Oncogene (2002) 21, 4266 ± 4276 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Calgranulins S100A8 and S100A9 are negatively regulated by glucocorticoids in a c-Fos-dependent manner and overexpressed throughout skin carcinogenesis Christoer Gebhardt1, Ute Breitenbach2, Jan Peter Tuckermann3, Bernd Thilo Dittrich1, Karl Hartmut Richter1 and Peter Angel*,1 1Deutsches Krebsforschungszentrum, Division of Signal Transduction and Growth Control, 69120 Heidelberg, Germany The two calgranulins S100A8 and S100A9 were found to the S100 protein family of highly homologous low be dierentially expressed at sites of acute and chronic molecular weight calcium binding proteins (reviewed in¯ammation. Here we have employed the phorbol ester- by Donato, 2001). The calgranulins S100A8 and induced multistage skin carcinogenesis protocol in mice S100A9 can form a noncovalent heterodimer protein to determine the expression of both genes in in¯amed complex called calprotectin, which antagonizes the skin and in skin tumors. We show that expression is monomer functions (Newton and Hogg, 1998). coordinately induced by the phorbol ester TPA in Calgranulins are characterized by cell type-speci®c epithelial cells as well as in®ltrating leukocytes. By expression in cells of epithelial, myeloid and endothe- comparing S100A8 and S100A9 mRNA levels in wild lial origin and accumulation at sites of acute and type and c-Fos de®cient mice (c-fos7/7) we found that chronic in¯ammation (e.g. rheumatoid arthritis, cystic expression is negatively regulated by c-Fos/AP-1. ®brosis, psoriasis, allergic dermatitis, in¯ammatory Glucocorticoids, which exhibit potent anti-in¯ammatory bowel diseases) (reviewed by Donato, 2001). Recently and anti-tumor promoting activities repressed TPA- a functional interaction of the calgranulin family mediated S100A8 and S100A9 induction in wild type, member S100A12 with the receptor for advanced but not in c-fos7/7 mice, thus identifying both genes as glycation end products (RAGE) was discovered the ®rst examples of AP-1 target genes whose repression (Hofmann et al., 1999). RAGE was found to be of TPA-induced transcription by glucocorticoids depends involved in in¯ammation (Hofmann et al., 1999), in on c-Fos. Finally, we show that enhanced expression is tumor growth and metastasis (Taguchi et al., 2000), in not restricted to the initial TPA-induced in¯ammatory wound healing (Goova et al., 2001), and the pathology response but is observed at all stages of skin carcinogen- of certain diseases such as diabetes and Alzheimer's esis. These data identify S100A8 and S100A9 as novel, disease (reviewed by Schmidt et al., 2000). tumor-associated genes and may point to an as yet The calgranulins S100A8 and S100A9 are currently unrecognized function of both genes in the development attracting much interest because of their wide range of of epithelial skin tumors. possible intracellular as well as extracellular functions Oncogene (2002) 21, 4266 ± 4276. doi:10.1038/sj.onc. (reviewed by Donato, 2001). For example, both 1205521 proteins have been suggested to aect alteration of the cytoskeleton and cell shape, signal transduction, Keywords: AP-1; epidermis; in¯ammation; papilloma; exclusive carrying of arachidonic acids (Kerkho et tumor promoter al., 1999), and modulation of intracellular calcium (Schafer and Heizmann, 1996). Moreover, S100A8 and S100A9 have been described to be involved in Introduction in¯ammatory conditions (Odink et al., 1987; Brandt- zaeg et al., 1987). Approximately 45% of the S100A8 (MRP-8; CP-10; calgranulin A; cystic ®brosis cytoplasmic protein fraction of neutrophils is com- antigen), S100A9 (MRP-14; calgranulin B) and prised of S100A8 and S100A9 underlining their S100A12 (calgranulin C; EN-RAGE) are members of importance in in¯ammation (Edgeworth et al., 1991). The release of these small proteins via a TPA (12-O- tetradecanoyl-13-phorbolacetate)-stimulated tubulin- dependent pathway (Rammes et al., 1997) into the *Correspondence: P Angel, Deutsches Krebsforschungszentrum, Division of Signal Transduction and Growth Control, Im extracellular space supports the importance of their Neuenheimer Feld 280, 69120 Heidelberg, Germany; extracellular functions which includes potent chemo- E-mail: [email protected] tactic activity, triggering of local in¯ammation, Current addresses: 2Beiersdorf AG, Hamburg, Germany; 3Division of regulation of adhesivity, transendothelial migration Molecular Biology of the Cell I, Deutsches Krebsforschungszentrum, of leukocytes and striking antimicrobial properties 69120 Heidelberg, Germany Received 10 October 2001; revised 6 March 2002; accepted 26 (Kerkho et al., 1998; Passey et al., 1999a; Steinbakk March 2002 et al., 1990). Expression of calgranulins S100A8/S100A9 