Delayed Wound Healing and Epidermal Hyperproliferation in ... - Core

ORIGINAL ARTICLE

Delayed Wound Healing and Epidermal Hyperproliferation in Mice Lacking JunB in the Skin Lore Florin1, Julia Knebel1, Paola Zigrino2, Birgitta Vonderstrass1, Cornelia Mauch2, Marina Schorpp-Kistner1, Axel Szabowski1 and Peter Angel1 The cutaneous response to injury and stress comprises a temporary change in the balance between epidermal proliferation and differentiation as well as an activation of the immune system. Soluble factors play an important role in the regulation of these complex processes by coordinating the intercellular communication between keratinocytes, fibroblasts, and inflammatory cells. In this study, we demonstrate that JunB, a member of the activator protein-1 transcription factor family, is an important regulator of cytokine expression and thus critically involved in the cutaneous response to injury and stress. Mice lacking JunB in the skin develop normally, indicating that JunB is neither required for cutaneous organogenesis, nor homeostasis. However, upon wounding and treatment with the phorbol ester 12-O-decanoyl-phorbol-13-acetate, JunB-deficiency in the skin likewise resulted in pronounced epidermal hyperproliferation, disturbed differentiation, and prolonged inflammation. Furthermore, delayed tissue remodelling was observed during wound healing. These phenotypic skin abnormalities were associated with JunB-dependent alterations in expression levels and kinetics of important mediators of wound repair, such as granulocyte macrophage colony-stimulating factor, growthregulated protein-1, macrophage inflammatory protein-2, and lipocalin-2 in both the dermal and epidermal compartment of the skin, and a reduced ability of wound contraction of mutant dermal fibroblasts in vitro. Journal of Investigative Dermatology (2006) 126, 902–911. doi:10.1038/sj.jid.5700123; published online 26 January 2006

INTRODUCTION Formation and maintenance of the skin epithelium, which seals the body from the outside world, are obtained through the tightly controlled balance between proliferation and terminal differentiation of keratinocytes. As the epidermis is a frequent target of tissue damage as well as chemical and mechanical stress, this dynamic equilibrium has perpetually to be adapted to meet the actual requirements. After cutaneous wounding, keratinocytes acquire an activated state in which proliferation is favored over differentiation in order to replenish the lost material and rapidly close the site of injury (for a review, see Martin, 1997; Singer and Clark, 1999). Subsequently, tissue architecture within the healing neo-epidermis has to be re-established, which is achieved by the switch from keratinocyte proliferation to the apoptosislike program of terminal differentiation. The temporally controlled sequential events occurring during skin regenera1

Division of Signal Transduction and Growth Control, Deutsches Krebsforschungszentrum, Heidelberg, Germany and 2Department of Dermatology and Venerology, University of Cologne, Cologne, Germany Correspondence: Professor Peter Angel, Division of Signal Transduction and Growth Control, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. E-mail: [email protected] Abbreviations: AP-1, activator protein-1; ECM, extracellular matrix; Gro-1, growth-regulated protein-1; K10, Keratin 10; Lcn-2, lipocalin-2; Mip-2, macrophage inflammatory protein-2; TPA, 12-O-decanoyl-phorbol-13-acetate Received 20 April 2005; revised 24 October 2005; accepted 31 October 2005; published online 26 January 2006

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tion and the cutaneous stress response rely on complex interactions of many different cell types including keratinocytes, fibroblasts, and immune cells. Their concerted actions are coordinated through the mutual exchange of soluble cytokines, most of which are not expressed in healthy skin but are rapidly induced under conditions of tissue challenge (for a review, see Gillitzer and Goebeler, 2001; Werner and Grose, 2003). There is considerable evidence that the transcription factor activator protein-1 (AP-1) plays an important role in the skin: involvement in keratinocyte differentiation was suggested as AP-1 activity is induced during this process (Rutberg et al., 1996) and binding sites have been mapped within the promoters of genes encoding keratinocyte-specific proteins such as keratins as well as the differentiation markers involucrin, profilaggrin, loricrin, and transglutaminase (for a review, see Eckert et al., 1997; Angel et al., 2001). In fibroblasts, expression of extracellular matrix (ECM), components such as col1a2, laminin B1 and g2, fibronectin, and vimentin appears to be controlled by AP-1 (Angel et al., 2001; Vogt, 2001) and this list could be further elongated in recent studies using large-scale gene expression profiling (Leaner et al., 2003; Nowinski et al., 2004; Florin et al., 2004b). Moreover, various cytokines, such as heparinbinding epidermal growth factor, granulocyte macrophage colony-stimulating factor (GM-CSF), keratinocyte growth factor (KGF) and TGF-b, and proteins involved in cutaneous wound healing, for example, decorin, tenascin-C, several & 2006 The Society for Investigative Dermatology

L Florin et al. Wound Healing in JunB Mutant Mice

RESULTS COL1A2-Cre junB mice overcome embryonic lethality

To investigate the role of JunB in skin development, homeostasis, and during tissue regeneration, we generated conditional JunB-deficient mice to circumvent the lethality of junB/ embryos (Schorpp-Kistner et al., 1999). Mice harboring floxed junB alleles (junBf/f; Kenner et al., 2004) or carrying a single floxed and one inactivated junB-Neo allele (junBf/; Figure 1a) were crossed with COL1A2-Cre mice, where Cre expression is driven by the Collagen type (I)-

