Kimberly M. Newkirk

Cutaneous wound reepithelialization is compromised in mice lacking functional Slug (Snai2)

BACKGROUND Keratinocytes at wound margins undergo partial epithelial to mesenchymal transition (EMT). Based on previous in vitro and ex vivo findings, Slug (Snai2), a transcriptional regulator of EMT in development, may play an important role in this process. OBJECTIVES This study was designed to validate an in vivo role for Slug in wound healing. METHODS Excisional wounds in Slug null and wild type mice were examined histologically at 6, 24, 48, and 72h after wounding; reepithelialization was measured and immunohistochemistry for keratins 8, 10, 14, and 6 and E-cadherin was performed. In 20 Slug null and 20 wild type mice exposed three times weekly to two minimal erythemal doses of UVR, the development of non-healing cutaneous ulcers was documented. Ulcers were examined histologically and by immunohistochemistry. RESULTS The reepithelialization component of excisional wound healing was reduced 1.7-fold and expression of the Slug target genes keratin 8 and E-cadherin was increased at wound margins in Slug null compared to wild type mice. In contrast, no differences in expression of keratins 10 or 14 or in markers of proliferation K6 and Ki-67 were observed. Forty per cent of Slug null mice but no wild type mice developed non-healing cutaneous ulcers in response to chronic UVR. Keratinocytes at ulcer margins expressed high levels of keratin 8 and retained E-cadherin expression, thus resembling excisional wounds. CONCLUSION Slug is an important modulator of successful wound repair in adult tissue and may be critical for maintaining epidermal integrity in response to chronic injury.

Slug/Snai2 Is a Downstream Mediator of Epidermal Growth Factor Receptor-Stimulated Reepithelialization

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.

Ultraviolet Radiation Stimulates Expression of Snail Family Transcription Factors in Keratinocytes

The related zinc finger transcription factors Slug and Snail modulate epithelial mesenchymal transformation (EMT), the conversion of sessile epithelial cells into migratory fibroblast‐like cells. EMT occurs during development, wound healing, and tumor progression. Growth factors, acting through mitogen‐activated protein kinase (MAPK) cascades, regulate expression of Slug and Snail. Expression of Snail family transcription factors appears to be elevated in UVR‐induced murine squamous cell carcinomas (SCC). We report here that ultraviolet radiation (UVR), which activates MAPK cascades, also stimulates Snail and Slug expression in epidermal keratinocytes. UVR exposure transiently elevated Slug and Snail mRNA expression in human keratinocytes in vitro and mouse epidermis in vivo. This induction was mediated, at least in part, through the ERK and p38 MAPK cascades, as pharmacological inhibition of these cascades partially or completely blocked Slug and Snail induction by UVR. On the other hand, UVR induction of Slug and Snail was enhanced by inhibition of JNK. Slug appears to play a functional role in the acute response of keratinocytes to UVR, as UVR induction of keratin 6 in the epidermis of Slug knockout mice was markedly delayed compared to wild‐type mice. Slug and Snail are known to regulate molecules important in the cytoskeleton, intercellular adhesion, cell motility, and apoptosis, thus it seems probable that transiently or persistently elevated expression of these factors fosters the progression of UVR‐induced SCC.

Ultraviolet Radiation-Induced Corneal Degeneration in 129 Mice

Ultraviolet radiation (UVR) is a risk factor for the development of ocular disease in humans, including acute photokeratitis, chronic corneal spheroidal degeneration, and cataract formation. This report describes the ocular lesions seen in 21 mice chronically exposed to UVR as part of a skin carcinogenicity study. All globes were affected to varying degrees. The primary lesion, not previously reported in UVR-exposed mice, was marked loss of keratocytes relative to age-matched controls. Secondary lesions included corneal stromal thinning, keratoconus, corneal vascularization and fibrosis, keratitis, globe rupture, and phthisis bulbi. In addition, more than 90% of UVR-exposed and unexposed lenses had evidence of cataract formation; this is the first report of the occurrence of spontaneous cataracts in 129 mice. In a subsequent study, apoptotic cells were identified histologically and by cleaved caspase 3 immunoreactivity in the corneal epithelium and, less commonly, in the corneal stroma after acute UVR exposure. Based on this finding, we propose that the loss of keratocytes observed in the chronic study was due to UVR-induced apoptosis.

