Inactivation of Autophagy in Keratinocytes Reduces Tumor Growth in Mouse Models of Epithelial Skin Cancer.

Autophagy is a ubiquitous degradation mechanism, which plays a critical role in cellular homeostasis. To test whether autophagy suppresses or supports the growth of tumors in the epidermis of the skin, we inactivated the essential autophagy gene Atg7 specifically in the epidermal keratinocytes of mice (Atg7∆ep) and subjected such mutant mice and fully autophagy-competent mice to tumorigenesis. The lack of epithelial Atg7 did not prevent tumor formation in response to 7, 12-dimethylbenz(a)anthracene (DMBA) as the initiator and 12-O tetradecanoylphorbol-13-acetate (TPA) as the promoter of tumor growth. However, the number of tumors per mouse was reduced in mice with epithelial Atg7 deficiency. In the K5-SOS EGFRwa2/wa2 mouse model, epithelial tumors were initiated by Son of sevenless (SOS) in response to wounding. Within 12 weeks after tumor initiation, 60% of the autophagy-competent K5-SOS EGFRwa2/wa2 mice had tumors of 1 cm diameter and had to be sacrificed, whereas none of the Atg7∆ep K5-SOS EGFRwa2/wa2 mice formed tumors of this size. In summary, the deletion of Atg7 reduced the growth of epithelial tumors in these two mouse models of skin cancer. Thus, our data show that the inhibition of autophagy limits the growth of epithelial skin tumors.

Cell death induced autophagy contributes to terminal differentiation of skin and skin appendages.

In the adult mammalian skin, cells are constantly renewing, differentiating and moving upward, to finally die in a yet not fully understood manner. Here, we provide evidence that macroautophagy/autophagy has a dual role in the skin. In addition to its known catabolic protective role as an evolutionary conserved upstream regulator of lysosomal degradation, we show that autophagy induced cell death (CDA) occurs in epithelial lineage-derived organs, such as the inter-follicular epidermis, the sebaceous- and the Harderian gland. By utilizing GFP-LC3 transgenic and ATG7-deficient mice, we show that CDA is initiated during terminal differentiation at a stage when the cells have become highly resistant to apoptosis. In these transitional cells, the Golgi compartment expands, which accounts for the formation of primary lysosomes, and the nucleus starts to condense. During CDA a burst of autophagosome formation is observed, first the endoplasmic reticulum (ER) is phagocytosed followed by autophagy of the nucleus. By this selective form of cell death, most of the cytoplasmic organelles are degraded, but structural proteins remain intact. In the absence of autophagy, consequently, parts of the ER, ribosomes, and chromatin remain. A burst of autophagy was stochastically observed in single cells of the epidermis and collectively in larger areas of ductal cells, arguing for a coordinated induction. We conclude that autophagy is an integral part of cell death in keratinocyte lineage cells and participates in their terminal cell fate.Abbreviations: Atg7: autophagy related 7; BECN1: beclin 1; CDA: cell death-induced autophagy; Cre: Cre-recombinase; DAPI: 4',6-diamidino-2-phenylindole; ER: endoplasmatic reticulum; GFP: green fluorescent protein; HaGl: haderian gland; IVL: involucrin; KRT14: keratin 14; LD: lipid droplet; LSM: laser scanning microscope; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PN: perinuclear space; RB: residual body; rER: rough endoplasmatic reticulum; SB: sebum; SG-SC: stratum granulosum - stratum corneum; SGl: sebaceous gland; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labelling.

Suppression of autophagy dysregulates the antioxidant response and causes premature senescence of melanocytes.

Autophagy is the central cellular mechanism for delivering organelles and cytoplasm to lysosomes for degradation and recycling of their molecular components. To determine the contribution of autophagy to melanocyte (MC) biology, we inactivated the essential autophagy gene Atg7 specifically in MCs using the Cre-loxP system. This gene deletion efficiently suppressed a key step in autophagy, lipidation of microtubule-associated protein 1 light chain 3 beta (LC3), in MCs and induced slight hypopigmentation of the epidermis in mice. The melanin content of hair was decreased by 10-15% in mice with autophagy-deficient MC as compared with control animals. When cultured in vitro, MCs from mutant and control mice produced equal amounts of melanin per cell. However, Atg7-deficient MCs entered into premature growth arrest and accumulated reactive oxygen species (ROS) damage, ubiquitinated proteins, and the multi-functional adapter protein SQSTM1/p62. Moreover, nuclear factor erythroid 2-related factor 2 (Nrf2)-dependent expression of NAD(P)H dehydrogenase, quinone 1, and glutathione S-transferase Mu 1 was increased, indicating a contribution of autophagy to redox homeostasis in MCs. In summary, the results of our study suggest that Atg7-dependent autophagy is dispensable for melanogenesis but necessary for achieving the full proliferative capacity of MCs.

