Id Proteins: Novel Targets of Activin Action, Which Regulate Epidermal Homeostasis

Home , ID1, ID2, MAX (gene)

Oncogene (2006) 25, 2070–2081 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Id proteins: Novel targets of activin action, which regulate epidermal homeostasis

D Rotzer1, M Krampert1, S Sulyok1,3, S Braun1, H-J Stark2, P Boukamp2 and S Werner1

1Institute of Cell Biology, Department of Biology, ETH Zu¨rich, Ho¨nggerberg, Zu¨rich, Switzerland and 2German Cancer Research Center, Department Genetics of Skin Carcinogenesis, Heidelberg, Germany

Activin is a member of the transforming growth factor b Vale, 1993; Tsuchida et al., 2004). In addition, activins (TGF-b) family, which plays a crucial role in skin bind to the soluble glycoprotein follistatin that inhibits morphogenesis and wound healing. To gain insight into activin action (Nakamura et al., 1990). the underlying mechanisms of action, we searched for Recent studies provided evidence for a role of activin activin-regulated genes in cultured keratinocytes. One of in skin morphogenesis, repair, and disease. Thus, the identified target genes encodes Id1, a negative transgenic mice overexpressing the activin bA chain in regulator of helix–loop–helix transcription factors. We the epidermis were characterized by dermal fibrosis and show that Id1, Id2, and Id3 are strongly downregulated by by a hyperthickened epidermis (Munz et al., 1999). After activin in keratinocytes in vitro and in vivo. To determine skin injury, activin bA and bB mRNAs and activin A the role of Id1 in keratinocyte biology, we generated protein levels increase significantly (Hu¨ bner et al., 1996; stable HaCaT keratinocyte cell lines overexpressing this Cruise et al., 2004), suggesting an important role of protein. Our results revealed that enhanced levels of Id1 activin in wound repair. Indeed, transgenic mice over- do not affect proliferation of keratinocytes in monoculture expressing the activin antagonist follistatin in the under exponential culture conditions or in response to epidermis are characterized by strongly delayed wound activin or TGF-b1. However, in three-dimensional orga- healing (Wankell et al., 2001a). By contrast, mice notypic cultures, Id1-overexpressing HaCaT cells formed overexpressing activin in the epidermis showed en- a hyperthickened and disorganized epithelium that was hanced granulation tissue formation and re-epitheliali- characterized by enhanced keratinocyte proliferation, zation after skin injury (Munz et al., 1999). These abnormal differentiation, and an increased rate of findings demonstrate that aberrant expression and apoptosis. These results identify an important function activity of activin are associated with abnormal skin of Id1 in the regulation of epidermal homeostasis. development and impaired wound healing and suggest Oncogene (2006) 25, 2070–2081. doi:10.1038/sj.onc.1209230; that activin regulates migration, proliferation, and published online 14 November 2005 differentiation of many cell types in the skin, including keratinocytes. This was confirmed by our recent results, Keywords: epidermis; skin; TGF-b; wound which demonstrated that activin receptor signaling in keratinocytes is important for hair follicle morphogenesis and wound re-epithelialization (Bamberger et al., 2005). In addition, a role of activin in the regulation of keratinocyte proliferation and differentiation was found Introduction in in vitro studies with keratinocyte monocultures, where a weak inhibitory effect of activin on keratinocyte Activins are members of the transforming growth factor proliferation and induction of keratinocyte differentia- b (TGF-b) superfamily that exist as homo- or hetero- tion by this factor was demonstrated (Shimizu et al., dimers consisting of bA and bB chains. The major forms 1998; Seishima et al., 1999). In addition, activin was of activin are activin A (bAbA), activin B (bBbB), and shown to stimulate keratinocyte migration (Zhang et al., activin AB (bAbB) (Massague, 1990). Activin activities 2005). are mediated by heteromeric receptor complexes, con- To gain insight into the mechanisms of activin action sisting of type I (ActRIA/Alk2, ActRIB/Alk4, and in the skin, we searched for genes that are regulated by Alk7) and type II (ActRII and ActRIIB) receptors, activin A in cultured keratinocytes. One of the activin which are all serine/threonine kinases (Mathews and target genes that we identified encodes Id1. Id proteins (Id1–Id4) are negative regulators of basic helix–loop– Correspondence: Professor S Werner, Institute of Cell Biology, ETH helix (bHLH) transcription factors. They promote cell Zurich, Ho¨ nggerberg, HPM D42, CH-8093 Zu¨ rich, Switzerland. growth, inhibit differentiation, and play critical roles in E-mail: [email protected] development and cancer (Lasorella et al., 2001). 