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• W1970843614 abstract "UV radiation from the sun activates both the membrane death receptor and the intrinsic or mitochondrial apoptotic signaling pathways in epidermal keratinocytes, triggering apoptosis and affording protection against skin cancer formation. We have investigated the involvement of caspase-9 in the UV death effector pathway in human keratinocytes, since this is the initiating caspase in the mitochondrial pathway required for UV-induced apoptosis in some, but not all, cell types. UV radiation triggered activation of caspase-3, caspase-9, and caspase-8 with similar kinetics, although the rank order of activation was caspase-3 > caspase-9 > caspase-8. Inhibition of caspase-9 with either the peptide inhibitor benzyloxycarbonyl-Leu-Glu(OCH3)-His-Asp(OCH3)-fluoromethyl ketone, or expression of a catalytically inactive caspase-9 by retroviral transduction, protected normal keratinocytes from UV-induced apoptosis. HaCaT keratinocytes harboring mutant p53 alleles were also protected from UV-induced apoptosis by the dominant negative caspase-9. The dominant negative caspase-9 blocked UV-induced activation of caspase-3, caspase-9, and caspase-8, and also protected cells from the loss of mitochondrial membrane potential. In contrast, the dominant negative caspase-9 did not protect from anti-Fas-induced apoptosis or caspase activation. These results identify caspase-9 as the critical upstream caspase initiating apoptosis by UV radiation in human keratinocytes, the relevant cell type for this important environmental carcinogen. UV radiation from the sun activates both the membrane death receptor and the intrinsic or mitochondrial apoptotic signaling pathways in epidermal keratinocytes, triggering apoptosis and affording protection against skin cancer formation. We have investigated the involvement of caspase-9 in the UV death effector pathway in human keratinocytes, since this is the initiating caspase in the mitochondrial pathway required for UV-induced apoptosis in some, but not all, cell types. UV radiation triggered activation of caspase-3, caspase-9, and caspase-8 with similar kinetics, although the rank order of activation was caspase-3 > caspase-9 > caspase-8. Inhibition of caspase-9 with either the peptide inhibitor benzyloxycarbonyl-Leu-Glu(OCH3)-His-Asp(OCH3)-fluoromethyl ketone, or expression of a catalytically inactive caspase-9 by retroviral transduction, protected normal keratinocytes from UV-induced apoptosis. HaCaT keratinocytes harboring mutant p53 alleles were also protected from UV-induced apoptosis by the dominant negative caspase-9. The dominant negative caspase-9 blocked UV-induced activation of caspase-3, caspase-9, and caspase-8, and also protected cells from the loss of mitochondrial membrane potential. In contrast, the dominant negative caspase-9 did not protect from anti-Fas-induced apoptosis or caspase activation. These results identify caspase-9 as the critical upstream caspase initiating apoptosis by UV radiation in human keratinocytes, the relevant cell type for this important environmental carcinogen. The induction of programmed cell death, or apoptosis, by UV radiation is an important protective mechanism from neoplastic transformation for the skin. UV radiation from the sun is the main environmental carcinogen responsible for the formation of basal and squamous cell carcinomas, the most common human cancer types (1Kraemer K.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11-14Crossref PubMed Scopus (341) Google Scholar). Epidermal keratinocytes are efficiently protected from the mutagenic effect of UV by undergoing apoptosis; however, prior sun exposure, which causes mutations in the p53 tumor suppressor gene, or expression of antiapoptotic proteins such as Bcl-2, prevents apoptosis and leads to increase skin cancer incidence (2Rodriguez-Villanueva J. Greenhalgh D. Wang X.J. Bundman D. Cho S. Delehedde M. Roop D. McDonnell T.J. Oncogene. 1998; 16: 853-863Crossref PubMed Scopus (70) Google Scholar, 3Pena J.C. Fuchs E. Thompson C.B. Cell Growth Differ. 1997; 8: 619-629PubMed Google Scholar, 4Ziegler A. Leffell D.J. Kunala S. Sharma H.W. Gailani M. Simon J.A.X.H. A. Baden H.P. Shapiro P.E. Bale A.E. Brash D.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4216-4220Crossref PubMed Scopus (673) Google Scholar, 5Ziegler A. Jonason A.S. Leffell D.J. Simon J.A. Sharma H.W. Kimmelman J. Remington L. Jacks T. Brash D.E. Nature. 1994; 372: 773-776Crossref PubMed Scopus (1352) Google Scholar, 6Li G. Tron V. Ho V. J. Invest. Dermatol. 1998; 110: 72-75Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 7Jiang W. Ananthaswamy H.N. Muller H.K. Kripke M.L. Oncogene. 