The E3 ligase Itch knockout mice show hyperproliferation
and wound healing alteration
Alessandro Giamboi-Miraglia1, Francesca Cianfarani2, Caterina Cattani2, Anna Maria Lena1, Valeria Serra1, Elena Campione3, Alessandro Terrinoni4, Giovanna Zambruno2, Teresa Odorisio4, Nicola Di Daniele5, Gerry Melino1,6and Eleonora Candi1,4
1 Department of Experimental Medicine and Surgery, University of ‘Tor Vergata’, Rome, Italy
2 Molecular and Cell Biology Laboratory, Istituto Dermopatico dell’Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico (IDI-IRCCS), Rome, Italy
3 Department of Dermatology, University of ‘Tor Vergata’, Rome, Italy
4 Biochemistry Laboratory, Istituto Dermopatico dell’Immacolata-Istituto di Ricovero e Cura a Carattere Scientifico (IDI-IRCCS), Rome, Italy 5 Department of Systems Medicine, Hypertension and Nephrology Unit, University of ‘Tor Vergata’, Rome, Italy
6 MRC Toxicology Unit, Leicester, UK
Keywords
Itch E3 ubiquitin ligase; keratinocytes; p63; wound healing
Correspondence
E. Candi, University of ‘Tor Vergata’, Department of Experimental Medicine and Surgery, Via Montpellier 1, 00133 Rome, Italy
Fax: +39 06 7259 6977 Tel: +39 06 7259 6487 E-mail: candi@uniroma2.it
G. Melino, Medical Research Council Toxicology Unit, Hodgkin Building University of Leicester, PO Box 138, Lancaster Road, Leicester LE1 9HN, UK
Fax: +44 (0)116 252 5616 Tel: +44 (0)116 252 5551 E-mail: gm89@le.ac.uk
(Received 19 June 2015, revised 20 August 2015, accepted 9 September 2015) doi:10.1111/febs.13514
The HECT-type E3 ubiquitin ligase Itch is absent in the non-agouti-lethal 18H or Itchy mice, which develop a severe immunological disease. Several of the known Itch substrates are relevant for epidermal development and homeostasis, such as p63, Notch, c-Jun and JunB. By analysing Itchy mice before the onset of immunological alterations, we investigated the contribu-tion of Itch in skin development and wound healing. Itchy newborn mice manifested hyperplastic epidermis, which is not present in adulthood. Itch/ cultured keratinocytes showed overexpression of proliferating markers and increased capability to proliferate, migrate and to repair a scratch injury in vitro. These data correlated with improved in vivo wound healing in Itchy mice, at late time points of the repair process when Itch is physiologically upregulated. Despite healing acceleration, epidermal remod-elling was delayed in the scars of Itch/ mice, as indicated by enhanced epidermal thickening, keratinocyte proliferation and keratin 6 expression, and retarded keratin 14 polarization to the basal layer. Itch/keratinocyte prolonged activation was not associated with increased immune cell persis-tence in the scars. Our in vitro and in vivo results indicate that Itch plays a role in epidermal homeostasis and remodelling and this feature does not seem to depend on immunological alterations.
Introduction
The ubiquitin E3 ligase Itch, also known as atrophin-1 interacting protein 4 (AIP4), is a monomeric enzyme which belongs to the E6-AP carboxy terminus (HECT)
type family of E3 ligases. At the structural level, Itch includes an N-terminal Ca2+-dependent phospholipid-binding C2 domain, a multiple protein–protein
interac-Abbreviations
AIP4, atrophin-1 interacting protein 4; BrdU, 5-bromo-20-deoxyuridine; DAPI, 40,6-diamidino-2-phenylindole; E, embryonic day; HECT, E6-AP carboxy terminus; IL, interleukin; K, keratin; wt, wild type.
tion WW domain and a C-terminal HECT domain [1]. Similarly to other HECT-type E3 ligases, Itch WW interacting domains commonly recognize the Pro-rich PPXY (PY) consensus sequence, although they also interact with phospho-Ser/phospho-Thr followed by a Pro residue. In addition, atypical interactions with unre-lated modular domains in the substrate [2–5] or adaptor proteins [6–9] have been described. The agouti locus, which identifies the Itch gene, has been genetically dis-rupted, providing an excellent tool to investigate its function [10]. The non-agouti-lethal 18H or Itchy mice display severe immune and inflammatory defects and manifest a persistent scratching of the skin [1]. On the C57BL/10 background, Itch deficiency results in sponta-neous development of a late onset (after 6 months) and progressively lethal systemic autoimmune-like disease, characterized by lympho-proliferation in the spleen, lymph nodes and medulla of the thymus and by chronic pulmonary interstitial inflammation. Death occurs at 6– 8 months of age, and it is probably caused by hypoxia [11]. The Itchy null mice exhibit a higher level of TH 2-dependent Ig G1 and E subtypes than their normal counterpart, possibly due to an increased production of TH2 cytokines [e.g. interleukin-4 (IL-4) and IL-5] [12]. Itch typically regulates the stability of both transmem-brane receptors through canonical mono-ubiquitylation or multi-ubiquitylation, and intracellular substrates through poly-ubiquitylation, driving them to lysosomal and proteasomal degradation, respectively. Proteolysis-independent ubiquitylation events have also been ascribed to the E3 activity of Itch [5,13]. Interestingly, a number of Itch substrates (c-Jun, JunB, p63, Notch, Gli1) are transcription factors controlling epidermal proliferation and keratinocyte differentiation [2,12,14– 18]. It is therefore likely that Itch-mediated degradation of some or all of these proteins would have a regulative role in skin biology and skin homeostasis. In particular, the hypothesis that Itch directly regulates the protein stability of crucial protein for the homeostasis of the epidermis, such as c-Jun and JunB [19,20], p63 [21–26], Notch [27–29], Gli, strongly suggests that Itch is a cru-cial regulator of the expansion of the basal keratinocyte. In the present investigation we have tested this hypothe-sis, taking advantage of the genetic disruption of the Itch locus. Accordingly, here, we have characterized the epidermal phenotype of Itch/ mice. The results indi-cate that Itch contributes in skin development and wound healing, independently of the immunological phenotype. Although we could not establish the role of each individual substrate, the data shown suggest that modulation of Itch enzymatic activity has a role in skin homeostasis and remodelling.
Results
Functional role of Itch in epidermis
To define the in vivo functional role of Itch in epider-mis development, and in particular in controlling pro-liferation and differentiation pathways, we investigated Itch/ dorsal skin morphology and proliferation in newborn and adult mice. The apparent gross morphol-ogy of the Itch/epidermis was similar to that of the wild type (wt), in both newborn and adult mice. How-ever, the epidermis of the Itch/ dorsal skin was sig-nificantly thicker in newborn animals compared with wt controls (Fig. 1A,B); this difference was not main-tained in 2-month-old mice, as found in dorsal skin (Fig. 1A, lower panels). To investigate if skin thicken-ing in Itch/newborn mice was due to an increase of the proliferation rate or to a delay in differentiation, we performed a 4 h 5-bromo-20-deoxyuridine (BrdU) pulse-chase labelling in wt and Itch/ skin at embry-onic day (E) 18.5. The results obtained clearly showed a significant increase, from 19% to 33%, of BrdU incorporation in Itch/ skin compared to wt (Fig. 1C,D). Immunofluorescence and confocal analy-sis of keratin 14 (K14), marker of proliferating cells, confirmed that the basal layer, which is the prolifera-tive compartment of the tissue, is expanded (Fig. 1E). As for differentiation markers, involucrin and loricrin are expressed in a very similar manner between wt and Itch/ mice, while K10 appears to be more expressed in Itch/ skin. When epidermal differentia-tion markers were analysed in the skin of adult mice, no difference could be observed between Itch/ and wt mice (data not shown). To define the functional role of Itch during skin development, we performed whole-mount assay for skin permeability in wt and Itch/ embryos at E16.5, E17.5 and E18.5, using toluidine blue dye. No differences were observed between Itch wt and Itch/ embyos on skin barrier formation (Fig. 1F); as a consequence, the out-inner and inner-out barrier function are not affected, as assessed by Lucifer yellow and biotinylated reagent injection (Fig. 1G,H). To define the role of Itch in controlling keratinocyte differentiation, we performed a set of experiments using primary keratinocytes iso-lated from newborn mice induced to differentiate upon calcium addition to the medium for 1 and 3 days. The results obtained ex vivo are coherent with what is observed in vivo. Indeed, the proliferation marker K14 decreased more slowly during calcium-induced differ-entiation in Itch/ keratinocytes compared to wt, while K10 was expressed at a higher level in Itch/
keratinocytes induced to differentiate (Fig. 2A,B). This was also confirmed by western blot (Fig. 2C,D). Col-lectively, these results indicate that Itch loss increases proliferation in vivo, resulting in thicker epidermis. However, this hyperplastic epidermis is present only in embryonic and newborn skin, but not in adult skin, suggesting that Itch function in controlling ker-atinocyte proliferation is confined to skin develop-ment. Results also show that Itch loss does not alter
keratinocyte differentiation capabilities and skin bar-rier formation.
