@article{house_gray_owen_jima_smart_hall_2023, title={C/EBP beta deficiency enhances the keratinocyte innate immune response to direct activators of cytosolic pattern recognition receptors}, volume={29}, ISSN={["1753-4267"]}, DOI={10.1177/17534259231162192}, abstractNote={ The skin is the first line of defense to cutaneous microbes and viruses, and epidermal keratinocytes play a critical role in preventing infection by viruses and pathogens through activation of the type I interferon (IFN) response. Using RNAseq analysis, here we report that the conditional deletion of C/EBPβ transcription factor in mouse epidermis (CKOβ mice) resulted in the upregulation of IFNβ and numerous keratinocyte interferon-stimulated genes (ISGs). The expression of cytosolic pattern recognition receptors (cPRRs), that recognize viral RNA and DNA, were significantly increased, and enriched in the RNAseq data set. cPRRs stimulate a type I IFN response that can trigger cell death to eliminate infected cells. To determine if the observed increases in cPRRs had functional consequences, we transfected CKOβ primary keratinocytes with the pathogen and viral mimics poly(I:C) (dsRNA) or poly(dA:dT) (synthetic B-DNA) that directly activate PRRs. Transfected CKOβ primary keratinocytes displayed an amplified type I IFN response which was accompanied by increased activation of IRF3, enhanced ISG expression, enhanced activation of caspase-8, caspase-3 and increased apoptosis. Our results identify C/EBPβ as a critical repressor of the keratinocyte type I IFN response, and demonstrates that the loss of C/EBPβ primes keratinocytes to the activation of cytosolic PRRs by pathogen RNA and DNA to induce cell death mediated by caspase-8 and caspase-3. }, number={1-2}, journal={INNATE IMMUNITY}, author={House, John S. and Gray, Sophia and Owen, Jennifer R. and Jima, Dereje D. and Smart, Robert C. and Hall, Jonathan R.}, year={2023}, month={Jan}, pages={14–24} }
 @article{kaur_barnes_pan_detwiler_liu_mahn_hall_messenger_you_piehler_et al._2021, title={TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA}, volume={49}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkab1142}, abstractNote={Abstract}, number={22}, journal={NUCLEIC ACIDS RESEARCH}, author={Kaur, Parminder and Barnes, Ryan and Pan, Hai and Detwiler, Ariana C. and Liu, Ming and Mahn, Chelsea and Hall, Jonathan and Messenger, Zach and You, Changjiang and Piehler, Jacob and et al.}, year={2021}, month={Dec}, pages={13000–13018} }
 @article{tam_hall_messenger_jima_house_linder_smart_2019, title={C/EBP beta suppresses keratinocyte autonomous type 1 IFN response and p53 to increase cell survival and susceptibility to UVB-induced skin cancer}, volume={40}, ISSN={["1460-2180"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-85083447649&partnerID=MN8TOARS}, DOI={10.1093/carcin/bgz012}, abstractNote={Abstract}, number={9}, journal={CARCINOGENESIS}, author={Tam, Hann W. and Hall, Jonathan R. and Messenger, Zachary J. and Jima, Dereje D. and House, John S. and Linder, Keith and Smart, Robert C.}, year={2019}, month={Sep}, pages={1099–1109} }
 @article{messenger_hall_jima_house_tam_tokarz_smart_2018, title={C/EBPβ deletion in oncogenic Ras skin tumors is a synthetic lethal event}, volume={9}, ISSN={2041-4889}, url={http://dx.doi.org/10.1038/S41419-018-1103-Y}, DOI={10.1038/s41419-018-1103-y}, abstractNote={Abstract}, number={11}, journal={Cell Death & Disease}, publisher={Springer Science and Business Media LLC}, author={Messenger, Zachary J. and Hall, Jonathan R. and Jima, Dereje D. and House, John S. and Tam, Hann W. and Tokarz, Debra A. and Smart, Robert C.}, year={2018}, month={Oct} }