1999; 18: 4247-4253Crossref PubMed Scopus (149) Google Scholar). Understanding the molecular and cellular regulation of UV-induced apoptosis is thus a major focus of skin carcinogenesis research. UV radiation has multiple cellular targets that trigger different signaling cascades leading to apoptosis. UV radiation is a DNA-damaging agent that activates a p53-dependent apoptotic response (1Kraemer K.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11-14Crossref PubMed Scopus (341) Google Scholar, 5Ziegler A. Jonason A.S. Leffell D.J. Simon J.A. Sharma H.W. Kimmelman J. Remington L. Jacks T. Brash D.E. Nature. 1994; 372: 773-776Crossref PubMed Scopus (1352) Google Scholar). DNA-damaging agents, such as UV, activate the intrinsic death effector pathways that perturb mitochondrial structure and function, leading to the release of cytochrome c (8Denning M.F. Wang Y. Alkan S. Nickoloff B.J. Qin J.Z. Cell Death Differ. 2002; 9: 40-52Crossref PubMed Scopus (170) Google Scholar). Cytosolic cytochrome c forms an apoptosome complex with APAF-1, dATP, and the initiator procaspase-9 to cause activation of caspase-9 and trigger subsequent effector caspase activation (9Zou H., Li, Y. Liu X. Wang X. J. Biol. Chem. 1999; 274: 11549-11556Abstract Full Text Full Text PDF PubMed Scopus (1803) Google Scholar, 10Saleh A. Srinivasula S.M. Balkir L. Robbins P.D. Alnemri E.S. Nat. Cell Biol. 2000; 2: 476-483Crossref PubMed Scopus (740) Google Scholar, 11Stennicke H.R. Deveraux Q.L. Humke E.W. Reed J.C. Dixit V.M. Salvesen G.S. J. Biol. Chem. 1999; 274: 8359-8362Abstract Full Text Full Text PDF PubMed Scopus (419) Google Scholar, 12Renatus M. Stennicke H.R. Scott F.L. Liddington R.C. Salvesen G.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14250-14255Crossref PubMed Scopus (372) Google Scholar). The importance of this intrinsic pathway in UV-induced keratinocyte apoptosis has been supported by the ability of Bcl-2 family members or survivin to inhibit apoptosis induced by UV exposure and increased sensitivity to apoptosis by Bcl-xL antisense or in Bcl-2 null keratinocytes (2Rodriguez-Villanueva J. Greenhalgh D. Wang X.J. Bundman D. Cho S. Delehedde M. Roop D. McDonnell T.J. Oncogene. 1998; 16: 853-863Crossref PubMed Scopus (70) Google Scholar, 3Pena J.C. Fuchs E. Thompson C.B. Cell Growth Differ. 1997; 8: 619-629PubMed Google Scholar, 13Rossiter H. Beissert S. Mayer C. Schon M.P. Wienrich B.G. Tschachler E. Kupper T.S. Cancer Res. 2001; 61: 3619-3626PubMed Google Scholar, 14Grossman D. Kim P.J. Blanc-Brude O.P. Brash D.E. Tognin S. Marchisio P.C. Altieri D.C. J. Clin. Invest. 2001; 108: 991-999Crossref PubMed Scopus (197) Google Scholar, 15Taylor J.K. Zhang Q.Q. Monia B.P. Marcusson E.G. Dean N.M. Oncogene. 1999; 18: 4495-4504Crossref PubMed Scopus (84) Google Scholar, 16Gillardon F. Moll I. Meyer M. Michaelidis T.M. Cell Death. Differ. 1999; 6: 55-60Crossref PubMed Scopus (23) Google Scholar). UV also causes ligand-dependent and -independent clustering and activation of membrane death receptors such as Fas or tumor necrosis factor α (17Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (432) Google Scholar, 18Rehemtulla A. Hamilton C.A. Chinnaiyan A.M. Dixit V.M. J. Biol. Chem. 1997; 272: 25783-25786Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 19Schwarz A. Bhardwaj R. Aragane Y. Mahnke K. Riemann H. Metze D. Luger T.A. Schwarz T. J. Invest. Dermatol. 1995; 104: 922-927Abstract Full Text PDF PubMed Scopus (251) Google Scholar, 20Sheikh M.S. Antinore M.J. Huang Y. Fornace A.J., Jr. Oncogene. 1998; 17: 2555-2563Crossref PubMed Scopus (114) Google Scholar, 21Leverkus M. Yaar M. Gilchrest B.A. Exp. Cell Res. 1997; 232: 255-262Crossref PubMed Scopus (173) Google Scholar, 22Leverkus M. Yaar M. Eller M.S. Tang L. Gilchrest B.A. J. Invest. Dermatol. 1998; 110: 353-357Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 23Hill L.L. Ouhtit A. Loughlin S.M. Kripke M.L. Ananthaswamy H.N. Owen-Schaub L.B. Science. 2000; 285: 898-900Crossref Scopus (224) Google Scholar). The role of the death receptor pathway in UV apoptosis in keratinocytes has also been supported by irradiation of cells at 10 °C to inhibit receptor clustering and by expressing a dominant negative FADD to prevent coupling of death receptors to initiator caspases such as caspase-8. Both of these approaches to block death receptor signaling provided partial protection from UV-induced apoptosis in keratinocytes (17Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (432) Google Scholar, 18Rehemtulla A. Hamilton C.A. Chinnaiyan A.M. Dixit V.M. J. Biol. Chem. 1997; 272: 25783-25786Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). The role of caspase-9 in UV-induced apoptosis is very cell type-dependent. Embryonic stem cells and fibroblasts require caspase-9 for UV apoptosis, whereas thymocytes and splenocytes lacking caspase-9 are still sensitive to UV (24Hakem R. Hakem A. Duncan G.S. Henderson J.T. Woo M. Soengas M.S. Elia A.X. Pompa J.L. Kagi D. Khoo W. Potter J. Yoshida R. Kaufman S.A. Lowe SW Penninger J.M. Mak