Ets1 in Squamous Neoplasia Figure 3. Ets1 expression causes hyperplastic and dysplastic changes in the skin epidermis. Microscopic features of the cutaneous changes in (A) dorsal trunk, (B) ear and (C) tail skin of BT mice upon Ets1 induction show epidermal and sebaceous hyperplasia, hyperkeratosis, and parakeratosis, keratin pearls (black arrowheads), vascularization, leukocytic infiltration and dermal enclosures within the epidermal downgrowths (white arrowheads). (D) High power view of the dorsal epidermis demonstrating the presence of several mitotic figures including some in the suprabasal layers (black arrowheads). Reactive leukocytic infiltration is also evident (asterisk). (E) Further magnification of supra-basal mitoses in lesions of the dorsal skin in BT mice. Some of the mitoses exhibit abnormal, tripolar mitotic spindles (blue arrowheads). doi:10.1371/journal.pone.0004179.g003 These results were confirmed by Western blotting (Figure 4D and data not shown). The reduced expression of loricrin, filaggrin and involucrin in skin of BT mice likely results in abnormal barrier function in the affected epidermis. In summary, these studies indicate that epidermal differentiation of keratinocytes in BT mice is disrupted with an expansion of keratinocyte populations expressing early differentiation markers and a reduction in keratinocyte populations expressing late differentiation markers. Keratinocytes in epithelia expressing Ets1 undergo enhanced proliferation and induce the growth of local blood vessels As described above, the epidermis of BT mice expressing Ets1 exhibits a dramatic hyperproliferation accompanied by increased numbers of mitotic figures. Several characteristic marker proteins are associated with hyper-proliferative epidermis, but are not normally expressed in the interfollicular epidermis. For instance, the expression of keratin 6 (K6) is normally limited to the hair follicles of mice, but is expressed at high levels in interfollicular epidermis in a variety of hyperplastic and dysplastic conditions. Consistent with this, there was dramatic upregulation of K6 in the interfollicular epidermis of BT mice (Figure 5A). Similar upregulation of K17 was also observed, another keratin that is characteristically expressed by hyperplastic and dysplastic epider- mis (data not shown). The enhanced expression of K6 and K17 in BT epidermis suggested the possibility that there might be altered expression of proteins involved in regulating the proliferation of epidermal stem or transit-amplifying cells. One such protein that has been shown to be critical for epidermal stem cell maintenance is the transcription factor p63 [38]. Immunostaining demonstrated that there was a striking increase in the cell population that labeled with antibody against DN isoform of p63 (the predominant isoform expressed in skin epidermis) in BT epidermis as compared to control (Figure 5B). Some of these p63+ cells were found in suprabasal layers of BT epidermis, whereas they are only found in the basal layer of wild-type controls. Increased numbers of p63+ cells have also been found in human squamous cell cancers [39,40] and high levels of p63 expression are correlated with a poor patient prognosis [41,42]. To more directly test the effects of Ets1 on the proliferation of epidermal keratinocytes, we stained them with antibodies specific for PCNA and Ki67 (Figure 5C–D), proteins expressed only in cells that have entered the cell cycle. The BT epidermis contained many more PCNA+ and Ki67+ cells than did control epidermis, confirming enhanced epidermal proliferation in this tissue. Although the Ets1 transgene was expressed in the more differentiated layers of the epidermis (upper spinous and granular layers), the PCNA and Ki67 labeling was mainly associated with less differentiated layers (basal and lower spinous layers). This observation suggests that expression of Ets1 may not directly induce cell proliferation, but rather induce changes in the differentiation program of the suprabasal cells that cause the basal cells to undergo a dramatic proliferative response. It is possible that the increased proliferation may reflect a secondary response to the compromised barrier function of the epidermis. In addition, some suprabasal keratinocytes also labeled with anti- bodies to PCNA and Ki67 indicating a supra-basal proliferation. While collecting samples of epidermis from BT mice for processing, we noted increased vascularization of the affected skin as evidenced by the presence of numerous, large blood vessels in the dermal tissue (Figure 6A). These vessels were also apparent in hematoxylin and eosin stained sections of the skin epidermis PLoS ONE | www.plosone.org 5 January 2009 | Volume 4 | Issue 1 | e4179