Jun signalling in the epidermis: From developmental defects to psoriasis and skin tumors

The International Journal of Biochemistry & Cell Biology 38 (2006) 1043–1049

Signalling networks in focus

Jun signalling in the epidermis: From developmental defects to psoriasis and skin tumors Rainer Zenz ∗∗ , Erwin F. Wagner ∗ Research Institute of Molecular Pathology (I.M.P.), Dr. Bohr-Gasse 7, A-1030 Vienna, Austria Received 28 September 2005; received in revised form 16 November 2005; accepted 21 November 2005 Available online 20 December 2005

Abstract The Jun proteins Jun, JunB and JunD are core members of activator protein-1 (AP-1), a dimeric transcription factor complex consisting of homo- and heterodimers of the Jun, Fos, activating transcription factor (ATF) and musculoaponeurotic fibrosarcoma (Maf) families. Growth factors, hormones and a variety of environmental stresses activate mitogen activated protein kinase (MAPK) cascades that enhance Jun/AP-1 activity, e.g. through phosphorylation thereby regulating cell proliferation, differentiation, transformation and/or apoptosis. Embryonic lethality of various AP-1 knock-outs, e.g. for Jun, JunB, Fra-1 and Fra-2 largely prevented functional studies in vivo. Therefore, conditional knock-out strategies, in particular for the epidermis, have become an important model to study the regulation and function of AP-1 subunits in physiological and pathological processes in vivo. Jun is regarded as a positive regulator of keratinocyte proliferation/differentiation during development and in skin cancer through its direct transcriptional effect on epidermal growth factor receptor (EGFR) expression. In contrast, JunB can antagonize proliferation of keratinocytes and hematopoietic stem cells. Furthermore, it has been demonstrated in patient’s samples and an inducible mouse model that downregulation of JunB/AP-1 in keratinocytes is one initiating event in the aetiology of psoriasis which is characterized by increased cell proliferation and deregulated cytokine expression. © 2006 Published by Elsevier Ltd. Keywords: Jun; AP-1; Skin; Epidermis; Keratinocyte; Conditional; Inducible; Knock-out; EOB; Papilloma; Cancer; EGFR; Psoriasis; S100a8; S100a9; T cell; TNF signalling

Signalling network facts • Jun, Fos, ATF and Maf proteins form the core family of the dimeric AP-1 transcription factor. • AP-1 can be regulated by dimer composition, transcription, post-translational modification and interaction with other proteins.

Abbreviations: bZip, basic leucine zipper; EGFR, epidermal growth factor receptor; EGF, epidermal growth factor; GM-CSF, granulocytemacrophage colony stimulating factor; G-CSF, granulocyte colony stimulating factor; HB-EGF, heparin-binding epidermal growth factor; JNKs, Jun N-terminal kinases, JNK1, JNK2 and JNK3; KGF, keratinocyte growth factor; LPS, lipopolysaccharide; RTK, receptor tyrosine kinase; Rag2, recombination activating gene 2; Ref-1, redox factor 1 (also AP endonuclease; a DNA repair enzyme); SOS, son of sevenless; TGF-␣, transforming growth factor ␣; TCFs, ternary complex factors, a subgroup of the ETS protein family; TGF-␤, transforming growth factor ␤; VEGF, vascular endothial growth factor ∗ Corresponding author. Tel.: +43 1 79730 888; fax: +43 1 79893 90. ∗∗ Corresponding author. Present address: Ludwig Boltzmann Institute for Cancer Research (LBI-CR), W¨ ahringer Straße 13A, Vienna, Austria. E-mail addresses: [email protected] (R. Zenz), [email protected] (E.F. Wagner). 1357-2725/$ – see front matter © 2006 Published by Elsevier Ltd. doi:10.1016/j.biocel.2005.11.011