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integrins, and matrix metalloproteinases harbor AP-1 sites within their promoters (for a review, see Benbow and Brinckerhoff, 1997; Angel et al., 2001; Yates and Rayner, 2002). The transcription factor AP-1 consists of a variety of homoand heterodimers, which are built up by members of the Jun (c-Jun, JunB, JunD), Fos (c-Fos, FosB, Fra1, Fra2), and ATF families (AFT2, ATF3, ATF4, ATFa; Shaulian and Karin, 2002). In response to a multitude of extracellular stimuli or activating oncogenes, AP-1 regulates transcription of different sets of target genes depending on its subunit composition and the cellular context (Whitmarsh and Davis, 1996; MechtaGrigoriou et al., 2001). Although the use of genetically engineered mice has provided considerable insight into the specific functions of individual AP-1 subunits (Jochum et al., 2001; Hess et al., 2004), little is known about the specific role of junB in the skin. During embryonic skin formation, junB expression starts around E17.5 and was localized in dermal cells and in basal and granular layers of the epidermis (Wilkinson et al., 1989; Rutberg et al., 1996; Mehic et al., 2005). Functional studies in organotypic skin equivalents in vitro revealed, that junB in dermal fibroblasts modulates proliferation and differentiation of the keratinocyte epithelium in trans through paracrine mesenchymal–epithelial interactions (Szabowski et al., 2000). Loss-of-function approaches in vivo, however, have been hampered as conventional gene-targeting results in early embryonic lethality due to defective placental vascularization (Schorpp-Kistner et al., 1999). However, junB/Ubi-junB mice, which express a junB transgene during embryogenesis under the control of the human Ubiquitin-C promoter (SchorppKistner et al., 1999; Hartenstein et al., 2002), survive but were reported to develop a chronic myeloid leukemia (CML)-like myeloproliferative disease (Passegue et al., 2001, 2004). Epiblast-specific junB deletion – with the limitation of possible Cre mosaicism (Tallquist and Soriano, 2000; Hayashi and McMahon, 2002) – likewise rescued embryonic lethality (Kenner et al., 2004). Yet, detailed analyses of the skin of JunB-deficient mice have not been performed. In this study, we describe the generation of conditional JunB knockout mice using Cre/lox-mediated deletion of junB in tissues expressing Collagen (I)-alpha2-Cre and report on the characterization of their phenotype during skin development and homeostasis as well as during the cutaneous response to injury and stress.

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Figure 1. Ubiquitous junB deletion and growth retardation in conditional junB knockout mice. (a) Schematic representation of mutant junB alleles, comprising the junB-Neo fusion (), the floxed (f) and deleted (D) loci. The junB coding sequence is represented by an open box, the start site of transcription is indicated by an arrow; loxP sites are shown as triangles. Positions of the probe used for Southern hybridization and the primers for genotyping (B1, B6, B16, B49) are indicated. A, AccI; E, EcoR1; H, HindIII; P, PstI; S, SmaI; X, XhoI; Xb, XbaI. (b) Southern blot analysis showing Cre-mediated ubiquitous junB deletion. A 176 bp PstI/HindIII probe was used to detect the genomic junB locus digested with PstI. (c) Genomic PCR confirming complete recombination of the junB allele in the whole skin and in the bone of junBD/D mice. Positions of oligonucleotide primers are depicted in (a). (d) Semiquantitative RT-PCR on RNA isolated from the dermis and epidermis of junB mutant and control animals following TPA treatment. Collagen type I-alpha2 (co1a2) was used as control for tissue separation. cDNA levels were standardized to hprt quantities. (e) Immunofluorescence staining of JunB in TPA-treated skin of control and junB mutant animals, nuclei were counterstained using Hoechst dye. The residual unspecific signal in the dermis of junBD/D mice originates from enucleated erythrocytes. Bar ¼ 100 mm.

alpha2 promoter (Florin et al., 2004a) starting at E8.5–9.5 (Adamson and Ayers, 1979; Leivo et al., 1980; Khillan et al., 1986). Conditional COL1A2-Cre junB mutant mice (junBD/D) were born with Mendelian frequencies and survived to adulthood. Mutant mice differed from their littermates by their shaggy fur as well as reduced size and body weight (Figure S1) reminiscent of animals displaying strongly reduced junB expression in bone, such as junB/Ubi-junB (Hess et al., 2003) and Mox2-Cre junBf/f mice (Kenner et al., 2004). As we never observed any differences between Cre junBD/D and junBD/ mice, the mutants were comparably used for the experiments. www.jidonline.org

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Assessment of the extent of junB recombination in various organs of conditional junB knockout mice by Southern blot analysis revealed deletion of the floxed junB allele in all organs examined (Figure 1b). Furthermore, ubiquitous junB excision was confirmed by the more sensitive PCR method with only minor amounts of the unrecombined junB locus being detectable in the spleen, thymus, and liver (Figure 1c). Recombination of the junB allele was also detected in peripheral blood lymphocytes and neutrophil granulocytes (data not shown). In the skin, the junB gene was completely recombined in the epidermis and dermis (Figure 1b and c). Accordingly, neither junB transcripts (Figure 1d) nor JunB protein (Figure 1e) were detectable in mutant mice, even after successive applications of the phorbol ester 12-O-decanoylphorbol-13-acetate (TPA) confirming that both, fibroblasts and keratinocytes of the skin were junB-deficient. Normal skin development and homeostasis in the absence of junB