3Current address: Department of Dermatology, University of Ulm, D-89081 Ulm, Germany. Expression of Id proteins is increased in a large variety Received 19 July 2005; revised 5 October 2005; accepted 7 October 2005; of human tumors, including cutaneous squamous cell published online 14 November 2005 carcinoma and basal cell carcinoma (Langlands et al., Id proteins in epidermal homeostasis D Rotzer et al 2071 2000; Chaturvedi et al., 2003; Sikder et al., 2003). In Since the regulation of Id1 expression by TGF-b addition, enhanced levels of Id1 were detected in occurs in a cell-type-specific manner (Chambers et al., biopsies from lesioned psoriatic skin, suggesting a 2003; Kurisaki et al., 2003), we tested if this is also the regulatory role of this protein family in keratinocyte case for activin. Therefore, we treated primary human proliferation and differentiation (Bjorntorp et al., 2003). dermal fibroblasts with either activin A or TGF-b1 for Id genes are known targets of TGF-b and BMP different time periods and subjected the cell lysates to signaling, and they are regulated in a cell-type-specific Western blotting. In contrast to keratinocytes, we manner by these factors (Korchynskyi and ten Dijke, observed a strong upregulation of Id1 protein expres- 2002; Kurisaki et al., 2003; Kowanetz et al., 2004). sion in dermal fibroblasts after 3 h of activin or TGF-b1 However, their physiological roles in the TGF-b/BMP treatment (Figure 1c). Expression subsequently de- signaling pathways are still poorly understood. Here, we clined, but was still higher compared to controls within show that activin A suppresses the expression of Id1, 5–8 h after addition of either activin or TGF-b1. Thus, Id2, and Id3 in keratinocytes at the mRNA and protein activin-mediated Id1 expression indeed occurs in a cell- level in vitro and in vivo, and we provide evidence for an type-specific manner. This result was reproduced in an important regulatory function of Id1 in skin home- independent experiment (data not shown). ostasis. Reduced expression of id1, id2, and id3 genes in tail skin of transgenic mice overexpressing activin in the epidermis Results To determine if the three Id family members are also regulated by activin in vivo, we performed RPAs with Regulation of Id1, Id2, and Id3 expression in HaCaT cells RNAs from tail skin of transgenic mice that overexpress by activin A and TGF-b1 the activin bA chain under the control of the K14 To identify activin-regulated genes in keratinocytes, we promoter in the epidermis (Munz et al., 1999). RNAs hybridized a cDNA array with radioactively labeled from wild-type littermates were used as a control. Tail cDNA from quiescent HaCaT cells and from cells that skin from four mice per genotype was pooled for each had been treated for 5 or 8 h with activin A. Among the experiment. Indeed, we found reduced levels of id1, id2, 588 genes represented on this filter, only three appeared and id3 mRNAs in tail skin of transgenic mice compared to be regulated by activin A. One of them encodes Id1, a to control mice. Figure 1d shows the result of one negative regulator of bHLH transcription factors representative experiment, which was reproduced at (Benezra et al., 1990). To verify this regulation and to least once with independent RNA samples from addi- determine, if other Id family members are also regulated tional tail biopsies. by activin, we treated quiescent human HaCaT kerati- Activin-mediated downregulation was also observed nocytes with activin A for different time periods. Since on the protein level for Id3 by immunofluorescence TGF-b is known to regulate id genes in different cell staining of tail skin sections from activin-overexpressing types (Ling et al., 2002; Kang et al., 2003; Kurisaki and wild-type animals. In normal tail skin, we found et al., 2003), we used TGF-b1 as a positive control. As strong signals for Id3 in keratinocytes of the epidermis shown by RNase protection assay (RPA) (Figure 1a), and the hair follicles (Figure 1e, left panel). In tail skin id1, id2, and id3 mRNA levels were downregulated from activin transgenic mice, this keratinocyte staining within 5–8 h of treatment with either activin A or TGF- was almost completely absent, whereas the weak signal b1. The time course and extent of downregulation were in the underlying dermis remained (middle panel). This different for the two TGF-b family members. In the case suggests that activin also suppresses Id3 expression in of id1, the TGF-b1- and activin-induced downregulation keratinocytes in vivo. The specificity of the staining was was preceded by a transient upregulation between 1 and confirmed by preincubation of the antibody with the 3 h after addition of the factor (Figure 1a and data not immunization peptide (right panel). shown). Taken together, these results demonstrate that over- To confirm the activin-mediated Id1 regulation at the expression of activin is associated with downregulation protein level, total cell lysates from activin A- or TGF- of Id1 levels in vivo, indicating that high levels of activin b1-treated cells were subjected to Western blot analysis. can indeed regulate Id1 expression not only in vitro but Consistent with the results obtained at the RNA level, also in vivo. an initial upregulation of Id1 expression was observed within the first hour after activin- or TGF-b1 treatment Expression of Id1, Id2, and Id3 in healing skin wounds (data not shown), followed by a strong downregulation We next addressed the question if endogenous activin of this protein within 3–8 h (Figure 1b). To verify that can also regulate id expression in vivo. For this purpose, this effect is not a starvation artefact, lysates were also we analysed the expression of these transcriptional prepared from cells, which had been incubated in serum- regulators during the healing process of full-thickness free medium in the absence of activin. The latter excisional mouse wounds (Figures 2 and 3), since activin displayed high levels of Id1 protein, and no down- is highly expressed between day 1 and day 7 of wound regulation was observed upon starvation (Figure 1b). repair (Hu¨ bner et al., 1996). The time points that we This result was reproduced in several independent chose for the expression analysis are characteristic for experiments (data not shown). the three different phases of repair. In the early