 @article{duke_thompson_ihrie_taylor-just_ash_shipkowski_hall_tokarz_cesta_hubbs_et al._2018, title={Role of p53 in the chronic pulmonary immune response to tangled or rod-like multi-walled carbon nanotubes}, volume={12}, ISSN={["1743-5404"]}, DOI={10.1080/17435390.2018.1502830}, abstractNote={Abstract The fiber-like shape of multi-walled carbon nanotubes (MWCNTs) is reminiscent of asbestos, suggesting they pose similar health hazards when inhaled, including pulmonary fibrosis and mesothelioma. Mice deficient in the tumor suppressor p53 are susceptible to carcinogenesis. However, the chronic pathologic effect of MWCNTs delivered to the lungs of p53 heterozygous (p53+/−) mice has not been investigated. We hypothesized that p53+/− mice would be susceptible to lung tumor development after exposure to either tangled (t-) or rod-like (r-) MWCNTs. Wild-type (p53+/+) or p53+/− mice were exposed to MWCNTs (1 mg/kg) via oropharyngeal aspiration weekly over four consecutive weeks and evaluated for cellular and pathologic outcomes 11-months post-initial exposure. No lung or pleural tumors were observed in p53+/+ or p53+/− mice exposed to either t- or rMWCNTs. In comparison to tMWCNTs, the rMWCNTs induced the formation of larger granulomas, a greater number of lymphoid aggregates and greater epithelial cell hyperplasia in terminal bronchioles in both p53+/− and p53+/+ mice. A constitutively larger area of CD45R+/CD3+ lymphoid tissue was observed in p53+/− mice compared to p53+/+ mice. Importantly, p53+/− mice had larger granulomas induced by rMWCNTs as compared to p53+/+ mice. These findings indicate that a combination of p53 deficiency and physicochemical characteristics including nanotube geometry are factors in susceptibility to MWCNT-induced lymphoid infiltration and granuloma formation.}, number={9}, journal={NANOTOXICOLOGY}, author={Duke, Katherine S. and Thompson, Elizabeth A. and Ihrie, Mark D. and Taylor-Just, Alexia J. and Ash, Elizabeth A. and Shipkowski, Kelly A. and Hall, Jonathan R. and Tokarz, Debra A. and Cesta, Mark F. and Hubbs, Ann F. and et al.}, year={2018}, month={Oct}, pages={975–991} }
 @article{hall_messenger_tam_phillips_recio_smart_2015, title={Long noncoding RNA lincRNA-p21 is the major mediator of UVB-induced and p53-dependent apoptosis in keratinocytes}, volume={6}, ISSN={["2041-4889"]}, DOI={10.1038/cddis.2015.67}, abstractNote={Abstract}, journal={CELL DEATH & DISEASE}, author={Hall, J. R. and Messenger, Z. J. and Tam, H. W. and Phillips, S. L. and Recio, L. and Smart, R. C.}, year={2015}, month={Mar} }
 @article{hall_bereman_nepomuceno_thompson_muddiman_smart_2014, title={C/EBPα regulates CRL4Cdt2-mediated degradation of p21 in response to UVB-induced DNA damage to control the G1/S checkpoint}, volume={13}, ISSN={1538-4101 1551-4005}, url={http://dx.doi.org/10.4161/15384101.2014.962957}, DOI={10.4161/15384101.2014.962957}, abstractNote={The bZIP transcription factor, C/EBPα is highly inducible by UVB and other DNA damaging agents in keratinocytes. C/EBPα-deficient keratinocytes fail to undergo cell cycle arrest in G1 in response to UVB-induced DNA damage and mice lacking epidermal C/EBPα are highly susceptible to UVB-induced skin cancer. The mechanism through which C/EBPα regulates the cell cycle checkpoint in response to DNA damage is unknown. Here we report untreated C/EBPα-deficient keratinocytes have normal levels of the cyclin-dependent kinase inhibitor, p21, however, UVB-treated C/EBPα-deficient keratinocytes fail to up-regulate nuclear p21 protein levels despite normal up-regulation of Cdkn1a mRNA levels. UVB-treated C/EBPα-deficient keratinocytes displayed a 4-fold decrease in nuclear p21 protein half-life due to the increased proteasomal degradation of p21 via the E3 ubiquitin ligase CRL4Cdt2. Cdt2 is the substrate recognition subunit of CRL4Cdt2 and Cdt2 mRNA and protein levels were up-regulated in UVB-treated C/EBPα-deficient keratinocytes. Knockdown of Cdt2 restored p21 protein levels in UVB-treated C/EBPα-deficient keratinocytes. Lastly, the failure to accumulate p21 in response to UVB in C/EBPα-deficient keratinocytes resulted in decreased p21 interactions with critical cell cycle regulatory proteins, increased CDK2 activity, and inappropriate entry into S-phase. These findings reveal C/EBPα regulates G1/S cell cycle arrest in response to DNA damage via the control of CRL4Cdt2 mediated degradation of p21.}, number={22}, journal={Cell Cycle}, publisher={Informa UK Limited}, author={Hall, Jonathan R and Bereman, Michael S and Nepomuceno, Angelito I and Thompson, Elizabeth A and Muddiman, David C and Smart, Robert C}, year={2014}, month={Oct}, pages={3602–3610} }