Histological examination of embryonic back skin at E17.5 (Figure 2a), and tail skin of 6-week-old adult mice (Figure 2b)

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revealed normal tissue development in junBD/D mice. No differences in the overall skin architecture of junB mutant and control mice were observed and the distribution of hair follicles as well as the number and differentiation of epithelial layers was not disturbed. Obviously, in junBD/D mice, JunBdeficiency in both fibroblasts and keratinocytes did neither impair skin development nor homeostasis, suggesting that JunB might either not be required for these processes or that its deletion can be functionally compensated. TPA-induced hyperproliferation

In 3D-co-cultures of fibroblasts and keratinocytes, we could demonstrate that the loss of JunB in fibroblasts modulates keratinocyte growth through paracrine mechanisms resulting in disturbed epithelial differentiation and hyperplasia (Szabowski et al., 2000). However, these in vitro skin equivalents do not reflect tissue homeostasis, but rather represent a model system for wound healing and thus skin challenge, where AP-1 activity is induced. To investigate whether also in vivo the cutaneous response to stress was affected by the loss of JunB, we topically treated the skin of junBD/D and control mice with the phorbol ester TPA, a strong inducer of AP-1 activity (Angel and Karin, 1991; Young et al., 1999; Gallucci et al., 2000). In junBf/D control mice, three successive applications of TPA in intervals of 48 hours resulted in a gain in epidermal thickness (Figure 3) with a concomitant increase in Ki67positive proliferating keratinocytes in basal and suprabasal layers in comparison to untreated animals. However, TPAtreated skin of junB mutant mice was even thicker, palpably hardened, and the number of Ki67-positive cells in suprabasal layers of the epidermis was further increased. Moreover, the irregular signal of the keratinocyte differentiation marker Keratin 10 (K10) indicated disturbances of the differentiation process. Furthermore, in the dermis of junBD/D mice, infiltration of neutrophils in response to TPA was enhanced, whereas the amount of invading macrophages appeared

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Figure 2. Normal skin development and homeostasis in the absence of junB. (a) Histological sections of embryonic back skin (E17.5) of control (junBf/D) and junB mutant mice (junBD/D). (b) Examination of tissue architecture in the tail skin of 6-week-old junBD/D and control animals. Sections were stained with hematoxylin/eosin (H&E) and immunofluorescence for the epidermal marker proteins K10 and Keratin 6 was performed. Bar ¼ 100 mm.

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Figure 3. Analysis of TPA-induced hyperproliferation in junBD/D mice. Histological examination of solvent (control) and TPA-treated back skin of conditional junB knockout (junBD/D) and control mice (junBf/D) by staining for Ki67 and K10 to visualize keratinocyte proliferation and differentiation, respectively. Staining of neutrophils demonstrated an increased inflammatory response in TPA-treated skin of junB mutant mice. Bar ¼ 100 mm.


similar in mutant and control animals (Figure 3 and data not shown). Thus, treatment of junBD/D mice led to increased keratinocyte hyperproliferation and irregular differentiation, thus recapitulating the phenotype of in vitro skin equivalents containing junB/ fibroblasts, and enhanced inflammation. Delayed tissue remodelling in wounded skin of junBD/D mice

In contrast to topical application of phorbol esters, wounding of the skin represents a physiological stimulus which rapidly induces AP-1 activity (Martin and Nobes, 1992; Shirai et al., 2001; Gangnuss et al., 2004). As cutaneous wound healing is a complex and dynamic process consisting of various sequential phases (Martin, 1997; Singer and Clark, 1999; Gillitzer and Goebeler, 2001), the wound repair was carefully analyzed over all stages of healing in order to elucidate the consequences of JunB deletion with respect to quality and the kinetics of tissue regeneration (Figure 4). Full-thickness excisional wounds were generated in the back skin of junB mutant and control mice and the healing process was analyzed at days 1, 2, 5, 7, 10, 14, 21, and 28 post-injury (nX5 for each time point and genotype). As already visible macroscopically, wound healing in junBD/D mice was markedly delayed compared to those of control littermates (Figure 4). Histological examination revealed normal keratinocyte migration into the wound field and subsequent fusion of epithelial leading edges in both wildtype and junBD/D wounds occurred comparably at days 5–7 (Figure 5a). Yet, wounds of junB mutant mice progressed markedly later into the final remodelling phase, which is characterized by an involution of the hyperplastic neoepi-

dermis, wound contraction, and dermal collagen reorganization, and did not normalize until day 28. Instead, mutant epidermis remained hyperplastic for up to 3 weeks (Figure 5b) and the volume of the granulation tissue stayed nearly constant for at least 4 weeks, as demonstrated by measuring the area between the flanking hair follicles (Figures 4 and 5c). An enlargement of the granulation tissue might be explained by alterations in the composition and remodelling of the ECM because of differences in the number and/or activity of myofibroblasts (Tomasek et al., 2002). When we stained sections with picrofuchsin (Figure S2a) and estimated the ratio of total collagen versus highly organized collagen fibers, no major differences in collagen organization were observed between wounds of knockout mice and their control littermates. Moreover, the overall cell density within the granulation tissue was not significantly different between wild-type and mutant mice and staining of wounds with asmooth muscle actin, a marker for differentiated myofibroblasts, at days 5–7 (Figure S2b and data not shown) did not reveal obvious differences in the amount and distribution of a-smooth muscle actin-positive contractile cells. To directly compare the ability of this type of cells to reorganize and contract ECM, primary fibroblasts were isolated from the skin