Oncogene Id proteins in epidermal homeostasis D Rotzer et al 2072

Figure 1 Activin A-mediated regulation of Id1, Id2, and Id3 mRNAs and proteins in HaCaT keratinocytes and activin- overexpressing transgenic mice. (a) HaCaT keratinocytes were rendered quiescent by serum starvation and subsequently treated with activin A or TGF-b1 for different time periods. Nontreated cells were used as controls (control). Total cellular RNA from these cells was analysed by RPA for the presence of id mRNAs. As a loading control, 1 mg of each RNA sample was loaded on a 1% agarose gel and stained with ethidium bromide (shown below the RPA). (b) Western blotting was performed with protein lysates of untreated (control) or activin A- or TGF-b1- treated HaCaT keratinocytes using anti-Id1 and anti-b-actin antibodies. (c) Western blotting was performed with protein lysates of untreated (control) or activin A- or TGF-b1- treated primary human dermal fibroblasts using anti- Id1 and anti-b-actin antibodies. (d) Total RNAs from pools of tail skin of four activin bA-overexpressing and four wild-type littermates were analysed by RPA for the expression of id and gapdh mRNAs. The ratios of id and gapdh signal intensities were determined and are depicted in bar graphs. The values obtained with control RNAs were arbitrarily set as 1. (e) Immunofluorescence staining for Id3 on frozen tail skin sections from wild-type (wt) and transgenic activin bA-overexpressing (tg) animals. As a control for unspecific antibody binding, the Id3 antibody was preincubated with the immunization peptide (IP). E, epidermis; D, dermis; HF, hair follicle.

inﬂammatory phase, neutrophils and macrophages 3 and day 7 after injury. Id1 mRNA was regulated become activated and enter the wound area (days 1– differently compared to id2 and id3, since its level of 3). The second phase is characterized by the formation expression was low in unwounded skin and increased of new tissue and comprises granulation tissue forma- transiently early after wounding, starting within 1–3 h tion and re-epithelialization (days 3–7). The third phase after injury (data not shown). Thereafter, it returned to is the phase of tissue remodeling (starting around day 7) basal levels (Figure 2). At 14 days after injury, wounds (Martin, 1997; Singer and Clark, 1999). Overviews of were completely re-epithelialized, but still contained a wounds in phases 2 and 3 are schematically depicted in cell-rich granulation tissue. At this stage of repair, id2 Figure 3, top panel. As shown in Figure 2, id2 and id3 and id3 mRNA levels were as high as in unwounded mRNA levels were slightly downregulated between day skin, whereas id1 mRNA levels increased again.

Oncogene Id proteins in epidermal homeostasis D Rotzer et al 2073 skin 1d 3d 5d 7d 14d

id1

gapdh

3 2.5 2 1.5 1 0.5 0 skin 1d 3d 5d 7d 14d

skin 1d 3d 5d 7d 14d

id2

gapdh

1.5

0.5

0 skin 1d 3d 5d 7d 14d Figure 3 Id proteins are predominantly expressed in basal keratinocytes of the hyperproliferative wound epidermis. Frozen skin 1d 3d 5d 7d 14d sections from 5-day and 14-day wounds of BALB/c mice were subjected to immunofluorescence staining using antibodies directed against Id1, Id2 or Id3 (red). Nuclei were counterstained with id3 DAPI (blue). Id-positive nuclei appear in pink (arrowheads). The corresponding stages of wound healing are schematically illustrated above the immunofluorescence pictures. The wound areas shown in the immunofluorescence stainings are marked with rectangles. gapdh Magnification: Â 200. E, epidermis; GT, granulation tissue; HE, hyperproliferative epithelium.