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• Different AP-1 dimers control transcriptional activation or suppression of a variety of genes involved in the regulation of proliferation, differentiation, apoptosis and transformation. • An auto-regulatory loop from EGF/-R to Jun-dependent EGFR expression is important for keratinocyte differentiation and skin tumor development. • MAPK/RTK (receptor tyrosine kinase) regulates Fos/AP-1 activity via ERK1,2 and Jun/AP-1 activity via JNK. 1. Introduction Activator protein-1 (AP-1) is a dimeric transcription factor complex that comprises members of the Jun, Fos, activating transcription factor (ATF) and musculoaponeurotic fibrosarcoma (Maf) protein families (Eferl & Wagner, 2003). Fos and Jun family proteins function as dimeric transcription factors that bind to AP-1 binding sites in the promoter and enhancer regions of numerous mammalian genes (Curran & Franza, 1988). Jun proteins form both homodimers and heterodimers with Fos proteins, whereas Fos proteins require heterodimerization to bind DNA (Chinenov & Kerppola, 2001). Despite the high degree of structural homology, the different members of the Jun and Fos families exhibit significant differences in DNA-binding and transcriptional activation suggesting specific functions in gene regulation for individual AP-1 dimers (Shaulian & Karin, 2002). AP-1 acting downstream of evolutionarily conserved signalling pathways, such as MAPK, TGF-␤ and Wnt, is one of the key factors that translate external stimuli both into short- and long-term changes of gene expression. AP1 activity is induced by a myriad of agents from growth factors, neurotransmitters, polypeptide hormones to bacterial and viral infections as well as by a variety of physical and chemical stresses. These stimuli activate mitogen activated protein kinase (MAPK) cascades that enhance AP-1 activity, e.g. through phosphorylation of distinct substrates (Chang & Karin, 2001). A functional role for AP-1 components in the epidermis of the skin has been suggested for differentiation, carcinogenesis, UV-response, photo-aging and wound repair (Angel, Szabowski, & Schorpp-Kistner, 2001). Here, we will discuss recent discoveries regarding the functions of Jun proteins, in particular in skin biology. These experiments demonstrate that Jun proteins are important regulators of keratinocyte proliferation/differentiation and cytokine production, which play important roles not only in inflammatory diseases such as psoriasis but also in skin tumor formation. 2. Cascades In a given cell, AP-1 activity is regulated by a broad range of physiological and pathological

stimuli, including cytokines, growth factors, stress signals as well as oncogenic stimuli, which lead to activation of MAPK signalling. Regulation of AP-1 can be achieved at different levels by changes in transcription of genes encoding AP-1 subunits, by controlling the stability of the mRNAs, by posttranslational processing, turnover and modification by phosphorylation of AP-1 proteins, and by specific interaction between AP-1 proteins and other transcription factors and cofactors (Hess, Angel, & SchorppKistner, 2004). The expression of Fos is induced by ternary complex factors (TCFs), which are activated by the extracellular-signalling-regulated kinase (ERK) MAPKs. Subsequently, Fos and myocyte-enhancer factor 2 (MEF2) transcription factors induce Jun expression. Once the AP-1 complexes are present in larger amounts, JNK and p38 kinases increase the transactivation potential of Jun and ATF proteins by phosphorylation. The mechanism of post-translational control is extensively documented in the case of mitogen- and cellular-stressinduced hyperphosphorylation of Jun through the Jun N-terminal kinase (JNK) cascade (Karin, Liu, & Zandi, 1997). Activated by a MAPK cascade, JNKs translocate to the nucleus, where they phosphorylate Jun within its N-terminal transactivation domain (residues Ser63 and Ser73) and thereby enhance its transactivation potential (reviewed in (Hess et al., 2004). The JNKs also phosphorylate and potentiate the activity of JunD and ATF-2. In contrast, the kinases that regulate the activity of Fos are less well defined. Potential candidates are a poorly defined Fos-related kinase (FRK) (Deng & Karin, 1993) and ERK (Chen, Abate, & Blenis, 1993). Additional kinases such as casein kinase II, glycogen synthase kinase 3␤ or RSK2 can phosphorylate Fos and Jun proteins thereby regulating their transactivation potential and DNA-binding activity (Eferl & Wagner, 2003). Importantly, RSK2 is essential for efficient c-Fos dependent osteosarcoma development (David et al., 2005). DNA-binding of the Jun–Fos heterodimer is also modulated by reduction–oxidation of a single conserved cysteine residue in the DNA-binding domains of the two proteins (Abate, Patel, Rauscher, & Curran, 1990). Reduction of oxidized Jun and Fos by redox factor 1 (Ref-1) stimulates sequence-specific AP-1 DNA-binding activity (Xanthoudakis & Curran, 1996).

