Enhanced contact hypersensitivity and antiviral immune responses in vivo by keratinocyte-targeted overexpression of IL-15

IL-15 is involved in lymphocyte homeostasis. To investigate the role of IL-15 in the skin in vivo, mice were generated that overexpress IL-15 in keratinocytes, resulting in increased IL-15 protein levels in the skin but not elevated IL-15 serum concentrations. Keratin 14 (K14)-IL-15 transgenic (tg) mice showed increased contact hypersensitivity (CHS) responses. Transfer of primed wild-type (wt) and tg T cells into naive wt or tg recipients indicated that skin-derived IL-15 enhanced the induction but not the elicitation phase of CHS. Tg mice could be sensitized even by suboptimal hapten concentrations. Accordingly, Langerhans cells (LC) from tg skin were identified as potent allostimulators, suggesting the involvement of IL-15-stimulated LC in the induction of adaptive immunity. Overexpression of IL-15 also strengthened innate immunity since tg mice infected with human HSV type I developed significantly smaller HSV skin lesions. In addition, tg mice resisted re-infection with HSV more effectively than wt mice did, which was associated with an elevated anti-HSV Ab production. Accordingly, injection of serum from re-infected tg mice protected naive recipients significantly from epicutaneous HSV infection, indicating that anti-HSV Ab produced by tg mice play an important role in resistance in vivo. Together, our results show that overexpression of IL-15 in the epidermis enhances both innate and adaptive cutaneous immunity.     

Unexpected reduction of skin tumorigenesis on expression of cyclin-dependent kinase 6 in mouse epidermis

Cyclin-dependent kinases (CDKs) 4 and 6 are important regulators of the G(1) phase of the cell cycle, share 71% amino acid identity, and are expressed ubiquitously. As a result, it was assumed that each of these kinases plays a redundant role regulating normal and neoplastic proliferation. In previous reports, we have described the effects of CDK4 expression in transgenic mice, including the development of epidermal hyperplasia and increased malignant progression to squamous cell carcinoma. To study the role of CDK6 in epithelial growth and tumorigenesis, we generated transgenic mice carrying the CDK6 gene under the keratin 5 promoter (K5CDK6). Similar to K5CDK4 mice, epidermal proliferation increased substantially in K5CDK6 mice; however, no hyperplasia was observed. CDK6 overexpression also triggered keratinocyte apoptosis in interfollicular and follicular epidermis as a compensatory mechanism to override aberrant proliferation. Unexpectedly, CDK6 overexpression results in decreased skin tumor development compared with wild-type siblings. The inhibition in skin tumorigenesis was similar to that previously reported in K5-cyclin D3 mice. Furthermore, biochemical analysis of the K5CDK6 epidermis showed preferential complex formation between CDK6 and cyclin D3, suggesting that this particular complex plays an important role in tumor restraint. These studies provide in vivo evidence that CDK4 and CDK6 play a similar role as a mediator of keratinocyte proliferation but differ in apoptosis activation and skin tumor development.     

The PSORS1 locus gene CCHCR1 affects keratinocyte proliferation in transgenic mice