T.W. Cell. 1998; 94: 339-352Abstract Full Text Full Text PDF PubMed Scopus (1171) Google Scholar). Given this cell type specificity, it is critical to evaluate the role of caspase-9 in UV-induced apoptosis in relevant cell types. To this end, we have evaluated the role of caspase-9 in UV apoptotic signaling in human keratinocytes using peptide and dominant negative inhibitors of caspase-9. We have also assayed the activity of multiple caspase following UV exposure to better understand the relationships among different caspases in the caspase activation cascade. Our results indicate that caspase-9 activation is a major determinant of UV-induced keratinocyte apoptosis independent of p53 status and reveals potential positive feedback regulation between caspase-3 and caspase-8. For Western detection and immunofluorescence staining of procaspase-9, rabbit polyclonal antibody 9502 (Cell Signaling, Beverly, MA) was used. For Western analysis of procaspase-9 processing, the cleavage-specific rabbit polyclonal antibody 9501S (Cell Signaling) was used. Protein kinase Cδ was detected with rabbit polyclonal antibody sc-937 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). For induction of apoptosis in keratinocytes via activation of Fas receptor, 5 μg/ml of cyclohexamide (CHX 1The abbreviations used are: CHXcyclohexamideCHAPS3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acidDTTdithiothreitolD/Ndominant negativePKCδprotein kinase CδAFCamino-4-trifluoromethylcoumarinZ-LEHD-FMKbenzyloxycarbonyl-Leu-Glu(OCH3)-His-Asp(OCH3)-fluoromethyl ketone ; Sigma) and an anti-Fas IgM antibody at 100 ng/ml (CH11; Upstate Biotechnology, Inc., Lake Placid, NY) were used. For caspase assays, the fluorogenic substrate Ac-DEVD-AFC (for caspase-3), Ac-IETD-AFC (for caspase-8), and Ac-LEHD-AFC (for caspase-9) (Enzyme Systems Products, Livermore, CA) were prepared as 20 mm stock in Me2SO. cyclohexamide 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid dithiothreitol dominant negative protein kinase Cδ amino-4-trifluoromethylcoumarin benzyloxycarbonyl-Leu-Glu(OCH3)-His-Asp(OCH3)-fluoromethyl ketone Normal human epidermal keratinocytes were isolated from neonatal foreskin following routine circumcision, as previously described (25Mitra R. Nickoloff B. Leigh I. Watt F. Keratinocyte Methods. Cambridge University Press, Cambridge, UK1994: 17-19Google Scholar, 26Denning M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). After isolation, the cells were cultured in Medium 154 CF (Cascade Biologics, Inc., Portland, OR) containing 0.07 mm CaCl2, human keratinocyte growth supplement, and penicillin/streptomycin/amphotericin B (Medium 154) until they reached 40–50% confluence. For expansion, the cells were trypsinized with 0.03% trypsin, 0.01% EDTA and plated in Medium 154. Each experiment was performed on the cells isolated from a single foreskin and were used at passages 1–2. The immortalized human keratinocyte cell line HaCaT, which has two mutant p53 alleles, was kindly provided by Dr. Norbert Fusening (German Cancer Research Center, Heidelberg, Germany) and was also grown in Medium 154 (27Boukamp P. Petrussevska R.T. Breitkreutz D. Hornung J. Markham A. Fusenig N.E. J. Cell Biol. 1988; 106: 761-771Crossref PubMed Scopus (3493) Google Scholar, 28Lehman T.A. Modali R. Boukamp P. Stanek J. Bennett W.P. Welsh J.A. Metcalf R.A. Stampfer M.R. Fusenig N. Rogan E.M. Harris C.C. Carcinogenesis. 1993; 14: 833-839Crossref PubMed Scopus (391) Google Scholar). Keratinocytes were irradiated with a Panelite Unit (Ultralite Enterprises, Inc., Lawrenceville, GA) equipped with four UVB bulbs (FS36T12/UVB-VHO), which have the majority of their output in the UVB range (∼65%), with minor output in the UVA and UVC wavelengths (∼34 and 1%, respectively). The cells were exposed with the dish lids removed, with a 30 mJ/cm2 dose requiring about 1 min of exposure. The UV dose was monitored with an International Light Inc. (Newburyport, MA) radiometer fitted with a UVB detector. For experiments with the caspase-9 peptide inhibitor, 10 μmZ-LEHD-FMK (catalog no. FK-022; Enzyme System Products, Livermore, CA), was added to cells immediately after UV exposure. For anti-Fas induction of apoptosis, the cells were pretreated with the protein synthesis inhibitor CHX at 5 μg/ml for 2 h before anti-Fas antibody was added at 100 ng/ml to the medium. CHX pretreatment was necessary to get significant induction of apoptosis in keratinocytes (29Qin J.Z. Chaturvedi V. Denning M.F. Choubey D. Diaz M.O. Nickoloff B.J. J. Biol. Chem. 1999; 274: 37957-37964Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 30Qin J.Z. Bacon P. Chaturvedi V. Nickoloff B.J. J. Invest. Dermatol. 