Itch loss enhances p63, c-Jun, JunB and Notch expression in keratinocytes
Next, we decided to investigate whether loss of Itch is sufficient to alter the expression of Itch substrates rele-vant for skin formation and homeostasis (Fig. 3A,B).
0 5 10 15 20 25 30 35 40
Rel. % of BrdU pos cells
0 10 20 30 40 50 60 * * Epidermis thickness (µm)
Keratin 14 Keratin 10 Involucrin Loricrin
Itch+/+ Itch–/– Itch+/+ Itch–/– Itch+/+ Itch–/– Itch+/+ Itch–/– Keratin 5 BrdU Keratin 5 BrdU
E16.5 E17.5 E18.5 E18.5 60 days Itch+/+ Itch–/– Itch+/+ Itch–/– Itch +/+ Itch –/– Itch+/+ Itch–/– Itch+/+ Itch–/– Itch+/+ Itch–/– A B C E F G H D
Fig. 1. Morphological analysis ofItch/ mice epidermis. (A) Haematoxylin and eosin staining of dorsal skin sections of newborn mice (upper panels) and two 60-day-old mice. (B) Quantification of newborn mice epidermis thickness. The results represent the mean SD of six mice from a representative experiment (*P < 0.05). (C) BrdU staining (red) on the dorsal skin section of E18.5 mouse. Staining of K5 is shown in green. Nuclei were counterstained with DAPI (blue). (D) Quantification of BrdU-positive cells in the epidermis. BrdU-positive cells in the epidermis were counted over a linear distance of 0.6 mm. The results represent the mean SD of six mice from a representative experiment (*P < 0.05). All the scale bars represent 40lm. (E) Confocal immunostaining of dorsal skin sections of newborn mice. Sections were stained (green) with antibodies against differentiated basal (K14) and upper (K10, involucrin, loricrin) layer markers. Nuclei were counterstained with DAPI (blue). One representative experiment is shown. The scale bars represent 40lm. (F) Photographs of mouse embryos stained with 0.1% toluidine blue. Mice were time-mated and embryos were isolated at the indicated embryonic days. (G) Confocal immunostaining of dorsal skin sections of newborn mice stained with Lucifer yellow (green). (H) Confocal immunostaining of dorsal skin sections of newborn mice injected with a biotinylation reagent and then stained with streptavidin (red). Nuclei in (G) and (H) were counterstained with DAPI (blue). All the scale bars represent 40lm.
We performed ex vivo experiments on isolated wt and Itch/ keratinocytes induced to differentiate by cal-cium addition (1.2 mM) to the culture medium for 1, 3 and 5 days. The results indicate that the substrates analysed (p63, c-Jun, JunB) are expressed 20–40% more compared to wt keratinocytes (Fig. 3A,B); inter-estingly, upon calcium addition, they are degraded at a similar rate, suggesting that alternative mechanisms are engaged by calcium during differentiation, including microRNAs. To confirm this, we decided to investigate whether Itch loss is sufficient to alter the expression of Itch substrates relevant for skin formation and home-ostasis in vivo (Fig. 4A,B). Confocal immunostaining of dorsal skin sections of newborn mice showed that all the substrates analysed (p63, c-Jun, JunB) were signifi-cantly upregulated in Itch/epidermis compared with wt epidermis. The expression of the Itch substrates was evaluated measuring p63-, c-Jun- and JunB-positive cells over the total nuclei (P< 0.05). Notch expression was indirectly evaluated by determining Hes-1 mRNA levels (Fig. 4C). Real-time PCR analysis indicated that at mRNA level there is no difference in expression between wt and Itch/ epidermis (Fig. 4D). Overall, the results obtained indicate that the E3 ligase Itch par-ticipates in controlling the expression of p63, c-Jun, JunB steady-state levels during keratinocyte differentia-tion, in ex vivo and in vivo conditions.
Itch loss promotes cutaneous wound healing To further investigate the role of Itch in skin home-ostasis, we analysed the effect of Itch loss in skin
repair, a process in which keratinocytes acquire an activated phenotype and start to proliferate and migrate to re-epithelialize the injured skin. Wound healing was initially analysed in vitro by a scratch assay performed on monolayers of confluent primary keratinocytes isolated from newborn Itch/ mice and control mice. Itch/ keratinocytes showed improved ability to fill the gap of scrape injuries compared to wt cells (Fig. 5A). Indeed, the cell-free area was signif-icantly reduced in Itch/ monolayers at 24 and 48 h after scraping (24 h, wt, 76.1% 8.9%, Itchy, 51.2% 17.7%; 48 h, wt, 20.0% 13.3%, Itchy, 10.1% 12.1%). Concordantly, Itch/ keratinocyte proliferation, assessed by BrdU incorporation, and migration, evaluated by a modified Boyden chamber assay, were significantly increased in keratinocytes iso-lated from Itch/ mice with respect to their wt coun-terpart (Fig. 5B,C). We then analysed wound healing in vivo in 8-week-old mice. This analysis revealed that this process was improved in Itch/ mice compared to wt controls (Fig. 5D). Wound area was constantly, although not significantly, smaller at early time points after wounding; starting from day 8, an increase in such difference was observed, reaching a significant value at day 10 (wt, 32.1% 2.8% of initial area; Itch/, 16.0% 5.6%; P < 0.01). This result sug-gests that Itch loss improves healing at late time points of the repair process. The analysis of Itch expression during wound healing on wt mice, as both mRNA and protein, indicated a progressive increase reaching maximal levels at around 15 days post-injury (Fig. 5E,F). More precisely, real-time PCR analysis
0 1 2 3 4 5 6 0 1 3 Keratin 10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 Relative expression Keratin 14
*
*
0 100 200 300 400 500 Relative expression ** Keratin 10 Keratin 10 116 - Involucrin Days of diff. Itch+/+ 80 - 60 - 48 - Itch Itch–/– Itch+/+ Itch–/– β-actin 0 1 3 0 1 3 Days A.U.*
*
Days 0 1 3 0 1 3 0 1 3 0 1 3 A B C DFig. 2. Epidermal differentiation inItch/ keratinocytes. (A), (B) Levels of epidermal proliferation (K14) and differentiation (K10) marker mRNAs evaluated by real-time qPCR at the indicated time points after induction of differentiation in primary mouse keratinocytes. Itch+/+are shown in blue;Itch/are shown in red. Results are shown as mean SD from three independent experiments (*P < 0.05, **P < 0.001). (C) Levels of epidermal differentiation proteins (involucrin and K10) evaluated by western blot. Actin levels were measured as loading control. One representative experiment of three is shown. (D) Average densitometry of the western blots (*P < 0.05).
indicated that Itch levels increased by twofold at day 3 compared to the amount detected in non-lesional skin (day 0), and continued to increase to more than four times by day 15; a progressive decrease then occurred (Fig. 5E). Western blot on proteins extracted from wounds revealed that Itch expression started to increase above the basal amount at day 5, reached maximal levels from day 15 until day 21, and returned to the amount found in non-lesional skin by day 60 post-injury (Fig. 5F). These data are in agreement with the observed effects of Itch loss in promoting wound closure at late time points of the healing process.