The CCHCR1 gene (Coiled-Coil alpha-Helical Rod protein 1) within the major psoriasis susceptibility locus PSORS1 is a plausible candidate gene for the risk effect. We have previously generated transgenic mice overexpressing either the psoriasis-associated risk allele CCHCR1*WWCC or the normal allele of CCHCR1. All transgenic CCHCR1 mice appeared phenotypically normal, but exhibited altered expression of genes relevant to the pathogenesis of psoriasis, including upregulation of hyperproliferation markers keratins 6, 16 and 17. Here, we challenged the skin of CCHCR1 transgenic mice with wounding or 12-O-tetradecanoyl-13-acetate (TPA), treatments able to induce epidermal hyperplasia and proliferation that both are hallmarks of psoriasis. These experiments revealed that CCHCR1 regulates keratinocyte proliferation. Early wound healing on days 1 and 4 was delayed, and TPA-induced epidermal hyperproliferation was less pronounced in mice with the CCHCR1*WWCC risk allele than in mice with the normal allele or in wild-type animals. Finally, we demonstrated that overexpression of CCHCR1 affects basal keratinocyte proliferation in mice; CCHCR1*WWCC mice had less proliferating keratinocytes than the non-risk allele mice. Similarly, keratinocytes isolated from risk allele mice proliferated more slowly in culture than wild-type cells when measured by BrdU labeling and ELISA. Our data show that CCHCR1 may function as a negative regulator of keratinocyte proliferation. Thus, aberrant function of CCHCR1 may lead to abnormal keratinocyte proliferation which is a key feature of psoriatic epidermis.     

The adaptor protein FADD protects epidermal keratinocytes from necroptosis in vivo and prevents skin inflammation

Epidermal keratinocytes provide an essential structural and immunological barrier forming the first line of defense against potentially pathogenic microorganisms. Mechanisms regulating barrier integrity and innate immune responses in the epidermis are important for the maintenance of skin immune homeostasis and the pathogenesis of inflammatory skin diseases. Here, we show that epidermal keratinocyte-restricted deficiency of the adaptor protein FADD (FADD(E-KO)) induced severe inflammatory skin lesions in mice. The development of skin inflammation in FADD(E-KO) mice was triggered by RIP kinase 3 (RIP3)-mediated programmed necrosis (termed necroptosis) of FADD-deficient keratinocytes, which was partly dependent on the deubiquitinating enzyme CYLD and tumor necrosis factor (TNF)-TNF receptor 1 signaling. Collectively, our findings provide an in?vivo experimental paradigm that regulation of necroptosis in keratinocytes is important for the maintenance of immune homeostasis and the prevention of chronic inflammation in the skin.     

Activin controls skin morphogenesis and wound repair predominantly via stromal cells and in a concentration-dependent manner via keratinocytes

The transforming growth factor-beta family member activin is a potent regulator of skin morphogenesis and repair. Transgenic mice overexpressing activin in keratinocytes display epidermal hyper-thickening and dermal fibrosis in normal skin and enhanced granulation tissue formation after wounding. Mice overexpressing the secreted activin antagonist follistatin, however, have the opposite wound-healing phenotype. To determine whether activin affects skin morphogenesis and repair via activation of keratinocytes and/or stromal cells, we generated transgenic mice expressing a dominant-negative activin receptor IB mutant (dnActRIB) in keratinocytes. The architecture of adult skin was unaltered in these mice, but delays were observed in postnatal pelage hair follicle morphogenesis and in the first catagen-telogen transformation of hair follicles. Although dnActRIB-transgenic mice showed slightly delayed wound re-epithelialization after skin injury, the strong inhibition of granulation tissue formation seen in follistatin-transgenic mice was not observed. Therefore, although endogenous activin appeared to affect skin morphogenesis and repair predominantly via stromal cells, overexpressed activin strongly affected the epidermis. The epidermal phenotype of activin-overexpressing mice was partially rescued by breeding these animals with dnActRIB-transgenic mice. These results demonstrate that activin affects both stromal cells and keratinocytes in normal and wounded skin and that the effect on keratinocytes is dose-dependent in vivo.     