2001; 117: 898-907Abstract Full Text Full Text PDF PubMed Google Scholar). Before making the protein extract, floating cells were collected and combined with cells growing on the dish and washed two times with cold phosphate-buffered saline. The cells were lysed in 2× caspase lysis buffer: 25 mm HEPES-NaOH, pH 7.4, 10% sucrose, 0.1% CHAPS, 2 mm EDTA, 5 mmdithiothreitol. Cell lysates were spun for 3 min in a microcentrifuge, and a Bradford protein assay was performed on the supernatant. 50 μg of total protein was mixed with 2× caspase assay buffer: 25 mm HEPES-NaOH, pH 7.4, 5 mm DTT and a 100 μm concentration of one of the following caspase fluorogenic substrates: Ac-DEVD-AFC (for caspase-3), Ac-IETD-AFC (for caspase-8), and Ac-LEHD-AFC (for caspase-9). After incubation at 37 °C for 3 h, the fluorometric detection of cleaved AFC product was performed on a CytoFluor Multi-Well Plate Reader Series 4000 (PerSeptive Biosystems) using a 400-nm excitation filter and a 530-nm emission filter. For preparation of the AFC calibration curve, 80 μm free AFC was diluted in the caspase assay buffer without substrate to give 1.6, 3.2, and 4.8 μmol of free AFC, and fluorescence was measured on the fluorometer. For the in vitro caspase-8 activation assays, 50 ng of caspase-3 (catalog no. 201-038-C005; Alexis Biochemicals, San Diego, CA), 50 μg of total protein and 100 nm caspase-9 Z-LEHD-FMK peptide inhibitor were used. The caspase-9 dominant negative (D/N) cDNA containing a mutation of Cys287 to Ser, (C287S) cloned in EcoRI/XhoI sites of pMARX IVrPuro (31Fearnhead H.O. Rodriguez J. Govek E.E. Guo W. Kobayashi R. Hannon G. Lazebnik Y.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13664-13669Crossref PubMed Scopus (160) Google Scholar) was a gift from Dr. Yuri A. Lazebnik (Cold Spring Harbor, NY). For construction of the episomal retroviral expression vector containing the caspase-9 D/N cDNA, two primers (cgf, 5- AGCTCGGATCCACTAGTAACGGCCGCC-3 containing a BamHI site (underlined), and cgf2, 5-ATAGTTAGCGGCCGCATTAAGTTTAAACGGGCCCTC-3, containing a NotI site (underlined)) were used to amplify the cDNA, and the PCR product after restriction digestion and purification was cloned into the BamHI/NotI sites of the retroviral vector LZRS-Linker (32Kinsella T.M. Nolan G.P. Hum. Gene Ther. 1996; 7: 1405-1413Crossref PubMed Scopus (672) Google Scholar). After cloning, the insert was verified by sequencing, and the presence of the C287S mutation was confirmed. For production of retrovirus, the LZRS-caspase-9 D/N DNA was transfected into the Phoenix-Ampho retroviral packaging cell line using calcium phosphate as described (30Qin J.Z. Bacon P. Chaturvedi V. Nickoloff B.J. J. Invest. Dermatol. 2001; 117: 898-907Abstract Full Text Full Text PDF PubMed Google Scholar). Packaging cells were selected and expanded in the presence of 1 μg/ml puromycin, and virus was harvested from confluent dishes cultured for 24–48 h at 32 °C. For infection, keratinocytes were plated in six-well dishes at 105/well, and the following day viral supernatant was added in the presence of 4 μg/ml polybrene (hexadimethrine bromide; Sigma). The cells were infected by spinning the plates at 300 ×g for 1 h at 32 °C, and the viral supernatants were replaced with fresh Medium 154. The day after infection, cells were washed two times with phosphate-buffered saline and fed with Medium 154. For the caspase assay, infected cells were expanded for 2 days by plating them in p60 dishes. For immunofluorescence, cells were grown on glass coverslips, and 1 day after infection, they were washed with phosphate-buffered saline and fixed in −20 °C acetone/methanol (1:1) for 10 min. The cells were stained with human caspase-9 primary antibody (9502; Cell Signaling) diluted in phosphate-buffered saline (1:250) with normal goat serum (1:20 dilution) for 1 h at room temperature, washed with FA buffer (Difco), and incubated with secondary fluorescein isothiocyanate-conjugated antibody at 1:40 dilution. Cells were washed in FA buffer and mounted in 40% polyvinyl alcohol (molecular weight 300–70,000) in glycerol containing 100 mg/ml 1,4-diazabicyclo octane to reduce fading of the fluorescence. The cells were viewed with an Olympus AX80 fluorescence microscope. Apoptosis was routinely measured by determining DNA content of cells by propidium iodide staining and flow cytometry, as previously described (26Denning M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). Briefly, cells were trypsinized, fixed with ethanol, and stained with propidium iodide before being analyzed on a Coulter Epics XL-MCL flow cytometer. Cells with DNA content less than the G1 amount of untreated cells were considered apoptotic. Mitochondria membrane potential was measured by rhodamine 123 fluorescence (33Shapiro H.M. Methods. 