Proteomic Profiling Identifies an UV-Induced Activation of Cofilin-1 and Destrin in Human Epidermis
2005; Elsevier BV; Volume: 124; Issue: 4 Linguagem: Inglês
10.1111/j.0022-202x.2005.23597.x
ISSN1523-1747
AutoresPaul J. Hensbergen, Astrid E. Alewijnse, Johanna Kempenaar, R.C. van der Schors, Crina I.A. Balog, Andre M. Deelder, Gerrit J Beumer, Maria Ponec, Cornelis P. Tensen,
Tópico(s)Cellular Mechanics and Interactions
ResumoThe human skin is the only line of defense against UV radiation. A series of responses to protect the skin are induced by UV radiation. In this study, a proteomic approach was used to study these responses. We have performed high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of (solar simulated) UV-exposed reconstructed skin equivalents as well as native skin. Differentially expressed proteins were processed for mass spectrometric analysis, when consistent differences were observed in all individual human skin equivalents. In addition to proteins known to be involved in UV responses (HSP27, MnSOD, and PDX-2), we identified two novel proteins that were downregulated following UV exposure. Further analysis revealed that these proteins were the phosphorylated forms of the actin cytoskeleton modulators cofilin-1 and destrin. The de-phosphorylation of cofilin-1 was confirmed using western blotting of UV-exposed skin equivalents and ex vivo skin protein extracts. In conclusion, our study indicates the potency of a proteomic approach to study UV-induced changes in a tissue culture system mimicking human skin as well as excised human skin. The human skin is the only line of defense against UV radiation. A series of responses to protect the skin are induced by UV radiation. In this study, a proteomic approach was used to study these responses. We have performed high-resolution two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) analysis of (solar simulated) UV-exposed reconstructed skin equivalents as well as native skin. Differentially expressed proteins were processed for mass spectrometric analysis, when consistent differences were observed in all individual human skin equivalents. In addition to proteins known to be involved in UV responses (HSP27, MnSOD, and PDX-2), we identified two novel proteins that were downregulated following UV exposure. Further analysis revealed that these proteins were the phosphorylated forms of the actin cytoskeleton modulators cofilin-1 and destrin. The de-phosphorylation of cofilin-1 was confirmed using western blotting of UV-exposed skin equivalents and ex vivo skin protein extracts. In conclusion, our study indicates the potency of a proteomic approach to study UV-induced changes in a tissue culture system mimicking human skin as well as excised human skin. two-dimensional polyacrylamide gel electrophoresis mass spectrometry One of the important functions of the human skin is protection against solar radiation. Overexposure to UV radiation induces a multitude of acute and delayed defensive responses that provide protection against further UV damage. The molecular and biochemical mechanisms that lead to these responses are, however, not fully understood and have only recently become the focus of concerted studies. Immediate responses to UV radiation are derived (at least in part) from UV-generated reactive oxidants (free radicals and reactive oxygen species), which can be growth factors and cytokine receptors as well as intracellular signalling cascades (Heck et al., 2004Heck D.E. Gerecke D.R. Vetrano A.M. Laskin J.D. Solar ultraviolet radiation as a trigger of cell signal transduction.Toxicol Appl Pharmacol. 2004; 195: 288-297Crossref PubMed Scopus (105) Google Scholar). Cellular protection against reactive oxidants is provided by the intracellular enzymatic anti-oxidant defense system, which includes catalase, superoxide dismutase, and glutathione peroxidase (Shindo et al., 1993Shindo Y. Witt E. Packer L. Antioxidant defense mechanisms in murine epidermis and dermis and their responses to ultraviolet light.J Invest Dermatol. 1993; 100: 260-265Abstract Full Text PDF PubMed Google Scholar). For ethical and safety reasons, extensive and detailed in vivo studies on the UV-induced processes in human skin are not feasible to perform. Therefore, a considerable amount of the work has been undertaken to develop a skin model, which would allow screening of the skin responses to potentially harmful environmental conditions. The development of suitable in vitro cultured skin analogues accelerated during recent years, showing remarkable improvement in the quality and properties of the skin substitutes. Human skin substitutes are three-dimensional cell-culture systems kept at an air–liquid interface. They consist of epidermal cells seeded on an appropriate substrate, either cellular (fibroblast-populated) or acellular such as inert filters or de-epidermized dermis (Ponec, 