In situ reduction of oxidative damage, increased cell turnover, and delay of mitochondrial injury by overexpression of manganese superoxide dismutase in a multistage skin carcinogenesis model

To study early subcellular pathologic changes of tumorigenesis in mouse skin and possible modulation by overexpression of the mitochondrial antioxidant enzyme manganese superoxide dismutase (MnSOD), skin keratinocytes from nontransgenic (Ntg) and transgenic (TgH) mice overexpressing MnSOD topically treated with one dose of 7,12-dimethylbenz(a)anthracene (DMBA) and a subsequent dose of 12-O-tetradecanoylphorbol 13-acetate (TPA) were analyzed in situ for levels of MnSOD and the oxidative damage product 4-hydroxy-2-nonenal (4HNE)-modified proteins using specific antibodies and immunogold electron microscopy. At all selected time points analyzed after TPA treatment, there was more MnSOD immunoreactive protein in mitochondria of keratinocytes of TgH mice than Ntg mice. Compared with untreated groups, there was a large increase in 4HNE-modified proteins at 6-24 h after TPA treatment, and this increase was larger in Ntg than TgH mice. Indices of mitosis and apoptosis of keratinocytes were greater in DMBA/TPA-treated TgH than Ntg mouse skin. Mitochondrial injury detected by transmission electron microscopy was delayed in keratinocytes of TgH compared with Ntg mice. The present study demonstrated that overexpression of MnSOD not only protected cells from oxidative damage, but also affected cell turnover kinetics. Thus, previously identified reduction in papilloma formation observed in TgH mice is correlated with mitochondrial events.     

Epidermal expression of the truncated prelamin A causing Hutchinson-Gilford progeria syndrome: effects on keratinocytes, hair and skin

Hutchinson-Gilford progeria syndrome (HGPS) is an accelerated aging disorder caused by point mutation in LMNA encoding A-type nuclear lamins. The mutations in LMNA activate a cryptic splice donor site, resulting in expression of a truncated, prenylated prelamin A called progerin. Expression of progerin leads to alterations in nuclear morphology, which may underlie pathology in HGPS. We generated transgenic mice expressing progerin in epidermis under control of a keratin 14 promoter. The mice had severe abnormalities in morphology of skin keratinocyte nuclei, including nuclear envelope lobulation and decreased nuclear circularity not present in transgenic mice expressing wild-type human lamin A. Primary keratinocytes isolated from these mice had a higher frequency of nuclei with abnormal shape compared to those from transgenic mice expressing wild-type human lamin A. Treatment with a farnesyltransferase inhibitor significantly improved nuclear shape abnormalities and induced the formation of intranuclear foci in the primary keratinocytes expressing progerin. Similarly, spontaneous immortalization of progerin-expressing cultured keratinocytes selected for cells with normal nuclear morphology. Despite morphological alterations in keratinocyte nuclei, mice expressing progerin in epidermis had normal hair grown and wound healing. Hair and skin thickness were normal even after crossing to Lmna null mice to reduce or eliminate expression of normal A-type lamins. Although progerin induces significant alterations in keratinocyte nuclear morphology that are reversed by inhibition of farnesyltransferasae, epidermal expression does not lead to alopecia or other skin abnormalities typically seen in human subjects with HGPS.     

Increased expression of beta6-integrin in skin leads to spontaneous development of chronic wounds

Integrin alphavbeta6 is an epithelial cell-specific receptor that is not normally expressed by resting epithelium but its expression is induced during wound healing. The function of alphavbeta6-integrin in wound repair is not clear. In the present study, we showed that beta6-integrin expression was strongly up-regulated in the epidermis in human chronic wounds but not in different forms of skin fibrosis. To test whether increased beta6-integrin expression plays a role in abnormal wound healing we developed four homozygous transgenic mouse lines that constitutively expressed human beta6-integrin in the epithelium. The mice developed normally and did not show any histological abnormalities in the skin. The rate of experimental skin wound closure was unaltered and the wounds healed without significant scar formation. However, during breeding program 16.1 to 27.0% of transgenic mice developed spontaneous, progressing fibrotic chronic ulcers. None of the wild-type animals developed these lesions. The chronic lesions had areas with severe fibrosis and numerous activated macrophages and fibroblasts expressing transforming growth factor (TGF)-beta. The level of TGF-beta1 was significantly increased in the lesions as compared with normal skin. The findings suggest that increased alphavbeta6-integrin in keratinocytes plays an active part in abnormal wound healing possibly through a mechanism involving increased activation of TGF-beta.     