Itch loss delays epithelial remodelling
Having found an improvement in wound closure in Itchy mice, the effect of Itch loss on keratinocyte func-tion at the wound site was investigated by histomor-phometric analysis. Keratinocyte activation was analysed on wound specimens collected from Itch/ mice and wt controls at day 5 after injury, correspond-ing to the active proliferative phase when re-epithelial-ization occurs and Itch starts to be upregulated, and in the scars at day 21, when keratinocytes are reverting to normal differentiation and Itch is expressed at high-est levels.
Keratinocyte proliferation was analysed by immun-odetecting the proliferative nuclear marker Ki67. A comparable percentage of stained nuclei were detected in Itch/ mice and in wt controls in day 5 wound specimens (wt, 28.1% 9.7%; Itch/, 26.3% 9.3%). As for scar analysis, a significant increase in epithelial thickness in Itch/ mice compared to wt controls was observed (Fig. 6A,B). In parallel, by Ki67 immunohistochemistry an increased number of proliferating keratinocytes in the basal compartment of Itch/ epidermis was found (Fig. 6C). These data suggest that keratinocytes of Itchy mice manifest a more activated phenotype at day 21 compared to those of wt controls. To confirm this observation, the expressions of K6, a marker of hyperproliferative activated keratinocytes strongly expressed in the migrating epithelial tongue during wound healing, and K14, that labels specifically basal keratinocytes in non-lesional skin but is also expressed by suprabasal keratinocytes during wound healing [30], were investi-gated by immunofluorescence. K6 staining was stron-ger and more diffuse in the scar epidermis of Itchy mice (Fig. 6D). As for K14 expression, it was con-fined to keratinocytes of the basal layer in wt mice, while in Itch/ skin it was still expressed in supraba-sal keratinocytes (Fig. 6D). These data confirmed that, although wound healing was improved in Itch/ mice, Itch/ keratinocytes remained activated for a longer period and manifested a delay in reverting to nor-mal differentiation. Since Itch loss was associated with altered immunological response and the immune status strongly regulates epidermal homeostasis [31,32], to evaluate whether the observed delayed recovery of homeostasis of Itch/ keratinocytes could be due to enhanced inflammation, the presence of immune cells was evaluated by immunohistochemistry in the scars of Itchy mice and wt controls. T cells, macrophages and neutrophils were detected with antibodies recognizing CD3, F4/80 and myeloperoxidase, respectively. The
A.U. A.U. p63 cJun JunB β-actin p63 cJun JunB Itch+/+ Itch Itch–/– Itch+/+ Itch–/– 80 - 43 - 48 - 43 - 116 - 0 1 3 5 0 1 3 5 0 0.5 1 1.5 2 0 1 3 5 0 0.5 1 1.5 2 0 1 3 5 0 0.5 1 1.5 0 1 3 5
*
*
*
* A BFig. 3. Expression of Itch substrates inItch/ keratinocytes. (A) Levels of Itch substrates evaluated in ex vivo conditions by western blot at the indicated time points after induction of differentiation in primary mouse keratinocytes isolated from wt (Itch+/+) andItch/keratinocytes. Actin levels were measured as loading control. One representative experiment of three is shown. (B) Average densitometry of the western blots (*P < 0.05).
three cell types were only slightly, and not significantly, more expressed in the scars of Itch/mice compared to the wt counterpart (Fig. 6E). Finally, the analysis of p63, Notch and c-Jun, performed by immunofluores-cence on scar specimens, confirmed that all three Itch substrates were more expressed in the scars of Itchy mice compared to wt controls (Fig. 7). These data sug-gest that prolonged Itch/keratinocyte activation after wound closure is independent of chemokines and growth factors released by immune cells.
Discussion
The development and the homeostasis of the epidermis is a very complex biological model [33–37] involving a peculiar mechanism of cell death [38], under intense research activity [39–43] also for its wound healing implications [44–49]. In the current work, we have investigated the phenotype of Itch/ mice at the level of the epidermis. We show that loss of Itch affects skin development and epidermal remodelling upon wound healing. In detail, in the absence of Itch expression,
c-Jun positive cells/field
c-Jun DAPI
JunB positive cells/field
JunB DAPI p63 DAPI Notch DAPI Itch+/+ Itch+/+ Itch–/– Itch+/+ Itch–/– Itch+/+ Itch–/– Itch–/– 25 * * * 20 15 10 5 0 0 5 10 15 20 25 30 35 40 45 p63 positive cells/field 05 10 15 20 25 30 35 40 45
Fold over control
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6
Fold over control
0
0 1 3 Days p63 Notch cJun JunB
5 10 15 * A B C D
Fig. 4. Expression of Itch substrates in Itch/mice. (A) Confocal immunostaining
of dorsal skin section of newborn mice. Sections were stained (red) with antibodies against the E3 ubiquitin ligase Itch substrates. Nuclei were
counterstained with DAPI (blue). (B) Quantification of expression of Itch substrates in the epidermis. For p63, c-Jun and JunB, positive cells were counted over the total nuclei. We could not quantify Notch staining; as indirect evidence of Notch increases inItch/ mice, we determined the expression of Hes-1 in the samples described in Fig. 3. We observed a significant increase of Hes-1 mRNA (C) indicating increased Notch expression activity. Itch+/+are shown in blue;Itch/are shown in red. The results represent the mean SD of three mice from a representative experiment (*P < 0.05). The scale bars represent 40lm. (D) Itch depletion does not affect mRNA levels of p63, Notch, c-Jun and JunB in epidermis. mRNAs were extracted by dorsal skin biopsies of newborn mice. Real-time PCRs were performed to evaluate and compare wt andItch/ epidermis (four mice each). Results indicate that inItch+/+andItch/ epidermis the mRNA levels of Itch substrates are comparable. Primers are available upon request. Values are expressed as mean SD of four mice for each time point.
basal keratinocytes manifest a higher proliferative rate in association with increased expression levels of the Itch substrates p63, c-jun, JunB. DNp63 [50–53], a member of the p53 family [54–59], is a major substrate of Itch that is responsible for maintaining the basal layer proliferative potential [16,60]. The loss ofDNp63 results in the absence of the epidermis as well as of other stratified epithelia, with aplasia of multiple ectodermal appendages (tooth, hair, nails) and limb defects. Indeed, p63 and Itch are co-expressed in normal epidermis, being predominantly distributed in the basal and the upper layer, respec-tively [14,15]. In Itch/ mice we found an increased steady state of p63 protein levels, both at E18.5 and in the scar, concomitant with an increased proliferation in vivo, as detected by the increases in BrdU incorpo-ration. Altogether, these findings strongly indicate that Itch physiologically controls p63 steady-state protein levels in vivo and, as a consequence, the capacity of the transcription factor to direct the expansion of the basal compartment of the epidermis. Interestingly, an
altered expression of p63 was also detected in patients affected by psoriasis, suggesting that Itch may be involved in this skin pathology as well.
