Knockout Skin Research Articles

Immunomodulatory Effects of Histone Variant H2A.J in Ionizing Radiation Dermatitis

Histone variant H2A.J is associated with premature senescence after ionizing radiation (IR) and modulates senescence-associated secretory phenotype (SASP). Using constitutive H2A.J knock-out mice, the role of H2A.J was investigated in radiation dermatitis. H2A.J wild-type (WT) and knock-out (KO) mice were exposed to moderate or high IR doses (≤20 Gy, skinfold IR). Radiation-induced skin reactions were investigated up to 2 weeks post-IR at macroscopic and microscopic levels. H2A.J and other senescence markers, as well as DNA damage and proliferation markers, were studied by immunohistochemistry, immunofluorescence, and electron microscopy. After high-dose IR, protein-coding transcriptomes were analyzed by RNA sequencing, immune cell infiltration by flow cytometry, and gene expression by reverse transcription polymerase chain reaction in (non-) irradiated WT versus KO skin. In WT skin, epidermal keratinocytes showed time- and dose-dependent H2A.J accumulation after IR exposure. Unexpectedly, stronger inflammatory reactions with increased epidermal thickness and progressive hair follicle loss were observed in irradiated KO versus WT skin. Clearly more radiation-induced senescence was observed in keratinocyte populations of KO skin after moderate and high doses, with hair follicle stem cells being particularly badly damaged, leading to follicle atrophy. After high-dose IR, transcriptomic analysis revealed enhanced senescence-associated signatures in irradiated KO skin, with intensified release of SASP factors. Flow cytometric analysis indicated increased immune cell infiltration in both WT and KO skin; however, specific chemokine-mediated signaling in irradiated KO skin led to more neutrophil recruitment, thereby aggravating radiation toxicities. Increased skin damage in irradiated KO skin led to hyperproliferation, abnormal differentiation, and cornification of keratinocytes, accompanied by increased upregulation of transcription-factor JunB. Lack of radiation-induced H2A.J expression in keratinocytes is associated with increased senescence induction, modulation of SASP expression, and exacerbated inflammatory skin reactions. Hence, epigenetic H2A.J-mediated gene expression in response to IR regulates keratinocyte immune functions and plays an essential role in balancing the inflammatory response during radiation dermatitis.

Epithelial-Macrophage Crosstalk Initiates Sterile Inflammation in Embryonic Skin.

Macrophages are highly responsive to the environmental cues and are the primary responders to tissue stress and damage. While much is known about the role of macrophages during inflammatory disease progression; the initial series of events that set up the inflammation remains less understood. In this study, we use next generation sequencing (NGS) of embryonic skin macrophages and the niche cells - skin epithelia and stroma in the epidermis specific knockout of integrin beta 1 (Itgβ1) model to uncover specific roles of each cell type and identify how these cell types communicate to initiate the sterile inflammatory response. We demonstrate that while the embryonic skin fibroblasts in the Itgβ1 knockout skin are relatively inactive, the keratinocytes and macrophages are the critical responders to the sterile inflammatory cues. The epidermis expresses damage associated molecular patterns (DAMPs), stress response genes, pro-inflammatory cytokines, and chemokines that aid in eliciting the inflammatory response. The macrophages, in-turn, respond by acquiring enhanced M2-like characteristics expressing ECM remodeling and matrisome signatures that exacerbate the basement membrane disruption. Depletion of macrophages by blocking the CSF1 receptor (CSF1R) results in improved basement membrane integrity and reduced ECM remodeling activity in the KO skin. Further, blocking the skin inflammation with celecoxib reveals that the acquired fate of macrophages in the KO skin is dependent on its interaction with the epidermal compartment through COX2 dependent cytokine production. Taken together, our study highlights a critical crosstalk between the epithelia and the dermal macrophages that shapes macrophage fate and initiates sterile inflammation in the skin. The insights gained from our study can be extrapolated to other inflammatory disorders to understand the early events that set up the disease.

Serration pattern analysis as a practical adjunct tool for categorization of subepidermal autoimmune blistering diseases.