CDK2 activation in mouse epidermis induces keratinocyte proliferation but does not affect skin tumor development

It has been widely assumed that elevated CDK2 kinase activity plays a contributory role in tumorigenesis. We have previously shown that mice overexpressing CDK4 under control of the keratin 5 promoter (K5CDK4 mice) develop epidermal hyperplasia and increased susceptibility to squamous cell carcinomas. In this model, CDK4 overexpression results in increased CDK2 activity associated with the noncatalytic function of CDK4, sequestration of p21(Cip1) and p27(Kip1). Furthermore, we have shown that ablation of Cdk2 reduces Ras-Cdk4 tumorigenesis, suggesting that increased CDK2 activity plays an important role in Ras-mediated tumorigenesis. To investigate this hypothesis, we generated two transgenic mouse models of elevated CDK2 kinase activity, K5Cdk2 and K5Cdk4(D158N) mice. The D158N mutation blocks CDK4 kinase activity without interfering with its binding capability. CDK2 activation via overexpression of CDK4(D158N), but not of CDK2, resulted in epidermal hyperplasia. We observed elevated levels of p21(Cip1) in K5Cdk2, but not in K5Cdk4(D158N), epidermis, suggesting that CDK2 overexpression elicits a p21(Cip1) response to maintain keratinocyte homeostasis. Surprisingly, we found that neither CDK2 overexpression nor the indirect activation of CDK2 enhanced skin tumor development. Thus, although the indirect activation of CDK2 is sufficient to induce keratinocyte hyperproliferation, activation of CDK2 alone does not induce malignant progression in Ras-mediated tumorigenesis.     

Overexpression of Bcl-2 protects from ultraviolet B-induced apoptosis but promotes hair follicle regression and chemotherapy-induced alopecia

Hair follicle (HF) growth and regression is an exquisitely regulated process of cell proliferation followed by massive cell death and is accompanied by cyclical expression of the apoptosis regulatory gene pair, Bcl-2 and Bax. To further investigate the role of Bcl-2 expression in the control of hair growth and keratinocyte apoptosis, we have used transgenic mice that overexpress human Bcl-2 in basal epidermis and in the outer root sheath under the control of the human keratin-14 promoter (K14/Bcl-2). When irradiated with ultraviolet B (UVB) light, K14/Bcl-2 mice developed about 5-10-fold fewer sunburn cells (ie, apoptotic keratinocytes) in the basal layer of the epidermis, compared to wild-type mice, whereas cultures of primary keratinocytes from transgenic mice were completely resistant to UVB-induced histone formation, at doses that readily induced histone release from wild-type cells. K14/Bcl-2 mice show no alteration of neonatal hair follicle morphogenesis or of the onset of the first wave of HF regression (catagen). However, compared to wild-type controls, K14/Bcl-2 mice subsequently displayed a significant acceleration of spontaneous catagen progression. During chemotherapy-induced alopecia, follicular dystrophy was promoted in K14/Bcl-2 mice. Thus, although K14-driven overexpression of Bcl-2 protected murine epidermal keratinocytes from UVB-induced apoptosis, it surprisingly promoted catagen- and chemotherapy-associated keratinocyte apoptosis.     

CCL27-transgenic mice show enhanced contact hypersensitivity to Th2, but not Th1 stimuli