A second major substrate of Itch are the Notch receptors that have also been implicated in controlling the balance between proliferative basal cells and termi-nally differentiating suprabasal keratinocytes by com-mitting basal progenitors to a spinous cell fate [61]. In normal epidermis, Notch1–3 receptors are primarily expressed in the suprabasal layers, where they nega-tively regulate the transcription of genes typically expressed in the basal layer, i.e. the proliferative com-partment; moreover they activate early genes of the keratinocyte differentiation programme. On the other hand, Notch receptors control the switch between basal and spinous layer; they are expressed in the suprabasal layer and are responsible for inhibition of the expression of the basal-layer-specific genes and simultaneously they promote the expression of spi-nous-layer-specific genes. The spinous layer expansion, evaluated by K10 increased expression, observed in the
0 20 40 60 80 100 0 2 4 6 8 10 12 14 16 * % initial area % initial area 24 h 48 h 0 10 20 30 40 50 60 70 80 90 * * Itch–/– Itch+/+ Itch–/– Itch+/+ Itch–/– Itch+/+ Itch–/– Itch+/+ Itch–/– Itch+/+ 0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 * Relative BrdU incorporation 0 5 10 15 20 25 Cell n./field * 0 1 2 3 4 5 0 1 3 5 7 9 13 15 21 24
Days after wounding Days after wounding
Days after wounding
Fold increase 10 0 2 4 6 8 0 3 5 7 9 15 21 30 60 Fold increase 0 3 5 7 9 15 21 30 60 Itch 37- 100 - A B C D E F GAPDH Fig. 5. Wound healing inItch/mice and
Itch expression during wound healing. (A) Scratch assay on confluent keratinocytes isolated fromItch/mice and wt controls (Itch+/+) (*P < 0.05). (B) BrdU
incorporation to assess proliferation of cultured primary keratinocytes isolated from Itch knockout mice (Itch/) and wt controls (Itch+/+) (*P < 0.05). (C) Keratinocyte migration assessedex vivo by a modified Boyden chamber assay (*P < 0.05). The results shown for in vitro assays represent the mean SD of three mice from representative experiments. (D) Analysis of wound closure. Values are expressed as mean SD of three independent experiments with six mice each (*P < 0.05). (E) Real-time PCR to detect Itch mRNA during wound healing. Values are expressed as mean SD of three mice for each time point. (F) Western blot for Itch during wound healing. GAPDH levels were evaluated as loading control. The results reported in the histograms represent the mean SD of two experiments on different wound extracts.
E18.5 Itchy mice epidermis could be due to an increased expression and activity of Notch receptors; therefore, Itch-mediated degradation of Notch would thus contribute to regulate spinous cell fate specifica-tion and keratinocyte differentiaspecifica-tion. This is also sup-ported by the transgenic mouse model generated by Blanpain and co-workers, in which the expression of activated Notch in the epidermis resulted in expansion of the spinous layer [61]. Interestingly, the spinous layer expansion does not alter skin barrier functions, similarly to Itch/ mice. Together, both p63 and
Notch could be responsible for the hyperproliferative epidermis during skin development and in scar forma-tion observed in the Itch/mice.
A third relevant Itch substrate is the AP-1 transcrip-tion factor that consists of different combinatranscrip-tions (ho-modimer and/or heterodimers) of the Fos proteins
K6 K6
K14 K14
Mean cell n./field
0 5 10 15 20 25 30 35 T cells Macrophages 0 5 10 15 20 25 30 35 0 1 2 3 4 5 6 7 8 Neutrophils % Ki67+ cells 0
**
5 10 15 20 25 30 35 0 50 100 150 200 250 300 % thickness Itch–/– Itch+/+ Itch–/– Itch+/+ Itch–/–Itch+/+ Itch+/+ Itch–/– Itch+/+ Itch–/– Itch–/– Itch+/+ Itch–/– Itch+/+ Itch–/– Itch+/+
*
A B C D E Itch+/+ Itch–/–Itch+/+ Itch–/– Itch+/+ Itch–/– Itch+/+ Itch–/–
p63/Dapi p63/Dapi Notch/Dapi Notch/Dapi c-Jun/Dapi c-Jun/Dapi
*
p63 Fold over ctr c-Jun*
Notch*
3 2 1 0 3 2 1 0 3 2 1 0 A BFig. 7. Expression of Itch interactors in the scars of Itch/ mice and in wt controls. (A) Confocal immunostaining of dorsal skin section of newborn Itch/ and wt mice (Itch+/+). Sections were stained with antibodies against p63 (red), Notch (red) and c-Jun (green). Nuclei were counterstained with DAPI (blue). The scale bars represent 40lm. (B) Fluorescence intensity was evaluated analysing five slides obtained from three wt andItch/mice. *P < 0.05 Fig. 6. Analysis of the scar ofItch/mice. (A) Epithelial thickness is reduced in the scars of Itchy mice (Itch/) at day 21 compared to controls (Itch+/+). The yellow bars indicate average thickness. The scale bars represent 40lm. (B) Quantification of epithelial thickness in scar epidermis. Thickness is expressed as a percentage value with respect to measures obtained in the epidermis surrounding the wound. The results represent the mean SD of 10 mice (*P < 0.05). (C) Quantification of cells expressing Ki67 in the epidermis of scars. Values are expressed as a percentage of positive nuclei over the total number of nuclei analysed. The results represent the mean SD of 10 mice (**P < 0.01). (D) Confocal immunostaining of scar sections. Sections were stained (green) with antibodies against the keratinocyte activation marker K6 and the differentiated basal marker K14. Nuclei were counterstained with DAPI (blue). The scale bars represent 40lm. (E) Quantification of immune cell types in the dermal compartment of the scars. T cells, macrophages and neutrophils were detected by immunohistochemistry with an CD3, an F4/80 or an anti-myloperoxidase antibody, respectively. Values are expressed as mean number of positive cells per 409 field. The results represent the mean SD of five mice.
(c-Fos, FosB, Fra-1 and Fra-2) and Jun proteins (c-Jun, JunB and JunD) [62]. In vitro loss or gain of function experiments and in vivo knockout/transgenic mice have shown that AP-1, and its members, have important roles in many biological processes including proliferation, differentiation, apoptosis and tumour formation [63]. The AP-1 complex is very important during skin development and keratinocyte differentia-tion [64], and indeed many genes affecting skin home-ostasis are under AP-1 control; these include transglutaminase 1 [65], keratins (K1, K5, K14, K17) [66,67], involucrin [68], loricrin [69] and profillagrin [70]. Skin-specific c-Jun knockout has demonstrated that it plays an important role in regulating keratinocyte prolif-eration and tumour formation [71,72]. While epidermis-specific, JunB knockout did not show any phenotype; epidermis-specific deletion of both c-Jun and JunB resulted in skin lesions resembling psoriasis [73]. This, and other studies, demonstrated that expression and/or deletion of AP-1 proteins in epidermis is critically impli-cated in altering cytokine expression, resulting in distur-bance of skin homeostasis and in chronic skin disease. From the data obtained, unfortunately, we were not able to evaluate the distinct contribution of c-Jun and JunB in the phenotype described; this is probably due to the limited increase of these specific AP-1 compo-nents in the skin as a consequence of Itch depletion.
As discussed above, we have analysed all these sub-strates and their effect in Itch/ mice, and the results demonstrate that Itch-mediated substrate ubiquityla-tion is important for regulating keratinocyte activaubiquityla-tion and, consequently, for epidermal remodelling in young mice as well as in wound healing [74,75]. Conversely, Itch depletion does not seem to significantly affect epi-dermal homeostasis of adult skin in normal conditions, indicating that active self-regulatory mechanisms are able to compensate the absence of Itch.
In conclusion, although we could not strictly estab-lish the role of each individual substrate in the Itch/ skin phenotype, this study indicates that modulation of Itch enzymatic activity may have a role in skin pathogenesis, such as psoriasis [76–78], and/or in tissue remodelling upon injury in normal and pathological conditions.
Material and methods
Mice
Non-agouti-lethal 18H mice were originally generated through a radiation-induced chromosomal inversion and characterized as having a disrupted gene that was later ter-med Itch [10]. Heterozygous breeding pairs were set up,
and from the F1 generation offspring Itch+/+ (wild type C57BL/10) and Itch/ breeding colonies were established. Cross-breeding experiments were performed using itchy mice from the breeding colonies and wt C57BL/10 mice purchased from the Jackson Laboratory (Bar Harbor, ME, USA). C57BL/10 mice were used as wt controls. The analy-sis of Itch expression during wound healing was performed on 8-week-old CD1 mice (Charles River Italia, Calco, Italy). Animal experiments were approved by the Care and Use Committee of the University of Rome ‘Tor Vergata’.
Cell culture
Primary keratinocytes were isolated according to Yuspa and colleagues from the skin of newborn mice [79]. Briefly, the skin was floated overnight on trypsin/EDTA at 4°C. Trypsinization allows separation of dermis from epidermis. Primary keratinocytes were isolated from the latter and pla-ted on collagen-coapla-ted dishes in medium supplemenpla-ted with 0.05 mMCaCl2. Cells were induced to differentiate by
add-ing 1.2 mMCaCl2to the culture medium.