Serration pattern analysis helps in the classification of subepidermal autoimmune blistering disorders; more precisely, it helps to differentiate epidermolysis bullosa acquisita from other subepidermal autoimmune blistering disorders. Most of the published reports of this tool have come from a single center. The objectives of the study were to study the utility of serration pattern analysis in classifying subepidermal autoimmune blistering disorders. Seventy five cases of subepidermal autoimmune blistering disorders were enrolled in this prospective study. A three millimeter punch biopsy was taken from the perilesional skin or mucosa for direct immunofluorescence; indirect immunofluorescence was carried out using salt-split skin. Subclassification of subepidermal autoimmune blistering disorders was done based on direct immunofluorescence, indirect immunofluorescence on salt-split skin, indirect immunofluorescence using knockout skin and serration pattern analysis findings. Indirect immunofluorescence was positive in 68 cases; 14 cases showed a dermal staining pattern while the rest showed either an epidermal or a combined pattern. All patients with epidermal or combined staining patterns showed "n" serrated pattern on direct immunofluorescence. Nine patients with dermal staining on indirect immunofluorescence also revealed an "n" serration pattern on direct immunofluorescence indicating the diagnosis of anti-p200 pemphigoid, and the rest showed a "u" serrated pattern. Three patients with negative indirect immunofluorescence showed "u" serration on direct immunofluorescence while the rest showed "n" serration. ELISA and immunoblotting could not be performed due to resource constraints. Based on indirect immunofluorescence and serration pattern analysis, classification of the majority of patients with subepidermal autoimmune blistering disorders was possible in our study. Pattern recognition is a cost-effective tool and can be easily learnt. It is recommended to be practiced in all laboratories where facilities for advanced immunological diagnosis are unavailable.

Sequestosome 1/p62 enhances chronic skin inflammation

The molecular control of inflammation and epidermal thickening in skin lesions of patients with atopic dermatitis (AD) is not known. Sequestosome 1/p62 is a multifunctional adapter protein implicated in the control of key regulators of cellular homeostasis, such as proinflammatory and mechanistic target of rapamycin signaling. We sought to determine whether p62 plays a role in the cutaneous and systemic manifestations of an AD-like mouse model. AD-like skin lesions were induced by deletion of JunB/AP-1, specifically in epidermal keratinocytes (JunBΔep). The contribution of p62 to pathological changes was determined by inactivation of p62 in JunBΔepp62-/- double knockout mice. Expression of p62 was elevated in skin lesions of JunBΔep mice, resembling upregulation of p62 in AD and psoriasis. When p62 was inactivated, JunBΔep-associated defects in the differentiation of keratinocytes, epidermal thickening, skin infiltration by mast cells and neutrophils, and the development of macroscopic skin lesions were significantly reduced. p62 inactivation had little effect on circulating cytokines, but decreased serum IgE. Signaling through mechanistic target of rapamycin and natural factor kappa B was increased in JunBΔep but not in JunBΔepp62-/- double knockout skin, indicating an important role of p62 in enhancing these signaling pathways in the skin during AD-like inflammation. Our results provide the first invivo evidence for a proinflammatory role of p62 in skin and suggest that p62-dependent signaling pathways may be promising therapeutic targets to ameliorate the skin manifestations of AD and possibly psoriasis.

225 Desmoglein 1 deficiency in knockout mice impairs epidermal barrier formation and results in a psoriasis-like gene signature in E18.5 embryos