 @article{thompson_zhu_hall_house_ranjan_burr_he_owens_smart_2011, title={C/EBP alpha Expression Is Downregulated in Human Nonmelanoma Skin Cancers and Inactivation of C/EBP alpha Confers Susceptibility to UVB-Induced Skin Squamous Cell Carcinomas}, volume={131}, ISSN={["0022-202X"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-79956039252&partnerID=MN8TOARS}, DOI={10.1038/jid.2011.31}, abstractNote={Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G1 checkpoint, and diminished or ablated expression of C/EBPα results in G1 checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB. Human epidermis is routinely subjected to DNA damage induced by UVB solar radiation. Cell culture studies have revealed an unexpected role for C/EBPα (CCAAT/enhancer-binding protein-α) in the DNA damage response network, where C/EBPα is induced following UVB DNA damage, regulates the G1 checkpoint, and diminished or ablated expression of C/EBPα results in G1 checkpoint failure. In the current study we observed that C/EBPα is induced in normal human epidermal keratinocytes and in the epidermis of human subjects exposed to UVB radiation. The analysis of human skin precancerous and cancerous lesions (47 cases) for C/EBPα expression was conducted. Actinic keratoses, a precancerous benign skin growth and precursor to squamous cell carcinoma (SCC), expressed levels of C/EBPα similar to normal epidermis. Strikingly, all invasive SCCs no longer expressed detectable levels of C/EBPα. To determine the significance of C/EBPα in UVB-induced skin cancer, SKH-1 mice lacking epidermal C/EBPα (CKOα) were exposed to UVB. CKOα mice were highly susceptible to UVB-induced SCCs and exhibited accelerated tumor progression. CKOα mice displayed keratinocyte cell cycle checkpoint failure in vivo in response to UVB that was characterized by abnormal entry of keratinocytes into S phase. Our results demonstrate that C/EBPα is silenced in human SCC and loss of C/EBPα confers susceptibility to UVB-induced skin SCCs involving defective cell cycle arrest in response to UVB. CCAAT/enhancer-binding protein-α DNA damage response immunohistochemical keratin 5 minimum erythemic dose phosphate-buffered saline squamous cell carcinoma 12-O-tetradecanoylphorbol-13-acetate The epidermis is routinely subject to DNA damage by UVB, which is considered to be the principal carcinogenic component of sunlight. Exposure to UVB results in DNA damage in the form of cyclobutane pyrimidine dimers, 6–4 photoproducts, DNA strand breaks, and DNA crosslinks (Brash, 1997Brash D.E. Sunlight and the onset of skin cancer.Trends Genet. 1997; 13: 410-414Abstract Full Text PDF PubMed Scopus (259) Google Scholar; de Gruijl et al., 2001de Gruijl F.R. van Kranen H.J. Mullenders L.H. UV-induced DNA damage, repair, mutations and oncogenic pathways in skin cancer.J Photochem Photobiol B. 2001; 63: 19-27Crossref PubMed Scopus (385) Google Scholar). If not repaired or if misrepaired, this DNA damage can result in mutations in the genome, and can ultimately contribute to the development of skin cancers (Brash et al., 1991Brash D.E. Rudolph J.A. Simon J.A. et al.A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma.Proc Natl Acad Sci USA. 1991; 88: 10124-10128Crossref PubMed Scopus (1643) Google Scholar). Solar radiation is responsible for >1,000,000 nonmelanoma skin cancer cases per year in the United States, and these cases account for 40% of all new cancer cases diagnosed each