CCL27 is one of the CC chemokines produced by epidermal keratinocytes and is suggested to be involved in the pathogenesis of inflammatory skin diseases. To clarify the contribution of CCL27 in skin inflammation, we created transgenic C57BL/6 mice that constitutively produce CCL27 in epidermal keratinocytes. These mice had high serum CCL27 levels and did not show any phenotypical change. Thus we stimulated these mice with various reagents by single and repeated application. Interestingly, only contact hypersensitivity to repeated application with fluorescein isothiocyanate was significantly enhanced in transgenic mice compared to non-transgenic mice. Under this condition, the numbers of inflammatory cells, CCR10-positive cells, CCR4-positive cells and cutaneous lymphocyte-associated antigen-positive cells were increased, and IL-4 mRNA expression was higher in the lesional skin of transgenic mice. Increased number of mast cells and higher serum IgE levels, which were similar to atopic dermatitis, were also observed. These results indicated that CCL27 modified inflammation by attracting CCR10-positive and CCR4-positive cells into the lesional skin, and may participate in the pathogenesis of Th2-shifted skin diseases such as atopic dermatitis.     

Differential tumor biology effects of double-initiation in a mouse skin chemical carcinogenesis model comparing wild type versus protein kinase Cepsilon overexpression mice

Our previous studies showed that protein kinase Cepsilon (PKCepsilon) verexpression in mouse skin resulted in metastatic squamous cell carcinoma (SCC) elicited by single 7,12-dimethylbenz(a)anthracene (DMBA)-initiation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promotion in the absence of preceding papilloma formation as is typically observed in wild type mice. The present study demonstrates that double-DMBA initiation modulates tumor incidence, multiplicity, and latency period in both wild type and PKCepsilon overexpression transgenic (PKCepsilon-Tg) mice. After 17 weeks (wks) of tumor promotion, a reduction in papilloma multiplicity was observed in double- versus single-DMBA initiated wild type mice. Papilloma multiplicity was inversely correlated with cell death indices of interfollicular keratinocytes, indicating decreased papilloma formation was caused by increased cell death and suggesting the origin of papillomas is in interfollicular epidermis. Double-initiated PKCepsilon-Tg mice had accelerated carcinoma formation and cancer incidence in comparison to single-initiated PKCepsilon-Tg mice. Morphologic analysis of mouse skin following double initiation and tumor promotion showed a similar if not identical series of events to those previously observed following single initiation and tumor promotion: putative preneoplastic cells were observed arising from hyperplastic hair follicles (HFs) with subsequent cancer cell infiltration into the dermis. Single-initiated PKCepsilon-Tg mice exhibited increased mitosis in epidermal cells of HFs during tumor promotion.     

UV-B radiation induces macrophage migration inhibitory factor-mediated melanogenesis through activation of protease-activated receptor-2 and stem cell factor in keratinocytes

UV radiation indirectly regulates melanogenesis in melanocytes through a paracrine regulatory mechanism involving keratinocytes. Protease-activated receptor (PAR)-2 activation induces melanosome transfer by increasing phagocytosis of melanosomes by keratinocytes. This study demonstrated that macrophage migration inhibitory factor (MIF) stimulated PAR-2 expression in human keratinocytes. In addition, we showed that MIF stimulated stem cell factor (SCF) release in keratinocytes; however, MIF had no effect on the release of endothelin-1 or prostaglandin E2 in keratinocytes. In addition, MIF had no direct effect on melanin and tyrosinase synthesis in cultured human melanocytes. The effect of MIF on melanogenesis was also examined using a three-dimensional reconstituted human epidermal culture model, which is a novel, commercially available, cultured human epidermis containing functional melanocytes. Migration inhibitory factor induced an increase in melanin content in the epidermis after a 9-day culture period. Moreover, melanin synthesis induced by UV-B stimulation was significantly down-regulated by anti-MIF antibody treatment. An in vivo study showed that the back skin of MIF transgenic mice had a higher melanin content than that of wild-type mice after 12 weeks of UV-B exposure. Therefore, MIF-mediated melanogenesis occurs mainly through the activation of PAR-2 and SCF expression in keratinocytes after exposure to UV-B radiation.     