2002Ponec M. Skin constructs for replacement of skin tissues for in vitro testing.Adv Drug Deliv Rev. 2002; 54: S19-S30Crossref PubMed Scopus (99) Google Scholar). Under these conditions, the epidermal cells are nourished by diffusion of nutrients upwards through the underlying substrate. Exposure of epidermal cells to the air is crucial for the build-up of the multi-layered fully differentiated tissue with an intact stratum corneum. One of the main advantages of these three-dimensional skin cultures is that their tissue architecture, expression of specific differentiation-related markers, and epidermal lipid profile are highly similar to those found in in vivo skin tissue. The presence of a functional stratum corneum enables the exposure of the reconstructed epidermis to UV irradiation at doses comparable to human solar exposure. Because keratinocytes are the primary cells that are naturally exposed to UV and they have to protect the rest of the body, others have hypothesized that keratinocyte responses to UV will be unique and not just resemble those from another stress (Li et al., 2001Li D. Turi T.G. Schuck A. Freedberg I.M. Khitrov G. Blumenberg M. Rays and arrays: The transcriptional program in the response of human epidermal keratinocytes to UVB illumination.FASEB J. 2001; 15: 2533-2535Crossref PubMed Scopus (109) Google Scholar). To identify these responses, gene expression profiling in UV-exposed keratinocyte monolayer cultures has been performed (Li et al., 2001Li D. Turi T.G. Schuck A. Freedberg I.M. Khitrov G. Blumenberg M. Rays and arrays: The transcriptional program in the response of human epidermal keratinocytes to UVB illumination.FASEB J. 2001; 15: 2533-2535Crossref PubMed Scopus (109) Google Scholar; Sesto et al., 2002Sesto A. Navarro M. Burslem F. Jorcano J.L. Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays.Proc Natl Acad Sci USA. 2002; 99: 2965-2970Crossref PubMed Scopus (155) Google Scholar). We have extended these approaches using a proteomic strategy for identifying changes in protein expression following UV exposure of human skin equivalents. For this purpose, reconstructed epidermis and excised human skin specimens were exposed to UV irradiation using a CLEO Natural lamp (Philips, Roosendaal, the Netherlands), mimicking solar UV radiation. Subsequently, epidermal proteins were isolated and analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Proteins that showed significant and reproducible changes in expression were characterized by mass spectrometry (MS). Apart from proteins known as oxidative stress markers, we describe a UV-induced dephosphorylation of the actin cytoskeleton proteins cofilin-1 and destrin. The dephosphorylation of these proteins was also found in UV-exposed ex vivo skin. To study the effect of UV irradiation in the solar band on the expression of epidermal proteins, reconstructed epidermis was exposed to Cleo natural lamps UV for 30 or 60 min (1.8 and 3.75 J per cm2, respectively). As expected from the dosage used, no significant morphological changes in reconstructed epidermis were observed in UV-exposed samples when compared with controls (Fig S1). Figure 1a shows a typical silver-stained 2D electrophoresis pattern of proteins isolated from reconstructed epidermis (control and UV-exposed) in the broad non-linear pH range 3–10. Four independent experiments were performed in duplicate with reconstructed epidermis generated with keratinocytes originating from four different skin donors. Using PD-Quest software (BIO-RAD Laboratories, Veenendaal, the Netherlands) on average about 1000 spots per gel were detected. The protein patterns showed high similarity in all 4 independent experiments performed. Differences in UV-induced protein expression profiles were considered as consistent in case the effect was reproducible with each of the 4 donors and in both duplicate experiments. This analysis revealed changes in 11 proteins (Figure 1b). These spots were further subjected to tryptic digestion and mass spectrometric analysis in order to identify the proteins. Following in gel tryptic digestion, a combination of peptide mass fingerprinting and MS/MS analysis was performed to analyze the 11 differentially UV-regulated proteins, seven of which could be identified (no unique peptide masses were observed for the other four proteins). For all details on the mass spectrometric identification, see Table S1. We identified HSP27 (spot SSP3308 and SSP4310), MnSOD (SSP7311), PDX-2 (SSP2201), and 60S acidic ribosomal phosphoprotein P0 (SSP4508) as proteins that appeared or were strongly increased after UV exposure of human skin equivalents. Two proteins were significantly down regulated following UV exposure of human skin equivalents (Figure 2). Spot SSP5207 decreased four to five times in UV-irradiated reconstructed epidermis. Peptide mass fingerprinting and MS/MS analysis identified this