Desmoglein 1 (Dsg1) is a desmosomal cadherin expressed in concert with a commitment to stratify and differentiate in multi-layered tissues. Its importance in epidermal function is underscored by human diseases such as SAM syndrome (Severe Skin Dermatitis, Allergies and Metabolic Wasting), caused by loss of function mutations. Dsg1 promotes differentiation and normal epidermal morphogenesis in human 3D epidermal models. To determine the systemic response to loss of Dsg1, we deleted the three, tandem a, b, and g genes in the mouse cadherin cluster using CRISPR/Cas9. Dsg1 knockout (KO) mice exhibit barrier impairment accompanied by peeling, denuded skin, and postnatal lethality. The basal desmosomal cadherin, desmoglein 3, is upregulated in Dsg1 KO skin, while differentiation associated proteins loricrin and filaggrin are decreased. Disrupted ZO1 localization, increased TEWL, and increased dye penetration are consistent with a defect in barrier formation. Whole transcriptome analysis of E18.5 skin was consistent with the observed alteration in differentiation and barrier formation, but also revealed an increase in pathways linked to psoriatic processes, including IL-17 signaling, MAPK activity, and ErbB signaling. There was significant overlap between the top 100 genes increased in human psoriasis lesions and genes increased in E18.5 Dsg1 KO animals. These data not only support an important role for Dsg1 in epidermal differentiation and barrier formation, they identify a potential role for Dsg1 in regulating inflammatory responses, with loss of Dsg1 in mouse embryo skin activating inflammatory pathways similar to those observed in patients with psoriasis.

Regulation of epidermal differentiation through KDF1-mediated deubiquitination of IKKα.

Progenitor cells at the basal layer of skin epidermis play an essential role in maintaining tissue homeostasis and enhancing wound repair in skin. The proliferation, differentiation, and cell death of epidermal progenitor cells have to be delicately regulated, as deregulation of this process can lead to many skin diseases, including skin cancers. However, the underlying molecular mechanisms involved in skin homeostasis remain poorly defined. In this study, with quantitative proteomics approach, we identified an important interaction between KDF1 (keratinocyte differentiation factor 1) and IKKα (IκB kinase α) in differentiating skin keratinocytes. Ablation of either KDF1 or IKKα in mice leads to similar but striking abnormalities in skin development, particularly in skin epidermal differentiation. With biochemical and mouse genetics approach, we further demonstrate that the interaction of IKKα and KDF1 is essential for epidermal differentiation. To probe deeper into the mechanisms, we find that KDF1 associates with a deubiquitinating protease USP7 (ubiquitin-specific peptidase 7), and KDF1 can regulate skin differentiation through deubiquitination and stabilization of IKKα. Taken together, our study unravels an important molecular mechanism underlying epidermal differentiation and skin tissue homeostasis.

Regulated in Development and DNA Damage Responses 1 Prevents Dermal Adipocyte Differentiation and Is Required for Hair Cycle–Dependent Dermal Adipose Expansion

Dermal white adipose tissue (dWAT) expansion is associated with important homeostatic and pathologic processes in skin. Even though mTOR/protein kinase B signaling is important for adipogenesis, the role of regulated development of DNA damage responses 1 (REDD1), a negative regulator of mTOR/protein kinase B, is poorly understood. Loss of REDD1 in mice resulted in reduction of body mass, total fat, size of gonadal white adipose tissue, and interscapular brown adipose tissue. Inguinal subcutaneous white adipose tissue and dWAT in REDD1 knockouts were expanded compared with wild type mice. Size and number of mature adipocytes in dWAT were also increased in adult REDD1 knockouts. This dWAT phenotype was established around postnatal day 18 and did not depend on the hair growth cycle. Numbers of adipocyte precursor cells were lower in REDD1 knockout skin. Invitro analysis revealed increased differentiation of skin-derived REDD1 knockout adipocyte precursor cells as indicated by higher lipid accumulation and increased adipogenic marker expression. 3T3L1 cells overexpressing REDD1 had decreased sensitivity to differentiation. Overall, our findings indicate that REDD1 silencing induced expansion of dWAT through hypertrophy and hyperplasia. This REDD1-dependent mechanism of adipogenesis could be used to preferentially target skin-associated adipose tissue for therapeutic purposes.

Matrix Metalloproteinase-3 is Key Effector of TNF-α-Induced Collagen Degradation in Skin.