RasGRP1 Transgenic Mice Develop Cutaneous Squamous Cell Carcinomas in Response to Skin Wounding : Potential Role of Granulocyte Colony-Stimulating Factor

Models of epidermal carcinogenesis have demonstrated that Ras is a critical molecule involved in tumor initiation and progression. Previously, we have shown that RasGRP1 increases the susceptibility of mice to skin tumorigenesis when overexpressed in the epidermis by a transgenic approach, related to its ability to activate Ras. Moreover, RasGRP1 transgenic mice develop spontaneous papillomas and cutaneous squamous cell carcinomas, some of which appear to originate in sites of injury, suggesting that RasGRP1 may be responding to signals generated during the wound-healing process. In this study, we examined the response of the RasGRP1 transgenic animals to full-thickness incision wounding of the skin, and demonstrated that they respond by developing tumors along the wounded site. The tumors did not present mutations in the H-ras gene, but Rasgrp1 transgene dosage correlated with tumor susceptibility and size. Analysis of serum cytokines showed increased levels of granulocyte colony-stimulating factor in transgenic animals after wounding. Furthermore, in vitro experiments with primary keratinocytes showed that granulocyte colony-stimulating factor stimulated Ras activation, although RasGRP1 was dispensable for this effect. Since granulocyte colony-stimulating factor has been recently associated with proliferation of skin cancer cells, our results may help in the elucidation of pathways that activate Ras in the epidermis during tumorigenesis in the absence of oncogenic ras mutations.The role of Ras activation in non-melanoma skin cancer is well-documented, both from analysis of human squamous cell carcinomas (SCC)¹ as well as from studies using mouse models of skin carcinogenesis.^2,3 In particular, the multistage carcinogenesis protocol on mouse skin has identified activating mutations in the ras proto-oncogene as the initiation event in skin neoplasms.⁴ Interestingly, whereas Ras mutations are prevalent in mouse models of skin carcinogenesis, they have only been identified in 12% to 46% of sporadic human SCC samples, despite the fact that Ras is activated in the majority of the human SCC.^1,5 This suggests that other mechanisms of Ras activation play a role in the human disease. For example, epidermal growth factor receptor overexpression by amplification is known to occur in human SCC,^6,7 and epidermal growth factor receptor could lead to the biochemical stimulation of Ras. In animal models, overexpression of epidermal growth factor receptor ligands like transforming growth factor-α,⁸ or a dominant form of the Ras exchange factor Sos of seven less (SOS),⁹ act as an initiation event in the epidermis, further demonstrating that alterations in Ras upstream signals could lead to tumorigenesis in the skin via wild-type Ras activation.Biochemical activation of Ras in keratinocytes can be triggered by various extracellular stimuli, but in all cases it requires the participation of exchange factors that catalyze the GDP–GTP exchange. The best studied GDP–GTP exchange factor is SOS1, which is activated in response to receptor tyrosine kinase activation.^10,11 In recent years, we have identified a new GDP–GTP exchange factor in epidermal keratinocytes, RasGRP1, and demonstrated that it can transduce the activation of Ras by the diacylglycerol analog and potent skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA).¹² The initial studies prompted us to investigate the role of RasGRP1 in the tumor promotion effects of phorbol esters in the multistage carcinogenesis protocol using a transgenic mouse model for overexpression of RasGRP1 under the keratin 5 promoter (K5.RasGRP1). Surprisingly, RasGRP1 overexpression did not increase the susceptibility to TPA in animals initiated by the carcinogen 7, 12-Dimethylbenz[a]anthracene (DMBA), but caused more malignant tumors than those observed in the wild-type mice, suggesting a role of RasGRP1 in tumor progression.¹³ This effect appeared strongly related to increases in active Ras, as RasGRP1 overexpression clearly leads to elevated Ras^GTP levels in mouse epidermal keratinocytes, both under basal as well as TPA stimulated conditions.¹³ Interestingly, the K5.RasGRP1 mice also produced tumors in response to TPA alone, implying that RasGRP1 overexpression can act as an initiation event. In addition to the response to chemical carcinogens and tumor promoters, we observed that the RasGRP1 transgenic colony developed spontaneous skin tumors over time, more frequently seen in animals housed in groups or with previous episodes of skin abrasion or injury.¹⁴ This response resembled that of the Tg.Ac mice, which express v-H-ras under the ζ-globin promoter and generate tumors in response to tumor promoting stimuli like wounding and phorbol esters in absence of carcinogenic/initiation events.^15,16The in vivo studies with the K5.RasGRP1 mice have implicated RasGRP1 as a novel link for Ras activation in epidermal keratinocytes and skin cancer. However, the signaling mechanisms contributing to RasGRP1 activation and spontaneous tumor formation, as well as the direct evidence for a role of skin wounding as a tumor promoter stimulus in the context of RasGRP1 overexpression, remained to be established. To address these questions, we have now characterized the tumorigenic response of the K5.RasGRP1 to full-thickness incision wounding of the skin. Our findings show that RasGRP1 overexpression in the epidermis conferred sensitivity to wounding-induced promotional stimuli. Furthermore, upon wounding, there was a significant increase in the levels of granulocyte colony-stimulating factor (G-CSF) in the circulation of transgenic mice compared with the wild-type animals. In vitro, G-CSF was able to stimulate Ras activation in keratinocytes in a very rapid fashion, although RasGRP1 appeared dispensable for this effect. Since G-CSF has been associated to the growth and progression of skin carcinoma cells, the data presented here may have implications for the understanding of RasGRP1-Ras signaling in skin tumor biology.     