Western blotting
Primary mouse keratinocytes were lysed in a buffer con-taining 100 mM Tris/HCl pH 8.5, 1% SDS, 20 mM DTT
and 5 mM EDTA. Total cell extracts were resolved on an
SDS/10% polyacrylamide gel and blotted onto a Hybond P poly(vinylidene difluoride) membrane (G&E Healthcare, Little Chalfont, UK). Membranes were blocked with PBST 5% non-fat dry milk, incubated with primary anti-bodies for 2 h at room temperature, washed and hybri-dized for 1 h at room temperature using the appropriate horseradish-peroxidase-conjugated secondary antibody (BioRad, Hercules, CA, USA). Detection was performed with an enhanced chemiluminescence kit (Perkin Elmer, Waltham, MA, USA). The antibodies used were polyclonal anti-MK5 (PRB-160P, Covance, Princeton, NJ, USA), poly-clonal anti-MK10 (PRB-159P, Covance), polypoly-clonal anti-in-volucrin (PRB-140C, Covance), monoclonal anti-p63 (MS-1084, Neomarkers, Portsmouth, NH, USA), monoclonal anti-c-Jun (610326, BD), monoclonal anti-JunB (SC-8051, Santa Cruz, Dallas, TX, USA), monoclonal anti-b-actin (A5441, Sigma Aldrich, Gillingham, Dorset, UK) and mon-oclonal anti-Itch (611198, BD, Franklin Lakes, NJ, USA).
Real-time PCR assay
RNA was extracted from primary mouse keratinocytes using the RNAeasy kit (Qiagen, Venlo, The Netherlands). A total of 500 ng of RNA was used for reverse transcrip-tion using the InPromII kit (Promega, Milano, Italy) and one-tenth of the reaction was used for PCR. Real-time PCR was performed by using the Platinum SYBR Green qPCRSuperMix UDG with Rox (Invitrogen, Waltham,
MA, USA), with an amplification programme as follows: one cycle of 95°C for 3 min and 40 cycles of 94 °C for 20 s and 60°C for 1 min. The reaction was followed by a melting curve protocol according to the specification of the ABI 7500 instrument (Applied Biosystems, Waltham, MA, USA). Primers used were as follows: mK5F 50 -tccagaacgc-cattgctgaag-30 and mK5R 50-ccgtagccagaagagacactgtttg-30 (for mouse K5); mK10F 50-aggctttggtggtggtggattc-30 and mK10R 50-gcattgtcagttgtcagggtgagg-30 (for mouse K10); mHes1F 50-atttgcctttctcatccccaacg-30 and mHes1R 50 -cagttccgccacggtctccacat-30 (for mouse Hes-1); mTG5F 50-cagcccaggagccagaag-30 and mTG5R 50 -ggcctcggcggacaac-30 (for mouse transglutaminase 5); mInvF 50 -tctccctcctgt-gagtttgtttgg-30 and mInvR 50-cagtgaagacctggcattgtgtagg-30 (for mouse involucrin). Mouse b-actin was used as house-keeping gene for quantity normalization, and primers used were mActF 50-tgtccctgtatgcctctggtcg-30 and mActR 50 -gaac-cgctcgttgccaatagtg-30. Relative quantification of gene expres-sion was calculated according to the method of 2-DDCT described in ABI User Bulletin no. 2 and the RQ software
version 1.3 of Applied Biosystems.
Confocal microscopy
Tissues were fixed in 4% paraformaldehyde and embedded in paraffin. After paraffin removal (Histolemon), 5lm thick sections were rehydrated in alcohol/distilled water. Micro-wave-assisted antigen retrieval was performed in 0.01M
sodium citrate (pH 6) for three cycles of 5 min (300 W) fol-lowed by cooling at 50°C and a final round of microwaving for 5 min, cooling and a rinse in PBS. Non-specific antigens were blocked by incubation in 10% goat serum in PBS for 2 h at 4°C. Sections were incubated overnight with the following primary antibodies: monoclonal anti-BrdU (NCL-BrdU, Novocastra, Nubloch, Germany), polyclonal anti-Ki67 (NCL-anti-Ki67p, Novocastra), polyclonal anti-MK14 (PRB-155P, Covance, Berkeley, CA, USA), polyclonal anti-MK10 (PRB-159P, Covance), polyclonal anti-MK6 169P, Covance), polyclonal anti-involucrin (PRB-140C, Covance), polyclonal anti-loricrin (PRB-145P, Cov-ance), monoclonal anti-p63 (MS-1084, Neomarkers), mono-clonal anti-c-Jun (SC-1694, Santa Cruz), monoclonal anti-JunB (sc-46, Santa Cruz), polyclonal anti-Notch (SC-6014, Santa Cruz). Sections were washed three times with PBS and incubated for 1 h with the secondary antibodies conjugated with Alexa Fluor 488 or 568 (Molecular Probes). Following two washes in PBS, the tissue sections were incu-bated for 5 min with 40,6-diamidino-2-phenylindole (DAPI) to reveal the nuclei. The tissue sections were then mounted using the Prolong Antifade Kit (Molecular Probes, Invitro-gen). Fluorescence was evaluated by confocal microscopy (Nikon, Tokyo, Japan, C1 on Eclipse TE200;EZC1 software)
fitted with an argon laser (488 nm excitation), an He/Ne laser (542 nm excitation) and UV excitation at 405 nm (DAPI staining) from a blue diode.
In vivo BrdU labeling
Pregnant mice were injected (intraperitoneally) with BrdU (50 mgkg1). For the cell proliferation assay, BrdU was delivered to pregnant mice 4 h before sacrifice. Three embryos for each genotype were analysed at E18.5.
Barrier function assay
For penetration assays, newborn mice were restrained in Petri dishes with their backs in contact with 1 mM Lucifer
yellow (Invitrogen) in PBS at 37 °C. After 1 h of incuba-tion, mice were sacrificed, and then the dorsal skin was fro-zen, sliced at a thickness of 5 lm and analysed by fluorescence microscopy. Toluidine blue dye staining of embryos was carried out as described [80]. In brief, embryos were dehydrated by incubations (1 min each) in 25%, 50% and 75% methanol/PBS followed by 1 min in 100% methanol. The embryos were then rehydrated with the same series of methanol solutions (1 min incubations), washed in PBS and stained for 1 min in 0.0125% toluidine blue O (Sigma)/PBS. After destaining in PBS, embryos were photographed. Epidermal inside-out barrier formation assay using the surface biotinylation technique was per-formed according to the method developed previously [81]. 50lL of 10 mgmL1 EZ-LinkTM
Sulfo-NHS-LC-Biotin (Pierce) in PBS containing 1 mM CaCl2 was injected into
the dermis on the back of the newborn mice. After 30 min incubation, the skin was taken out and embedded in OCT (Thermo Scientific, Cambridge, MA, USA). About 5lm thick frozen sections were fixed in 95% ethanol at 4°C for 30 min and then in 100% acetone at room temperature for 1 min. The sections were soaked in blocking solution for 30 min and incubated with Streptavidin Texas red (Onco-gene) for 30 min.
Analysis of wound closure
A 6 mm diameter full-thickness wound was performed on the dorsal midline of 8-week-old Itchy mice and age-matched wt controls, using a punch biopsy. The wound margin was traced on a transparent film put over the wound immediately after injury (day 0) and every second day thereafter. A blinded operator measured traced areas using a computer-assisted image analyser (Axiovision; Carl Zeiss, Jena, Germany). The rate of healing was expressed as the percentage of the area at day 0. Three independent experiments, each with five to six animals per group, were performed.
In vitro proliferation assay
The in vitro proliferation rate was determined by measuring BrdU incorporation with an ELISA kit (Roche, Indi-anapolis, IN, USA). Subconfluent keratinocytes were
trea-ted with 1lMBrdU for 16 h, and BrdU incorporation was
evaluated following the manufacturer’s instructions. Experi-ments were carried out in triplicate on three different cultures per genotype.
