been shown to contribute to squamous carcinogenesis [60,61]. Moreover, AP-1 transcription factors along with Ets proteins can exert a combinatorial control of expression of several genes that include importantly metalloproteinases. Matrix metalloproteinases appear to be chief effectors of Ets1 induced epithelial alterations [51]. Several MMPs have been shown to be direct targets of Ets proteins and are often co-regulated by AP- 1 proteins. Various classes of metalloproteinases such as collage- nases (MMPs 8 and 13), gelatinases (MMPs 2 and 9), stromelysins (MMPs 3, 10 and 11), metalloelastase (MMP12) and membrane- type MMPs (MMPs 14 and 16) are upregulated in Ets1 BT skin. This suggests that several biological processes that accompany Ets1 induced dysplasia could be attributed to the function of MMPs. We focused our attention on the most highly upregulated target gene, matrix metalloprotease 13 (MMP13). We demonstrate that Ets1 directly regulates expression of the MMP13 gene and can be detected at the promoter region. Interestingly, Ets1 was associated with the promoter region of MMP13 in both BT and control skin samples. This may suggest that Ets1 is constitutively associated with the promoter of this gene, but is inactive unless it becomes phosphorylated or that it requires the recruitment of an inducible co-factor to mediate full induction of the gene. - MMP enzymes including MMP13 are best known for their ability to degrade a wide variety of extracellular matrix substrates including collagen, fibronectin, laminin, perlecan, tenascin C and fibrillin [43,62]. By degrading extracellular matrix components, MMPs can promote the invasion of cells into the surrounding tissues. Moreover, degradation of extracellular matrix may lead to the release of latent growth factors and angiogenic factors, promoting tumor cell proliferation. Furthermore, numerous studies have detected degradation of key epithelial adhesion proteins, such as E-cadherin and desmogleins, by members of the MMP family [63,64,65,66,67,68], suggesting that MMP activity can result in diminished keratinocyte adhesion to neighboring cells. Together these activities of MMP proteins can promote keratinocyte proliferation, migration and invasion. -MMP proteins have the ability to promote dysplastic changes in tissues, even when they are expressed alone in the absence of other genetic changes or tumor inducing chemicals or UV radia- tion[63,69,70,71,72]. In addition, over-expression of matriptase, a protease that has been suggested to be an upstream activator of MMP3 [73], induces squamous cell carcinoma in the skin [52]. Thus, expression or activation of numerous MMP proteins can promote hyperplastic and neoplastic changes in epithelial tissues, supporting the idea that overexpression of MMPs may be important to the development of pre-neoplastic and neoplastic alterations in Ets1 BT mice. It is likely that MMPs cooperate with other genes induced by Ets1 overexpression (such as myc and AP-1 family members) to promote the dysplastic changes. The role of Ets1 in keratinocyte differentiation and future prospects In addition, to promoting proliferation and invasion of skin keratinocytes, Ets1 expression also blocked their terminal differentiation program. The block to terminal differentiation likely results in impaired barrier formation in the skin and is likely the cause of perinatal mortality for Ets1 BT mice that undergo transgene induction during embryonic development. The effect of Ets1 in blocking terminal differentiation may arise from its ability to interfere with the transcriptional programs that drive the differentiation process. For instance, we have recently shown that Ets1 physically interacts with the transcription factor Blimp-1 to inhibit its function [74]. Blimp-1 is important for the final differentiation step of keratinocytes from the granular layer to the Ets1 in Squamous Neoplasia cornified layer [75]. By interfering with Blimp-1 or other transcription factors, Ets1 may prevent their function and thereby block the terminal differentiation of the epidermis. In summary, using an inducible transgenic mouse model, we have demonstrated that expression of the Ets1 transcription factor leads to a dramatic hyperplastic and dysplastic phenotype in skin epithelium of young mice in the apparent absence of other genetic lesions. This represents the first report that Ets1 can play a major role in the early stages of the carcinogenic process. Together, these studies extend our understanding of the role of Ets1 in squamous cell tumors and suggest mechanisms by which Ets1 can promote cellular transformation and tumor progression. Our data described here strongly suggest that in a variety of human carcinomas, over- expression of Ets1 may not just be a prognostic indicator, but play an important causal role that can be exploited for targeted therapy. In the future, our transgenic model system can be used to induce Ets1 in any tissue or cell type by utilizing the appropriate tetracycline-transactivator lines, and hence will prove useful in understanding the role of Ets1 in regulating development, differentiation and oncogenic conversion in a variety of tissues. Materials and Methods Ethics Statement All mouse experiments were approved by the State University of New York at Buffalo IACUC committee and carried out in accordance with relevant national guidelines. Generation and analysis of transgenic mice The Ets1 doxycycline inducible construct was generated by cloning full length mouse Ets1 cDNA with a 59 HA tag into the pTRE-Tight plasmid (BD Biosciences Clontech). The inducible Ets1 construct was micro-injected into fertilized mouse oocytes derived from a mixed genetic background (C3Hf/HeR- os6C57BL/10 Rospd). Six transgenic lines were generated in these initial injections. The following primers were used to genotype the transgenic mice: forward (59-ATAGTTGT- GACCGCCTCACC -39) and reverse (59- GGGAGGTGTGG- GAGGTTTT-39). The founders were then crossed to INV-tTA mice (a kind gift from Dr. Julia Segre, National Human Genome Research Institute, Bethesda) to determine expression of the transgene. Inv-tTA mice were on an FVB/N genetic background and BT mice derived from these crosses were subsequently backcrossed to FVB/N mice for several generations. Antibodies Used The following primary antibodies were used for Western blotting, immunofluorescence and/or immunohistochemistry: rat monoclonal anti-HA (clone 3F10, Roche Applied Sciences), rabbit polyclonal anti-Ets1 (N-276, Santa Cruz Biotechnology), mouse monoclonal anti-GAPDH (clone 6C5, Chemicon International), goat polyclonal anti-MMP13 (W-16, Santa Cruz Biotechnology), mouse monoclonal anti-Ki67 (NCL-Ki67p, Novocastra), rat monoclonal anti-b4 integrin (CD104, clone 346-11A, BD Biosciences), rat monoclonal anti-PECAM1 (CD31, clone MEC- 13.3, BD Biosciences), rabbit polyclonal anti-laminin I (CL54851AP, Cedarlane Laboratories) and mouse monoclonal anti-PCNA (clone PC10, Dakocytomation). A rabbit polyclonal anti-DNp63 antibody (RR-14, developed in the laboratory of Dr. Satrajit Sinha) was also used. In addition, several polyclonal rabbit primary antibodies specific for keratinocyte marker proteins (keratin 1, keratin 5, keratin 6, keratin 10, keratin 14, involucrin and loricrin) were generous gifts of Dr. Julia Segre (National Human Genome Research Institute, National Institutes of PLoS ONE | www.plosone.org 12 January 2009 | Volume 4 | Issue 1 | e4179