Autophagy facilitates secretion and protects against degeneration of the Harderian gland.

The epithelial derived Harderian gland consists of 2 types of secretory cells. The more numerous type A cells are responsible for the secretion of lipid droplets, while type B cells produce dark granules of multilamellar bodies. The process of autophagy is constitutively active in the Harderian gland, as confirmed by our analysis of LC3 processing in GFP-LC3 transgenic mice. This process is compromised by epithelial deletion of Atg7. Morphologically, the Atg7 mutant glands are hypotrophic and degenerated, with highly vacuolated cells and pyknotic nuclei. The mutant glands accumulate lipid droplets coated with PLIN2 (perilipin 2) and contain deposits of cholesterol, ubiquitinated proteins, SQSTM1/p62 (sequestosome 1) positive aggregates and other metabolic products such as porphyrin. Immunofluorescence stainings show that distinct cells strongly aggregate both proteins and lipids. Electron microscopy of the Harderian glands reveals that its organized structure is compromised, and the presence of large intracellular lipid droplets and heterologous aggregates. We attribute the occurrence of large vacuoles to a malfunction in the formation of multilamellar bodies found in the less abundant type B Harderian gland cells. This defect causes the formation of large tertiary lysosomes of heterologous content and is accompanied by the generation of tight lamellar stacks of endoplasmic reticulum in a pseudo-crystalline form. To test the hypothesis that lipid and protein accumulation is the cause for the degeneration in autophagy-deficient Harderian glands, epithelial cells were treated with a combination of the proteasome inhibitor and free fatty acids, to induce aggregation of misfolded proteins and lipid accumulation, respectively. The results show that lipid accumulation indeed enhanced the toxicity of misfolded proteins and that this was even more pronounced in autophagy-deficient cells. Thus, we conclude autophagy controls protein and lipid catabolism and anabolism to facilitate bulk production of secretory vesicles of the Harderian gland.

Epidermal keratinocytes form a functional skin barrier in the absence of Atg7 dependent autophagy.

BACKGROUND: Cornification of keratinocytes involves the degradation of intracellular constituents which has led to the hypothesis that autophagy plays a role in this process. Mice, in which essential autophagy-related genes such as Atg7 are deleted systemically, die after birth and have not been characterized for potential epidermal defects. OBJECTIVE: This study tested whether autophagy is essential for epidermal barrier formation and function. METHODS: Atg7 was inactivated in epidermal keratinocytes by the Cre-loxP system under the control of the keratin K14 promoter (Atg7Δepi mice). Autophagic activity was detected using the GFP-microtubule-associated protein light chain 3 (GFP-LC3) reporter construct and Western blot analysis of LC3. Epidermal morphology was examined by histological and ultrastructural analyses, and barrier functions were assessed by dye diffusion and water loss assays. RESULTS: Suprabasal epidermal cells of normal mice contained GFP-LC3-labeled autophagosomes and epidermal lysates of these mice showed an excess of lipidated over non-lipidated LC3. These features of active autophagy were efficiently suppressed in Atg7Δepi epidermis. Atg7Δepi mice survived the perinatal period and were apparently healthy. Histologically, their epidermis was inconspicuous and ultrastructural analysis revealed no significant defect in cornification. There was however, an increase in the thickness of corneocytes in the back skin of mutant mice. Nevertheless, resistance to dye penetration into the skin and transepidermal water loss were normal in Atg7Δepi mice. CONCLUSION: This study demonstrates that autophagy is constitutively active in the epidermis but not essential for the barrier function of the skin.

Secretome of peripheral blood mononuclear cells enhances wound healing.

