hyperthermia-inducible protective response. J Invest Dermatol 103, 547-553 5. Sen, C. K. and Packer, L. (1996) Antioxidant and redox regulation of gene expression. FASEB J 10, 709-720