Inflammatory processes in the skin augment collagen degradation due to the up-regulation of matrix metalloproteinases (MMPs). The aim of the present project was to study the specific impact of MMP-3 on collagen loss in skin and its interplay with the collagenase MMP-13 under inflammatory conditions mimicked by the addition of the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Skin explants from MMP-3 knock-out (KO) mice or from transgenic (TG) mice overexpressing MMP-3 in the skin and their respective wild-type counterparts (WT and WTT) were incubated ex vivo for eight days. The rate of collagen degradation, measured by released hydroxyproline, was reduced (p < 0.001) in KO skin explants compared to WT control skin but did not differ (p = 0.47) between TG and WTT skin. Treatment with the MMP inhibitor GM6001 reduced hydroxyproline media levels from WT, WTT and TG but not from KO skin explants. TNF-α increased collagen degradation in the WT group (p = 0.0001) only. More of the active form of MMP-13 was observed in the three MMP-3 expressing groups (co-incubation with receptor-associated protein stabilized MMP-13 subforms and enhanced detection in the media). In summary, the innate level of MMP-3 seems responsible for the accelerated loss of cutaneous collagen under inflammatory conditions, possibly via MMP-13 in mice.

Deletion of the glucocorticoid receptor chaperone FKBP51 prevents glucocorticoid-induced skin atrophy.

FKBP51 (FK506-binding protein 51) is a known co-chaperone and regulator of the glucocorticoid receptor (GR), which usually attenuates its activity. FKBP51 is one of the major GR target genes in skin, but its role in clinical effects of glucocorticoids is not known. Here, we used FKBP51 knockout (KO) mice to determine FKBP51's role in the major adverse effect of topical glucocorticoids, skin atrophy. Unexpectedly, we found that all skin compartments (epidermis, dermis, dermal adipose and CD34+ stem cells) in FKBP51 KO animals were much more resistant to glucocorticoid-induced hypoplasia. Furthermore, despite the absence of inhibitory FKBP51, the basal level of expression and glucocorticoid activation of GR target genes were not increased in FKBP51 KO skin or CRISPR/Cas9-edited FKBP51 KO HaCaT human keratinocytes. FKBP51 is known to negatively regulate Akt and mTOR. We found a significant increase in AktSer473 and mTORSer2448 phosphorylation and downstream pro-growth signaling in FKBP51-deficient keratinocytes in vivo and in vitro. As Akt/mTOR-GR crosstalk is usually negative in skin, our results suggest that Akt/mTOR activation could be responsible for the lack of increased GR function and resistance of FKBP51 KO mice to the steroid-induced skin atrophy.

MiRNAs Are Required for Postinduction Stage Sweat Gland Development

miRNAs Are Required for Postinduction Stage Sweat Gland Development

Lhx2 is a direct NF-κB target gene that promotes primary hair follicle placode down-growth.

In the epidermis of mice lacking transcription factor nuclear factor-kappa B (NF-κB) activity, primary hair follicle (HF) pre-placode formation is initiated without progression to proper placodes. NF-κB modulates WNT and SHH signaling at early stages of HF development, but this does not fully account for the phenotypes observed upon NF-κB inhibition. To identify additional NF-κB target genes, we developed a novel method to isolate and transcriptionally profile primary HF placodes with active NF-κB signaling. In parallel, we compared gene expression at the same developmental stage in NF-κB-deficient embryos and controls. This uncovered novel NF-κB target genes with potential roles in priming HF placodes for down-growth. Importantly, we identify Lhx2 (encoding a LIM/homeobox transcription factor) as a direct NF-κB target gene, loss of which replicates a subset of phenotypes seen in NF-κB-deficient embryos. Lhx2 and Tgfb2 knockout embryos exhibit very similar abnormalities in HF development, including failure of the E-cadherin suppression required for follicle down-growth. We show that TGFβ2 signaling is impaired in NF-κB-deficient and Lhx2 knockout embryos and that exogenous TGFβ2 rescues the HF phenotypes in Lhx2 knockout skin explants, indicating that it operates downstream of LHX2. These findings identify a novel NF-κB/LHX2/TGFβ2 signaling axis that is crucial for primary HF morphogenesis, which may also function more broadly in development and disease.