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Figure 4. Wound healing is delayed in junBD/D mice. The healing process of 4 mm full-thickness excisional wounds in the back skin of junB mutant and control mice was followed over 28 days (n ¼ 3 per genotype and time point, 1–3 wounds of one animal were analyzed). Photomicrographs and histological sections demonstrated impaired wound healing and epidermal hyperproliferation following re-epithelialization in junBD/D mice. Wound edges are indicated by arrowheads. Bar ¼ 1 mm.


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Figure 5. Analysis of the wound healing phenotype. (a) Closure of wounds from junB mutant and control mice at days 2, 5, and 7 (n ¼ 5–7 wounds per genotype and time point) was judged microscopically and the number of open wounds was calculated in percent. (b, c) Thickness of the neo-epidermis (b) and granulation tissue volume (c) of junB mutant (black diamonds) and control wounds (open diamonds) were quantified and plotted as mean7 standard deviation. (d) Quantification of the cell density within the granulation tissue of control (white bars) and junB mutant wounds (black bars) at the indicated days after wounding. Three to five wounds were analyzed per genotype and time point, bars represent the mean7standard deviation. (e) Measurement of collagen gel contraction by primary junB mutant and control fibroblasts. Primary fibroblasts from adult junBD/D and control animals were cultured in collagen gels and at the indicated time points the gel surface area was measured (as the average of three random diameters of each gel). Contraction is indicated as percentage of the initial gel surface area, set arbitrarily as 100%, of quadruplicate gels. The results represent the mean7standard deviation of one representative experiment out of three.

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of newborn and 8-week-old mice and tested in 3D collagen I gels, which is regarded as an in vitro model to determine the reorganization of connective tissue during wound healing (Mauch et al., 1988; Tomasek and Akiyama, 1992). Indeed, dermal fibroblasts from newborn (data not shown) and adult mutant mice (Figure 5e) exhibited a significantly reduced capacity to contract collagen lattices, suggesting that in the absence of junB neither collagen remodelling nor myofibroblast differentiation is impaired, but rather defects in myofibroblast-dependent wound contraction might contribute to the phenotype of delayed wound healing in JunBdeficient mice. Loss of junB results in hyperproliferation of the neo-epidermis

In addition to these differences in wound contraction, the most prominent phenotype in junB mutant mice was the pronounced hyperplasia of the epidermis between days 7 and 21 post-wounding. In wild-type mice, fusion of the migrating epithelial edges after wounding was achieved between days 5 and 7. As shown in Figures 5b and 6a, the re-established

epidermis of control mice showed a mild hyperproliferative phenotype, which decreased around day 10 until epithelial thickness was reduced to nearly normal levels by day 14. By contrast, the neoepidermis of junBD/D wounds did not involute after day 7, was markedly thicker compared to controls by day 10, and the hyperplasia persisted at least until day 21 before reaching normal proportions by day 28 postwounding (Figures 4 and 5b). More detailed histological examination of wounds at day 7, when epidermal thickness was still comparable in JunB mutant and control mice, revealed that already at this stage, the neoepidermis of junBD/D mice was characterized by an increased number of proliferating keratinocytes in suprabasal layers as demonstrated by Ki67 staining (Figure 7a). Correspondingly, these cells did not express the keratinocyte differentiation marker K10, which was present only in the uppermost layers, whereas in control wounds, all suprabasal layers of the neo-epidermis were K10 positive. Consequently, 7 days later at day 14 post-wounding, the epithelium of junBD/D wounds was much thicker compared to

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Figure 6. Epidermal acanthosis and increased neutrophil invasion in junBD/D wounds. (a) Histological examination of wounds from junBD/D and control (junBf/D) mice 2 weeks after injury indicates marked epidermal hyperthickening in junBD/D wounds. Keratin 6, K10, and Loricrin were used to mark hyperproliferation, early, and late keratinocyte differentiation, respectively. Co-staining using anti-CD68 and anti-neutrophils antibodies revealed enhanced neutrophil infiltration but equal numbers of macrophages in wounds of junBD/D mice compared to controls. Bar ¼ 100 mm. (b) Quantification of inflammatory cells in 14-day-old wounds of control (white bars) and junB mutant mice (black bars). The bars represent the mean7standard deviation of at least three wounds from different animals per time point and genotype.