Detection of human papillomavirus DNA in feline premalignant and invasive squamous cell carcinoma

Squamous cell carcinoma (SCC) is the most common malignant cutaneous and oral neoplasm of cats. Papillomavirus (PV) DNA has been identified in a proportion of feline Bowenoid in situ carcinomas (BISCs), cutaneous SCCs and a single oral SCC, but its exact role in the pathogenesis remains unknown. In humans, it has been suggested that ultraviolet (UV) light and human PV (HPV) may act as cofactors in cutaneous SCC carcinogenesis. Little is known about the influence of UV light on PV prevalence in feline cutaneous lesions, including actinic keratosis (AK). Additionally, PV prevalence in noncutaneous feline lesions, including oral SCC, is largely not known. This study aimed to determine the presence of PV in 84 cats with premalignant and invasive SCC from cutaneous and noncutaneous sites using polymerase chain reaction and to investigate an association with UV light. Papillomaviral DNA was amplified from two of 12 cases of AK, seven of 22 BISCs, nine of 39 cutaneous SCCs and two of 35 non-cutaneous SCCs. Of the PV DNA sequenced, 50% was most similar to HPV of the genus Betapapillomavirus, while the other 50% was most similar to Felis domesticus PV type 2. Exposure to UV was not associated with an increase in PV for cutaneous SCC. The results of this study suggest that in the cat, HPV DNA may be detectible within a higher percentage of squamous lesions than previously demonstrated, UV exposure may not be a confounder for PV presence, and noncutaneous lesions may have a low prevalence of PV.

Immunohistochemical analysis of ocular hemangiomas and hemangiosarcomas in dogs

PURPOSE To determine if molecular markers typically associated with ultraviolet exposure could be detected in canine ocular hemangiomas (HA) and hemangiosarcomas (HSA). METHODS Paraffin-embedded samples of canine ocular HA (n = 6) and HSA (n = 6) were examined for the presence of p53, p21, p16, cyclin D, PCNA, pAkt, telomerase, and estrogen receptor (ER)-alpha using immunohistochemistry. RESULTS p53 and cyclin D protein were not detected in any of the canine HA or HSA samples. The majority of the HA and HSA were negative for both p21 and telomerase. pAkt immunoreactivity was absent in one HA, one HSA, but was present in five HA and five HSA. All of the HA or HSA samples were strongly positive for p16 and PCNA. ERalpha was expressed in all of the samples examined; there was more intense staining in the HSA samples compared to the HA samples. CONCLUSIONS Results from this study describe the protein expression, via immunohistochemistry, that might be altered in UV exposure in HA and HAS formation. p53 may not play an important role in tumor development; rather, in the tumors examined, expression of cell cycle regulators independent of the p53 pathway appear central in HA and HSA formation and progression. In addition, this study finds that ERalpha may be involved in promoting the invasive behavior associated with HSA.

The acute cutaneous inflammatory response is attenuated in Slug-knockout mice

We previously reported ultraviolet radiation (UVR) induction of Slug, a Snail family zinc-finger transcription factor, in the epidermis of mice; we now report that Slug-knockout mice are, unexpectedly, more resistant to sunburn than wild-type mice. There was a marked difference between the cutaneous inflammatory response in the skin of Slug-knockout and wild-type mice from 12 h to 1 week following a single exposure to 3 minimal erythemal doses of UVR. Slug-knockout mice showed a much reduced immediate increase in skin thickness and neutrophil infiltration compared to wild-type mice. However, there were as many or more intraepidermal T cells, dermal mast cells, and dermal blood vessels in the UVR-exposed skin of Slug-knockout mice as in the skin of wild-type mice. Differences in cytokine and chemokine expression following UVR appeared to account for at least some differences between the genotypes in cutaneous inflammatory response. Despite the reported antiapoptotic and antiproliferative role for Slug in some cell types, we observed little difference between the genotypes in UVR-induced keratinocyte apoptosis or proliferation. Our findings indicate an unexpected but important role for Slug in the acute cutaneous inflammatory response to UVR.