Non-healing skin ulcers are often resistant to most common therapies. Treatment with growth factors has been demonstrated to improve closure of chronic wounds. Here we investigate whether lyophilized culture supernatant of freshly isolated peripheral blood mononuclear cells (PBMC) is able to enhance wound healing. PBMC from healthy human individuals were prepared and cultured for 24 hours. Supernatants were collected, dialyzed and lyophilized (SEC(PBMC)). Six mm punch biopsy wounds were set on the backs of C57BL/6J-mice and SEC(PBMC) containing emulsion or controls were applied daily for three days. Morphology and neo-angiogenesis were analyzed by H&E-staining and CD31 immuno-staining, respectively. In vitro effects on diverse skin cells were investigated by migration assays, cell cycle analysis, and tube formation assay. Signaling pathways were analyzed by Western blot analysis. Application of SEC(PBMC) on 6 mm punch biopsy wounds significantly enhanced wound closure. H&E staining of the wounds after 6 days revealed that wound healing was more advanced after application of SEC(PBMC) containing emulsion. Furthermore, there was a massive increase in CD31 positive cells, indicating enhanced neo-angiogenesis. In primary human fibroblasts (FB) and keratinocytes (KC) migration but not proliferation was induced. In endothelial cells (EC) SEC(PBMC) induced proliferation and tube-formation in a matrigel-assay. In addition, SEC(PBMC) treatment of skin cells led to the induction of multiple signaling pathways involved in cell migration, proliferation and survival. In summary, we could show that emulsions containing the secretome of PBMC derived from healthy individuals accelerates wound healing in a mouse model and induce wound healing associated mechanisms in human primary skin cells. The formulation and use of such emulsions might therefore represent a possible novel option for the treatment of non-healing skin ulcers.

Increased sensitivity of histidinemic mice to UVB radiation suggests a crucial role of endogenous urocanic acid in photoprotection.

Urocanic acid (UCA) is produced by the enzyme histidase and accumulates in the stratum corneum of the epidermis. In this study, we investigated the photoprotective role of endogenous UCA in the murine skin using histidinemic mice, in which the gene encoding histidase is mutated. Histidase was detected by immunohistochemistry in the stratum granulosum and stratum corneum of the normal murine skin but not in the histidinemic skin. The UCA content of the stratum corneum and the UVB absorption capacity of aqueous extracts from the stratum corneum were significantly reduced in histidinemic mice as compared with wild-type mice. When the shaved back skin of adult mice was irradiated with 250 mJ cm(-2) UVB, histidinemic mice accumulated significantly more DNA damage in the form of cyclobutane pyrimidine dimers than did wild-type mice. Furthermore, UVB irradiation induced significantly higher levels of markers of apoptosis in the epidermis of histidinemic mice. Topical application of UCA reversed the UVB-photosensitive phenotype of histidinemic mice and increased UVB photoprotection of wild-type mice. Taken together, these results provide strong evidence for an important contribution of endogenous UCA to the protection of the epidermis against the damaging effects of UVB radiation.

Knockdown of filaggrin impairs diffusion barrier function and increases UV sensitivity in a human skin model.

Loss-of-function mutations in the filaggrin gene are associated with ichthyosis vulgaris and atopic dermatitis. To investigate the impact of filaggrin deficiency on the skin barrier, filaggrin expression was knocked down by small interfering RNA (siRNA) technology in an organotypic skin model in vitro. Three different siRNAs each efficiently suppressed the expression of profilaggrin and the formation of mature filaggrin. Electron microscopy revealed that keratohyalin granules were reduced in number and size and lamellar body formation was disturbed. Expression of keratinocyte differentiation markers and the composition of lipids appeared normal in filaggrin-deficient models. The absence of filaggrin did not render keratins 1, 2, and 10 more susceptible to extraction by urea, arguing against a defect in aggregation. Despite grossly normal stratum corneum morphology, filaggrin-deficient skin models showed a disturbed diffusion barrier function in a dye penetration assay. Moreover, lack of filaggrin led to a reduction in the concentration of urocanic acid, and sensitized the organotypic skin to UVB-induced apoptosis. This study thus demonstrates that knockdown of filaggrin expression in an organotypic skin model reproduces epidermal alterations caused by filaggrin mutations in vivo. In addition, our results challenge the role of filaggrin in intermediate filament aggregation and establish a link between filaggrin and endogenous UVB protection.