year in the United States (American Cancer Society, 2008American Cancer SocietyCancer Facts & Figures 2008.in: 2008http://www.cancer.org/Research/CancerFactsFigures/cancer-facts-figures-2008Google Scholar). To maintain genome integrity and to prevent heritable mutations that lead to cancer, cells respond to DNA damage produced by intrinsic or environmental factors by engaging the DNA damage response (DDR) network. This network entails signaling pathways involving cell cycle checkpoints, DNA repair, transcription programs, and apoptosis. Cell cycle checkpoints can occur in any phase of the cell cycle and are characterized as a pause in the cell cycle that allows time for the repair of damaged DNA. Defective checkpoints can contribute to genome instability and cancer pathogenesis (Kastan and Bartek, 2004Kastan M.B. Bartek J. Cell-cycle checkpoints and cancer.Nature. 2004; 432: 316-323Crossref PubMed Scopus (2028) Google Scholar). C/EBPα (CCAAT/enhancer-binding protein-α) is one of the six members of the C/EBP family of basic leucine zipper transcription factors (Ramji and Foka, 2002Ramji D.P. Foka P. CCAAT/enhancer-binding proteins: structure, function and regulation.Biochem J. 2002; 365: 561-575Crossref PubMed Google Scholar; Johnson, 2005Johnson P.F. Molecular stop signs: regulation of cell-cycle arrest by C/EBP transcription factors.J Cell Sci. 2005; 118: 2545-2555Crossref PubMed Scopus (221) Google Scholar). C/EBPα has been established as a tumor-suppressor gene in human acute myeloid leukemia (Pabst et al., 2001Pabst T. Mueller B.U. Zhang P. et al.Dominant-negative mutations of CEBPA, encoding CCAAT/enhancer binding protein-alpha (C/EBPalpha), in acute myeloid leukemia.Nat Genet. 2001; 27: 263-270Crossref PubMed Scopus (705) Google Scholar). Additionally, there is circumstantial evidence for its function as a tumor suppressor based on diminished C/EBPα expression in a multitude of human tumor types including liver (Xu et al., 2001Xu L. Hui L. Wang S. et al.Expression profiling suggested a regulatory role of liver-enriched transcription factors in human hepatocellular carcinoma.Cancer Res. 2001; 61: 3176-3181PubMed Google Scholar), lung (Halmos et al., 2002Halmos B. Huettner C.S. Kocher O. et al.Down-regulation and antiproliferative.Cancer Res. 2002; 62: 528-534PubMed Google Scholar), breast (Gery et al., 2005Gery S. Tanosaki S. Bose S. et al.Down-regulation and growth inhibitory role of C/EBPalpha in breast cancer.Clin Cancer Res. 2005; 11: 3184-3190Crossref PubMed Scopus (76) Google Scholar), endometrial (Takai et al., 2005Takai N. Kawamata N. Walsh C.S. et al.Discovery of epigenetically masked tumor suppressor genes in endometrial cancer.Mol Cancer Res. 2005; 3: 261-269Crossref PubMed Scopus (63) Google Scholar), and head and neck squamous cell carcinomas (SCCs; Bennett et al., 2007Bennett K.L. Hackanson B. Smith L.T. et al.Tumor suppressor activity of CCAAT/e.Cancer Res. 2007; 67: 4657-4664Crossref PubMed Scopus (70) Google Scholar). The traditional view of C/EBPα in cell biology involves its role in cellular differentiation and metabolism (Roesler, 2001Roesler W.J. The role of C/EBP in nutrient and hormonal regulation of gene expression.Annu Rev Nutr. 2001; 21: 141-165Crossref PubMed Scopus (81) Google Scholar); in cancer, the traditional view holds that it has a tumor-suppressor role, where the loss of expression/function results in an impaired differentiation commitment accompanied by deregulated proliferation (Schuster and Porse, 2006Schuster M.B. Porse B.T. C/EBPalpha: a tumour suppressor in multiple tissues?.Biochim Biophys Acta. 2006; 1766: 88-103PubMed Google Scholar; Koschmieder et al., 2009Koschmieder S. Halmos B. Levantini E. et al.Dysregulation of the C/EBPalpha differentiation pathway in human cancer.J Clin Oncol. 