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In addition, DNA-binding of AP-1 is also influenced by cofactors like Jun activation domain-binding protein 1 (Jab1), which interacts with Jun and JunD, but not with JunB (Claret, Hibi, Dhut, Toda, & Karin, 1966). Interestingly, thioredoxin (Trx) a cellular redox enzyme specifically interacts with and modulates the function of Jab1 indicating that reduction/oxidation of different AP-1 cascade components is involved in regulating proliferation and apoptosis at least in vitro (Hwang et al., 2004).

Table 1 Phenotypes of genetically modified Jun mice

3. Key molecules

Knock-out Jun

All three Jun proteins, Jun, JunB and JunD derive from an ancestral gene, which is conserved from worms to man (Mechta-Grigoriou et al., 2001). JunB has a lower homodimerization affinity and a much weaker AP1 binding affinity due to small amino acid changes in its bZip region (Deng & Karin, 1993). Whereas JunBmediated transactivation also requires synergistic interactions between multiple homodimers bound to adjacent sites, Jun does not need such interactions (Chiu, Angel, & Karin, 1989). As a consequence, Jun and JunB differ considerably in their ability to regulate target genes (Deng & Karin, 1993). Jun is an efficient activator of promoters containing a single AP-1-binding site; in contrast, JunB most efficiently activates promoters that contain repeated AP-1 sites and is able to antagonize Jun-mediated transactivation on a single AP-1 site. 4. Function Genetically modified mice and cells derived thereof have provided important insights into the biological functions of different Jun proteins (Table 1). Ectopic expression of Jun, JunD or JunB in transgenic mice does not result in an overt phenotype (Jochum, Passegue, & Wagner, 2001), although targeted over-expression of JunB in T lymphocytes interferes with the differentiation of T helper cells (Li, Tournier, Davis, & Flavell, 1999). Expression of JunD under the control of the ubiquitin C promoter in mice reduces the number of peripheral T and B cells further indicating a role of Jun proteins in the regulation of the immune system (Meixner, Karreth, Kenner, & Wagner, 2004). Jun and JunB are essential proteins for embryonic development, whereas JunD is required postnatally. Mice lacking Jun die between embryonic day (E) 12.5 and E14.5 of development and show defects in liver development and heart morphogenesis (Eferl & Wagner, 2003). Inactivation of JunB results in embryonic lethality around E9.5, caused by impaired vasculogenesis and angiogenesis in the extra-embryonal

Phenotype Transgene/promoters Jun/H2 None

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Affected organs and cell types None

JunB/Ubi-C JunB/CD4

None Enhanced TH-2 maturation

None Thymus, CD4 thymocytes

JunD/Ubi-C

Peripheral T and B cells reduced

Lymphocytes

Embryonic lethal E12.5

JunB

Embryonic lethal E10

JunD

Male sterility

Liver, heart, hepatoblasts, neural crest Extra-embryonic tissue, placenta Testis, spermatides

Rescues lethality of c-Jun k.o. until birth Rescues lethality of c-Jun k.o. until birth

Anterior eye structure, heart Anterior eye structure, heart

Knock-in Jun B for Jun JunD for Jun#

Conditional knock-out Impaired liver Jun∆li regeneration Scoliosis Jun∆chond Jun∆ep Eye open at birth, reduced tumors JunB∆/∆ Osteopenia, leukemia JunB∆ep#

Multiorgan disease

JunB/Jun∆ep*

Psoriasis, psoriatic arthritis

Hepatocyte Chondrocyte Keratinocyte Osteoblast, osteoclast, myeloid cells Keratinocyte, granulocyte, bone Skin, joint