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Figure 7. Delayed wound healing correlates with increased keratinocyte proliferation after epithelial closure of the wound and de-regulated expression of JunB-dependent cytokines. (a) Sections of 7-day-old wounds were stained with antibodies for Ki67, K10, and neutrophils to examine keratinocyte proliferation and differentiation as well as inflammation. (b) Cytokine expression during wound healing was measured by real-time PCR on RNA isolated from junBD/D and control wounds at days 0–10 after injury. Hprt was used as standard and relative mRNA levels compared to unwounded control skin () were calculated. All experiments were performed in triplicates, mean values7standard deviation are shown.


controls and displayed strong expression of Keratin 6, which is indicative for keratinocyte hyperproliferation (Figure 6a). Moreover, the area of cells positive for K10 and loricrin was markedly enlarged in wounds of JunB mutant animals compared to controls. However at this stage, the regular suprabasal onset of K10 expression was restored, suggesting that keratinocyte differentiation had normalized by this time point concomitant with proliferation being now restricted to the basal layer. Persistent inflammation in wounds lacking junB

We next examined, whether the delayed wound healing was correlated with an altered immune response. As in TPAtreated skin, immunofluorescence staining for CD68 revealed no difference in the infiltration of macrophages at any stage of wound repair (Figure 6a and b and data not shown). By contrast, the influx of neutrophils was initially delayed in junBD/D mice with reduced numbers of cells being detectable in wounds at 6 hours and 1 day post-wounding (data not shown). At days 2–10, the amount of neutrophils was comparable in wounds of mutant and control littermates (Figures 6b and 7a and data not shown). However, while their numbers decreased and only few cells were detected in the granulation tissue of controls at day 14, the level of neutrophils remained elevated in wounds of mutant mice (Figure 6a and b). Thus, the initially delayed infiltration was followed by a prolonged presence of neutrophils in the granulation tissue of junBD/D mice, indicating a longer lasting inflammation. Altered expression of cytokines in wounds of junB mutant mice

In organotypic co-cultures, epithelial hyperplasia could be attributed to the transcriptional de-repression and thus increased production of JunB-regulated paracrine epithelial growth factors and cytokines such as KGF and GM-CSF by the JunB-deficient feeder cells (Szabowski et al., 2000). We therefore determined whether expression of these genes was also de-regulated in vivo during wound healing in junBD/D mice (Figure 7). In addition, we investigated the expression of additional cytokines including the chemokines macrophage inflammatory protein-2 (Mip-2/CXCL2), growth-regulated protein-1 (Gro-1/KC/Mgsa/CXCL1), and the pro-inflammatory cytokine lipocalin-2 (Lcn-2), which we recently identified as JunB target genes by microarray analysis of IL-1-treated wildtype and junB/ fibroblasts (Florin et al., 2004b). Quantitative RT-PCR on cDNAs prepared from control and junBD/D wounds at different stages of healing revealed that expression of gm-csf, mip2, gro1, and lcn2 was induced at day 1 after injury in control wounds. After reaching this maximum postwounding, mRNA levels decreased to nearly background levels after 5 days (Figure 7b). In the absence of JunB, basal level expression of gm-csf was significantly increased and highest induction of mip2, gro1, and lcn2 was shifted to later time points, thus correlating with reduced neutrophil invasion within the first day of healing. Importantly, after 48 hours, all four cytokines were hyperinduced in junB-deficient wounds and expression remained elevated for a prolonged period of time and did not decrease until day 10 after injury. By

contrast, no significant aberrations were observed in the expression of Kgf/Fgf7 (data not shown) and TGF-b1 (Figure 7b). Normal levels of TGF-b1, which is implicated in the regulation of matrix deposition and wound contraction by promoting myofibroblast differentiation (Tomasek et al., 2002), also agreed with normal collagen organization and abundance of a-smooth muscle actin expressing fibroblasts in junBD/D wounds (Figure S2). In summary, we found a strongly extended window of elevated mRNA levels of four junB-dependent cytokines. As kgf and tgf-b1 did not follow this pattern, one could assume that these changes in cytokine expression are not a mere consequence of the decelerated wound healing. DISCUSSION In this study, we describe the skin phenotype of mice, which lack junB expression in the whole skin including dermal fibroblasts, epithelial keratinocytes, and leukocytes. These mice survive to adulthood and show a normal cutaneous architecture, but exhibited a dramatic skin phenotype in junB mutant mice in response to wounding or TPA treatment. Under these conditions, the skin of junBD/D mice was characterized by epidermal hyperproliferation, perturbed keratinocyte differentiation, and enhanced inflammation. These phenotypic alterations correlated with alterations in the expression levels and kinetics of different JunB-dependent epithelial growth factors, cytokines and chemoattractants, and a reduced ability of mutant dermal fibroblasts to contract collagen lattices in vitro. As JunB is essential for placental vascularization during early embryonic development (Schorpp-Kistner et al., 1999), conditional junB-deficient mice were generated. The efficient deletion of the junB gene in many organs of COL1A2-junB mice was surprising, as COL1A2-Cre expression is predominantly found in mesenchymal cells leading to cell typespecific deletion of floxed sequences, such as the flanking region of the b-gal gene in the ROSA26R mouse strain (Florin et al., 2004a). However, as described previously (Vooijs et al., 2001; Korets-Smith et al., 2004), floxed genes differ in the accessibility for Cre. The genomic locus of the junB, which, according to its transcriptional regulation of an immediate-early gene, is easily accessible and may therefore represent a class of gene loci, where only very low levels of Cre are required for efficient deletion. Regardless of the mechanism of deletion, COL1A2-Cre junB mice are viable but of reduced size and body weight, which most likely emerges as a consequence of the complete deletion of junB in the bone. Impaired JunB expression was shown to be associated with decreased proliferation of growth plate chondrocytes as well as defects in proliferation and differentiation of osteoblasts, which together resulted in a reduction of the overall bone mass (Hess et al., 2003; Kenner et al., 2004). In the in vitro wound healing model of organotypic cocultures, JunB activity in fibroblasts was shown to impact critically on proliferation and differentiation of keratinocytes (Szabowski et al., 2000). Accordingly, an obvious phenotype arose after challenge of the skin by the phorbol ester TPA or www.jidonline.org