2009; 27: 619-628Crossref PubMed Scopus (155) Google Scholar). However, recent studies indicate that the role of C/EBPα in cells/cancer is more complex and multifaceted than originally thought (Yoon and Smart, 2004Yoon K. Smart R.C. C/EBPalpha is a DNA damage-inducible p53-regulated mediator of the G1 checkpoint in keratinocytes.Mol Cell Biol. 2004; 24: 10650-10660Crossref PubMed Scopus (53) Google Scholar; Loomis et al., 2007Loomis K.D. Zhu S. Yoon K. et al.Genetic ablation of CCAAT/enhancer binding protein {alpha.Cancer Res. 2007; 67: 6768-6776Crossref PubMed Scopus (34) Google Scholar; Ranjan et al., 2009Ranjan R. Thompson E.A. Yoon K. et al.C/EBPalpha expression is partially regulated by C/EBPbeta in response to DNA damage and C/EBPalpha-deficient fibroblasts display an impaired G1 checkpoint.Oncogene. 2009; 28: 3235-3245Crossref PubMed Scopus (8) Google Scholar). Cell culture studies have revealed an unexpected role for C/EBPα in the DDR in keratinocytes where C/EBPα is induced following UVB-induced DNA damage, and it regulates the G1 checkpoint. Diminished or ablated expression of C/EBPα results in G1 checkpoint failure following UVB-induced DNA damage (Yoon and Smart, 2004Yoon K. Smart R.C. C/EBPalpha is a DNA damage-inducible p53-regulated mediator of the G1 checkpoint in keratinocytes.Mol Cell Biol. 2004; 24: 10650-10660Crossref PubMed Scopus (53) Google Scholar; Ranjan et al., 2009Ranjan R. Thompson E.A. Yoon K. et al.C/EBPalpha expression is partially regulated by C/EBPbeta in response to DNA damage and C/EBPalpha-deficient fibroblasts display an impaired G1 checkpoint.Oncogene. 2009; 28: 3235-3245Crossref PubMed Scopus (8) Google Scholar). In further support of a nonparadigmatic C/EBPα tumor-suppressor function, mice lacking C/EBPα in their epidermis do not display alterations in differentiation or proliferation and are susceptible to chemical carcinogen-induced skin tumorigenesis (Loomis et al., 2007Loomis K.D. Zhu S. Yoon K. et al.Genetic ablation of CCAAT/enhancer binding protein {alpha.Cancer Res. 2007; 67: 6768-6776Crossref PubMed Scopus (34) Google Scholar). Given the function of C/EBPα as a mediator of the G1 checkpoint in keratinocytes in response to UVB, it is possible that C/EBPα functions as a suppressor of UVB-induced tumorigenesis. Whereas C/EBPα expression is diminished in mouse skin SCCs (Oh and Smart, 1998Oh H.S. Smart R.C. Expression of CCAAT/enhancer binding proteins (C/EBP) is associated with squamous differentiation in epidermis and isolated primary keratinocytes and is altered in skin neoplasms.J Invest Dermatol. 1998; 110: 939-945Crossref PubMed Scopus (79) Google Scholar; Shim et al., 2005Shim M. Powers K.L. Ewing S.J. et al.Diminished expression of C/EBPalpha in skin carcinomas is linked to oncogenic Ras and reexpression of C/EBPalpha in carcinoma cells inhibits proliferation.Cancer Res. 2005; 65: 861-867PubMed Google Scholar), C/EBPα levels have not been examined in human skin precancerous and cancerous lesions. Therefore, the objectives of this study were: to examine the expression of C/EBPα in human skin precancerous and cancerous lesions, to characterize the response of C/EBPα to UVB in human keratinocytes and human skin; and to develop an in vivo SKH-1 mouse model to determine the in vivo physiological significance of C/EBPα in UVB-induced skin tumorigenesis. We examined the expression of C/EBPα in normal human epidermis, precancerous actinic keratoses, keratoacanthomas, SCCs in situ, and invasive SCCs as well as basal cell carcinomas. Immunohistochemical (IHC) staining for C/EBPα in human skin showed that C/EBPα was extensively expressed in the nuclei of nondividing keratinocytes of the suprabasal layers of the epidermis (Figure 1a). C/EBPα expression was also detected, although less frequently, in keratinocytes in the proliferative basal layer of epidermis. Actinic keratoses, a precancerous benign skin growth of which a small percentage progress to SCC, expressed levels of C/EBPα similar to normal epidermis (Figure 1a and b). Keratoacanthomas, once