Abstract A07: Knockdown laminin-511 expression blocked endlthelial cell function in vitro and angiogenesis in vivo knockout skin model

Abstract Laminin-511 (composed of alpha5, beta1 and gamma1 chains) is a major extracellular matrix component in the basement membrane of human skin dermal microvascular endothelial cells (HDMECs). In this study the functions of laminin-511 in angiogenesis were investigated using in vitro knockdown and in vivo knockout skin model. In the in vitro studies, normal primary HDMECs and RNA interfering technique (siRNA) to silence the gene expression strategy were used. A lentiviral-based delivery system was developed that expresses laminin alpha5 chain specific siRNA (pL1) or control LUC siRNA (pLUC). HDMECs transduced with Laminin alpha5 siRNA (HDMEC/pL1) or control (HDMEC/pLUC) were analyzed in cell attachment, migration and a tubule formation assays. The mRNA expressions of laminin alpha5 and integrins were examined by real-time RT-PCR. Focal adhesion kinase (FAK) activation was studied by Western blot. In the in vivo study, a knockout mouse skin angiogenesis model was used. Since laminin alpha5 homozygous knockout (-/-) is embryonic lethal in mouse, skin from laminin alpha5 (-/-) embryos was transplanted onto the back of nude mouse, excisional wounds were created and wound angiogenesis was analyzed. Our results demonstrated that knockdown laminin alpha5 expression significantly inhibited the ability of endothelial cells in attachment, migration and tubule formation in vitro and blood vessel growth in vivo. Cell adhesive ability of HDMEC/pL1 was reduced 60% during a 60 minute attachment assay. Cell mobility of HDMEC/pL1 was found remarkably decreased 83% and 71% in a 12-hour and 48-hour chamber migration assay respectively; and in consistence, cell migration rates in a scratch migration assay were 56% and 53% reduced in HDMEC/pL1 cells at 48 and 72 hours respectively. Moreover, depletion of laminin alpha5 prevented HDMEC from forming enclosed capillary-like tubular structure at later stage in a three-dimensional tubule formation assay. While, in vivo, homozygous laminin alpha5 knockout skin was associated with significantly reduced growth of new blood vessels. A simultaneous and significant down regulation of integrins alphaV and beta3 was found correlated with the laminin alpha5 knockdown and reduced rate of cell migration. Further, cells with laminin alpha5 knockdown failed FAK activation, FAK phosphorylation on tyrosine 397 changed little in HDMEC/pL1 cells while there was markedly increase in control cells. In conclusion, our data suggested a possible involvement of laminin-511 in integrin alphaV and beta3-dependent angiogenesis and blood vessel maturation. Our works revealed the important roles of laminin-511 in endothelial cell activities, which proved its significance for angiogenesis. Citation Format: Jie Li, Tengjiao Cui. Knockdown laminin-511 expression blocked endlthelial cell function in vitro and angiogenesis in vivo knockout skin model. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A07.

BCC or not: Sufu keeps it in check.

BCC or not: Sufu keeps it in check.

MicroRNA-205 controls neonatal expansion of skin stem cells by modulating the PI(3)K pathway.

Skin stem cells (SCs) are specified and rapidly expanded to fuel body growth during early development. However, molecular mechanisms that govern the amplification of skin SCs remain unclear. Here we report an essential role for miR-205, one of the most highly expressed miRNAs in skin SCs, in promoting neonatal expansion of these cells. Unlike most mammalian miRNAs, genetic deletion of miR-205 causes neonatal lethality with severely compromised epidermal and hair follicle growth. In the miR-205 KO skin SCs, phospho-Akt is significantly downregulated, and the SCs prematurely exit the cell cycle. In the hair follicle, this accelerates the transition of the neonatal SCs towards quiescence. We identify multiple miR-205 targeted negative regulators of PI3K signaling that mediate the repression of phospho-Akt and restrict the proliferation of SCs. Our findings reveal an essential role for miR-205 in maintaining the expansion of skin SCs by antagonizing negative regulators of PI3K signaling.