2000; 21: 271-279Crossref PubMed Scopus (138) Google Scholar). Cells were trypsinized and incubated for 20 min in 1 ml of room temperature medium containing 5 μmrhodamine 123. The cells were then washed and analyzed on a Coulter Epics XL-MCL flow cytometer for reduced rhodamine 123 fluorescence, indicating loss of mitochondrial membrane potential. The protein samples were loaded on 8.5 or 12.5% SDS-polyacrylamide gels and transferred to nitrocellulose membrane BA83 (Schleicher and Schuell). Protein bands were visualized with Ponceau S staining, and the membrane was blocked in 5% nonfat powdered milk in TBS (50 mm Tris, pH 7.5, 150 mm NaCl). The membrane was incubated with the primary antibody in 2.5% powdered milk in TBS, washed extensively with TBS, and then incubated with 1:1000 diluted secondary anti-rabbit or anti-mouse horseradish peroxidase-labeled antibody (AmershamBiosciences). The membrane was washed with TBS containing 0.05% Tween 20 for 1 h. Bands were visualized with ECL (Amersham Biosciences) according to the manufacturer's instructions. To determine the relative extent and kinetics of caspase activation in response to UV radiation, normal human keratinocytes were exposed to 30 mJ/cm2 UV light, and the activities of caspase-3, -8, and -9 measured using specific fluorogenic substrates. Fig. 1A shows the kinetics of these caspase activities. The induction of all three caspases begins 6–9 h after UV exposure. At 18 h after UV exposure, the activities of caspase-9, caspase-3, and caspase-8 were induced 4.8-, 21.7-, and 2.2-fold, respectively (Fig. 1A). Caspase-3 was the only effector caspase assayed and was activated to the greatest extent. Between the two initiator caspases (caspase-9 and caspase-8), caspase-9 activity increased more, suggesting that the intrinsic pathway plays a predominant role in UV–induced apoptosis. To determine the role of caspase-9 in UV-induced apoptosis, we used two approaches to block its activity. One was the treatment of cells with the specific irreversible peptide inhibitor Z-LEHD-FMK; the other was expressing a dominant-negative form of caspase-9. Fig. 1B shows that in keratinocytes treated with the caspase-9 peptide inhibitor, UV-induced apoptosis was inhibited 66%, as measured by sub-G1 DNA. Using a caspase-9 D/N retrovirus, we were able infect normal keratinocytes and detect expression of the dominant negative form of the caspase-9 protein in the majority of cells by immunofluorescence microscopy (Fig. 2A). Protein expression was confirmed by Western blot with the major 47-kDa procaspase-9 band detected (Fig. 2B). Higher molecular weight protein bands detected on the blot may represent aggregated caspase-9. Keratinocytes expressing the caspase-9 D/N appeared resistant to UV-induced cell death by morphology (Fig. 3A) and were 81% resistant to UV-induced apoptosis by sub-G1 DNA content (Fig. 4, A and B). Caspase-9 D/N-infected HaCaT cells, which have mutant p53 genes, show the same level of protection as normal keratinocytes (Figs.3B and 4C). These results indicate that caspase-9 activation is a required component of the UV death effector pathway in keratinocytes, independent of p53 status.Figure 3Caspase-9 D/N expression protects normal human keratinocytes and HaCaT cells from UV-induced morphological cell death. Keratinocytes were infected with either LZRS-Linker control virus or LZRS-caspase-9 D/N (LZRS-C9 D/N) virus. Two days after infection, cells were exposed to 30 mJ/cm2 of UV light. 18 h after UV exposure, cells were photographed. Note that the expression of caspase-9 D/N protein protected both normal keratinocytes (A) and HaCaT cells (B) from UV-induced cell death.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Caspase-9 D/N expression protects keratinocytes from UV-induced apoptosis.A, normal human keratinocytes infected with either LZRS-Linker or LZRS-caspase-9 D/N virus were exposed to 30 mJ/cm2 UV, and after 18 h cells were stained with propidium iodide before being analyzed on a flow cytometer. The DNA histograms show the percentage of apoptotic cell accumulation (Sub-G1 DNA). B, the bar graph shows data from four independent experiments performed on normal keratinocytes as described for A, which was normalized so that the percentage of UV-induced apoptosis was 100%. C, the bar graph shows data from three independent experiments performed on HaCaT cells as described for A. Error bars represent the S.D.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mitochondrial membrane potential is one indicator of cells undergoing the terminal phase of UV-induced apoptosis and is dependent on caspase activity (32Kinsella T.M. Nolan G.P. Hum. Gene Ther. 1996; 7: 1405-1413Crossref PubMed Scopus (672) Google Scholar, 34Adrain C. Creagh E.M. Martin S.J. EMBO J. 2001; 20: 6627-6636Crossref PubMed Scopus (362) Google Scholar). We have used rhodamine 123 as a molecular probe to assay the mitochondrial membrane potential in keratinocytes expressing caspase-9 D/N and exposed to UV. As shown in Fig. 5, normal keratinocytes and HaCaT cells are protected from losing mitochondrial membrane potential by caspase-9 D/N expression (p < 0.05 for both cell types). The