Cachexia and graft-vs.-host-disease-type skin changes in keratin promoter-driven TNF alpha transgenic mice.

Tumor necrosis factor alpha (TNF alpha) orchestrates a wide range of effects that combat severe infections in animals. At lower levels, TNF alpha plays an important protective role in stimulating chemotaxis and antimicrobial activity of neutrophils, macrophages, and eosinophils. During chronic illness, TNF alpha secretion can be elevated markedly, giving rise to cachexia, hemorrhage, necrosis and, ultimately, death. Although TNF alpha may mediate many of its effects through macrophages, 30% of TNF alpha injected into animals concentrates in the skin. In recent years, it has been shown that keratinocytes can be induced to synthesize TNF alpha. To explore the role of TNF alpha synthesis in keratinocytes, we used a keratin-14 (K14) promoter to target human TNF alpha expression in the epidermis and other stratified squamous epithelia of transgenic mice. Most mice expressing the K14-TNF alpha transgene stopped gaining weight within 1 week postbirth, and exhibited retarded hair growth. In the skin, adipose production was profoundly inhibited, whereas signs of fibrosis and immune infiltration were evident in the dermis. Over time, the epidermis exhibited an increased stratum corneum, as signs of necrosis began to appear in the skin. Within 3-5 weeks, the mice displayed features characteristic of cachexia and necrosis. Our results suggest that TNF alpha expression by keratinocytes not only plays a role in inflammatory and graft-versus-host-disease-like responses in the skin, but also in other tissues, apparently by virtue of stratified squamous epithelial-derived TNF alpha entering the bloodstream. Our results have enabled the first evaluation of many of the effects of TNF alpha in transgenic animals.     

Short tail with skin lesion phenotype occurs in transgenic mice with keratin-14 promoter-directed expression of mutant CXCR2