1.5

We subsequently localized Id proteins in normal and 1 wounded mouse skin by immunoﬂuorescence. Consistent with data obtained with human skin (Langlands et al., 0.5 2000; Bjorntorp et al., 2003; Chaturvedi et al., 2003), we found the three Id proteins predominantly in basal keratinocytes of tail skin from BALB/c mice (data not 0 shown). In 5d and 14d skin wounds, Id proteins were skin 1d 3d 5d 7d 14d expressed in cells of the granulation tissue as well as in the hyperproliferative epithelium, where nuclear as well Figure 2 Regulation of id1, id2, and id3 gene expression during wound healing. RNA was isolated from full-thickness excisional as cytoplasmic staining was observed (Figure 3). Ex- wounds of BALB/c mice at different time points after injury. Pools pression was strongest in basal and lower suprabasal of 16wounds from four mice were used for each time point. cells. Interestingly, the Id expression pattern showed an Samples of 20 mg RNA were analysed by RPA for the expression of inverse relationship to the one of activin that is id and gapdh mRNAs. The ratios of corresponding id and gapdh signal intensities were determined and are depicted in bar graphs. predominantly found in the upper, more differentiated The values obtained with RNA samples of unwounded skin were cells of the wound epidermis (Hu¨ bner et al., 1996; arbitrarily set as 1. Wankell et al., 2001b; SW, unpublished data).

Oncogene Id proteins in epidermal homeostasis D Rotzer et al 2074 Generation of Id1-overexpressing HaCaT cells gradually lost (data not shown). In the other clones, the To determine the importance of the activin-mediated levels of endogenous Id1 protein exceeded the levels of downregulation of Id1 for keratinocyte proliferation recombinant Id1-HA. These ratios between recombi- and differentiation, we generated stable HaCaT cell nant and endogenous Id1 were verified at the RNA level lines, which overexpress Id1 with a carboxyterminal by RPA using a probe that can distinguish between epitope of the influenza virus hemagglutinin (HA), mRNAs encoding endogenous id1 or recombinant id1- designated Id1-HA. Id1 was chosen for further analysis HA mRNAs (Figure 4a, right panel). In some clones, we because it is highly expressed in wounded and psoriatic found enhanced levels of endogenous Id1 protein in Id1- skin (see Figure 3 and Bjorntorp et al., 2003). Western HA-expressing clones, but this was not consistently blotting was performed with protein lysates from Id1- observed (Figure 4a left panel and data not shown). HA-transfected and vector-transfected (neo) cell clones. We next determined if the overexpression of Id1-HA In all clones, the levels of Id1-HA were similar to or is more pronounced under conditions where the expres- lower than the levels of endogenous Id1. The strongest sion of endogenous Id1 is downregulated, for example, expression of the recombinant protein was seen in clone upon treatment with TGF-b1. As expected, TGF-b1 #1, which showed equal levels of endogenous and treatment resulted in a downregulation of endogenous overexpressed protein under exponential growth condi- Id1. By contrast, expression of the CMV promoter- tions (Figure 4a, left panel). However, the expression driven id1-HA transgene was suppressed upon starva- levels of this clone declined upon culturing and freezing/ tion (0 and 8 h controls) and subsequently upregulated thawing, suggesting that the high-expressing cells are in response to TGF-b1 (Figure 4b for clone 1). Thus, the recombinant protein was indeed strongly overexpressed in TGF-b1-treated keratinocytes. a HaCaT Id1-HA HaCaT neo HaCaT Id1-HA 12 3neo Since Id expression is downregulated during differ- 123 1 23 entiation of other cell types, we determined if this is also Id1-HA Id1-HA- Id-1 the case for HaCaT keratinocytes. For this purpose, Id1- cells were grown to confluence and subsequently β-actin maintained in the same medium for several days. Under these conditions, partial differentiation of HaCaT h 5 keratinocytes is achieved (Ryle et al., 1989). In Western β13h β1 β18h GF- GF- b Co 0h T TGF- T Co 8h blot experiments, we indeed observed a differentiation- Id1-HA induced downregulation of Id1 (Figure 4c) that is Id-1 consistent with data obtained with primary keratinocytes (Hara et al., 1994; Langlands et al., 2000; Schaefer β-actin et al., 2001). In Id1-HA-expressing cells, the endogenous c Id1, but not the recombinant Id1-HA, was down- HaCaT: exp confl 3d 5d 6d 7d regulated during this density-dependent differentiation

α process (Figure 4c, lower panel). Compared to wild-type Id1 Id1 and vector-transfected HaCaT cells, downregulation

αβ-actin β-actin of endogenous Id1 was less pronounced in Id1-HA- expressing cells, possibly as a consequence of HaCaT/Id1-HA: exp. confl 4d 6d 7d 11d Id1-mediated inhibition of early differentiation (see αId1 Id1-HA below). Id1