protection was 77% for both normal keratinocytes and HaCaT cells. To determine whether caspase-9 D/N expression was blocking the activation of caspases in cells following UV exposure, we assayed caspase activity in UV-irradiated keratinocytes infected with either Linker or caspase-9 D/N virus. The proteolytic activities of caspase-3, -8, and -9 were more than 87% inhibited in keratinocytes expressing caspase-9 D/N protein (Fig. 6A), and the inhibition of each caspase was statistically significant (p < 0.01). Western blot analysis of caspase-9 D/N-infected keratinocytes showed that caspase-9 is cleaved following UV exposure (Fig. 6B) but not catalytically activated due to the C287S mutation (Fig. 6A). Cleaved caspase-9 was also detected in the caspase-9 D/N-expressing cells, and cleavage was increased further by UV (Fig. 6B). PKCδ is a downstream substrate of caspase-3 (26Denning M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 35Ghayur T. Hugunin M. Talanian R.V. Ratnofsky S. Quinlan C. Emoto Y. Pandey P. Datta R. Huang Y. Kharbanda S. Allen H. Kamen R. Wong W. Kufe D. J. Exp. Med. 1996; 184: 2399-2404Crossref PubMed Scopus (447) Google Scholar) and was not proteolytically processed in caspase-9 D/N-infected cells after UV irradiation (Fig. 6B, bottom panel). The caspase assays in Fig. 6A demonstrated inhibition of UV-induced caspase-8 activity in caspase-9 D/N-infected cells. This was unexpected, since caspase-8 is an initiator caspase, and its activation is not reported to be dependent on caspase-9. One possible explanation of this data is that caspase-3 may be able to activate procaspase-8, and the caspase-9 D/N protein blocked processing and activation of procaspase-3, thereby preventing activation of procaspase-8. To explore this possibility, we tested whether recombinant, catalytically active caspase-3 can activate procaspase-8 in vitro and whether this procaspase-8 activation can be blocked by caspase-9 inhibitors. As shown in Fig. 7, the addition of recombinant caspase-3 to normal protein extracts and to protein extracts from caspase-9 D/N-expressing keratinocytes activated caspase-8 2.8- and 2.3-fold, respectively. The presence of specific caspase-9 peptide inhibitor Z-LEHD-FMK or caspase-9 D/N protein in the assay did not affect the procaspase-8 activation by caspase-3. These results demonstrate that caspase-3 can directly or indirectly activate caspase-8 independent of caspase-9. These results also validate the specificity of our caspase assays, since no caspase-8 activity was detected in the recombinant caspase-3 preparation, and the peptide caspase-9 inhibitor (Z-LEHD-FMK) did not reduce the activity of caspase-8 (Ac-IETD-AFC cleavage). To determine whether caspase-9 D/N virus can protect from apoptosis induced by death receptor stimulation (30Qin J.Z. Bacon P. Chaturvedi V. Nickoloff B.J. J. Invest. Dermatol. 2001; 117: 898-907Abstract Full Text Full Text PDF PubMed Google Scholar), we used CHX plus an anti-Fas antibody (CH11) to induce apoptosis. Morphological examination of the cells expressing caspase-9 D/N protein and stimulated with anti-Fas showed no protection from apoptosis (data not shown). The level of apoptosis was also assayed by DNA staining and flow cytometry. As shown in Fig. 8A, although caspase-9 D/N protected keratinocytes from UV-induced apoptosis (81% inhibition), cells were not protected from anti-Fas-induced apoptosis by the caspase-9 D/N protein. We also measured the specific activities of caspases in this experiment. Treatment of keratinocytes with CHX and anti-Fas antibody caused 6.1-fold induction of caspase-3, 1.1-fold induction of caspase-8, and no induction of caspase-9 activity (Fig. 8B). All three caspase activities were unaffected by the presence of caspase-9 D/N for anti-Fas-induced apoptosis, in contrast to UV-induced apoptosis, where they were all inhibited (Figs.6A and 8B). Thus, the caspase-9 D/N protein does not block anti-Fas-induced apoptosis or caspase activation. We undertook this study to determine the involvement of caspase-9 activation in apoptosis induced by UV in human epidermal keratinocytes. UV initiates two major proapoptotic signaling pathways involving either death receptor activation, which couple to the activation of initiator caspases such as caspase-8, or DNA damage/cell stress, which activates the initiator caspase-9 via the intrinsic or mitochondrial pathway (36Thornberry N.A. Lazebnik Y. Science. 1998; 281: 1312-1316Crossref PubMed Scopus (6182) Google Scholar,37Green D.R. Reed J.C. Science. 