considered as terminally benign but now regarded and treated by many dermatologists as a malignant growth that can progress to SCC, expressed reduced levels of C/EBPα with 20% of keratoacanthomas no longer expressing detectable levels of C/EBPα (Figure 1a and b). Most striking, however, were the SCCs where the majority of both SCCs in situ (80%) and invasive SCCs (100%) no longer expressed detectable levels of C/EBPα (Figure 1a and b). Similarly, the IHC staining for C/EBPα was absent in 14/16 basal cell carcinoma cases (data not shown). Most human nonmelanoma skin cancers are caused by solar radiation, and UVB radiation is considered the most carcinogenic component of sunlight. To characterize the effects of UVB exposure on the expression of C/EBPα in human keratinocytes, we exposed subconfluent proliferating normal human epidermal keratinocytes to 5, 10, or 15mJcm2 UVB. Immunoblot analysis for C/EBPα revealed that the expression of C/EBPα protein was induced at all doses (Figure 2a). To characterize the effects of UVB exposure on the expression of C/EBPα in human skin in vivo, biopsies from UVB-treated human skin (1 minimum erythemic dose (MED) UVB) (N=5) were compared with biopsies from non-sun-exposed human skin (N=5) for C/EBPα expression. C/EBPα levels were increased throughout the epidermis of UVB-treated human skin as determined by the increased C/EBPα IHC nuclear staining intensity as well as by the overall statistically significant increase in the number of keratinocytes staining positively for C/EBPα (Figure 2b–d and Supplementary Figure S1 online). Although UVB treatment increased the percentage of C/EBPα-expressing keratinocytes in the spinous and granular nondividing suprabasal layers by 2.3-fold, we observed a 4.3-fold increase in the number of basal keratinocytes expressing C/EBPα (Figure 2b–d). Download .pdf (.66 MB) Help with pdf files Supplementary Figures S1 and S2 We treated SKH-1 hairless mice, a well-characterized mouse model frequently utilized to study the effects of UVB in skin in vivo (Benavides et al., 2009Benavides F. Oberyszyn T.M. VanBuskirk A.M. et al.The hairless mouse in skin research.J Dermatol Sci. 2009; 53: 10-18Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar), with UVB (50mJcm2) and, similar to human skin, we observed a significant increase in C/EBPα-expressing keratinocytes in the suprabasal and basal proliferative compartments of epidermis (Figure 3a). There was an ∼2-fold increase in the number of C/EBPα positively stained suprabasal cells, and a 3-fold increase in the number of C/EBPα positively stained basal keratinocytes (Figure 3b). To begin to determine the physiological significance of UVB-induced epidermal C/EBPα in vivo, we generated an epidermal-specific C/EBPα knockout SKH-1 mouse, hereafter referred to as conditional knockout (CKOα). CKOα mice were generated from C/EBPαfl/fl SKH-1 mice and keratin 5 (K5) Cre (K5Cre) SKH-1 mice where the K5 promoter directs Cre recombinase expression to the epidermis and other stratified squamous epithelia. As shown in Figure 3c, C/EBPα protein was not detectable in epidermal protein extracts prepared from CKOα mice. CKOα mice did not display any abnormal gross or morphological skin phenotype (data not shown). The most susceptible human population to UVB-induced damage is classified as type 1 according to the Fitzpatrick Classification Scale. These individuals have very fair and often freckled skin, have blond or red hair, always sun burn, and are highly susceptible to solar radiation-induced nonmelanoma skin cancer. The MED of UVB for this susceptible population is ∼20–25mJcm2. Therefore, we exposed CKOα and K5Cre control mice to 20mJcm2 UVB three times weekly to test whether the loss of C/EBPα in skin keratinocytes confers susceptibility to UVB-induced tumorigenesis. As shown in Figure 4a and b, CKOα mice were highly susceptibility to UVB-induced skin tumorigenesis as evidenced