α HA Id1-HA Id1 overexpression does not affect proliferation of HaCaT β αβ-actin -actin cells in monoculture Figure 4 Generation and characterization of Id1-HA-overexpres- We next determined whether Id1 overexpression affects sing HaCaT keratinocytes. (a) Left panel: Protein lysates from Id1- cell proliferation under exponential culture conditions HA-overexpressing (HaCaT/Id1-HA) and vector-transfected Ha- in serum-containing medium and/or in response to CaT keratinocytes (HaCaT/neo) were analysed by Western blotting TGF-b1 or activin A. Therefore, we performed 5- for the presence of recombinant Id1-HA and endogenous Id1 using 0 an anti-Id1 antibody that recognizes both proteins. Right panel: bromo-2 -deoxyuridine (BrdU) incorporation studies 20 mg total cellular RNA from HaCaT/Id1-HA and HaCaT/neo with different HaCaT/Id1-HA (n ¼ 5) and HaCaT/neo cells were analysed by RPA for the presence of id1 MRNAs using a (n ¼ 3) clones. However, no significant difference in the probe that can distinguish between endogenous id1 and id1-HA proliferation rate of these cells was observed in MRNA (b) Protein lysates of Id1-HA-overexpressing HaCaT cells monolayer culture (Figure 5a). Furthermore, we eval- were prepared at different time points after treatment with TGF-b1 (1 ng/ml) and subjected to Western blotting. (c) Western blot uated the effects of activin A and TGF-b1 on Id1-HA- analysis was performed with protein lysates from exponentially expressing HaCaT and control cells. Figure 5b shows growing and confluent Id1-HA-expressing (lower panel) and that the growth-inhibitory effect of TGF-b1 for control cells (upper panel) as well as from cells that were kept keratinocytes (Shipley et al., 1986) was not abrogated confluent for the indicated time points. The membrane was treated with the anti-Id1 antibody, then reprobed with an anti-HA by enhanced expression of Id1. Activin only showed a antibody for exclusive detection of the Id1-HA protein and again very weak antimitotic effect that was not influenced by reprobed with an antibody against b-actin as a loading control. Id1 overexpression.

Oncogene Id proteins in epidermal homeostasis D Rotzer et al 2075

ab70 120 ****** *** *** 60 100

50 80 40 w/o 18ng/ml Act 60 30 90ng/ml Act 1ng/ml TGF 20 40 percent of control (%)

percent of proliferating cells (%) 10 20

0 HaCaT Id1 HaCaT neo 0 (n=5) (n=3) HaCaT-Id1-1 HaCaT-Id1-2 HaCaT/neo1 HaCaT/neo2 Figure 5 Expression of Id1-HA does not affect proliferation of HaCaT cells in monolayer culture. (a) Proliferation of ﬁve different clones of Id1-HA-expressing HaCaT cells and three vector-transfected control clones under exponential growth conditions was analysed by BrdU-incorporation analysis. The percentage of BrdU positive cells is shown as a bar graph. (b) Id1-HA-expressing and vector-transfected cells were treated for 24 h with 18 or 90 ng/ml activin A or 1 ng/ml TGF-b1, respectively, and subsequently incubated with BrdU. Proliferating cells were determined in triplicate wells by BrdU incorporation analysis and values are given as the percentage of the value obtained with nontreated cells. These data were reproduced in two independent experiments with the same cell clones as well as with two different clones. ***Po0.001.