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wounding. Both stimuli, which are known triggers of AP-1 activity (Angel and Karin, 1991; Gallucci et al., 2000), induced epidermal hyperproliferation, impaired keratinocyte differentiation, and enhanced inflammatory response. Notably, hyperproliferation and irregular keratinocyte differentiation have similarly been observed in organotypic co-cultures in vitro (Szabowski et al., 2000). Here, JunB–deficiency, only in fibroblasts, was sufficient to evoke abnormal epithelialization through de-regulated expression of paracrine-acting factors. On the other hand, exclusive deletion of junB in epidermal keratinocytes using K5-Cre mice also resulted in increased epithelial thickness after TPA treatment (R. Zenz and E.F. Wagner, personal communication), suggesting that the absence of junB in the keratinocytes itself might induce the expression of genes, which participate cell-autonomously to the observed phenotype. Based on these data and our finding that upon TPA treatment expression profiles of JunB target genes, such as gro1 and gm-csf, are altered both in the dermal and epidermal compartment (Figure S3), it is tempting to speculate that JunB is involved in the control of epithelial architecture both through cell-autonomous functions as well as disturbance of the dermal–epidermal interaction. As suggested by the enhanced inflammation, leukocytes, which are found in high numbers in the wound granulation tissue as well as in TPA-treated skin, are likely to participate in the establishment of the skin phenotype of JunB mutant mice. Although not included in the co-culture system and thus possibly not strictly required for the JunB-dependent development of epidermal hyperplasia, infiltrating neutrophils contribute in vivo substantially to the expression of chemokines and epidermal growth factors (for a review, see Schroder, 1995; Gillitzer and Goebeler, 2001; Grazul-Bilska et al., 2003). In junBD/D mice, circulating lymphocytes as well as neutrophil granulocytes harbor the deleted junB allele (data not shown). Thus, the prolonged presence of neutrophils in wounds as well as their increased abundance in TPAtreated skin might either be explained by junB-dependent changes in immune cell behavior or be the consequence of an altered microenvironment resulting from JunB deficiency in the dermis and epidermis. To gain insight into the molecular mechanism, we analyzed the expression of various chemoattractants and known players in cutaneous wound healing such as Mip-2, Gro-1, and Lcn-2 (Fahey, III et al., 1990; Nanney et al., 1995; Florin et al., 2004b, respectively), which were either directly or indirectly repressed by JunB. Accordingly, expression of the cytokines Mip-2, Gro-1, Lcn-2, and GM-CSF was also altered in vivo in junBD/D mice, showing enhanced basal levels in unchallenged skin (gm-csf), a stronger induction in TPA-treated skin (Figure 3b) and a delayed, but prolonged and increased expression during wound repair (Figure 6). Mip-2/CXCL2 and Gro-1/KC/Mgsa/CXCL1 belong to the CXC-family of small inducible cytokines (Balkwill, 2004) and are potent mediators of neutrophil migration and chemotaxis (Schroder, 1995), and overexpression of either Gro-1 in keratinocytes (Lira et al., 1994) or Mip-2 in gut epithelial cells (Ohtsuka et al., 2001) was found to induce leukocyte infiltration in the respective tissues. As both cytokines show 908