CXCR2 plays an important role during cutaneous wound healing. Transgenic mice were generated using the keratin-14 promoter/enhancer to direct expression of wild-type human CXCR2 (K14hCXCR2 WT) or mutant CXCR2, in which the carboxyl-terminal domain (CTD) was truncated at Ser 331 and the dileucine AP-2 binding motif was mutated to alanine (K14hCXCR2 331T/LL/AA/IL/AA). Our results indicate that K14hCXCR2WT transgenic mice exhibited a normal phenotype, while K14hCXCR2 331T/LL/AA/IL/AA transgenic mice were born with tails of normal length, but three to eight days after birth their tails degenerated, leaving only a short tail stub. The tissue degeneration in the tail started between caudal somites with degeneration of bone and connective tissue distal to the constriction, which was replaced with stromal tissue heavily infiltrated with inflammatory cells. The tail lesion site revealed coagulation in enlarged vessels and marked edema that eventually led to loss of the distal tail. Moreover, 66% of the mice exhibited focal skin blemishes and inflammation that exhibited an increase in the number of sebaceous glands and blood vessels, enlargement of the hair follicles due to increased number of keratinocytes, reduction in the connective tissue content, and a thickening of the epidermis. Furthermore, immunohistochemical staining of the epidermis from tail tissue in the transgenic mice indicated a loss of the cell adhesion markers E-cadherin and desmoplakin. These data suggest that keratinocyte expression of a CTD mutant of CXCR2 has effects on homeostasis of the connective tissue in the tail, as well as the maintenance of the epidermis and its appendages.     

Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice.

Reproducible multi-stage progression to invasive squamous carcinoma of the epidermis has been achieved in transgenic mice expressing the HPV16 early-region genes, including the E6/E7 oncogenes, under the control of the human keratin-14 promoter/enhancer. Although 100% of K14-HPV16 transgenic animals develop hyperplastic and/or dysplastic lesions in several inbred backgrounds, including C57BL/6, BALB/c, and SSIN/SENCAR, only mice backcrossed into the FVB/n background progress to malignant squamous cell carcinomas of two pathological grades, well differentiated and moderate/poorly differentiated (WDSC or MPDSC, respectively), each displaying characteristic patterns of malignant behavior. WDSCs typically arise within the epidermis of the ear and invade deeply into the underlying dermis but fail to metastasize, whereas MPDSCs develop on the chest and truncal skin and invariably metastasize to regional lymph nodes. The transition to the malignant state, in 21% of FVB/n transgenic mice, is characterized by alteration of the repertoire of keratin intermediate filament proteins expressed within neoplastic epidermis, such that WDSCs maintain expression of keratins common to terminally differentiating stratified keratinocytes (K10), whereas MPDSCs are distinguished from WDSCs by activation of embryonic and mucosal keratins (K13, K8, and K19). Precursor hyperplastic and dysplastic lesions are characterized by a progressively increased proliferative index, striking morphological alterations in keratinocyte cell-cell and cell-matrix interactions, and extensive remodeling of the underlying dermal stroma. Remarkably, this extensive stromal remodeling, which may facilitate both angiogenesis and eventual tumor cell invasion, develops early at the dysplastic stage in all animals well before malignant conversion.     

Facilitated wound healing by activation of the Transglutaminase 1 gene

Transglutaminase 1 (TGase 1) is a Ca(2+)-dependent enzyme which catalyzes epsilon-(gamma-glutamyl)lysine cross-linking of substrate proteins such as involucrin and loricrin to generate the cornified envelope at the cell periphery of the stratum corneum. We have shown that disruption of the TGase 1 gene in mice results in neonatal lethality, absence of the cornified envelope, and impaired skin barrier function. Based on the importance of TGase 1 in epidermal morphogenesis, we have now assessed its role in wound healing. In neonatal mouse skin, TGase 1 mRNA as well as keratin 6alpha was induced in the epidermis at the wound edges as early as 2 hours after injury and that expression continued in the migrating epidermis until completion of re-epithelialization. The TGase 1 enzyme co-localized on the plasma membrane of migrating keratinocytes with involucrin, but not with loricrin, which suggests the premature assembly of the cornified envelope. Similar injuries to TGase 1 knockout mouse skins grafted on athymic nude mice showed substantial delays in wound healing concomitant with sustained K6alpha mRNA induction. From these results, we suggest that activation of the TGase 1gene is essential for facilitated repair of skin injury.