Id1-overexpressing HaCaT cells differentiate abnormally cells of the basal and the suprabasal layers of cultures in organotypic cultures derived from HaCaT/Id1-HA clones (Figure 6b, left Finally, we determined if Id1 overexpression affects panels). Consistent with this expression pattern of Id1- keratinocyte differentiation. Since HaCaT cells differ- HA, we found Id1 immunoreactivity in the basal layer entiate similarly to primary keratinocytes in three- of vector-transfected and Id1-HA-expressing cells, dimensional organotypic cultures (Schoop et al., 1999; whereas suprabasal Id1-positive nuclei were only seen Maas-Szabowski et al., 2003), we established such in Id1-HA cultures (Figure 6b, right panels). cultures using either vector-transfected or HaCaT-Id1- In control cultures, the a6-integrin subunit is re- HA cells. Figure 6shows staining of cultures from one stricted to the basolateral side of basal keratinocytes representative HaCaT/neo and one HaCaT/Id1-HA (Figure 6c). In these cells it forms a dimer with the b4 clone. In total, we tested five Id1-HA clones, and three subunit, and these a6b4 integrins are a component of clones of vector-transfected cells and we analysed two hemidesmosomes that connect basal cells to the base- clones of each genotype in two independent experiment membrane (Watt, 2002). In Id1-HA-expressing ments. cells, a more diffuse a6-integrin staining was observed, After 14 days of cultivation, vector-transfected with staining around the basal cells and punctuate HaCaT cells formed a multilayered, partially differen- staining in a few suprabasal cells. A less-organized tiated epidermis-like epithelium. After 4 weeks, the staining compared to control cells was also seen for the epithelium was fully differentiated and consisted of well- basement membrane marker laminin-5 (Figure 6d), organized basal, spinous, and granular layers, and a further reflecting the disorganized basal cell layer and parakeratotic cornified layer (Figure 6a). By contrast, the invasion of cells into the collagen gel. No epithelial– the organotypic cocultures with Id1-HA-overexpressing mesenchymal transition occurred in the epithelium of HaCaT cells showed severe abnormalities. As shown in Id1-HA cells as revealed by the lack of the mesenchymal the hematoxylin/eosin-stained sections (Figure 6a), Id1- marker protein vimentin in these cells (data not HA clones formed a hyperplastic epithelium after 2–4 shown). weeks of culture. The time point of maximal hyperplasia To analyse the degree of differentiation of Id1- varied among the Id1-HA cultures and was particularly overexpressing HaCaT cells in more detail, we stained early in cultures of the clone with the highest expression sections with antibodies against characteristic early and level (data not shown). Id1-HA cultures showed an late differentiation markers (shown for the 4-week increase in the number of viable cell layers, but a thinner cultures in Figure 6e and f). Whereas expression of the cornified layer. Furthermore and in contrast to the early differentiation marker keratin 10 (K10) started in control cultures, the Id1-HA cells had invaded the the first suprabasal layer of control cultures, we found collagen gel, leading to a disorganized border between an irregular distribution of K10 in Id1-HA cultures collagen gel and epithelium. (Figure 6e, and data not shown). In particular, the cells, To verify the expression of Id1-HA in the epithelium which had invaded into the collagen gel, did not express of organotypic cultures, we performed immunofluores- K10. In addition, the number of K10-positive layers was cence staining using an antibody directed against the also increased in most of the cultures. Most surprisingly, HA-epitope. We found specific signals (pink nuclei) in the late differentiation markers loricrin and filaggrin

Oncogene Id proteins in epidermal homeostasis D Rotzer et al 2076

Figure 6 Id1 overexpression affects proliferation, differentiation, and apoptosis of HaCaT keratinocytes in organotypic cultures. Cells were cultured in three-dimensional organotypic cultures for 14 days (a, g, h, upper panel) or 28 days (a, g, h, lower panel and (b–f)), embedded in parafﬁn or tissue freezing medium and sectioned. Sections were stained with (a) hematoxylin/eosin or immunostained with antibodies to the HA epitope (b, left panel), Id1 (b, right panel), a6-integrin (c), laminin-5 (d), keratin 10 (green) and keratin 14 (red) (e), loricrin (f), or cleaved caspase-3 (h). In (b), (f), and (h), nuclei were counterstained with Hoechst (blue). K, keratinocytes; F, ﬁbroblasts. In (b), the basement membrane is marked with a dashed line. (g) Cultures were incubated in BrdU-containing medium for 2 h before harvesting. Frozen sections were stained with an antibody directed against BrdU, followed by peroxidase staining, and counterstained with hematoxylin. Bars represent 100 mm. In (g) and (h), the panels at the right-hand side show the results of the quantitative analysis of BrdU- or cleaved caspase-3-positive cells, respectively. Four pictures from each of three cultures per clone were counted. *Po0.05, ***Po0.001.

(Figure 6f and data not shown), which are character- Taken together, our results reveal that a small istically expressed in the uppermost living cell layers, enhancement in the levels of Id1 affects proliferation, were present throughout the epithelium in a patchy differentiation, and apoptosis of keratinocytes grown in distribution. organotypic cultures. This suggests that normal epider- We next determined if the hyperplasia of the mis formation depends on a tight regulation of the levels epidermal layer results in part from enhanced prolifera- of Id1. tion. As shown in Figure 6g, the proliferation rate of basal keratinocytes was slightly increased in Id1-HA cultures after 2 weeks (upper panel), but returned to normal levels after 4 weeks (lower panel). In all cultures, Discussion BrdU-positive cells were predominantly seen in the basal layer, although a few cells in the suprabasal layers of Id proteins: novel targets of activin action in keratinocytes Id1-HA cultures had also incorporated BrdU. and ﬁbroblasts Finally, we determined the rate of apoptosis in the Activin is an important regulator of skin homeostasis different organotypic cultures by staining for cleaved and wound repair. However, there is little knowledge caspase-3 (Figure 6h). As expected, we found caspase-3- about the mechanisms underlying activin action in the positive cells in the most upper layer of all cultures, skin, and the activin target genes are largely unknown. reﬂecting the disassembly of the cellular components Here we show that activin suppresses the expression of during terminal differentiation. However, the number of id genes in keratinocytes in vitro. Most interestingly, apoptotic cells within the epithelium was strongly downregulation of these genes was also seen in enhanced in 2-week and 4-week Id1-HA cultures. In keratinocytes of activin-overexpressing transgenic mice, particular, a large number of cells in the lower suggesting that id genes are also targets of activin action suprabasal layers stained positive for caspase-3 in these in vivo. This hypothesis is further supported by the cultures. inverse relationship between activin and Id expression in

