Table 1. Gene expression profiles in WT versus BT mice. Gene Name Common Name Fold change in expression Flt1 Kdr Tie1 Tek Angpt2 Nos3 Itgav Cdh1 Cdh5 Cdkn1a Cdkn2a Cdc2l6 Trp53 Mdm2 Jun Junb Fos Myc Ddit3 Lmo2 Vim Bcl2l1 Bcl2 Casp1 Fasl Tnf Pthlh Met Plau Serpinb5 Mmp2 Mmp3 Mmp8 Mmp9 Mmp10 Mmp11 Mmp12 Mmp13 Mmp14 Mmp15 Mmp16 Mmp17 Mmp19 Timp1 2.4 1.67 0.71 0.57 1.67 2 1.36 0.85 0.81 4.5 6.78 0.87 1.2 1.23 22.0 7.26 9.0 2.24 1.02 0.83 0.74 2.25 0.6 1.52 0.08 5.5 0.59 0.95 0.64 1.39 4.2 3.8 43.1 7.4 41.6 13.8 770 2.3 0.9 8.0 0.5 0.9 5.3 151.2 Ets1 in Squamous Neoplasia keratinocytes, except under conditions of wound healing, dysplasia or neoplasia [44,45,46,47,48]. Moreover, MMP13 expression is considered to be a poor prognostic indicator in SCC [49,50]. Based on these observations as well as the fact that MMP13 was the most highly-upregulated gene among those we tested, we focused our further analyses on this putative Ets1 target. We performed immunostaining for MMP13 in BT and control mouse skin and found that MMP13 was highly expressed in the BT skin, while there was no staining in the control samples (Figure 9A). This is in agreement with the real time PCR data and also confirms that upregulation of MMP13 is seen in the keratinocyte compartment (although some staining of the dermal compartment was also detected). Expression of MMP13 was particularly evident in the invasive front formed by the proximal portions of hair follicles and sebaceous glands and in the cytoplasmic compartments of hyperplastic keratinocytes. Upregu- lation of MMP13 expression was confirmed by Western blotting (Figure 9B). The MMP13 promoter contains three PEA3 motifs (PEA3 sequences are potential Ets binding motifs) and Ets1 has already been shown to bind to one of these sequences by EMSA analysis [16,48]. These observations suggest that MMP13 is likely to be a direct target of Ets1. To confirm that Ets1 can transactivate the MMP13 promoter, we co-transfected Ets1 and luciferase reporter gene constructs harboring various lengths of MMP13 promoter into a mouse keratinocyte cell line. The reporter gene constructs chosen for these studies have been previously described [48] and include a 660 bp fragment of the mouse MMP13 promoter fused to luciferase (p660) as well as several MMP13 promoter deletions: a 391 bp fragment (p391), a 91 bp fragment (p91) and a 66 bp fragment (p66). Ets1 dramatically transactivated both the 660 bp and 391 bp MMP13 promoter constructs by approximately 30- fold in these cells (Figure 9C), suggesting that the relevant regulatory elements needed for Ets1 transactivation were con- tained within the first 391 bp of the promoter. In contrast, Ets1 was significantly less effective in transactivating the 91 and 66 bp MMP13 promoters (2–3 fold stimulation of activity by Ets1). These observations suggest that the important Ets1 responsive elements are localized between 291 and 2391 bp of the MMP13 promoter. This was confirmed by testing the activity of a mutant form of the p391 promoter (p391 del (PEA)) that contains a short deletion in one of the PEA3 motifs (the 2122 bp Ets binding site) (Figure 9C). The deletion construct exhibited responsiveness to Ets1 that was very similar to that of the p91 and p66 promoters. Thus, the Ets1 binding site at 2122 bp of the murine MMP13 promoter is essential for maximal transactivation capacity of Ets1. To confirm that Ets1 can associate with the MMP13 promoter in vivo, we performed chromatin immuno-precipitation (ChIP) experiments on dorsal mouse skin epidermis derived from BT and wild type mice using antibodies specific for Ets1 and for the HA- tag on the Ets1 transgene. In wild type mice, the anti-Ets1 antibody precipitated the MMP13 promoter, whereas the HA antibody did not (Figure 9D). In the BT sample, both anti-Ets1 and anti-HA antibodies pulled down the MMP13 promoter. These observations suggest that Ets1 is associated with the MMP13 promoter region in both wild type and BT mice, although the MMP13 gene is only expressed in the BT animals. Discussion VEGF-R1 VEGF-R2 Tie1 Tie2 Angiopoietin-2 eNOS av-Integrin E-Cadherin VE-Cadherin p21 p16 CDK11 p53 Mdm2 c-jun JunB c-fos C-myc GADD153 Lmo2 Vimentin Bcl-XL Bcl-2 Caspase-1 Fas Ligand TNF-a PTHrP c-met uPA Maspin Gelatinase A Stromelysin-1 Collagenase-2 Gelatinase B Stromelysin-2 Stromelysin-3 Macrophage metalloelastase Collagenase-3 Membrane type 1-MMP Membrane type 2-MMP Membrane type 3-MMP Membrane type 4-MMP Mmp19 Timp1 doi:10.1371/journal.pone.0004179.t001 MMP13 (also referred to as collagenase-3) is a protease that can cleave native, fibrillar collagens including Type IV collagen, a component of epithelial basement membranes [43]. Expression of MMP13 is very restricted in normal tissues and is not found in skin The Ets1 transcription factor regulates many important cellular processes including cellular growth and differentiation [51]. High level expression of the Ets1 oncogene has been linked previously to tumor progression, invasion and metastasis in squamous cell PLoS ONE | www.plosone.org 9 January 2009 | Volume 4 | Issue 1 | e4179