upregulated expression in the skin of junBD/D mice after TPA treatment (Figure S3), it seems likely that they might be causally related to the increased abundance of infiltrating neutrophils under these conditions (Figure 3). During wound healing, the expression kinetics of Mip-2 and Gro-1 is shifted in junB mutant animals, which correlates with the delayed appearance of neutrophils and may contribute to their prolonged persistence in the wound bed (Figure 6b). High levels of GM-CSF and the functional human homolog of Gro-1, IL-8, have been measured in hyperproliferative skin diseases (Gearing et al., 1990; Pastore et al., 1997) and both proteins were shown to exhibit a strong mitogenic activity for keratinocytes in vitro and/or in vivo (Olaniran et al., 1995; Rennekampff et al., 1997; Mann et al., 2001). Moreover, the JunB-dependent cytokine Lcn-2 was described to be strongly induced in hyperplastic psoriatic lesions (Mallbris et al., 2002). Thus, it is tempting to speculate that combined overexpression of several growth factors with mitogenic activity, such as Gro-1, GM-CSF (and possibly Lcn2) in challenged skin of junBD/D mice influence the balance of keratinocyte proliferation and differentiation and ultimately constitute a hyperplastic phenotype, which might not be achieved by enhanced expression of individual factors, such as GM-CSF in unchallenged skin of JunB mutant mice. Treatment of wounds with Gro-1 also led to a significant reduction in wound contraction, which was discussed to result either through a direct action of the chemokine on myofibroblasts or an indirect mechanism (Rennekampff et al., 1997). Thus, strongly enhanced and prolonged expression of Gro1 in wounded mutant skin (Figure 7) and in cultured fibroblasts (L. Florin and P. Angel, unpublished) may provide a functional link to the reduced ability of JunB-deficient fibroblasts to contract collagen lattices in vitro (Figure 5e). However, wound contraction depends on various parameters including expression of components of the ECM, receptors for ECM components, and proteases required for ECM remodelling (Tomasek et al., 2002) and many members of these protein families have been proposed to depend on AP-1 (Angel et al., 2001; Florin et al., 2004b). In summary, we demonstrated an important regulatory function of JunB in the cutaneous stress response and skin regeneration, which both require rapid changes in the transcriptional program. Obviously, based on genetic data, there is only little functional redundancy in the genetic programs controlled by either JunB or another AP-1 member, c-Jun. Studies in mice and Drosophila have established an essential role of c-Jun, as well as its upstream regulator JNK, as a positive regulator of EGFR-dependent signalling, promotion of epithelial cell migration and cytoskeletion rearrangement (Kockel et al., 2001; Li et al., 2003; Zenz et al., 2003; Xia and Karin, 2004). In contrast, our data imply that JunB can rather act as a negative regulator of keratinocyte proliferation via repression of critical cytokines and positively regulates fibroblast contraction by an as yet unknown mechanism. However, it is important to note that simultaneous, but not individual, deletion of c-Jun and JunB in keratinocytes of adult mice results in the spontaneous development of psoriasis. This is associated with an onset


of the two chemotactic proteins S100a8 and S100a9 before the disease (Zenz et al., 2005) supporting the concept of both antagonistic and cooperative functions of c-Jun and JunB in inflammatory responses of the skin. Taken together, our data show that the immediate-early transcription factor JunB regulates the cellular stress response by controlling a complex combination of growth factors and chemokines, which by both, autocrine and paracrine mechanisms modulate keratinocyte proliferation and differentiation as well as leukocyte chemotaxis during cutaneous inflammation and regeneration. MATERIALS AND METHODS Generation of mutant mice Mice carrying the floxed junB allele (junBflf) (Kenner et al., 2004) were bred with COL1A2-Cre transgenic mice (Florin et al., 2004a) to create COL1A2-Cre junBf/ þ mice. Alternatively, junBf/ mice harboring an inactive JunB-NEO fusion (Schorpp-Kistner et al., 1999) were used to obtain COL1A2-Cre junB þ / offspring. Conditional knockout mice (COL1A2-Cre junBf/ or f/f) were obtained in a second mating with homozygous junBf/f mice. COL1A2-Cre mice and junB floxed mice were backcrossed into C57BL/6 background for at least three generations.

Genotyping by Southern blot and PCR analysis For Southern blot analysis, genomic DNA of whole organs was prepared; the epidermis and dermis were separated by thermolysin treatment (Sigma, Deisenhofen, Germany). DNA (15 mg) was digested with PstI, yielding fragments of 2.6 kb for the floxed junB allele, 1.4 kb for the junB-Neo fusion, and 1.3 kb for the deleted junB locus. Bands were detected using a 176 bp PstI/HindIII fragment of the junB promoter as a probe. Primer sequences are listed in the Table S1.

TPA-induced hyperproliferation Age-matched 10 to 12-week-old junB mutant and control mice (n ¼ 4 per genotype) were shaved and, after 24 hours, treated three times every 48 hours with 10 nmol TPA/100 ml or solvent alone (acetone). Twenty-four hours after the last treatment, the skin was isolated and either fixed in 4% paraformaldehyde/phosphatebuffered saline at 41C overnight and embedded in paraffin for histological sections or taken for RNA analyses.

Wound healing experiments Four round full-thickness excisional wounds of 4 mm diameter were generated in the back skin of anesthetized female junBD/D mice (n ¼ 30) and control littermates (n ¼ 29) using sterile biopsy punches. The wounds were left untreated. The mice were killed at different time points after injury and wounds were either embedded for sectioning or excised including 2 mm of the epidermal margins for RNA isolation. Three animals and 3–7 wounds were analyzed per genotype and time point. Granulation tissue volume and epidermal thickness were measured using Adobes Photoshop software, quantification of total cell numbers, neutrophils and macrophages was performed using analySISs software. The animal experiments were performed in accordance with the guidelines of the ATBW (officials for animal welfare) and were approved by the Regierungspra¨sidium Karlsruhe, Germany.