protein spot as non-muscle cofilin-1. Using a similar approach, spot SSP6206 (Figure 2) was identified as destrin. Because of the novelty of UV-induced changes in cofilin-1 and destrin expression, we focused on these proteins in more detail. Close inspection of the position of the identified cofilin-1 and destrin spots on the 2D-PAGE gels revealed a large discrepancy between the theoretical pI (8.2 for cofilin-1 and 8.1 for destrin as determined from http://www.expasy.org/tools/pi_tool.html) and the experimental pI (≤7, Figure 1a), for both proteins. As it is known that protein phosphorylation in general lowers the pI of proteins, we hypothesized that the two proteins that were diminished following UV exposure were the phosphorylated forms of cofilin-1 and destrin. To identify the phosphorylation status of cofilin-1 and destrin, we took advantage of the fact that phosphorylated peptides have a tendency to bind to certain heavy metals, e.g., Ga3+ (Posewitz and Tempst, 1999Posewitz M.C. Tempst P. Immobilized gallium(III) affinity chromatography of phosphopeptides.Anal Chem. 1999; 71: 2883-2892Crossref PubMed Scopus (767) Google Scholar). Therefore, a tryptic digest of cofilin-1 was purified using immobilized gallium (III) affinity chromatography (Ga(III)-IMAC). This resulted in the selective enrichment of a cofilin fragment of m/z 613.2 [M+2H]2+. Subsequent MS/MS analysis showed that within this fragment, the serine at position 2 was phosphorylated (see supplemental information). This phosphorylated fragment corresponds to the N-terminal part of mature cofilin-1. In analogy to rat cofilin (Kanamori et al., 1995Kanamori T. Hayakawa T. Suzuki M. Titani K. Identification of two 17-kDa rat parotid gland phosphoproteins, subjects for dephosphorylation upon beta-adrenergic stimulation, as destrin- and cofilin-like proteins.J Biol Chem. 1995; 270: 8061-8067Crossref PubMed Scopus (39) Google Scholar), we found that the N-terminal methionine of human cofilin-1 is eliminated and the penultimate alanine is acetylated. Using a similar approach, the peptide fragments of the protein spot identified as destrin were purified using Ga(III)-IMAC. Also in this case, a serine phosphorylated (and alanine acetylated) N-terminal fragment of destrin was identified (Ac-ApSGVQVADEVCR, data not shown). All together, these experiments clearly demonstrate that the protein spots that were attenuated after UV exposure (Figure 2) correspond to the phosphorylated forms of cofilin-1 and destrin. To study the dose-dependency of the UV-induced effect in reconstructed human epidermis, skin equivalents were irradiated for 0, 30, and 60 min respectively, and the epidermal proteins were subsequently analyzed using 2D-PAGE. From the spots shown to be upregulated after UV exposure, Mn-SOD, 60S acidic ribosomal protein P0, and HSP27 were already upregulated after 30 min, whereas PDX-2 was not present after 30 min but only appeared after 1 h of UV exposure (data not shown). A clear concentration-dependent effect in response to UV irradiation for both phospho-cofilin-1 and phospho-destrin was observed (Figure 3). For phospho-cofilin-1, the decrease in relative spot intensity after 30 min exposure to UV did not significantly differ from the control. This was primarily due to a large inter-individual difference in the UV-induced response between the four donors resulting in a high SD. In two reconstructed epidermis cultures generated with keratinocytes originating from two of the donors, a significant decrease in phospho-cofilin-1 was already found after 30 min of UV exposure, whereas for the other two donors this effect appeared only after 1 h of UV irradiation. For phospho-destrin inter-individual variation was nearly absent and the overall expression level decreased from approximately 70% of the control value after 30 min of exposure to approximately 40% after 1 h of exposure (Figure 3). To investigate whether the levels of phospho-cofilin and destrin were also diminished after UV exposure of native human skin, 2D-PAGE analysis was performed on UV-exposed excised native human skin. We used higher UV doses in these experiments (3 and 4 h exposure) as initial experiments with lower dosages did not result in the upregulation of proteins involved in oxidative stress responses (MnSOD, PDX-2) observed in human HSE (see above), indicating that the UV transmittance in reconstructed human skin equivalents is different (e.g., more sensitive) in comparison with native human skin. In analogy with the results obtained with human skin equivalents, we also observed a strong downregulation of phospho-cofilin and phospho-destrin in UV-exposed excised human skin (Figure 4a). From the results described above, it can be inferred that de-phosphorylation of cofilin-1 and destrin would result in an increase in the non-phosphorylated forms of these proteins. As an alternative, also degradation