Abbreviations: Jun∆li , conditional knock-out mice lacking Jun in the liver, in chondrocytes, Jun∆chond , and in the epidermis, Jun∆ep ; Jun∆ep* , inducible conditional knock-out in the epidermis; #, unpublished work of the Wagner lab. Ubi-C, Ubiquitin C.

tissue (Schorpp-Kistner, Wang, Angel, & Wagner, 1999). Mice lacking JunD are viable but show reduced postnatal growth and exhibit multiple age-dependent defects in reproduction, hormone imbalance and impaired spermatogenesis (Thepot et al., 2000). Jun proteins exert antagonistic functions in biological processes such as oncogenic transformation and cell proliferation. Jun was shown to be primarily a positive regulator of cell proliferation; Jun-deficient fibroblasts have a marked proliferation defect in vitro (Schreiber et al., 1999; Wisdom, Johnson, & Moore, 1999) and proliferation of Jun-deficient hepatocytes is severely impaired during liver regeneration in vivo (Behrens et al., 2002). To fully promote cell cycle progression, Jun proteins can be activated by Jun-amino-terminal kinases (JNKs)

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Fig. 1. Signalling pathways depicting how cell surface receptors transduce the signals to Jun proteins in the nucleus and their effects on cell cycle control and target gene expression.

(Behrens, Sibilia, & Wagner, 1999); the activated Juncontaining AP-1 complex induces the transcription of positive regulators of cell cycle progression, such as cyclin D1 or represses negative regulators, such as the tumor suppressor p53 and the cyclin-dependent kinase inhibitor p16INK4A (Fig. 1). Moreover, it was recently shown that JunD reduces tumor angiogenesis by protecting cells from oxidative stress (Gerald et al., 2004). Conditional gene inactivation using the Cre–lox system enabled us to investigate the postnatal functions of Jun proteins in different organs (Eferl & Wagner, 2003). Mice lacking Jun in epidermal keratinocytes are born with open eyes (EOB) and eyelid cells show reduced expression of the epidermal growth factor receptor (EGFR) (Zenz et al., 2003). Similarly, mice carrying a hypomorphic EGFR mutant called waved-2 (wa2) and EGFR knock-out mice show skin defects and the EOB phenotype (Eferl & Wagner, 2003; Luetteke et al., 1994). Jun mutant primary keratinocytes exhibit a severe proliferation defect which can be rescued by conditioned medium or by addition of autocrine (EGF, HB-EGF, TGF-␣) or paracrine growth factors (KGF,

GM-CSF). Importantly, these keratinocytes also have reduced expression of EGFR and of its ligand HBEGF, thereby promoting induction of differentiation. These data demonstrate that Jun transcriptionally regulates EGFR, which following activation via MAPK leads to AP-1-dependent gene expression (Fig. 1). In addition, it has recently been shown that a ternary complex of phosphorylated Jun, the HMG-box transcription factor TCF4 and ␤-catenin potentiates WNT signalling during the development of intestinal tumors (Nateri, SpencerDene, & Behrens, 2005). JunB and JunD are often considered to be negative regulators of cell proliferation. Fibroblasts derived from JunB over-expressing mice show reduced proliferation, whereas JunD-deficient immortalized fibroblasts exhibit increased proliferation (Passegue & Wagner, 2000; Weitzman, Fiette, Matsuo, & Yaniv, 2000). However, primary JunD-deficient fibroblasts also show reduced proliferation indicating that JunD can both positively and negatively regulate cell cycle progression depending on the cellular context (Weitzman et al., 2000). JunB and JunD can change the Jun-mediated activation or repression of crucial regulators for cell cycle progression (Fig. 1). Over-expression of JunB seems to antagonize the Jun-mediated induction of cyclin D1 in fibroblasts (Bakiri, Lallemand, Bossy-Wetzel, & Yaniv, 2000) and JunD-deficient primary fibroblasts undergo premature senescence, which requires p53 and increased p19ARF expression (Weitzman et al., 2000). Besides this competitive inhibition of Jun-mediated gene activation, JunB-containing AP-1 complexes positively regulate the expression of cell cycle modulators that are independent of Jun such as the cyclin-dependent kinase inhibitor p16INK4a (Passegue & Wagner, 2000). Therefore, it is likely that Jun-containing complexes are the main contributors to AP-1 DNA-binding activity in highly proliferating tumors, whereas JunB and JunD are selectively down-regulated due to their anti-proliferative capacity. Transgenic mice specifically lacking JunB expression in the myeloid lineage develop a transplantable myeloproliferative disease eventually progressing to blast crisis, which resembles human chronic myeloid leukemia (CML) (Passegue, Jochum, Schorpp-Kistner, MohleSteinlein, & Wagner, 2001). The absence of JunB expression results in increased numbers of granulocyte progenitors, which display enhanced GM-CSF-mediated proliferation and extended survival associated with changes in the expression levels of the GM-CSF␣ receptor, the anti-apoptotic proteins Bcl2 and Bclxl, as well as the cell cycle regulator p16INK4a and Jun. Recently, JunB was shown to regulate the number of hematopoietic stem cells. JunB over-expression decreased the