Immunohistochemistry, immunofluorescence, and collagen-staining Paraffin sections (7 mm) of mouse back skin and cryosections of wounds were stained with hematoxylin and eosin or analyzed by immunological methods. Immunohistochemistry was performed using the following primary antibodies: JunB (C-11, #sc-8051 and N-17, #sc-46, Santa Cruz Biotechnology Inc., Santa Cruz, CA), Ki67 (Novocastra, Newcastle upon Type, UK), Keratin 6, K10 and Loricrin (BabCo, Freiburg, Germany), CD68 and Neutrophils (Serotec, Duesseldorf, Germany). Bisbenzimide dye (Hoechst No. 33258) was used for nuclear counterstaining. Collagen organization was estimated on cryosections (5 mm) of wounds that were stained with Picrosirius red (Junqueira et al., 1979) and imaged on a Zeiss Axioplan microscope. Images were captured using a Hamamatsu 3 CCD cooled camera (Hamamatsu, Hamamatsu City, Japan) and Simple PCI imaging software (Compix Imaging Systems, Pittsburgh, PA).

RNA isolation and RT-PCR Total RNA was isolated using TRIzols reagent (Invitrogen GmbH, Karlsruhe, Germany) according to standard protocols followed by treatment with DNAse I for 30 minutes at 371C. Quantitative differences in gene expression were determined by real-time RTPCR using the AbsoluteTM QPCR SYBRs Green Fluorescein Mix (ABgene, Surrey, UK) and a thermal cycler controlled by the MyiQ Real Time Detection System software (BioRad, Munich, Germany). All experiments were performed at least in triplicates. For semiquantitative RT-PCR, gene-specific fragments were obtained by linear phase PCR-amplification and standardized for hprt levels. Primer sequences are listed in the Table S1.

Collagen contraction assay Adult (12 weeks old) junB mutant and control animals were euthanized by decapitation. Back skin was removed and the dermis separated after incubation for 2 hours on thermolysin at room temperature. To isolate primary fibroblasts, the dermis was diced finely and incubated with 1 mg/ml Collagenase D (Boehringer Mannheim, Germany) for 2–3 hours at 371C. Tissue debris was removed by centrifugation and collected fibroblasts were cultured in DMEM (Invitrogen, Karlsruhe, Germany) containing 20% FCS (PAA Laboratories, Co¨lbe, Germany), 2 mM glutamine, and antibiotics (Seromed-Biochrom, Berlin, Germany). The fibroblasts were used between passages 2 and 4 for experiments. Three-dimensional collagen gels were prepared as described previously (Mauch et al., 1988). Briefly, type I collagen (3 mg/ml, Vitrogen) and 10 DMEM were combined in a 10:1 ratio and neutralized by the addition of 0.1 M NaOH. Fibroblasts were seeded into collagen gels at a density of 2.5 105 cells/ml and incubated at 371C. After 2 hours incubation, gels were gently detached from the plates and contraction monitored over time. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank Jochen Hess for carefully proofreading manuscript, Bernd Dittrich for help in creating the punch wounds, Melanie Sator-Schmitt and Sibylle Teurich for technical assistance, Rainer Zenz and Erwin F. Wagner for sharing unpublished results. This work was supported by the Deutsche

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Forschungsgemeinschaft (An 182/8-2) and by the Research Training Network (RTN) Program of the European Community as well as the Koeln Fortune Program of the Faculty of Medicine grant 10/2004 (P. Zigrino) and Deutsche Forschungsgemeinschaft through the SFB 589 at the University of Cologne (C. Mauch)

Grazul-Bilska AT, Johnson ML, Bilski JJ, Redmer DA, Reynolds LP, Abdullah A et al. (2003) Wound healing: the role of growth factors. Drugs Today (Barc) 39:787–800

SUPPLEMENTARY MATERIAL

Hayashi S, McMahon AP (2002) Efficient recombination in diverse tissues by a tamoxifen-inducible form of Cre: a tool for temporally regulated gene activation/inactivation in the mouse. Dev Biol 244:305–18

Table S1. Oligonucleotide sequences used for PCR analyses. Figure S1. COL1A2-Cre x junB mice show a reduced body size and lower body weights. (a) A 3 month-old conditional junB mutant (junBD/D) mouse and a control littermate are shown. (b) Reduced body weight of male and female junB mutant (black dots) compared to controls (open circles). Larger circles indicate the mean values of control (n ¼ 12) and mutant animals (n ¼ 11). Figure S2. Analysis of the granulation tissue (a) Collagen was stained with picrofuchsin to examine matrix organization as measured by the distribution of total collagen (BF, brightfield) versus highly organized collagen fibres (PC, phase contrast). Scale bar ¼ 100 mm. (b) Immunohistochemical staining for aSMA detected comparable amounts of myofibroblasts in wounds of junBD/D mice and controls at day 5. Scale bar ¼ 100 mm. Lower panel: Higher magnification of a-SMA positive cells. Scale bar ¼ 20 mm Figure S3. Contribution of the dermal and epidermal compartment to cytokine expression. (a) Semi-quantitative RT-PCR and (b) real-time PCR on RNA from separated dermis and epidermis of untreated () and TPA treated ( þ , three consecutive applications every 48 hours) control (n ¼ 2) and junB mutant mice (n ¼ 4) revealed both dermal and epidermal cells as source of deregulated expression of JunB dependent cytokines. Collagen type I-alpha2 (col1a2) was used as control for tissue separation. cDNA levels were standardized to hprt quantities. Error bars in (b) indicate the mean7SEM of triplicate experiments.

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