of phospho-cofilin and destrin could explain the obtained results. The basal amount of non-phosphorylated cofilin-1, for example, is very high and is located on a region of a 2D-PAGE gel (>pH=8.0) where the focusing is in general very poor. Using the 2D-PAGE approach, it was therefore difficult to determine whether cofilin-1 was de-phosphorylated or degraded. To discriminate between these two possibilities, western blot analysis was performed using both an antibody specific for phospho-cofilin-1 and one for total cofilin-1. In line with the findings obtained with the 2D-PAGE experiments, a strong downregulation of phospho-cofilin-1 was found after solar-simulated UV exposure of human skin equivalents and ex vivo human skin (Figure 4b). But no apparent difference in the total amount of cofilin-1 was observed. Therefore, we conclude that the fraction of phospho-cofilin-1 within the total cofilin-1 pool is highly decreased after UV exposure without any detectable loss (degradation) of cofilin-1. This is highly indicative of an active cofilin-1 de-phosphorylation process. Due to its barrier function, the skin is our first and only line of defense that has to protect the human body from the harmful effects of UV radiation. The clinical and histological effects of UV damage are well known but the molecular mechanisms that cause them are still not fully understood. Various approaches have been used to examine the effect of UV exposure onto human skin. To investigate the UVB-induced modulation of gene expression in epidermal keratinocytes,Li et al., 2001Li D. Turi T.G. Schuck A. Freedberg I.M. Khitrov G. Blumenberg M. Rays and arrays: The transcriptional program in the response of human epidermal keratinocytes to UVB illumination.FASEB J. 2001; 15: 2533-2535Crossref PubMed Scopus (109) Google Scholar used cDNA microarrays in combination with conventional (submerged) monolayer keratinocyte cultures. It was found that UV regulated the expression of at least 198 genes and the response could be grouped into three waves (early, intermediate, and late). To the UV-regulated genes belong transcription factors, signal transduction proteins, cytoskeletal proteins, growth factors, cytokines, chemokines, components of the cornified envelope, and mitochondrial proteins. Using oligonucleotide microarrays, the number of identified regulated transcripts in cultured human keratinocytes upon UVB exposure was increased to 539 (Sesto et al., 2002Sesto A. Navarro M. Burslem F. Jorcano J.L. Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays.Proc Natl Acad Sci USA. 2002; 99: 2965-2970Crossref PubMed Scopus (155) Google Scholar), further illustrating the complexity of the keratinocyte response to the UVB radiation. Although these approaches have provided valuable information on the UV-induced transcriptional responses in keratinocytes, they do not necessarily reflect changes in protein expression, certainly not when it comes to protein modifications. Furthermore, the above-mentioned experiments were performed using high UVB dosages on monolayer cultures without any protection from the stratum corneum. In this study, we used a proteomic approach (2D-PAGE in combination with MS) to identify novel proteins that respond to solar simulated UV irradiation in reconstructed epidermis and excised human skin. This proteomic technique was previously used for large-scale protein expression analysis in cultured human keratinocytes (Celis et al., 1995Celis J.E. Rasmussen H.H. Gromov P. et al.The human keratinocyte two-dimensional gel protein database (update 1995): Mapping components of signal transduction pathways.Electrophoresis. 1995; 16: 2177-2240Crossref PubMed Scopus (109) Google Scholar), differential protein expression analysis in human skin equivalents upon application of a skin irritant (Boxman et al., 2002Boxman I.L. Hensbergen P.J. Van Der Schors R.C. Bruynzeel D.P. Tensen C.P. Ponec M. Proteomic analysis of skin irritation reveals the induction of HSP27 by sodium lauryl sulphate in human skin.Br J Dermatol. 2002; 146: 777-785Crossref PubMed Scopus (40) Google Scholar), and more recently to create a reference proteome map of epidermal and subepidermal expressed proteins in murine skin (Huang et al., 2003Huang C.M. Foster K.W. DeSilva T. et al.Comparative proteomic profiling of murine skin.J Invest Dermatol. 