1998; 281: 1309-1312Crossref PubMed Google Scholar). Due to the selectivity for the initiator caspase-9 and caspase-8 activation by either the mitochondrial or death receptor apoptotic pathway, respectively (38Sun X.M. MacFarlane M. Zhuang J. Wolf B.B. Green D.R. Cohen G.M. J. Biol. Chem. 1999; 274: 5053-5060Abstract Full Text Full Text PDF PubMed Scopus (780) Google Scholar), we analyzed caspase activation during UV-induced apoptosis in human keratinocytes. The activities of caspase-3, caspase-8, and caspase-9 were all induced with similar kinetics following UV irradiation, with significant increases in activity detected 6–9 h after exposure (Fig. 1A). Caspase-3 was activated by far the greatest, and we attribute its large activation to its being an effector caspase at the end of a caspase cascade that amplifies the proteolytic activation of caspases. Caspase-8 consistently had the highest basal activity; however, caspase-9 was activated more than caspase-8 by UV radiation, suggesting a stronger upstream signal for caspase-9 activation. It is unclear why there is a delay of up to 6 h before caspase activation, but this delay is consistent with a series of events, such as gene transcription or apoptosome complex formation, occurring prior to activation of the caspase cascade. The delayed onset of caspase activation may also reflect the relatively slow zymogen activation of procaspase-9 relative to procaspase-8 (39Salvesen G.S. Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (773) Google Scholar). Taken together, the delayed activation of caspases and the greater activation of caspase-9 relative to caspase-8 suggest that UV primarily signaling through the intrinsic pathway rather than the death receptor pathway. The dramatic inhibition of UV-induced apoptosis by both the Z-LEHD-FMK peptide caspase-9 inhibitor (Fig. 1B) and the caspase-9 D/N (Figs. 3, 4, and 8A) strongly suggest that caspase-9 is the dominant upstream caspase in UV keratinocyte apoptosis. The caspase-9 D/N virus afforded >80% protection from UV-induced apoptosis (Figs.4, B and C, and 8A) but did not protect from anti-Fas apoptosis (Fig. 8A), demonstrating high efficacy and specificity. Transient transfection of a dominant negative FADD into keratinocytes to preferentially block the death receptor signaling resulted in only modest (30–40%) protection from UV-induced apoptosis (17Aragane Y. Kulms D. Metze D. Wilkes G. Poppelmann B. Luger T.A. Schwarz T. J. Cell Biol. 1998; 140: 171-182Crossref PubMed Scopus (432) Google Scholar). The caspase-9 D/N blocked many UV-induced apoptotic endpoints, including morphological cell death (Fig. 3), sub-G1 DNA (Figs. 4 and 8A), loss of mitochondrial membrane potential (Fig. 5), caspase activation (Fig. 6A), and cleavage of death substrates (Fig. 6B). None of the endpoints measured were blocked during anti-Fas-induced apoptosis (Fig. 8). The ability of the caspase-9 D/N to block UV apoptosis in HaCaT cells indicates that this pathway is p53-independent, at least in cell culture. This is relevant to skin photocarcinogenesis, where many premalignant lesions have mutant p53 and are thought to be relatively resistant to UV apoptosis. While UV radiation triggered activation of caspase-3, -9, and -8, the expression of caspase-9 D/N protein led to almost complete inhibition of all three caspase activities induced by UV, consistent with data from cell-free extracts demonstrating caspase-9-dependent activation of multiple caspases (Fig. 6A) (40Slee E.A. Harte M.T. Kluck R.M. Wolf B.B. Casiano C.A. Newmeyer D.D. Wang H.G. Reed J.C. Nicholson D.W. Alnemri E.S. Green D.R. Martin S.J. J. Cell Biol. 1999; 144: 281-292Crossref PubMed Scopus (1687) Google Scholar). Western blot analysis of protein used in the caspase assay with an antibody specific for cleaved caspase-9 revealed the cleavage of procaspase-9 after UV in Linker virus-infected cells and in both unexposed and UV-exposed caspase-9 D/N-expressing cells (Fig. 6B). Despite the cleavage of procaspase-9 detected in all LZRS-C9 D/N virus-infected keratinocytes, caspase-9 activity was not observed, verifying the catalytic inactivity of the C287S mutant caspase-9 (Fig. 6A). We demonstrated that caspase-9 D/N was selective for the intrinsic death effector pathway by its inability to block apoptosis induced by CHX/anti-Fas (Fig. 8A). The inability of caspase-9 D/N to block anti-Fas-induced apoptosis was not due to inhibition of caspase-9 D/N protein synthesis by CHX, since CHX did not reduce the levels of caspase-9 D/N protein significantly over the course of these experiments (data not shown). Although CHX/anti-Fas treatment induced caspase-3 activation, which was not blocked by caspase-9 D/N expression, we did not detect activation of either caspase-8 or caspase-9 (Fig. 8B). The lack of caspase-8 activation is surprising, since Fas is coupled to caspase-8 via the adaptor protein FADD (41Ashkenazi A. Dixit V.M. Science. 