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frequency of long-term hematopoietic stem cells (LTHSC), while JunB inactivation specifically expands the number of LT-HSC and granulocyte/macrophage progenitors resulting in chronic myeloproliferative disorder (Passegue, Wagner, & Weissman, 2004). These results demonstrate a stem cell-specific role for JunB in normal and leukemic hematopoiesis and identify JunB as a potential tumor suppressor gene. However, it is worth pointing out that in bone cells JunB acts as a positive regulator in the osteoblast and osteoclast lineage (Kenner et al., 2004). 5. Associated pathologies and therapeutic implications 5.1. Skin tumors and Jun expression The role of Jun in skin tumor formation was analyzed in tumor-prone K5-SOS-F transgenic mice. Skin papillomas are induced with very high efficiency in K5-SOS-F transgenic mice and are strictly dependent on a functional EGFR (Mechta-Grigoriou et al., 2001; Sibilia et al., 2000). Moreover, K5-SOS-F transgenic mice harboring an allele of Jun (JunAA) that cannot be phosphorylated by JNK show a reduction in tumor mass suggesting that activation-dependent target genes of Jun are critically involved in the development of skin papillomas (Jochum et al., 2001). In the absence of Jun, tumor formation is strongly inhibited with reduced expression of EGFR in basal keratinocytes. Thus, Jun is a key transcriptional regulator of EGFR and HB-EGF and controls keratinocyte proliferation and skin tumor formation (Zenz et al., 2003). 5.2. Psoriasis and the role of Jun proteins Human JunB (19p13.2) is localized in the psoriasis susceptibility region PSORS6 (19p13) and is known to regulate cell proliferation, differentiation, stress responses and cytokine expression in various organs. Interestingly, the expression of JunB is greatly reduced in lesional skin of severe psoriasis and intermediately expressed in mild psoriasis (Zenz et al., 2005). Epidermis-specific, inducible knock-out of JunB alone did not reveal any signs of a psoriasis-like skin disease in mice. However, inducible epidermis-specific deletion of both JunB and Jun results within 2 weeks in alterations of the hairless skin closely resembling the lesions of psoriatic patients. All double mutant mice show a strong phenotype with scaly plaques affecting primarily ears, paws and tail, and less frequently the hairy back skin. The hallmarks of psoriasis are present, such