2003; 121: 51-64Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). In this study, we focused on proteins that were differentially expressed in UV-exposed human skin equivalents derived from four different donors to rule out donor-to-donor inconsistencies. Thus, the detected changes are likely to reflect a common phenomenon in (solar-simulated) UV-exposed skin. This method resulted in the identification of 11 proteins, seven of which could be characterized using MS. The reason why we were unable to identify any unique peptide masses from the other four spots is most likely due to the sensitivity of the mass spectrometer and/or ineffective/unfavorable tryptic digestion. It is important to note that the number of differentially expressed proteins would increase dramatically (>50) if changes not meeting our strict criterion (taking into account only those proteins that were consistently up- or downregulated in all four donors) had been included. Because it is well known that UV induces oxidative stress responses in human skin (Schallreuter and Wood, 2001Schallreuter K.U. Wood J.M. Thioredoxin reductase—its role in epidermal redox status.J Photochem Photobiol B. 2001; 64: 179-184Crossref PubMed Scopus (87) Google Scholar; Sander et al., 2004Sander C.S. Chang H. Hamm F. Elsner P. Thiele J.J. Role of oxidative stress and the antioxidant network in cutaneous carcinogenesis.Int J Dermatol. 2004; 43: 326-335Crossref PubMed Scopus (342) Google Scholar), we expected to find proteins that are involved in these processes. Indeed, we identified HSP27, MnSOD, and PDX-2 to be consistently upregulated following UV exposure demonstrating the validity of our approach. In line with our findings,Sesto et al., 2002Sesto A. Navarro M. Burslem F. Jorcano J.L. Analysis of the ultraviolet B response in primary human keratinocytes using oligonucleotide microarrays.Proc Natl Acad Sci USA. 2002; 99: 2965-2970Crossref PubMed Scopus (155) Google Scholar, using oligonucleotide microarrays, found an increase in MnSOD and heat shock protein (including HSP27) gene expression. But no difference in the expression level of PDX-2 was found in this study, suggesting that the use of different model systems and UV spectra/dosages influences experimental outcome. In addition to the above-mentioned group of proteins, we show a UV-induced de-phosphorylation of cofilin-1 and destrin in human epidermis. Both proteins belong to actin depolymerizing factor (ADF)/cofilin family of proteins of the actin cytoskeleton modulators. This is a family of conserved, widespread, small (15–18 kDa) actin-binding proteins (Moon and Drubin, 1995Moon A. Drubin D.G. The ADF/cofilin proteins: Stimulus-responsive modulators of actin dynamics.Mol Biol Cell. 1995; 6: 1423-1431Crossref PubMed Scopus (222) Google Scholar) playing an important role in actin polymerization and, as proposed recently, also in directed migration of cells (Nishita et al., 2002Nishita M. Aizawa H. Mizuno K. Stromal cell-derived factor 1alpha activates LIM kinase 1 and induces cofilin phosphorylation for T-cell chemotaxis.Mol Cell Biol. 2002; 22: 774-783Crossref PubMed Scopus (114) Google Scholar; Ghosh et al., 2004Ghosh M. Song X. Mouneimne G. Sidani M. Lawrence D.S. Condeelis J.S. Cofilin promotes actin polymerization and defines the direction of cell motility.Science. 2004; 304: 743-746Crossref PubMed Scopus (529) Google Scholar). In the presence of ADF/cofilin proteins the actin filaments shorten, which in turn affects the cell motility and morphology. Interestingly, the actin binding activity of cofilin-1 and destrin is negatively regulated by phosphorylation of a single conserved serine residue in the N-terminal region. Therefore, the observed de-phosphorylation of cofilin-1 and destrin directly reflects activation of these two proteins. We used western blot analysis to further confirm cofilin-1 de-phosphorylation instead of cofilin-1 degradation. As mentioned above, a predicted shift to an increase in total cofilin-1 protein could not be demonstrated, mainly because the amount of total cofilin-1 in comparison with phospho-cofilin-1 is already very high. Therefore, the relative increase in total cofilin-1 as a result of phospho-cofilin-1 dephosphorylation is only small, whereas the relative decrease in phospho-cofilin-1 is large. This might raise the question about the biological significance of this contribution of the dephosphorylated cofilin to the total pool of active cofilin already present. It has been suggested, however, that only cofilin-1 molecules that are activated by de-phosphorylation are available as F-actin severing molecules, whereas the un-phosphorylated cofilin-1 molecules already present within the cell are in complex with G-actin (Moriyama et al., 1996Moriyama K. Iida K. Yahara I. Phosphorylation of Ser-3 of cofilin regulates its essential function on actin.Genes Cells. 1996; 1: 73-86Crossref PubMed Scopus (302) Google Scholar) and therefore not accessible. Thus, the instantaneous increase in active cofilin might be crucial for reorganization of the actin cytoskeleton. Several stresses and stimuli other than UV have been shown to be implicated in the de-phosphorylation of cofilin-1 and destrin (Yahara et al., 1996Yahara I. Aizawa H. Moriyama K. et al.A role of cofilin/destrin in reorganization of actin cytoskeleton in response to stresses and cell stimuli.Cell Struct Funct. 