Influence of Interferon-γ Deficiency in Immune Tolerance Induced by Male Islet Transplantation

BackgroundTraditionally, interferon-γ (IFN-γ) was regarded as a pro-inflammatory cytokine, however, recent reports suggested role of IFN-γ in immune tolerance. In our previous report, we could induce tolerance to male antigen (HY) just by male islet transplantation in wild type C57BL/6 mice without any immunological intervention. We tried to investigate the influence of IFN-γ deficiency on tolerance induction by male islet transplantation.MethodsTo examine the immunogenicity of male tissue in the absence of IFN-γ, we transplanted male IFN-γ knock-out (KO) skin to female IFN-γ KO mice. Next, we analyzed male IFN-γ KO islet to streptozotocin-induced diabetic female IFN-γ KO mice. And, we checked the functionality of grafted islet by graft removal and insulin staining.ResultsAs our previous results in wild type C57BL/6 mice, female IFN-γ KO mice rejected male IFN-γ KO skin within 29 days, and did not reject male IFN-γ KO islet. The maintenance of normal blood glucose level was dependent on the presence of grafted male islet. And the male islet recipient did not reject 2nd challenge of male islet graft also.ConclusionDeficiency of IFN-γ does not have influence on the result of male skin graft and male islet transplantation. Conclusively, male islet transplantation induced T cell tolerance is not dependent on the presence of IFN-γ.

Corneodesmosin gene ablation induces lethal skin-barrier disruption and hair-follicle degeneration related to desmosome dysfunction

Corneodesmosin (CDSN) is specific to desmosomes of epithelia undergoing cornification, mainly the epidermis and the inner root sheath of the hair follicles. CDSN nonsense mutations are associated with hypotrichosis simplex of the scalp, a rare disease that leads to complete baldness in young adults. CDSN displays adhesive properties, mostly attributable to its N-terminal glycine-rich domain, and is sequentially proteolyzed as corneocytes migrate towards the skin surface. K14-promoter driven Cre-mediated deletion of Cdsn in mice resulted in neonatal death as a result of epidermal tearing upon minor mechanical stress. Ultrastructural analyses revealed a desmosomal break at the interface between the living and cornified layers. After grafting onto nude mice, knockout skin showed a chronic defect in the epidermal permeability barrier. The epidermis was first hyperproliferative with a thick cornified layer, then, both the epidermis and the hair follicles degenerated. In adults, Cdsn deletion resulted in similar histological abnormalities and in a lethal barrier defect. We demonstrate that Cdsn is not essential for skin-barrier formation in utero, but is vital throughout life to preserve this barrier by maintaining desmosome integrity. The strong adhesive function that the protein confers on corneodesmosomes also seems necessary for maintaining the architecture of the hair follicle.

Conditional targeting of plectin in prenatal and adult mouse stratified epithelia causes keratinocyte fragility and lesional epidermal barrier defects

Plectin, a widespread intermediate filament-based cytolinker protein capable of interacting with a variety of cytoskeletal structures and plasma membrane-bound junctional complexes, serves essential functions in maintenance of cell and tissue cytoarchitecture. We have generated a mouse line bearing floxed plectin alleles and conditionally deleted plectin in stratified epithelia. This strategy enabled us to study the consequences of plectin deficiency in this particular type of tissues in the context of the whole organism without plectin loss affecting other tissues. Conditional knockout mice died early after birth, showing signs of starvation and growth retardation. Blistering was observed on their extremities and on the oral epithelium after initial nursing, impairing food uptake. Knockout epidermis was very fragile and showed focal epidermal barrier defects caused by the presence of small skin lesions. Stratification, proliferation and differentiation of knockout skin seemed unaffected by epidermis-restricted plectin deficiency. In an additionally generated mouse model, tamoxifen-induced Cre-ER(T)-mediated recombination led to mice with a mosaic plectin deletion pattern in adult epidermis, combined with microblister formation and epidermal barrier defects. Our study explains the early lethality of plectin-deficient mice and provides a model to ablate plectin in adult animals which could be used for developing gene or pharmacological therapies.