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as a strongly thickened epidermis with prominent rete ridges, thickened keratinized upper layers (hyperkeratosis) with parakeratosis (nucleated keratinocytes in the cornified layer) and increased subepidermal vascularization. Intra-epidermal T cells, epidermal micro-abscesses and the typical inflammatory cell infiltrate consisting of neutrophiles are seen along with increased numbers of macrophages in the dermis. A large number of cytokines, chemokines and transcription factors, which have been proposed to contribute to the pathogenesis of psoriasis, are found to be deregulated (Zenz et al., 2005). Moreover, arthritic lesions strongly reminiscent of psoriatic arthritis seen in 5–40% of psoriasis patients are observed with 100% penetrance. Inflammatory infiltrates are present in the joint regions along with massive bone destruction and periostitis. The analysis of early molecular events in the aetiology of the phenotype as well as deletion of JunB and Jun in cultured keratinocytes identified two chemotactic proteins, S100a8 and S100a9 as targets of JunB/Jun (Fig. 1). These proteins are potential mediators in psoriasis, since their genes are located in the psoriasis susceptibility region PSORS4 (1q12). Both proteins are potent stimulators of neutrophiles and passive immunization against S100a8/a9 inhibited neutrophil migration in response to LPS injection (Vandal et al., 2003). Thus, JunB/Jundependent deregulation of S100a8/a9 proteins appears to be one important early molecular event in the development of psoriasis. For several years it has been discussed whether psoriasis represents a fundamental disorder of the skin or the immune system. Strong genetic evidence against an absolute requirement of T cells in the initial development of psoriasis is provided by the deletion of JunB and Jun in a Rag2-deficient background. Under these conditions, histological analyses revealed all hallmarks of psoriasis, although the skin phenotype was milder when compared to T cell-competent controls. In addition, a similar chemokine/cytokine profile was observed in Rag2-deficient mice lacking JunB and Jun indicating that T cells are of minor importance in the development of the disease. We envisage that the role of T cells could be to amplify the initial inflammatory response. Interestingly, only small epidermal lesions at the paws were observed and the inflammation in the joint region was strongly reduced indicating a central role of T cells in the pathogenesis of the arthritic lesions. This represents the first mouse model recapitulating most of the histological and molecular characteristics of this important human disease and will be highly suitable for future preclinical studies ultimately aimed at understanding and curing psoriasis.

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6. Conclusions and outlook Although the different members of the Jun transcription factor family are derived from an ancestral gene, they have acquired different, sometimes even antagonistic roles. These proteins have evolved to execute very specific, cell-context-dependent functions during development and in pathological situations. We have observed that Jun is often an activator of cell proliferation and functions as an oncogene in liver and skin cancer, whereas JunB or JunD can take on opposite roles. It is now evident that Jun proteins are also important in inflammatory diseases, since they regulate the expression of growth factors, cytokines and chemotactic proteins. When environmental or microbial infections perturb the balance of Jun/AP-1 expression, e.g. in the skin, the altered expression pattern can affect not only the neighboring cells but also distant organs such as bone, joints and haematopoiesis. In this way, Jun/AP-1 proteins in the epidermis of the skin can be regarded as biosensors controlling homeostatic mechanisms and exhibiting endocrine-like functions. AP-1 has many more functions to be discovered, as shown in a recent paper demonstrating that AP-1 and the molecular clock mediate leptin-dependent sympathetic regulation of bone formation (Fu, Patel, Bradley, Wagner, & Karsenty, 2005). Therefore, a better understanding of Jun/AP-1 signalling might allow us to keep cells intact when desired, also to kill them when necessary and to develop strategies to fight diseases such as inflammation and cancer. Using more sophisticated inducible mouse models, together with studies in human cells, we are certain to learn more about the exciting functions of Jun/AP-1 proteins in different cell types and organs, about their regulation and the target genes they control to exploit this knowledge for modern therapeutic approaches in health and disease. Acknowledgements We are very grateful to Drs. Latifa Bakiri and Peter Hasselblatt for critical comments on the manuscript, Hannes Tkadletz for help with the illustrations. The IMP is funded by the Boehringer Ingelheim and this work was supported by grants from the Austrian Research Foundation (NFN S94) and the Research Training Network (RTN) program of the European Community. References Abate, C., Patel, L., Rauscher, F. J., 3rd, & Curran, T. (1990). Redox regulation of fos and jun DNA-binding activity in vitro. Science, 249, 1157–1161.

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