by decreased latency (18 weeks), increased tumor incidence (85 vs. 25%), and a 6-fold increase in tumor multiplicity. Histological analysis of all skin tumors at the termination of experiment revealed that the majority of tumors (∼90%) in both genotypes were SCCs (in situ or invasive). Most importantly, we observed that 48% of SCCs in CKOα mice were invasive SCCs compared with 15% in the UVB-treated K5Cre control mice (Figure 4c). Invasive carcinomas were identified by severe dysplasia to anaplastic growth, marked atypia in all cell layers, and most importantly, invasion through the panniculus carnosus and/or basement membrane (Supplementary Figure S2 online). To demonstrate that these tumors were the result of UVB treatment and not advanced age, 8 CKOα mice were held for 52 weeks and no skin tumors of any kind were detected. Collectively, these results indicate that loss of C/EBPα confers susceptibility to UVB-induced skin cancer at a biologically relevant dose and loss of C/EBPα accelerates skin tumor progression. To determine whether the ablation of epidermal C/EBPα alters the ability of keratinocytes to undergo a cell cycle arrest in response to UVB treatment in vivo in mouse skin, we treated K5Cre control mice and CKOα mice with UVB, and then examined the number of actively replicating keratinocytes post-UVB by measuring the incorporation of the nucleotide analog BrdU (1hour BrdU pulse before skin collection). As shown in Figure 5a, UVB-treated control mice (K5Cre) displayed a significant cell cycle arrest; at 4hours post-UVB treatment, there was ∼60% decrease in the number of BrdU-positive S-phase basal keratinocytes in the epidermis and this decrease was sustained at 6 and 10hours post-UVB (Figure 5a). At 12hours post-UVB, the number of BrdU-positive S-phase basal keratinocytes returned to untreated control levels (data not shown). Although UVB-treated CKOα mice displayed a similar cell cycle arrest as UVB-treated control mice at 4hours post-UVB treatment, this inhibition was not sustained and cells resumed their progression in the cell cycle prematurely. At 6hours post-UVB, the number of BrdU-positive S-phase cells was significantly increased and by 10hours post-UVB treatment, there was a 3-fold increase in BrdU-positive S-phase basal keratinocytes in CKOα epidermis compared with UVB-treated control mice. Representative examples of BrdU-positive S-phase staining at 10hours post-UVB in K5Cre and CKOα mouse epidermis are shown in Figure 5a (right panel). These results indicate that the loss of C/EBPα in epidermis results in an impaired cell cycle arrest in response to UVB in vivo. To further investigate the role of C/EBPα in UVB-induced cell cycle checkpoints in epidermis in vivo, we utilized an in vivo model of cell cycle regulation involving the induction of synchronous cell proliferation induced by the potent mitogen, 12-O-tetradecanoylphorbol-13-acetate (TPA) (Foley et al., 1993Foley J. Ton T. Maronpot R. et al.Comparison of proliferating cell nuclear antigen to tritiated thymidine as a marker of proliferating hepatocytes in rats.Environ Health Perspect. 1993; 101: 199-205Crossref PubMed Scopus (96) Google Scholar; Kim et al., 1997Kim T.W. Porter K.L. Foley J.F. et al.Evidence that mirex promotes a unique population of epidermal cells that cannot be distinguished by their mutant Ha-ras genotype.Mol Carcinog. 1997; 20: 115-124Crossref PubMed Scopus (9) Google Scholar; Rodriguez-Puebla et al., 1998Rodriguez-Puebla M.L. Robles A.I. Johnson D.G. et al.Synchronized proliferation induced by 12-O-tetradecanoylphorbol-13-acetate treatment of mouse skin: an in vivo model for cell cycle regulation.Cell Growth Differ. 