1999; 281: 1305-1308Crossref Scopus (5169) Google Scholar). Thus, the mechanism of caspase-3 activation following anti-Fas treatment in our system is unclear, but it may involve activation of other potential initiator caspases such as caspase-10, which contains two death effector domains homologous to those found in caspase-8 and could potentially substitute for caspase-8 (42Cohen G.M. Biochem. J. 1997; 326: 1-16Crossref PubMed Scopus (4146) Google Scholar). As shown in Fig. 6A, expression of caspase-9 D/N also blocked the induction of caspase-8 activity by UV. This was unexpected, since the caspase-9 D/N should not interfere with caspase-8 activation via death receptors and did not prevent anti-Fas-induced apoptosis (Fig. 8A). One possible explanation is that the caspase-8 activation seen following UV exposure is not due to death receptor activation but results from activation of other caspases that are dependent on caspase-9, such as caspase-3. To explore this possibility, we determined whether procaspase-8 could be activated by recombinant catalytically active caspase-3 in vitro (Fig. 7). Activation of caspase-8 activity by recombinant caspase-3 in vitro was 2–3-fold in our experiments and was not blocked by the caspase-9 peptide inhibitor or in lysates from caspase-9 D/N-expressing cells. Theoretically, caspase-8 (isoform A) has a potential caspase-3 cleavage site at position DEAD398↓, and cleavage at this site would generate fragments in good agreement with the sizes of known caspase-8 cleaved products (18 and 43 kDa) (43Grenet J. Teitz T. Wei T. Valentine V. Kidd V.J. Gene (Amst.). 1999; 226: 225-232Crossref PubMed Scopus (82) Google Scholar). The data in Fig. 7also validate the specificity of our caspase assays, since no caspase-8 activity (Ac-IEDT-AFC cleavage) was detected in the recombinant caspase-3-alone assay (second bar), and the caspase-9 inhibitory peptide Z-LEHD-FMK did not inhibit caspase-8 activity. In some cells, cross-talk exists between the death receptor and mitochondrial apoptotic pathways. For example, cleavage of BID by caspase-8 can promote cytochrome c release (44Li H. Zhu H. Xu C.J. Yuan J. Cell. 1998; 94: 491-501Abstract Full Text Full Text PDF PubMed Scopus (3798) Google Scholar), and mitochondria are required for amplification of caspase-8-initiated apoptosis (45Kuwana T. Smith J.J. Muzio M. Dixit V. Newmeyer D.D. Kornbluth S. J. Biol. Chem. 1998; 273: 16589-16594Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). In keratinocytes, death receptor to mitochondria cross-talk may not be functional, since Bcl-2 and Bcl-xL do not prevent TRAIL-induced apoptosis (46Bacon P. Panella J. Denning M. Nickoloff B.J. J. Invest. Dermatol. 2000; 114: 763Google Scholar) and our caspase-9 dominant negative did not block anti-Fas apoptosis (Fig. 8). By integrating the available data, we propose the UV apoptotic signaling pathway outlined in Fig. 9. UV radiation activates primarily the mitochondrial or intrinsic apoptotic pathway, resulting in activation of procaspase-9, whereas activation of procaspase-8 via death receptors is a relatively minor pathway. The procaspase-9 activation may be mediated by apoptosome complex formation, since cytochrome c release is an early, caspase-independent event in UV apoptotic signaling (47Du C. Fang M., Li, Y., Li, L. Wang X. Cell. 2000; 102: 33-42Abstract Full Text Full Text PDF PubMed Scopus (2941) Google Scholar). Once activated, the initiator caspases cleave and activate effector caspases, such as procaspase-3, which cleave a large number of death substrates, including PKCδ. As shown in Fig. 7, active caspase-3 may also be able to directly or indirectly activate caspase-8. The proteolytic activation of PKCδ is involved in triggering loss of mitochondrial membrane potential and apoptosis in keratinocytes (8Denning M.F. Wang Y. Alkan S. Nickoloff B.J. Qin J.Z. Cell Death Differ. 2002; 9: 40-52Crossref PubMed Scopus (170) Google Scholar,26Denning M.F. Wang Y. Nickoloff B.J. Wrone-Smith T. J. Biol. Chem. 1998; 273: 29995-30002Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), and the data in Fig. 6B indicate that PKCδ cleavage also requires caspase-9 activation. In summary, these studies establish that procaspase-9 activation is required for the activation of other caspases by UV radiation and thus has a role in triggering caspase-dependent apoptotic events, such as loss of mitochondrial membrane potential and DNA fragmentation in human keratinocytes. Furthermore, caspase-9 is a major determinant of UV-induced apoptosis in keratinocytes with mutant p53 and thus may have potential as a therapeutic target for triggering apoptosis in premalignant actinic keratosis that harbor mutant p53. We thank all members of the Skin Cancer Research Program for help with this project, in particular Drs. Brian J. Nickoloff and Jian-Zhong Qin. We also thank Drs. Paul Khavari and Garry P. Nolan for providing the LZRS retroviral vector and Phoenix-Ampho packaging cells. We are especially grateful to Dr. Yuri A. Lazebnik for providing the caspase-9 dominant negative cDNA and a caspase-9 antibody." @default.
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