Migration assay
Keratinocyte migration was analysed using Boyden cham-bers (Neuroprobe, Gaithersburg, MD, USA) equipped with 8 mm pore diameter polycarbonate filters (Nuclepore, Whatman, Clifton, NJ, USA) coated with 0.125 mgmL1 of murine collagen type VI (BD). Five hundred thousand cells were suspended in Cnt-57 keratinocyte medium (CELLnTEC, Bern, Switzerland) and loaded into the upper compartment of the Boyden chambers. Analysis was per-formed after 16 h of incubation. Experiments were carried out in triplicate on three different cultures per genotype.
Scratch assay
Confluent monolayers of cultured primary keratinocytes obtained from newborn Itch/ mice and age-matched C57BL/10 mice were scratched with the tip of a P-200 pip-ette to create a uniform cell-free zone in each well. Cellular debris was removed by washing with PBS. Wounded mono-layers were observed at different time points after injury and pictures were taken with a digital camera at 0, 24 and 48 h. The residual gap area between keratinocyte edges was measured at 24 and 48 h with a computer-assisted analysis system (Axiovision; Carl Zeiss) and expressed as a percent-age of the initial scratched area (0 h). Experiments were performed in duplicate on six mice per genotype.
Wound histomorphometric analysis
Full-thickness excisional wounds made in the dorsum of 8-week-old Itchy and wt mice were harvested 5, 8 and 21 days after injury. Mice were housed individually during the healing period. For histological analysis the wounds surrounded by 1 mm of the epithelial margins were dis-sected, fixed overnight in 4% paraformaldehyde in PBS and embedded in paraffin. Sections (5lm) from the middle of the wound were stained with haematoxylin and eosin.
Epidermal hyperplasia in the scars was evaluated by measuring 10 different points along the epithelium covering the scar (scar thickness, S) and three different points in the non-wounded epidermis (epidermal thickness, E) at both specimen margins. Values were expressed as S/E9 100. For proliferation analysis, sections were treated with a
rab-bit polyclonal serum Ki67 antigen (NCLK67p,
Novocastra), diluted 1 : 800. Ki67-positive cells were evalu-ated in 10 fields per section (1009 magnification) by two blinded observers, by counting the number of stained nuclei of the epithelial tongues, at day 5 and day 8 after
wound-ing, or of the epidermis in scars, and were expressed as a percentage of the positive cell nuclei counted in the same areas. For the analysis of immune cells in the scars, immunohistochemical detection on paraffin sections was performed by using the following antibodies: for T cells, a polyclonal anti-human CD3 (A-0452, Dako; Glostrup, Denmark), diluted 1 : 100; for neutrophils, a polyclonal anti-human myeloperoxidase (A-0398; Dako), diluted 1 : 400; for macrophages, a monoclonal anti-mouse F4/80 antibody (MCA407R; Serotec, Puchheim, Germany), diluted 1 : 500. Two blinded observers independently analysed the dermal compartment for CD3-, F4/80- or myeloperoxidase-positive cell density by computer-assisted image analysis (Axioskop, Carl Zeiss) in 10 non-contiguous, randomly selected fields, at 409 magnification. Values are expressed as mean number of positive cells per field.
Statistical analysis
All numerical data are presented as mean standard devi-ation (SD). Statistical evaludevi-ation was performed using an unpaired Student’s t test. The mean values were considered significantly different when the probability of the differ-ences of that magnitude fell below 5% (P< 0.05).
Conflict of interest
The authors state no conflict of interest.
Acknowledgements
Research described in this article has been supported by AIRC grant (2013IG 13387) to EC. Partially sup-ported by Min. Salute (ric. Oncologica 26/07), ‘Istituto Dermopatico dell’Immacolata’ (RF0 c.73, RF07c.57, RF08c.15, .57) to GM.
Author contribution
AG-M: performed in vitro, in vivo experiment on dif-ferentiation. FC, CC, TO: performed experiments on wound and repair. AML, VS, AT: performed confocal analysis. NDD, GZ, GM: analyzed data. EC: planned experiments, analyzed data, wrote the paper.
References
1 Schwarz SE, Rosa JL & Scheffner M (1998) Characterization of human hect domain family
members and their interaction with UbcH5 and UbcH7. J Biol Chem273, 12148–12154.
2 Qiu L, Joazeiro C, Fang N, Wang H-Y, Elly C, Altman Y, Fang D, Hunter T & Liu YC (2000) Recognition and ubiquitination of Notch by Itch, a
hect-type E3 ubiquitin ligase. J Biol Chem 275, 35734–35737.
3 Marchese A, Raiborg C, Santini F, Keen JH, Stenmark H & Benovic JL (2003) The E3 ubiquitin ligase AIP4 mediates ubiquitination and sorting of the G protein-coupled receptor CXCR4. Dev Cell5, 709–722. 4 Rossi M, Rotblat B, Ansell K, Amelio I, Caraglia M,
Misso G, Bernassola F, Cavasotto CN, Knight RA, Ciechanover A et al. (2014) High throughput screening for inhibitors of the HECT ubiquitin E3 ligase ITCH identifies antidepressant drugs as regulators of autophagy. Cell Death Dis5, e1203.
5 Wegierski T, Hill K, Schaefer M & Walz G (2006) The HECT ubiquitin ligase AIP4 regulates the cell surface expression of select TRP channels. EMBO J25, 5659–5669.
6 Courbard JR, Fiore F, Adela€ıde J, Borg JP, Birnbaum D & Ollendorff V (2002) Interaction between two ubiquitin-protein isopeptide ligases of different classes, CBLC and AIP4/ITCH. J Biol Chem277, 45267–45275.
7 Oberst A, Malatesta M, Aqeilan RI, Rossi M, Salomoni P, Murillas R, Sharma P, Kuehn MR, Oren M, Croce CM et al. (2007) The Nedd4-binding partner 1 (N4BP1) protein is an inhibitor of the E3 ligase Itch. Proc Natl Acad Sci USA104, 11280–11285.
8 Mazza D, Infante P, Colicchia V, Greco A, Alfonsi R, Siler M, Antonucci L, Po A, De Smaele E, Ferretti E et al.(2013) PCAF ubiquitin ligase activity inhibits Hedgehog/Gli1 signaling in p53-dependent response to genotoxic stress. Cell Death Differ20, 1688–1697. 9 Khedgikar V, Kushwaha P, Gautam J, Verma A,
Changkija B, Kumar A, Sharma S, Nagar GK, Singh D, Trivedi PK et al. (2013) Withaferin A: a
proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone. Cell Death Dis4, e778.
10 Perry WL, Hustad CM, Swing DA, O’Sullivan TN, Jenkins NA & Copeland NG (1998) The itchy locus encodes a novel ubiquitin protein ligase that is disrupted in 18H mice. Nat Genet18, 143–146. 11 Melino G, Gallagher E, Aqeilan RI, Knight R,
Peschiaroli A, Rossi M, Scialpi F, Malatesta M, Zocchi L, Browne G et al. (2008) Itch: a HECT-type E3 ligase regulating immunity, skin and cancer. Cell Death Differ 15, 1103–1112.
12 Fang D, Elly C, Gao B, Fang N, Altman Y, Joazeiro C, Hunter T, Copeland N, Jenkins N & Liu YC (2002) Dysregulation of T lymphocyte function in itchy mice: a role for Itch in TH2 differentiation. Nat Immunol3, 281–287.
13 Bai Y, Yang C, Hu K, Elly C & Liu YC (2004) Itch E3 ligase-mediated regulation of TGF-beta signaling by modulating smad2 phosphorylation. Mol Cell15, 825–831.
14 Rossi M, Aqeilan RI, Neale M, Candi E, Salomoni P, Knight RA, Croce CM & Melino G (2006) The E3 ubiquitin ligase Itch controls the protein stability of p63. Proc Natl Acad Sci USA103, 12753–12758. 15 Candi E, Rufini A, Terrinoni A, Dinsdale D, Ranalli
M, Paradisi A, De Laurenzi V, Spagnoli LG, Catani MV, Ramadan S et al. (2006) Differential roles of p63 isoforms in epidermal development: selective genetic complementation in p63 null mice. Cell Death Differ13, 1037–1047.