Oncogene Id proteins in epidermal homeostasis D Rotzer et al 2077

Figure 6 continued

the hyperproliferative wound epidermis (Hu¨ bner et al., abolished the antimitotic effect of TGF-b (Kowanetz 1996, and this study), and indicates that endogenous et al., 2004). activin also regulates the levels of Id proteins. Thus, in Strong Id1 overexpression is detrimental for keratinocytes addition to TGF-b (Ling et al., 2002; Kretschmer et al., To determine if Id1 affects keratinocyte proliferation, 2003; Siegel et al., 2003; Kowanetz et al., 2004), differentiation, and/or apoptosis, we generated stable activin was identified as a novel regulator of Id gene HaCaT cell lines overexpressing Id1. Surprisingly, we expression. only obtained cell lines that express the recombinant Similar to TGF-b, activin has opposite effects on Id1-HA protein at similar or lower levels compared to keratinocytes and fibroblasts. It slightly reduces kerati- the endogenous Id1. This finding is consistent with nocyte proliferation (Shimizu et al., 1998; Seishima reports from other investigators who failed to over- et al., 1999), but enhances growth of cultured fibroblasts express Id1 in HaCaT cells (Langlands et al., 2000) and (Ohga et al., 1996; Yamashita et al., 2004). The different suggests that enhanced levels of this protein are responses of fibroblasts and keratinocytes to activin and detrimental. This hypothesis is further supported by TGF-b are reflected by the opposite regulation of Id our results, which revealed a higher rate of apoptosis of expression in both cell types, suggesting that the up- or the Id1-overexpressing clones in organotypic cultures as downregulation of Id proteins by activin/TGF-b could well as by preliminary findings with the clone with the mediate – at least in part – the effects of these factors on highest Id1 expression level. The latter showed a higher proliferation of fibroblasts and keratinocytes. Consis- apoptotic rate in response to staurosporine treatment or tent with this hypothesis, Id1 proteins are required for UVB irradiation, and the high-expressing cells in this G1 progression of human fibroblasts (Hara et al., 1994), culture were lost upon passaging (data not shown). whereas overexpression of Id2 and Id3 in HaCaT cells Consistent with our findings, stable transfection of

