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Many peptide growth factors, including EGFR ligands, accelerate wound reepithelialization in vivo and in vitro . Furthermore, EGFR expression is transiently increased at wound margins, suggesting an active role for this receptor in wound repair. During reepithelialization of cutaneous wounds, keratinocytes display a phenotypic plasticity resembling aspects of epithelial–mesenchymal transformation. The transcription factor Slug/Snai2 is a regulator of epithelial–mesenchymal transformation during development, and we previously reported that Slug expression is elevated in keratinocytes bordering cutaneous wounds in vivo , ex vivo , and in vitro . In this study we provide evidence that Slug expression is necessary for an EGFR-stimulated reepithelialization response. Epidermal growth factor (EGF) induces Slug expression and the response to EGFR activation is more robust than to other receptor tyrosine kinase ligands. EGFR-stimulated reepithelialization is highly dependent on Slug, as demonstrated by the absence of EGF-stimulated outgrowth in explants derived from Slug null mice. In vitro reepithelialization stimulated by ectopic Slug expression was not impaired by an inhibitor of EGFR catalytic activity, suggesting that Slug is a downstream mediator of this EGFR-stimulated response. Our findings provide evidence that Slug is an essential component of the pathway leading to EGFR-mediated epithelial outgrowth.

Prostaglandin E2 (PGE2) upregulation in response to UV light exposure is a significant factor in the development of non-melanoma skin cancer. It is known that PGE2 signals via the E prostanoid receptors, EP1-4, but the role that each receptor plays in skin carcinogenesis is unclear. Immunohistochemical analysis of EP receptor staining in unirradiated and UVB-exposed SKH-1 mouse skin demonstrated the localization of EP1 and EP2 to the plasma membrane of differentiated epidermal keratinocytes. In contrast, the EP3 receptor localized to the basal layer of the epidermis in unirradiated skin and throughout the epidermis in UVB-exposed skin. In unirradiated skin, cytoplasmic EP4 staining was seen throughout the epidermis, in dermal leukocytes, and in vascular endothelium. However, UVB exposure resulted in relocalization of the EP4 receptor to the plasma membrane of keratinocytes, with no change in the dermal staining pattern. In tumors isolated from UVB-exposed mice, EP1 and EP2 staining was detected in the more differentiated cells surrounding keratin pearls, whereas EP3 and EP4 were detectable throughout the tumors. Differential expression of the EP receptors suggests that each receptor may play a distinct role in skin tumor development.

To assess the premise that genetically determined differences in susceptibility to UV-induced immunosuppression are reflected in UV carcinogenesis, we investigated UV skin cancer induction in two strains of reciprocal F1 hybrid mice CB6F1 males with high susceptibility to UV immunosuppression and a BALB/c X-chromosome and B6CF1 males with low susceptibility to UV immunosuppression and a C57BL/6 X-chromosome. Four experimental groups comprising both strains treated three times weekly with two UV regimens (daily doses incremented from 2.25 to 6 or 4.5 to 12 kJ per m2) were monitored for skin tumor development. Survival without a skin tumor differed over the four groups (p< 0.0001) and differed according to UV regimen within each strain (p

Mounting evidence suggests that macrophage migration inhibitory factor (MIF) may serve as an important link between chronic inflammation and cancer development. The proinflammatory and proangiogenic activities of MIF position it as a potentially important player in the development and progression of nonmelanoma skin cancer (NMSC). To assess the role of MIF in the development and progression of NMSC, we exposed MIF(-/-) BALB/c mice to acute and chronic ultraviolet B (UVB) irradiation. Our studies demonstrate that MIF(-/-) BALB/c mice have a significantly diminished acute inflammatory response to UVB exposure compared to wild-type mice, as measured by myeloperoxidase activity, dermal neutrophil infiltration, and edematous response. Relative to wild-type mice, MIF(-/-) mice also show significantly lower vascular endothelial growth factor (VEGF) concentrations in whole skin and significantly lower 8-oxo-dG adduct concentrations in epidermal DNA following UVB exposure. Furthermore, MIF(-/-) mice showed significant increases in p53 activity, epidermal thickness, and epidermal cell proliferation following acute UVB insult. In response to chronic UVB exposure, MIF(-/-) mice showed a 45% reduction in tumor incidence, significantly less angiogenesis, and delayed tumor progression when compared to their wild-type counterparts. These data indicate that MIF plays an important role in UVB-induced NMSC development and progression.

