VILIP-1 is expressed in the central nervous system, where it regulates cAMP levels, cell signaling and differentiation 
. VILIP-1 is also widely expressed in sites outside the nervous system such as human heart, lung, liver and testis and moderately expressed in ovary, kidney, spleen and pancreas, suggesting that VILIP-1 might be required for the maintenance of tissue homeostasis in different organs 
. Given the central role of VILIP-1 as a calcium sensor in mediating cAMP response, deregulation of VILIP-1 expression may cause abnormalities in various non-nervous tissues. In past reports, we have described that VILIP-1 expression is lost in chemically-induced mouse SCC 
. We have also shown that VILIP-1 plays a critical role in regulating the invasive/metastatic phenotype by decreasing cell proliferation and matrix degradation/tumor cell invasiveness through a cAMP mediated pathway 
. Furthermore, we have found that VILIP-1 is lost in aggressive SCCs of the human esophagus and lung suggesting a tumor suppressor function 
In this report we demonstrate that the transgenic expression of VILIP-1 targeted to the epidermis is able to decrease the baseline levels of cell proliferation and that this downregulation of epidermal proliferation is also very evident after short term treatment with the hyperplasiogenic tumor promoter TPA. The decrease in epidermal cell turnover is accompanied by an increased expression of products of keratinocyte differentiation, especially K1 and loricrin (Figure S1
). The main effect on cell proliferation and differentiation is attributable to the well known increase in cAMP levels in keratinocytes overexpressing VILIP-1, described in detail in our previous reports 
. VILIP-1 expression also induced astrocytic differentiation in C6 cells 
and is associated with increased squamous differentiation in human esophageal and lung tumors 
. In this context, it is noteworthy that Braunewell and Gundelfinger were able to demonstrate that differentiation is inducible using cAMP analogs 
. Similar effects of cAMP have been described in squamous cell lines and tissues in which cAMP enhanced the expression of keratins such as K1 and K10 
. Perhaps the most significant effect of VILIP-1 overexpression in epidermis is a decrease in MMP-9 activity. This was shown by us in VILIP-1 transfected cells 
and was considered a consequence of increased cAMP activity 
. In the present report we show that this decrease in primary keratinocytes derived from K5-VILIP-1 transgenic mice is accompanied by a significant increase in TIMP-1. This direct regulation of TIMP-1 levels by cAMP has been demonstrated in several different cells and tissue types 
and may be the principal mechanism of TIMP-1 induction by VILIP-1 overexpression in the transgenic mouse skin.
In order to evaluate whether VILIP-1 could modulate tumorigenesis and/or susceptibility to exogenous carcinogens we applied well known carcinogenesis protocols that are widely accepted as paradigms of epithelial carcinogenesis, i.e., the two stage carcinogenesis and the complete carcinogensis protocols of the mouse skin 
. The decreased keratinocyte proliferation and increased squamous differentiation patterns observed in transgenic epidermis have a direct correlation with our observations during skin carcinogenesis of K5-VILIP-1 mice that led to a decreased sensitivity to skin carcinogenesis. In both carcinogenesis protocols we observed a decrease of SCC multiplicity with respect to WT mice that was close to 49% in the two stage carcinogenesis experiment during the final weeks of the experiment (28-30 weeks) and approximately 33% in the complete carcinogenesis experiment at 36 to 40 weeks. In the two stage carcinogenesis experiment it was noteworthy that the ratio of SCC to papillomas was markedly decreased in K5-VILIP-1, indicating that the conversion rate was diminished in transgenic mice. In addition to this general decrease in the prevalence of SCC in K5-VILIP-1 mice, we observed a remarkable change in the distribution of SCCs of different histopathological grades at the final time-point of the experiments. In both carcinogenesis protocols the transgenic mice had a predominance of low grade SCCs over high grade SCCs when compared with the respective WT treated mice. This was clearly represented by the increase in SCC I, i.e., very well differentiated SCCs, that constituted 74% of all SCCs in K5-VILIP-1 mice versus only 49% of SCCs in WT mice treated with the two stage carcinogenesis protocol. In the complete carcinogenesis experiment, the difference was slightly lower, 69% in K5-VILIP-1 mice versus 49% in WT mice. These changes in the histopathological grades correlate well with our data on differentiation patterns seen in the transgenic epidermis. VILIP-1 overexpression increases epidermal keratinocyte differentiation in TPA treated skin and this tendency may influence the predominance of well differentiated SCCs in transgenic mice. Decreased cell proliferation could also be an important factor because quite frequently well differentiated SCCs not only have more advanced differentiation patterns than high grade SCCs but are also characterized by a relatively slower cell proliferation 
. This has also been seen in the tumors, especially in papillomas from transgenic K5-VILIP-1 animals, which showed a significant decrease in Ki67 labeling index with respect to the papillomas from WT mice. A similar tendency was seen in SCCs. Furthermore, MMP-9 expression in tumors from WT and transgenic animals showed the same pattern of MMP-9 activity seen in normal primary epidermal keratinocytes, i.e., tumors from transgenic origin showed less MMP-9 expression than that seen in tumors from WT mice treated with carcinogens.
Although, we evaluated local and distant metastases at the end of the experiments we did not find statistically significant differences (data not shown). This lack of differential metastatic abilities between SCCs from transgenic and WT mice is probably due to the fact that skin SCCs have relatively low and late metastatic potential 
. Furthermore, this inability to detect metastases is enhanced by the present bioethical guidelines that require culling animals that present with tumors larger than 10 mm diameter. The lack of significant metastases data notwithstanding, tumor grading evaluation indicated that the incidence of high grade SCCs, usually the most aggressive and invasive tumors, were decreased in K5-VILIP-1 transgenic mice. This data can be considered as evidence of decreased tumor progression in mice overexpressing this protein. This is supported by previous experiments demonstrating that overexpression of VILIP-1 in murine SCCs cell lines decreased invasion and migration 
In summary, transgenic VILIP-1 expression targeted to the epidermis results in decreased proliferation patterns that render the skin less susceptible to carcinogenesis. Furthermore, VILIP-1 overexpression attenuates the histotypes produced resulting in a slower conversion to overt malignancy in the two stage carcinogenesis protocol and a decreased prevalence of the most advanced high grade SCCs in both carcinogenesis protocols. These observations point to an inhibitory effect of VILIP-1 on tumor development, malignant conversion and on tumor progression.