1996; 21: 421-424Crossref PubMed Scopus (44) Google Scholar). In contrast, treatment of endothelial cells with angiogenesis inhibitors resulted in increased levels of phopho-cofilin (Keezer et al., 2003Keezer S.M. Ivie S.E. Krutzsch H.C. Tandle A. Libutti S.K. Roberts D.D. Angiogenesis inhibitors target the endothelial cell cytoskeleton through altered regulation of heat shock protein 27 and cofilin.Cancer Res. 2003; 63: 6405-6412PubMed Google Scholar). To date, little is known about common upstream mechanisms resulting in changes in the phosphorylation status of these proteins. Obviously, the net de-phosphorylation observed upon UV irradiation can result from either decreased kinase activity or increased phosphatase activity. The two primary enzymes involved in these processes are the kinase LIMK (Yang et al., 1998Yang N. Higuchi O. Ohashi K. et al.Cofilin phosphorylation by LIM-kinase 1 and its role in Rac-mediated actin reorganization.Nature. 1998; 393: 809-812Crossref PubMed Scopus (1000) Google Scholar) and the phosphatase Slingshot (Niwa et al., 2002Niwa R. Nagata-Ohashi K. Takeichi M. Mizuno K. Uemura T. Control of actin reorganization by Slingshot, a family of phosphatases that dephosphorylate ADF/cofilin.Cell. 2002; 108: 233-246Abstract Full Text Full Text PDF PubMed Scopus (503) Google Scholar); however, other potential kinase activities have also been described (Lian et al., 2000Lian J.P. Marks P.G. Wang J.Y. Falls D.L. Badwey J.A. A protein kinase from neutrophils that specifically recognizes Ser-3 in cofilin.J Biol Chem. 2000; 275: 2869-2876Crossref PubMed Scopus (20) Google Scholar; Toshima et al., 2001Toshima J. Toshima J.Y. Amano T. Yang N. Narumiya S. Mizuno K. Cofilin phosphorylation by protein kinase testicular protein kinase 1 and its role in integrin-mediated actin reorganization and focal adhesion formation.Mol Biol Cell. 2001; 12: 1131-1145Crossref PubMed Scopus (212) Google Scholar). Several upstream activators of LIMK and Slingshot have been described. LIMK is activated through the Rho/ROCK (Maekawa et al., 1999Maekawa M. Ishizaki T. Boku S. et al.Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase.Science. 1999; 285: 895-898Crossref PubMed Scopus (1207) Google Scholar) and Rac/PAK1 pathway, whereas it was recently shown that Slingshot can be activated through the PI3K activity (Nishita et al., 2004Nishita M. Wang Y. Tomizawa C. Suzuki A. Niwa R. Uemura T. Mizuno K. Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase Slingshot and its role for insulin-induced membrane protrusion.J Biol Chem. 2004; 279: 7193-7198Crossref PubMed Scopus (91) Google Scholar). Inhibition of the ROCK-signalling pathway through increased levels of cytoplasmic p21 has been described as a mechanism for reduced levels of phospho-cofilin in Ras-transformed cells. Interestingly, both p21 and PI3K are involved in the complex UV signalling process (Zhang et al., 2001Zhang Q.S. Maddock D.A. Chen J.P. et al.Cytokine-induced p38 activation feedback regulates the prolonged activation of AKT cell survival pathway initiated by reactive oxygen species in response to UV irradiation in human keratinocytes.Int J Oncol. 2001; 19: 1057-1061PubMed Google Scholar; Fotedar et al., 2004Fotedar R. Bendjennat M. Fotedar A. Role of p21WAF1 in the cellular response to UV.Cell Cycle. 2004; 3: 134-137Crossref PubMed Scopus (1) Google Scholar) where the irradiation spectrum and doses of UV are determining factors in comparing the data obtained from different experiments. In our study, we have chosen solar UV radiation of human skin equivalents, thus mimicking exposure to UV of the human skin. Reactive oxygen species are key mediators of many solar ultraviolet light-induced biological effects in the skin (Tyrrell, 1995Tyrrell R.M. Ultraviolet radiation and free radical damage to skin.Biochem Soc Symp. 1995; 61: 47-53Crossref PubMed Scopus (111) Google Scholar). This is in accordance with the observation that indeed proteins involved in the response to oxidative stress (MnSOD and PDX-2) were upregulated in our model system. In agreement with our findings, it is known that the chosen proteomic analysis (2D-PAGE in combination with MS) only partially covers the total spectrum of cellular proteins; in particular, highly acidic, basic, hydrophobic (membrane), and low-molecular-weight proteins are poorly represented on 2D-PAGE gels (Gygi et al., 2000Gygi S.P. Corthals G.L. Zhang Y. Rochon Y. Aebersold R. Evaluation of two-dimensional electrophoresis-based proteome analysis.Proc Natl Acad Sci USA. 2000; 97: 9390-9395Crossref PubMed Scopus (1169) Google Scholar). Moreover, secreted proteins were not analyzed. Additional experiments using differential extraction techniques (Wu and Yates, 2003Wu C.C. Yates J.R. The application of mass spectrometry to membrane proteomics.Nat Biotechnol. 