The HMGA1 proteins have a critical role in the process of carcinogenesis. They are overexpressed in most human malignant neoplasias, and the inhibition of their expression has been shown to prevent cell transformation and results in malignant cell death. To determine whether HMGA1 proteins are also required for in vivo carcinogenesis, we compared the tumour susceptibility of mice wild-type or knockout for the Hmga1 -null allele using a two-stage chemical skin carcinogenesis protocol. Hmga1 −/− mice exhibited a decreased number and a delayed onset of skin papillomas in comparison with wild-type mice. Moreover, the progression of skin papillomas to carcinomas was observed in only 5% of Hmga1 −/− compared to 18% of wild-type mice. These results suggest a lower susceptibility of Hmga1 −/− mice to skin carcinogenesis induced by chemical agents.

Snai2, encoded by the SNAI2 gene, has been shown to modulate epithelial-mesenchymal transformation (EMT), the conversion of sessile epithelial cells attached to adjacent cells and to the basement membrane into dissociated and motile fibroblastic cells. EMT occurs during development, wound healing, and carcinoma progression. Using Snai2-null mice (Snai2(lacZ)), we evaluated the role of Snai2 in UV radiation (UVR)-induced skin carcinogenesis. In chronically UVR-exposed nontumor-bearing skin from Snai2-null mice, inflammation and epidermal proliferation were decreased compared with wild-type (+/+) skin. Snai2-null mice had a consistently lower tumor burden than +/+ mice. In addition, null mice developed fewer aggressive spindle cell tumors, believed to arise from squamous cell carcinomas that have undergone EMT, than +/+ mice; however, the difference in tumor type distribution between the two genotypes was not statistically significant. No metastases were observed in either the +/+ or Snai2-null mice. Using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemistry, we showed that the spindle cell tumors in the Snai2-null mice demonstrated impaired EMT, as shown by decreased vimentin and increased cadherin 1 expression. This study confirms a role for Snai2 in EMT, but demonstrates that Snai2 expression is not required for the development or progression of UVR-induced skin tumors.

Slug (Snail2) is a member of the Snail family of zinc-finger transcription factors with regulatory functions in development, tissue morphogenesis, and tumor progression. Little is known about Slug in normal adult tissue; however, a role for Slug in the skin was suggested by our previous observations of Slug expression in normal murine keratinocytes and Slug induction at wound margins. To study the impact of Slug in the skin, we compared patterns of gene expression in epidermis from Slug-null and wild-type mice. A total of 139 genes had significantly increased, and 109 genes had significantly decreased expression in Slug knockout epidermis. Altered expression of selected genes in Slug knockout epidermis was validated by real-time PCR and immunohistochemistry. Previously reported Slug targets were identified, in addition to novel genes, including cytokeratins, adhesion molecules, and extracellular matrix components. Functional classification of altered gene expression was consistent with a role for Slug in keratinocyte development and differentiation, proliferation, apoptosis, adhesion, motility, as well as angiogenesis and response to environmental stimuli. These results highlight the utility of genetic models to study the in vivo impact of regulatory factors in unperturbed skin and suggest that Slug has significant activities in the adult epidermis.

Chronic exposure to UV light, the primary cause of skin cancer, results in the induction of high levels of cyclooxygenase-2 (COX-2) expression in the skin. The involvement of COX-2 in the carcinogenesis process is mediated by its enzymatic product, prostaglandin E(2) (PGE(2)). PGE(2) has been shown to have a variety of activities that can contribute to tumor development and growth. The effects of PGE(2) on different cell types are mediated by four E prostanoid (EP) receptors, EP(1)-EP(4). While recent studies have demonstrated the importance of EP(1) in the development of colon and breast cancer, the extent of EP(1) involvement in the cutaneous photocarcinogenesis process is unknown. This study found that topical treatment with celecoxib or the specific EP(1) antagonist ONO-8713 decreased acute UVB-induced inflammation in the skin and significantly reduced the number of tumors per mouse following 25 weeks of UVB exposure and topical treatment. This study suggests that drugs designed to block EP(1) may have the potential to be used as anti-inflammatory and/or chemopreventive agents that reduce the risk of skin cancer development.