Wound Healing in Tenascin-X Deficient Mice Suggests that Tenascin-X is Involved in Matrix Maturation Rather than Matrix Deposition

Tenascin-X (TNX) is an extracellular matrix glycoprotein whose absence in humans leads to a recessive form of Ehlers-Danlos Syndrome (EDS). TNX deficient patients have hypermobile joints and fragile skin, but unlike the classical type of EDS, no atrophic scars were observed. Anecdotal evidence suggested that wound healing in TNX deficient patients is abnormal, but no detailed study has been performed so far. To address the role of TNX in wound healing, we analyzed skin wound morphology and mechanical properties of scars in TNX knockout (KO) mice. Breaking strength of unwounded skin of KO mice is significantly lower (<50%) than that of wild-type (WT) mice. In the early stage of wound healing when TNX is hardly expressed in WT wounds (day 7), WT and KO skin are of similar strength. After 14 days, when TNX starts to be expressed at moderate levels in wounds of WT mice, the WT scars gain a further increase in breaking strength, whereas KO scars do not progress beyond the mechanical strength of uninjured KO skin. No obvious differences between KO and WT mice were noted in the rate of wound closure, or in expression of fibrillar collagens during wound healing. We conclude that TNX is unlikely to be involved in matrix deposition in the early phase of wound healing, but it is required in the later phase when remodeling and maturation of the matrix establishes and improves its biomechanical properties.

Hoxb13 Up-regulates Transglutaminase Activity and Drives Terminal Differentiation in an Epidermal Organotypic Model

Hox genes act to differentiate and pattern embryonic structures by promoting the proliferation of specific cell types. An exception is Hoxb13, which functions as a proapoptotic and antiproliferative protein during development of the caudal spinal cord and tail vertebrae and has also been implicated in adult cutaneous wound repair. The adult epidermis, which expresses several Hox genes including Hoxb13, is continually renewed in a program of growth arrest, differentiation, and a specialized form of apoptosis (cornification). Yet little is known about the function(s) of these genes in skin. Based on its role during embryogenesis, Hoxb13 is an attractive candidate to be involved in the regulation of epidermal differentiation. Here, we demonstrate that Hoxb13 overexpression in an adult organotypic epidermal model recapitulates actions of Hoxb13 reported in embryonic development. Epidermal cell proliferation is decreased, apoptosis increased, and excessive terminal differentiation observed, as characterized by enhanced transglutaminase activity and excessive cornified envelope formation. Overexpression of Hoxb13 also produces abnormal phenotypes in the epidermal tissue that resemble certain pathological features of dysplastic skin diseases. Our results suggest that Hoxb13 functions to promote epidermal differentiation, a critical process for skin regeneration and for the maintenance of normal barrier function.

CD8 T Cells Can Reject Major Histocompatibility Complex Class I-Deficient Skin Allografts

Following transplantation, recipient T cells can recognize and respond to donor antigens expressed directly on donor cells, and can respond to donor-derived peptides that have been processed and presented in the context of recipient MHC through the indirect pathway. Indirectly primed CD4+ T cells have been well studied in transplantation, but little information is available regarding whether indirectly primed CD8+ T cells participate in rejection. To address this, we placed MHC class I-deficient DbKb knockout skin grafts onto allogeneic H-2 k SCID recipients followed by adoptive transfer of purified H-2 k CD8+ T cells. The MHC class I-deficient grafts were rejected and only CD8+ T cells were detectable in the recipient lymphoid organs and in the skin grafts. Immunohistochemical analysis showed that CD8+ T cells were found in close proximity to vascular endothelial cells and to recipient infiltrating macrophages, suggesting specific interactions. The data demonstrate that cross-primed polyclonal CD8+ T cells can function as active participants in the effector phase of rejection. The findings confirm and extend previous studies using a monoclonal TCR transgenic T cell and shed light on mechanisms of acute and chronic graft injury that are potentially relevant to human transplant recipients.