2003; 21: 262-267Crossref PubMed Scopus (482) Google Scholar) and analysis of conditioned media in combination with either 2D-PAGE or direct (2D)-LC-MS analysis could fill this caveat in our approach. In conclusion, our study indicates the potency of a proteomic approach to study UV-induced changes in a tissue culture system mimicking human skin as well as excised human skin. Interestingly, in addition to the upregulation found for anti-oxidant enzymes we report a UV-induced change in the phosphorylation state of the actin cytoskeleton modulators cofilin-1 and destrin. All experiments were performed with institutional approval. Human breast skin obtained from plastic surgery (with informed consent of the donors) was processed and used for experiments within 24 h after surgery. The tissue was washed three times in buffer (Hanks' balanced salt solution without phenol red (HBBS)). The tissue was placed epidermal side up onto a sterile underlay pre-wetted with HBBS and stored until use (for a maximum of 16 h) at 4°C. Reconstructed human epidermis was generated on inert filter substrates, as previously described in detail (Ponec and Kempenaar, 1995Ponec M. Kempenaar J. Use of human skin recombinants as an in vitro model for testing the irritation potential of cutanecus irritants.Skin Pharmacol. 1995; 8: 49-59Crossref PubMed Scopus (99) Google Scholar). For irradiation we used CLEO Natural lamps (Philips), emitting a UV spectrum (4% UVB, 96 % UVA) that resembles that of mid-day sunlight during summer at 52°N, Amsterdam (de Winter et al., 2001de Winter S. Vink A.A. Roza L. Pavel S. Solar-simulated skin adaptation and its effect on subsequent UV-induced epidermal DNA damage.J Invest Dermatol. 2001; 117: 678-682Crossref PubMed Scopus (75) Google Scholar; Rijken et al., 2004Rijken F. Bruijnzeel P.L. Weelden H.H. Kiekens R.C. Responses of black and white skin to solar-simulating radiation: Differences in DNA photodamage, infiltrating neutrophils, proteolytic enzymes induced, keratinocyte activation, and IL-10 expression.J Invest Dermatol. 2004; 122: 1448-1455Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The MED (minimal erythema dose) for skin type II with this lamp ranges from 6 to 9 J per cm2- Waldman (Rijken et al., 2004Rijken F. Bruijnzeel P.L. Weelden H.H. Kiekens R.C. Responses of black and white skin to solar-simulating radiation: Differences in DNA photodamage, infiltrating neutrophils, proteolytic enzymes induced, keratinocyte activation, and IL-10 expression.J Invest Dermatol. 2004; 122: 1448-1455Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Cultures of reconstructed human epidermis were irradiated with 1.875 and 3.75 J per cm2 as determined by a Waldmann UV dosimeter (Waldmann Medizinintechniek, Schweningen, Germany), calibrated against the lamp. These cumulative doses were obtained using exposure periods of 30 and 60 min, respectively. Excised human skin samples were irradiated for periods of 3 and 4 h (resulting in a cumulative dose of 11.25 and 15 J per cm2, respectively). Control tissue samples were placed under the lamps but shielded from the UV light by an opaque sheet of cardboard. After irradiation, the tissue samples were placed on a stainless-steel grid and transferred into culture dishes filled with medium (DMEM supplemented with 100 IU per mL penicillin and 100 μg per mL streptomycin), the level of which was adjusted so that the tissue specimens were incubated at the air–liquid interface. Specimens were re-incubated for a recovery period of 16 h at 37°C and 5% CO2. Subsequently, a small tissue sample was taken from each specimen for morphological and immunohistochemical analyses. The remaining tissue was used to isolate epidermal proteins. Native skin samples were placed with the epidermal side down between two object slides on a heating plate at 60°C for 2 min. Reconstructed human epidermis could easily be removed from the filters mechanically using forceps. Isolated epidermis was immediately lysed in TRIzol (GibcoBRL/LifeTechnologies, Breda, the Netherlands) and stored at -80°C until use. Samples were fixed in 4% paraformaldehyde and processed for embedding in paraffin. Sections (5 μm) perpendicular to the skin surface were cut and stained with hematoxylin and eosin for light microscopic examination. Proteins were isolated as recommended by the supplier (TRIzol, GibcoBRL/LifeTechnologies) and as previously described (Boxman et al., 2002Boxman I.L. Hensbergen P.J. Van Der Schors R.C. Bruynzeel D.P. Tensen C.P. Ponec M. Proteomic analysis of skin irritation reveals the induction of HSP27 by sodium lauryl sulphate in human skin.Br J Dermatol. 