C Gebhardt et al 4267 S100A8-de®cient mouse embryos exhibiting an embryonic lethal phenotype have revealed non-redun- dant functions of S100A8 protein during fetal im- plantation (Passey et al., 1999b), and coordinate expression and secretion of S100A8 and S100A9 proteins have been observed during wound healing and in hyperthickened epithelium (Thorey et al., 2001). However, neither regulatory mechanisms of gene expression, nor the speci®c functions of the calgranu- lins S100A8 and S100A9 in in¯ammation-associated processes have yet been identi®ed conclusively. A frequently used model of acute in¯ammation is TPA- induced edema formation in the skin as the initial part of the chemically induced multistage skin carcinogenesis protocol (reviewed by Marks and FuÈ rstenberger, 1990; Bowden et al., 1994; Yuspa et al., 1996). Application of TPA to the skin causes swelling associated with increased vascular permeabil- ity and rapid in¯ux of neutrophil granulocytes and mononuclear cells into the skin. Cotreatment with glucocorticoids, which are in widespread medical use to inhibit in¯ammatory processes, interferes with both edema formation and the development of papilloma and carcinoma (Belman and Troll, 1972). Recently, we have applied PCR-based suppression subtraction hybridization to establish a cDNA library of TPA-inducible genes in mouse skin (Breitenbach et al., 2001). Two of these clones turned out to encode the calgranulins S100A8 and S100A9. Here we have determined cell type-speci®c expression of both genes in the initial TPA-induced in¯ammatory response and during various stages of skin carcinogenesis. Wild type and c-Fos de®cient mice (c-fos7/7) were used to establish the critical role of c-Fos in calgranulins S100A8 and S100A9 expression and repression by anti-in¯ammatory glucocorticoids. Results TPA coordinately induces calgranulins S100A8 and S100A9 expression in skin Figure 1 PKC-speci®c induction of calgranulins S100A8 and In an eort to identify genes in murine skin, which are S100A9 by TPA. (a) Induction of calgranulins S100A8 and transiently induced by tumor promoters, we isolated S100A9 by TPA in murine skin. TPA (10 nmol) in 100 ml acetone the two calgranulins S100A8 and S100A9 (Breitenbach was applied onto the backskin of C57BL/6 mice. After 0, 1, 4, 6 h et al., 2001). Induction kinetics revealed elevated mice were sacri®ced and RNA from skin was prepared. The level of calgranulins S100A8 and S100A9 transcripts was visualized by mRNA levels of S100A8 as well as S100A9 at 4 h Northern analysis. Rehybridization with a cDNA fragment of 18S after TPA treatment, whereas in the skin of control RNA was performed serving as loading control. One representa- treated animals only very low basal level expression of tive experiment out of three independent experiments using tissue both genes could be detected. Maximal levels of pooled from three mice is shown. (b) PKC-speci®c inhibitors suppress TPA-mediated induction of S100A8 and S100A9. PMK- induction (over 100-fold) were reached at 6 h, which R3 keratinocytes (Rennecke et al., 1999) were treated with returned to almost basal levels after 12 h (Figure 1a acetone (7), 10 nmol TPA (TPA), 100 nmol PKC-inhibitors and data not shown). Similar to the induction of Goedecke 6983 or 100 nmol Goedecke 6976 dissolved in acetone. S100A8 and S100A9 in vivo in skin, in the previously Fifteen min before TPA addition. Expression of SPRR2A was described immortalized mouse keratinocyte cell line chosen for comparison. Cells were harvested for RNA prepara- tion 6 h thereafter and analysed as described in (a) PMK-R3 (Rennecke et al., 1999) expression can be eciently induced by TPA (Figure 1b). These data show that expression of both genes is coordinately induced by TPA and follow similar kinetics as genes, such as MMP3 and MMP13 (Tuckermann et compared to previously described TPA-responsive al., 1999). Oncogene Expression of calgranulins S100A8/S100A9 C Gebhardt et al 4268 AP-1, c-Fos (Wang et al., 1992). Surprisingly, in PKC-specific inhibitors suppress TPA-induced comparison to the faint expression of S100A8 and upregulation of S100A8 and S100A9 S100A9 in acetone-treated control skin of wild type Distinct isoforms of the protein kinase C (PKC) family mice the expression levels of the calgranulins S100A8 represent the major `receptor' of the phorbol ester TPA and S100A9 in skin of c-fos7/7 mice were constitu- to initiate signaling pathways to the nucleus. To tively and coordinately enhanced in the acetone-treated identify the PKC isozymes that might be involved in backskin (Figure 2) as well as in untreated backskin the upregulation of calgranulins S100A8 and S100A9 (data not shown). Similar to the ®ndings in wild type by TPA we measured induction