16 Candi E, Schmidt R & Melino G (2005) The cornified envelope: a model of cell death in the skin. Nat Rev Mol Cell Biol6, 328–340.
17 Candi E & Melino G (2013) The human squamous epithelial cell envelope: the structural model by Peter M Steinert. Cell Death Differ20, 965–966.
18 Gao M, Labuda T, Xia Y, Gallagher E, Fang D, Liu YC & Karin M (2004) Jun turnover is controlled through JNK-dependent phosphorylation of the E3 ligase Itch. Science306, 271–275.
19 Macdonald JM, Doherty J, Hackett R & Freeman MR (2013) The c-Jun kinase signaling cascade promotes glial engulfment activity through activation of draper and phagocytic function. Cell Death Differ20, 1140–1148.
20 Tanigawa S, Lee CH, Lin CS, Ku CC, Hasegawa H, Qin S, Kawahara A, Korenori Y, Miyamori K, Noguchi M et al. (2013) Jun dimerization protein 2 is a critical component of the Nrf2/MafK complex
regulating the response to ROS homeostasis. Cell Death Dis4, e921.
21 Chari NS, Romano RA, Koster MI, Jaks V, Roop D, Flores ER, Teglund S, Sinha S, Gruber W, Aberger F et al.(2013) Interaction between the TP63 and SHH pathways is an important determinant of epidermal homeostasis. Cell Death Differ20, 1080–1088. 22 Chakravarti D, Su X, Cho MS, Bui NH, Coarfa C,
Venkatanarayan A, Benham AL, Flores Gonzalez RE, Alana J, Xiao W et al. (2014) Induced multipotency in adult keratinocytes through down-regulation ofDNp63 or DGCR8. Proc Natl Acad Sci USA111, E572–E581. 23 Paris M, Rouleau M, Puceat M & Aberdam D (2012)
Regulation of skin aging and heart development by TAp63. Cell Death Differ19, 186–193.
24 Tucci P, Agostini M, Grespi F, Markert EK, Terrinoni A, Vousden KH, Muller PA, D€otsch V, Kehrloesser S, Sayan BS et al. (2012) Loss of p63 and its microRNA-205 target results in enhanced cell migration and metastasis in prostate cancer. Proc Natl Acad Sci USA 109, 15312–15317.
25 Wang F, Liu X, Yang P, Guo L, Liu C, Li H, Long S, Shen Y & Wan H (2014) Loss of TACSTD2
contributed to squamous cell carcinoma progression through attenuating TAp63-dependent apoptosis. Cell Death Dis5, e1133.
26 Yallowitz AR, Alexandrova EM, Talos F, Xu S, Marchenko ND & Moll UM (2014) p63 is a
prosurvival factor in the adult mammary gland during post-lactational involution, affecting PI-MECs and ErbB2 tumorigenesis. Cell Death Differ21, 645–654. 27 Zhang X, Dong X, Wang H, Li J, Yang B, Zhang J &
Hua ZC (2014) FADD regulates thymocyte development at theb-selection checkpoint by modulating Notch signaling. Cell Death Dis5, e1273. 28 Christiansen AE, Ding T, Fan Y, Graves HK, Herz
HM, Lindblad JL & Bergmann A (2013) Non-cell autonomous control of apoptosis by ligand-independent Hedgehog signaling in Drosophila. Cell Death Differ 20, 302–311.
29 Cortegano I, Melgar-Rojas P, Luna-Zurita L, Siguero-Alvarez M, Marcos MA, Gaspar ML & De la Pompa JL (2014) Notch1 regulates progenitor cell proliferation and differentiation during mouse yolk sac
hematopoiesis. Cell Death Differ21, 1081–1094. 30 Usui ML, Underwood RA, Mansbridge JN, Muffley
LA, Cartes WG & Olerud JE (2005) Morphological evidence for the role of suprabasal keratinocytes in wound reepithelialisation. Wound Rep Reg13, 468–479. 31 Zaid A, Mackay LK, Rahimpour A, Braun A,
Veldhoen M, Carbone FR, Manton JH, Heath WR & Mueller SN (2014) Persistence of skin-resident memory T cells within an epidermal niche. Proc Natl Acad Sci USA111, 5307–5312.
32 Frauenstein K, Sydlik U, Tigges J, Majora M, Wiek C, Hanenberg H, Abel J, Esser C, Fritsche E, Krutmann J et al.(2013) Evidence for a novel anti-apoptotic pathway in human keratinocytes involving the aryl hydrocarbon receptor, E2F1, and checkpoint kinase 1. Cell Death Differ20, 1425–1434.
33 Dai D, Li L, Huebner A, Zeng H, Guevara E, Claypool DJ, Liu A & Chen J (2013) Planar cell polarity effector gene Intu regulates cell fate-specific differentiation of keratinocytes through the primary cilia. Cell Death Differ20, 130–138.
34 Dannappel M, Vlantis K, Kumari S, Polykratis A, Kim C, Wachsmuth L, Eftychi C, Lin J, Corona T,
Hermance N et al. (2014) RIPK1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis. Nature513, 90–94.
35 Dai W, Bai Y, Hebda L, Zhong X, Liu J, Kao J & Duan C (2014) Calcium deficiency-induced and TRP channel-regulated IGF1R-PI3K-Akt signaling regulates abnormal epithelial cell proliferation. Cell Death Differ 21, 568–581.
36 Driskell RR, Lichtenberger BM, Hoste E, Kretzschmar K, Simons BD, Charalambous M, Ferron SR, Herault Y, Pavlovic G, Ferguson-Smith AC et al. (2013) Distinct fibroblast lineages determine dermal architecture in skin development and repair. Nature 504, 277–281.
37 V€orsmann H, Groeber F, Walles H, Busch S, Beissert S, Walczak H & Kulms D (2013) Development of a human three-dimensional organotypic skin-melanoma spheroid model for in vitro drug testing. Cell Death Dis11, e719. 38 Melino G, Knight RA & Nicotera P (2005) How many
ways to die? How many different models of cell death?. Cell Death Differ12 (Suppl. 2), 1457–1462.
39 De Craene B, Denecker G, Vermassen P, Taminau J, Mauch C, Derore A, Jonkers J, Fuchs E & Berx G (2014) Epidermal Snail expression drives skin cancer initiation and progression through enhanced cytoprotection, epidermal stem/progenitor cell expansion and enhanced metastatic potential. Cell Death Differ21, 310–320.
40 Geserick P, Wang J, Feoktistova M & Leverkus M (2014) The ratio of Mcl-1 and Noxa determines ABT737 resistance in squamous cell carcinoma of the skin. Cell Death Dis5, e1412.
41 Ouyang H, Xue Y, Lin Y, Zhang X, Xi L, Patel S, Cai H, Luo J, Zhang M, Zhang M et al. (2014) WNT7A and PAX6 define corneal epithelium homeostasis and pathogenesis. Nature511, 358–361.
42 Yamamoto-Tanaka M, Makino T, Motoyama A, Miyai M, Tsuboi R & Hibino T (2014) Multiple pathways are involved in DNA degradation during keratinocyte terminal differentiation. Cell Death Dis5, e1181. 43 Vandenberghe M, Rapha€el M, Lehen’kyi V, Gordienko
D, Hastie R, Oddos T, Rao A, Hogan PG, Skryma R & Prevarskaya N (2013) ORAI1 calcium channel orchestrates skin homeostasis. Proc Natl Acad Sci U S A110, E4839–E4848.
44 Hiebert PR, Wu D & Granville DJ (2013) Granzyme B degrades extracellular matrix and contributes to delayed wound closure in apolipoprotein E knockout mice. Cell Death Differ20, 1404–1414.
45 Bossi F, Tripodo C, Rizzi L, Bulla R, Agostinis C, Guarnotta C, Munaut C, Baldassarre G, Papa G, Zorzet S et al. (2014) C1q as a unique player in angiogenesis with therapeutic implication in wound healing. Proc Natl Acad Sci USA 111, 4209–4214.