2002; 146: 777-785Crossref PubMed Scopus (40) Google Scholar). Protein concentrations were determined spectrophotometrically using the modified Bradford protein assay (Ramagli, 1999Ramagli L.S. Quantifying protein in 2-D PAGE solubilization buffers.Methods Mol Biol. 1999; 112: 99-103PubMed Google Scholar). A trace of bromophenol blue was added to the protein samples and 450 μg of the protein sample was loaded on 24 cm isoelectric focusing ready-made IPG strips with a non-linear gradient of pH 3–10 (Amersham Pharmacia Biotech, Roosendaal, the Netherlands). Rehydration of the IPG-strips was performed for 12 h at 30 V after which proteins were focused for 65,000 V h (IPGphor, Amersham Pharmacia Biotech). Prior to the second dimension, IPG strips were equilibrated in 1% dithiothreitol (wt/vol) followed by 2.5% iodoacetamide (wt/vol), both for 15 min in 50 mM Tris-HCl pH 8.8, 6 M urea, 30% glycerol, 2% SDS. After this procedure, the strips were placed on top of a 200 × 260 × 1.0 mm polyacrylamide gel (13% T homogenous, 2.6% C, 0.1% SDS, 375 mM Tris/HCl pH 8.8) and run for 1 h at 5 W per gel and at least 4 h at 15 W per gel (Ettan Dalt twelve Separation unit, Amersham Pharmacia Biotech). Proteins were visualized using silver or Coomassie G250 staining and analyzed by PD-Quest software version 7.0 (BIO-RAD Laboratories). Epidermal proteins from irradiated and control samples of reconstructed epidermis and native human skin were separated on a 10% SDS-PAGE gel and transferred onto nitrocellulose membranes. Non-specific binding was blocked by incubating the membranes with 3% wt/vol non-fat dry milk and 0.05% Tween-20 in TBS (TBST). Subsequently, membranes were incubated with a phospho-cofilin specific antibody (Cell Signaling, Beverly, Massachusetts, California) followed by the appropriate HRP-conjugated secondary antibody and visualized using a chemiluminescence kit (SuperSignal West Femto, Pierce, Rockford, Illinois). After stripping (Western Re-Probe buffer, Oncogene Research Products, San Diego, California) the same membrane was incubated using a total cofilin-specific antibody (Cytoskeleton, Denver, Colorado) followed by the appropriate HRP-conjugated secondary antibody and visualized as described above. Protein spots of interest were excised from Coomassie-stained gels, cut into small pieces, and dehydrated in 100% acetonitrile for 10 min. After removal of the acetonitrile, gel pieces were dried in a Speed-Vac (Savant Instruments, Holbrook, New York) and subsequently allowed to re-swell in a trypsin solution (20 ng per μL (Promega Benelux, Leiden, the Netherlands)) in 50 mM ammonium bicarbonate (pH 7.9) for 45 min on ice. Further incubation was performed overnight at 37°C. Peptide fragments were extracted for 20 min at RT using 100 μL 25 mM ammonium bicarbonate, followed two times by an extraction using 60 μL 0.1% trifluoric acid/50% acetonitrile. The extraction solutions were pooled, concentrated to approximately 30 μL (Speed-Vac, Savant Instruments), and desalted over Poros 50R2 (Applied Biosystems, Forster City, California). Peptides were eluted with 25% methanol/5% formic acid and measured by electrospray ionization MS (Waters Micromass Q-ToF, Milford, Massachusetts). For characterization by MALDI-ToF (Matrix Assisted Laser Desorption/Ionization-Time of Flight) MS (Ultraflex, Bruker, Germany), peptides were directly eluted with matrix solution (α-cyanocinnamic acid). All mass fingerprint and MS/MS data were searched against the human protein database using the Mascot program (Matrix Science, Boston, Massachusetts). Proteins identified with a score of higher than 50 were considered significant but always verified manually. To separate phosphorylated from non-phosphorylated peptides, tryptic digests of spots of interest were acidified to pH 3 using acetic acid and subsequently applied to a Gallium III affinity column (Pierce, Rockford, Illinois). The column was washed two times with a solution of 1% acetic acid and once with a solution of 0.1% acetic acid containing 10% acetonitrile. After a wash with water, phosphorylated peptides were eluted with 100 mM natriumdihydrogenphosphate (pH 9). Prior to MS, these fractions were desalted as described above. The following material is available from http://www.blackwellpublishing.com/products/journals/suppmat/JID/JID23597/JID23597sm.htm Figure S1 Morphological effect of solar simulated UV irradiation on reconstructed human epidermis on inert filters. Figure S2 Identification of the phosphorylation state of cofilin-1 A tryptic digest of cofilin-1 was purified using immobilized gallium (III) affinity chromatography. Table S1. Identification of differently expressed protein spots after UV expose of human skin equivalents We thank the Maurits en Anna de Kock foundation for financial support and Dr F. R. de Gruijl for advice on the use and calibration of the CLEO Natural lamp. ErrataJournal of Investigative DermatologyVol. 125Issue 6PreviewIn the article by Nazila Barahmani et al, "Interleukin-1 Receptor Antagonist Allele 2 and Familial Alopecia Areata" (J Invest Dermatol 118:335–337, 2002), the first author's name was spelled incorrectly. It is correct as appears above. The authors regret the error. Full-Text PDF Open Archive
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