Slug (Snai2) Expression during Skin and Hair Follicle Development

TO THE EDITOR Our previous investigations showed that Slug (Snai2), a member of the Snail family of developmental transcription factors, is expressed in unperturbed adult murine epidermis, where it regulates a wide variety of gene targets (Newkirk et al., 2008b). Slug expression is induced by a number of growth factors and environmental stimuli (Hudson et al., 2007; Kusewitt et al., 2009). Slug enhances cutaneous wound reepithelialization, skin tumor progression, and the sunburn response (Savagner et al., 2005; Newkirk et al., 2007, 2008a; Hudson et al., 2009). The present studies document Slug expression in embryonic and neonatal epidermis and hair follicles, and significant alterations in hair growth kinetics in Slug knockout mice during the first postnatal hair cycle, indicating a further contribution of Slug to the maintenance of skin homeostasis. Microwave antigen retrieval was performed in citrate buffer on deparaffinized E10–E17 CD1 mouse embryo sections (Zyagen, San Diego, CA). Slides were incubated overnight at 4 1C in monoclonal rabbit anti-Slug antibody (#9585, Cell Signaling, Danvers, MA) diluted 1:50, treated with Biocare Rabbit on Rodent HRP-Polymer (Biocare Medical, Concord, CA) and 3,30-diaminobenzidine chromagen, then counterstained with hematoxylin. Of the many anti-Slug antibodies we have tested, only this antibody consistently gives single bands of appropriate size on protein isolated from HaCaT human epidermal cells (data not shown). Keratin 14 immunostaining was performed similarly, applying first the primary antibody (Covance, Princeton, NJ) at a dilution of 1:500 for 30 minutes at room temperature, and then Envision Plus labeled polymer anti-rabbit-HRP (DAKO, Carpinteria, CA) for 30 minutes. In embryonic tissues, Slug staining was exclusively nuclear. On E10 and E11, Slug was expressed in scattered cells of the single-layered epidermis and in most of the underlying primitive mesenchymal cells, a time when keratin 14 immunoreactivity was seen in only a few cells (Figure 1). At the beginning of periderm formation at E12, when strong keratin 14 immunoreactivity was first observed, Slug was expressed in essentially all skin epithelial cells and in the underlying mesenchyme. As the epithelium underwent stratification, Slug and keratin 14 expression was progressively confined to basal keratinocytes, with Slug localization occurring earlier. Slug staining was observed in progressively fewer dermal cells as primitive mesenchymal cells matured. However, a substantial number of dermal cells continued to express Slug. In the placode, hair germ, and peg stages of hair follicle development, Slug expression was prominent in the thickened and invaginating epithelium, but was absent from the underlying mesenchymal cells that ultimately form the dermal papillae (Figure 1). In pigmented Slug-lacZ 129 mice, the Slug locus was inactivated by an inframe insertion of the b-galactosidase gene into the zinc finger coding region of the Slug gene (Jiang et al., 1998). Mice homozygous for the Slug-lacZ allele are functional Slug knockout animals, but heterozygous Slug knockout mice are phenotypically normal. Daily examination of newborn mice (Figure 2a) revealed that darkening of the skin occurred in all wild-type and heterozygous Slug knockout mice by postnatal days 2–3; however, darkening of the skin of Slug knockout neonates was not seen until postnatal days 4–7. In wild-type and heterozygous knockout mice, hair emergence occurred on postnatal days 5–7, but in knockout mice the emergence was delayed until days 8–10. Differences between knockout and wild-type/heterozygous mice were highly significant for both skin darkening and hair emergence (Po10 7 using the log-rank statistic). Skin darkening in pigmented neonatal mice occurs during early hair follicle growth and hair shafts emerge from follicles during mid to late follicle maturation (Muller-Rover et al., 2001). Thus, our findings suggest delayed hair follicle development in neonatal Slug knockout mice. Immunohistochemical analysis for Slug in 129 wild-type mice showed that Slug was expressed in many, but not all, basal keratinocytes at birth. As previously reported for adult epidermis (Parent et al., 2004), Slug-expressing keratinocytes were clustered around hair follicles. In developing follicles, Slug was stably expressed in the developing external root sheath, hair matrix cells, and some mesenchymal cells of the dermal papilla (Figure 2b). Most interfollicular epidermal cells were Slug-positive at 3 days after birth, but staining progressively declined. At 18 days after birth, follicles in catagen expressed little or no Slug, and Slug expression was also absent from the interfollicular epidermis. Early telogen follicles and interfollicular epidermis showed no Slug immunoreactivity. Localization of Slug was confirmed by immunohistochemical analysis for

Thyroid Neoplasia in Captive Raccoons (Procyon lotor)

Abstract Two adult, spayed, female raccoons were diagnosed with thyroid neoplasia. One raccoon had a palpable, left-sided, nonfunctional thyroid adenocarcinoma which was treated with a thyroidectomy twice with local recurrence both times. After the second recurrence, pulmonary metastases were identified. A third thyroidectomy was performed, and a vascular access port was placed for administration of intravenous doxorubicin. The raccoon developed pancytopenia and became anorexic after chemotherapy, and the owner elected humane euthanasia. The second raccoon had nonpalpable, bilateral, functional follicular thyroid adenomatous hyperplasia and was treated with a right thyroidectomy and a partial left thyroidectomy, leaving behind the grossly normal portion of the left thyroid. However, the animal was still hyperthyroid after surgery and was then successfully managed with topical methimazole gel. Thyroid pathology has been documented in raccoons in Europe, but is not reported in the United States. Thyroid neoplasia in raccoons can occur as a nonfunctional adenocarcinoma, as is commonly reported in dogs, or as a functional adenoma, as is commonly reported in cats. Raccoons with adenocarcinomas should be evaluated for pulmonary metastasis. Methimazole gel may be a viable treatment option for raccoons with hyperthyroidism.

Conjunctival xanthoma in a blue and gold macaw (Ara ararauna).

A 17-year-old female blue and gold macaw (Ara ararauna) presented for evaluation of a discreet, conjunctival mass of the OD. No other abnormalities were found on ophthalmic or physical examination. A heterophilic leukocytosis was present on the complete blood count, and elevated aspartate aminotransferase activity, creatinine kinase activity and cholesterol were present on the plasma biochemistry panel. Surgical removal of the mass was complete and no recurrence has occurred by six months after excision. Reduction of dietary fats was recommended to reduce serum cholesterol levels and reduce the likelihood of future occurrence of xanthomas in this bird.