46 Bourdeaux C, Lurquin C, Jacquemart I, Lethe B, Brasseur F, van Baren N, Baurain JF, Dyson J, Van Snick J, Uyttenhove C et al. (2014) Local
immunostimulation leading to rejection of accepted male skin grafts by female mice as a model for cancer immunotherapy. Proc Natl Acad Sci USA111, 3502–3507.
47 Hsu I, Parkinson LG, Shen Y, Toro A, Brown T, Zhao H, Bleackley RC & Granville DJ (2014) Serpina3n accelerates tissue repair in a diabetic mouse model of delayed wound healing. Cell Death Dis5, e1458. 48 Liu H, Duan Z, Tang J, Lv Q, Rong M & Lai R
(2014) A short peptide from frog skin accelerates diabetic wound healing. FEBS J281, 4633–4643.
49 Feng G, Hao D & Chai J (2014) Processing of CXCL12 impedes the recruitment of endothelial progenitor cells in diabetic wound healing. FEBS J281, 5054–5062.
50 Levine AJ, Tomasini R, McKeon FD, Mak TW & Melino G (2011) The p53 family: guardians of maternal reproduction. Nat Rev Mol Cell Biol12, 259–265.
51 M€uller M, Schleithoff ES, Stremmel W, Melino G, Krammer PH & Schilling T (2006) One, two, three– p53, p63, p73 and chemosensitivity. Drug Resist Updat 9, 288–306.
52 Tomasini R, Mak TW & Melino G (2008) The impact of p53 and p73 on aneuploidy and cancer. Trends Cell Biol18, 244–252.
53 Adamovich Y, Adler J, Meltser V, Reuven N & Shaul Y (2014) AMPK couples p73 with p53 in cell fate decision. Cell Death Differ21, 1451–1459.
54 Hoffman Y, Bublik DR, Pilpel Y & Oren M (2014) miR-661 downregulates both Mdm2 and Mdm4 to activate p53. Cell Death Differ21, 302–309. 55 Chen YC, Chan JY, Chiu YL, Liu ST, Lozano G,
Wang SL, Ho CL & Huang SM (2013) Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis. Cell Death Differ20, 732–743.
56 Amelio I, Inoue S, Markert EK, Levine AJ, Knight RA, Mak TW & Melino G (2015) TAp73 opposes tumor angiogenesis by promoting hypoxia-inducible factor 1a degradation. Proc Natl Acad Sci USA 112, 226–231.
57 Amelio I, Lena AM, Bonanno E, Melino G & Candi E (2013) miR-24 affects hair follicle morphogenesis targeting Tcf-3. Cell Death Dis4, e922.
58 Liu J, Zhang C, Wang XL, Ly P, Belyi V, Xu-Monette ZY, Young KH, Hu W & Feng Z (2014) E3 ubiquitin ligase TRIM32 negatively regulates tumor suppressor p53 to promote tumorigenesis. Cell Death Differ21, 1792–1804.
59 Kim J, Nakasaki M, Todorova D, Lake B, Yuan CY, Jamora C & Xu Y (2014) p53 induces skin aging by depleting Blimp1+ sebaceous gland cells. Cell Death Dis5, e1141.
60 Romano RA, Smalley K, Magraw C, Serna VA, Kurita T, Raghavan S & Sinha S (2012)DNp63 knockout mice reveal its indispensable role as a master regulator of epithelial development and differentiation. Development 139, 772–782.
61 Blanpain C, Lowry WE, Pasolli HA & Fuchs E (2006) Canonical notch signaling functions as a commitment switch in the epidermal lineage. Genes Dev20, 3022–3035.
62 Shaulian E & Karin M (2001) AP-1 in cell proliferation and survival. Oncogene20, 2390–2400.
63 Jochum W, Passegue E & Wagner EF (2001) AP-1 in mouse development and tumorigenesis. Oncogene20, 2401–2412.
64 Angel P, Szabowski A & Schorpp-Kistner M (2001) Function and regulation of AP-1 subunits in skin physiology and pathology. Oncogene20, 2413–2423. 65 Liew FM & Yamanishi K (1992) Regulation of
transglutaminase 1 gene expression by
12-O-tetradecanoylphorbol-13-acetate, dexamethasone, and retinoic acid in cultured human keratinocytes. Exp Cell Res202, 310–315.
66 Lu B, Rothnagel JA, Longley MA, Tsai SY & Roop DR (1994) Differentiation-specific expression of human keratin 1 is mediated by a composite AP-1/steroid hormone element. J Biol Chem269, 7443–7449. 67 Casatorres J, Navarro JM, Blessing M & Jorcano JL
(1994) Analysis of the control of expression and tissue specificity of the keratin 5 gene, characteristic of basal keratinocytes. Fundamental role of an AP-1 element. J Biol Chem269, 20489–20496.
68 Welter JF & Eckert RL (1995) Differential expression of the fos and jun family members c-fos, fosB, Fra-1, Fra-2, c-jun, junB and junD during human epidermal keratinocyte differentiation. Oncogene11, 2681–2687. 69 DiSepio D, Jones A, Longley MA, Bundman D,
Rothnagel JA & Roop DR (1995) The proximal promoter of the mouse loricrin gene contains a functional AP-1 element and directs keratinocyte-specific but not differentiation-keratinocyte-specific expression. J Biol Chem270, 10792–10799.
70 Jang SI, Steinert PM & Markova NG (1996) Activator protein 1 activity is involved in the regulation of the cell type-specific expression from the proximal promoter of the human profilaggrin gene. J Biol Chem271, 24105–24114. 71 Li G, Gustafson-Brown C, Hanks SK, Nason K,
Arbeit JM, Pogliano K, Wisdom RM & Johnson RS (2003) c-Jun is essential for organization of the epidermal leading edge. Dev Cell4, 865–877. 72 Zenz R, Scheuch H, Martin P, Frank C, Eferl R,
Kenner L, Sibilia M & Wagner EF (2003) c-Jun regulates eyelid closure and skin tumor development through EGFR signaling. Dev Cell4, 879–889. 73 Zenz R, Eferl R, Kenner L, Florin L, Hummerich L,
Mehic D, Scheuch H, Angel P, Tschachler E & Wagner EF (2005) Psoriasis-like skin disease and arthritis caused by inducible epidermal deletion of Jun proteins. Nature437, 369–375.
74 Choi YH, Kim YJ, Jeong HM, Jin YH, Yeo CY & Lee KY (2014) Akt enhances Runx2 protein stability by regulating Smurf2 function during osteoblast differentiation. FEBS J281, 3656–3666.
75 Bhattacharya S & Ghosh MK (2014) HAUSP, a novel deubiquitinase for Rb– MDM2 the critical regulator. FEBS J281, 3061–3078.
76 Sun P, Watanabe K, Fallahi M, Lee B, Afetian ME, Rheaume C, Wu D, Horsley V & Dai X (2014) Pygo2 regulatesb-catenin-induced activation of hair follicle stem/progenitor cells and skin hyperplasia. Proc Natl Acad Sci USA111, 10215–10220.
77 Riol-Blanco L, Ordovas-Montanes J, Perro M, Naval E, Thiriot A, Alvarez D, Paust S, Wood JN & Von Andrian UH (2014) Nociceptive sensory neurons drive interleukin-23-mediated psoriasiform skin inflammation. Nature510, 157–161.
78 KhmaladzeIa Kelkka T, Guerard S, Wing K, Pizzolla A, Saxena A, Lundqvist K, Holmdahl M, Nandakumar KS & Holmdahl R (2014) Mannan induces ROS-regulated, IL-17A-dependent psoriasis arthritis-like disease in mice. Proc Natl Acad Sci USA111, E3669–E3678.
79 Lichti U, Anders J & Yuspa SH (2008) Isolation and short-term culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. Nat Protoc3, 799–810.
80 Hardman MJ, Sisi P, Banbury DN & Byrne C (1998) Patterned acquisition of skin barrier function during development. Development125, 1541–1552.
81 Chen M, Marinkovich MP, Veis A, Cai X, Rao CN, O’Toole EA & Woodley DT (1997) Interactions of the amino-terminal noncollagenous (NC1) domain of type VII collagen with extracellular matrix components. A potential role in epidermal-dermal adherence in human skin. J Biol Chem272, 14516–14522.