1998; 9: 31-39PubMed Google Scholar). A single topical treatment of TPA resulted in synchronous entry of G1 keratinocytes into S phase as determined by BrdU-positive basal cells at 12hours post-TPA treatment and S-phase cells further increased at 14 and 16hours in control mice (1hour BrdU pulse before skin collection; Figure 5b). TPA-treated CKOα mice responded similarly and the numbers of S-phase cells were not significantly different from TPA-treated control mice at all time points (P>0.3, data not shown). To determine the effect of UVB on this entry into S phase, K5Cre and CKOα mice were treated with TPA, and 4hours later, a time when keratinocytes are still in G1, were either treated with UVB (50mJcm2) or left untreated. The number of BrdU-positive S-phase cells were then determined at 14hours post-TPA treatment and 10hours post-UVB. As shown in Figure 5c, TPA-treated K5Cre and CKOα mice not exposed to UVB displayed a 3-fold increase in the number of BrdU-positive S-phase keratinocytes. UVB treatment of TPA-treated mice resulted in dramatic decreases in the number of BrdU-positive S-phase cells in the epidermis of both genotypes compared with TPA treatment alone (Figure 5c). However, CKOα mice displayed ∼3 times more BrdU-positive S-phase cells than similarly treated K5Cre mice. Collectively, our findings suggest that the loss of epidermal C/EBPα results in an impaired cell cycle arrest involving the G1- to S-phase transition in response to UVB in vivo in mouse epidermis. Because p53 is an important player in the DDR and a key mediator of checkpoints following DNA damage, we wanted to determine whether the impaired cell cycle arrest at the G1- to S-phase transition observed in the CKOα mice following UVB treatment could be related to deficiencies in p53. We performed immunoblot analysis for p53 on epidermal lysates from both UVB-treated (50mJcm2) and untreated K5Cre and CKOα mice, and observed an induction in p53 protein in both genotypes following UVB treatment (Figure 5d). To further determine whether p53 was induced to a similar extent in both genotypes, IHC staining and quantitation of p53-positive basal cells were performed, and no statistically significant differences were found (Figure 5d). C/EBPα is abundantly expressed in the mouse and human epidermis (Swart et al., 1997Swart G.W. van Groningen J.J. van Ruissen F. et al.Transcription factor C/EBPalpha: novel sites of expression and cloning of the human gene.Biol Chem. 1997; 378: 373-379Crossref PubMed Scopus (29) Google Scholar; Maytin and Habener, 1998Maytin E.V. Habener J.F. Transcription factors C/EBP alpha, C/EBP beta, and CHOP (Gadd153) expressed during the differentiation program of keratinocytes in vitro and in vivo.J Invest Dermatol. 1998; 110: 238-246Crossref PubMed Scopus (119) Google Scholar; Oh and Smart, 1998Oh H.S. Smart R.C. Expression of CCAAT/enhancer binding proteins (C/EBP) is associated with squamous differentiation in epidermis and isolated primary keratinocytes and is altered in skin neoplasms.J Invest Dermatol. 1998; 110: 939-945Crossref PubMed Scopus (79) Google Scholar) and in this study we report that C/EBPα is induced by UVB in human epidermal keratinocytes and human epidermis. 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Our current results in CKOα mice demonstrate that the ablation of C/EBPα in mouse epidermis confers susceptibility to UVB-induced SCCs at a biologically relevant dose and results in the acceleration of skin cancer progression to invasive SCCs. Additionally, earlier studies showed that mice lacking C/EBPα in their epidermis are also more susceptible to chemical carcinogen-induced skin tumorigenesis, and tumors that develop in these mice exhibited an accelerated rate of malignant progression to invasive SCCs (Loomis et al., 2007Loomis K.D. Zhu S. Yoon K. et al.Genetic ablation of CCAAT/enhancer binding protein {alpha.Cancer Res. 2007; 67: 6768-6776Crossref PubMed Scopus (34) Google Scholar). Collectively, these results indicate an important role for the loss of C/EBPα in skin cancer progression induced by UVB radiation or chemical carcinogens. 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