The receptor for advanced glycation end-products (RAGE) increases in the human cataract and should correlate with increased DNA damage and proliferation of lens epithelial cells (LECs). The purpose of this study was to measure and immunolocalize RAGE in normal and cataractous canine LECs, and to determine whether there was a correlation between RAGE and DNA damage (gadd45), cell-cycle regulation (p21), and LEC proliferation (proliferating cell nuclear antigen, PCNA).Thirty-two anterior lens capsules from 22 dogs that underwent cataract surgery and 10 lenses from dogs with normal eyes were evaluated. Eleven of the cataractous lenses were from diabetic patients (n=16), and eleven were from patients with inherited cataracts (n=16). Standard immunohistochemical staining was performed using antibodies against RAGE, gadd45, p21, PCNA, alpha-smooth muscle actin, and TGF-beta. Immunostaining intensity for each antibody was given a score of 0-4+. Standard Western blot analysis on normal and cataractous lens capsules was performed using the same antibodies as in the immunohistochemical staining. Comparisons were also made based on age and sex. Real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed for RAGE.There was an increase in RAGE expression with age in normal LECs, but no significant difference was seen when normal adult LECs were compared to cataractous LECs. The stage of the cataract and the presence of LIU were not associated with a significant increase in RAGE expression. There was no age-dependent difference in the normal lenses for gadd45, p21, or PCNA. Significant up-regulation of p21 (P0.05) and PCNA (P0.05) was seen in diabetic cataracts compared to inherited cataracts.RAGE and PCNA expression did not increase with cataractogenesis, possibly due to overexpression associated with normal aging and constant exposure to oxidative stress from sunlight-related ultraviolet irradiation, respectively. However, p21 and PCNA increased in diabetic cataractogenesis suggesting cell cycle and proliferation dysregulation. This may be related to the rapid onset in this type of cataract compared with the more chronic and slower-to-develop inherited cataracts.

Successful cutaneous wound repair occurs in a series of tightly coordinated and overlapping steps: (1) inflammation and clot formation, (2) keratinocyte activation and migration, (3) remodeling of the basement membrane and extracellular matrix, and (4) dermal and epidermal maturation. During the final three stages of cutaneous wound healing, restoration of an intact epidermis occurs via a complex process termed reepithelialization. In this chapter, we focus on the process of wound reepithelialization, emphasizing the resemblance of reepithelialization to epithelial-mesenchymal transition (EMT) occurring during development and tumor progression. Based on the many morphologic and molecular similarities between the two processes, we propose that wound reepithelialization represents a partial and reversible EMT.

Solid organ transplant recipients have a 60-250-fold increased likelihood of developing sunlight-induced squamous cell carcinoma (SCC) compared with the general population. This increased risk is linked to the immunosuppressive drugs taken by these patients to modulate T cell function, thus preventing organ rejection. To determine the importance of T cells in the development of cutaneous SCC, we examined the effects of selectively depleting Skh-1 mice of systemic CD4+ or CD8+ T cells, using monoclonal antibodies, on ultraviolet B (UVB) radiation-induced inflammation and tumor development. Decreases in systemic CD4+ but not CD8+ T cells significantly increased and prolonged the acute UVB-induced cutaneous inflammatory response, as measured by neutrophil influx, myeloperoxidase activity, and prostaglandin E2 levels. Significantly more p53+ keratinocytes were observed in UVB-exposed CD4-depleted than in CD4-replete mice, and this difference was abrogated in mice depleted of neutrophils before UVB exposure. Increased acute inflammation was associated with significantly increased tumor numbers in CD4-depleted mice chronically exposed to UVB. Furthermore, topical treatment with the anti-inflammatory drug celecoxib significantly decreased tumor numbers in both CD4-replete and CD4-depleted mice. Our findings suggest that CD4+ T cells play an important role in modulating both the acute inflammatory and the chronic carcinogenic response of the skin to UVB.