Inactivation of VEGF in mammary gland epithelium severely compromises mammary gland development and function.

To investigate the role of the angiogenic cytokine vascular endothelial growth factor (VEGF) during pregnancy and lactation, we used mice in which VEGF had been inactivated in mammary gland epithelial cells. Pups born to mutant mothers failed to thrive, displaying little milk in their stomachs. However, when they were transferred to control mothers they developed normally. Investigation of the mammary gland morphology revealed that lobulo-alveolar expansion into the fat pad was not complete in lactating mutant glands, and an accumulation of fat globules was evident in their secretory epithelium. In contrast to control glands, lactating mutant glands failed to up-regulate mRNAs for genes involved in milk secretion. Blood vessel density was comparable in pregnant mice of both groups but was only half that of controls in lactating mutant mice. FITC-labeled albumin injected intravenously (i.v.) into lactating mice extravasated rapidly and accumulated in the mammary gland epithelial cells in control animals, but was almost completely retained within the vessels in the mutants. Injection of recombinant VEGF i.v. reversed this effect. These findings demonstrate that mammary epithelium-derived VEGF is partially dispensable for angiogenesis during pregnancy and lactation, and by regulating vascular function during lactation, this factor is crucial to mammary gland differentiation and milk production.

Caspase-14 but not caspase-3 is processed during the development of fetal mouse epidermis.

The activation of caspases is a central step in apoptosis and may also be critical for terminal differentiation of epidermal keratinocytes (KC). In particular, caspase-3 has been implicated in the differentiation of embryonic KC as well as in programmed cell death of KC, and caspase-14 has been suggested to function in the formation or homeostasis of the stratum corneum (SC). To test the putative roles of these proteases, we determined their expression level and activation status during development of fetal mouse epidermis. The level of procaspase-3 did not change significantly during epidermal development, and enzyme activation was undetectable at any timepoint investigated. Despite the lack of active caspase-3, the newly formed stratum granulosum and the regressing periderm contained cells positive in the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay, indicating that nuclear DNA was degraded without activation of caspase-3, thereby arguing against a proteolytic function of caspase-3 in embryonic KC differentiation. By contrast, caspase-14 increased in abundance from embryonic day 14.5 (E14.5) onwards and consistently localized to the suprabasal layers of fetal epidermis. The caspase-14 pro-enzyme was processed into its catalytic subunits, a step required for enzyme activity, on day E17.5, coinciding with SC formation. Thus, processing of procaspase-14 is not confined to air-exposed mature skin but also occurs during epidermal development in utero. In summary, this study demonstrates that caspase-14, but not caspase-3 activation coincides temporally and spatially with embryonic KC differentiation, suggesting a role for caspase-14 in terminally differentiated KC.

Loss of vascular endothelial growth factor a activity in murine epidermal keratinocytes delays wound healing and inhibits tumor formation.

The angiogenic cytokine vascular endothelial growth factor (VEGF)-A plays a central role in both wound healing and tumor growth. In the skin, epidermal keratinocytes are a major source of this growth factor. To study the contribution of keratinocyte-derived VEGF-A to these angiogenesis-dependent processes, we generated mice in which this cytokine was inactivated specifically in keratin 5-expressing tissues. The mutant mice were macroscopically normal, and the skin capillary system was well established, demonstrating that keratinocyte-derived VEGF-A is not essential for angiogenesis in the skin during embryonic development. However, healing of full-thickness wounds in adult animals was appreciably delayed compared with controls, with retarded crust shedding and the appearance of a blood vessel-free zone underneath the newly formed epidermis. When 9,12-dimethyl 1,2-benzanthracene was applied as both tumor initiator and promoter, a total of 143 papillomas developed in 20 of 23 (87%) of control mice. In contrast, only three papillomas arose in 2 of 17 (12%) of the mutant mice, whereas the rest merely displayed epidermal thickening and parakeratosis. Mutant mice also developed only 2 squamous cell carcinomas, whereas 11 carcinomas were found in seven of the control animals. These data demonstrate that whereas keratinocyte-derived VEGF-A is dispensable for skin vascularization under physiological conditions, it plays an important albeit nonessential role during epidermal wound healing and is crucial for the development of 9,12-dimethyl 1,2-benzanthracene-induced epithelial skin tumors.