Oncogene Id proteins in epidermal homeostasis D Rotzer et al 2078 rat embryo fibroblasts with Id2 resulted in apo- mobile phenotype (Martin, 1997). Schaefer et al. (2001) ptosis (Norton et al., 1998). Therefore, the extent of Id observed upregulation of Id1 in keratinocytes upon overexpression that can be achieved seems to be limited. detachment from their growth substratum. This and several reports on the crucial role of Id overexpression in tumor invasiveness and metastasis (Lin et al., 1999) Id1 is not required for the growth-suppressing effect of suggest an important influence of Id proteins not only TGF-b1 on proliferation, but also on the stimulation of cell Although the extent of Id1-HA overexpression was motility after skin injury. minor under normal culture conditions, significant We also found strong abnormalities in the differentia- overexpression was observed under conditions where tion pattern as well as in the rate of apoptosis of Id1-HA the endogenous protein was downregulated, such as in cell cultures. Thus, our results demonstrate that response to TGF-b1 or upon induction of differentia- enhanced levels of Id1 disturb the balance between tion. Therefore, these cell lines were suitable to keratinocyte proliferation and differentiation/apoptosis, determine the role of Id1 downregulation in the growth resulting in a hyperplastic, disorganized epidermis. suppressive effect of TGF-b and activin. However, These findings suggest that a tight regulation of the forced expression of Id1-HA in HaCaT cells did not levels of this transcriptional regulator is required for significantly affect the proliferation rate of keratinocytes epidermal homeostasis. Finally, our findings also in serum-containing medium and in response to activin suggest that Id1 induces invasive growth of keratino- and TGF-b1. This finding is consistent with results from cytes, since we observed a rather extensive ingrowth of Kang et al. (2003), who demonstrated that transient Id1-HA-expressing HaCaT cells into the dermal equiva- overexpression of Id1 in HaCaT cells does not diminish lent. As this phenotype is not unique for Id1 but is also the TGF-b cytostatic response. However, different observed with other HaCaT cell clones, which express results were obtained with overexpressed Id2 and Id3 genes that alter the differentiation process (PB and SW, in HaCaT cells. In this case, the growth-inhibiting effect unpublished data), it is tempting to speculate that with of TGF-b was abrogated (Kowanetz et al., 2004). In the altered differentiation, a program is induced that addition to the different regulation in cultured cells and causes activation of collagen-degrading enzymes and skin wounds, this finding suggests different functions of thus allows invasion into the dermal equivalent. Future Id1 as compared to the other Id proteins. experiments will address whether regulation of the expression of specific proteases such as collagenases A novel role of Id1 in epidermal homeostasis (matrix metalloproteases) or cathepsins may be another In addition to their effects on proliferation, Id proteins as yet unknown activity of Id1 in keratinocytes. potently suppress differentiation of various cell types (Ruzinova and Benezra, 2003). Therefore, we tested if Id1 also affects differentiation of keratinocytes. Since full differentiation of HaCaT cells can only be achieved Materials and methods in three-dimensional organotypic cultures (Schoop et al., 1999), we used this technology to determine the Cell culture and growth factor treatment of keratinocytes effect of Id1 overexpression on keratinocyte differentia- HaCaT keratinocytes (Boukamp et al., 1988) were grown to tion. Indeed, the epithelia formed by Id1-HA-expressing confluence in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, Deisenhofen, Germany) containing 1% penicillin/ cells were hyperthickened and disorganized, and under streptomycin and 10% fetal calf serum (FCS) (BioConcept these conditions, Id1 overexpression transiently en- Amimed, Allschwil, Switzerland). They were rendered quies- hanced the proliferation rate of keratinocytes. Thus, cent by serum starvation for 24–32 h and subsequently treated fibroblast-derived paracrine acting factors and/or the with fresh DMEM containing 1 ng/ml TGF-b1 (R&D Systems, three-dimensional architecture are obviously required Wiesbaden, Germany) or 18 ng/ml activin A (R&D Systems). for Id1-mediated stimulation of keratinocyte prolifera- These activin concentrations were reported to reduce kerati- tion. Interestingly, enhanced levels of Id1 were also seen nocyte proliferation and to induce their differentiation in the hyperplastic epidermis of psoriatic patients (Seishima et al., 1999), and they elicit maximal transcriptional (Bjorntorp et al., 2003), suggesting a functional role of responses in HaCaT cells (Werner et al., 2001). Cells were Id1 in the pathogenesis of this disease. In addition, high harvested at different time points after addition of the growth factor and used for RNA isolation or preparation of cell levels of Id proteins in the hyperproliferative wound lysates. epidermis may enhance proliferation and prevent pre- Primary human dermal fibroblasts from foreskin biopsies mature onset of early differentiation of wound kerati- were cultured in DMEM containing 1% penicillin/streptomy- nocytes, thereby contributing to wound re- cin and 10% FCS and treated with growth factors as described epithelialization. In suprabasal cells of the wound for HaCaT cells. Human dermal fibroblasts from adult trunk epidermis, high levels of activin and/or TGF-b (Frank skin were isolated from explant cultures of de-epidermized et al., 1996; Hu¨ bner et al., 1996) are likely to suppress Id dermis. They were expanded up to passage 3–4 in DMEM/10% expression, resulting in growth inhibition and induction FCS and cryo-preserved until used in organotypic cultures. of differentiation of these cells. During wound healing, keratinocytes from the border Organotypic cocultures of the lesion detach from the basement membrane and The preparation of the dermal equivalent based on type I repair the defect by transforming from a sessile into a collagen hydrogels has been described previously (Smola et al.,

Oncogene Id proteins in epidermal homeostasis D Rotzer et al 2079 1998; Stark et al., 1999; Maas-Szabowski et al., 2003). Briefly, scanned and analysed with the Scion Image software (Scion 80% (vol.) type I collagen in 0.1% acetic acid (4 mg/ml; rat tail Corporation, MD, USA). tendon) were mixed on ice with 10% (vol.) of 10 Â Hank’s buffered saline containing phenol red. After neutralizing with Preparation of protein lysates from HaCaT cells and fibroblasts 2 M NaOH, a fibroblast suspension in FCS was added under HaCaT cells were lysed in urea buffer (10 mM Tris [pH 8.0], stirring (1 Â 105 cells/ml, final concentration). This mixture was transferred into membrane filter inserts (Falcon no. 3901, 3 mm 9.5 M urea, 2 mM EDTA, 2 mM PMSF, 1 mM dithiothreitol). 1 pores, BD-Biosciences, Heidelberg, Germany), placed in deep Lysates were sonicated and centrifuged for 15 min at 4 C. well plates (BD-Biosciences no. 5467) and gelled at 371C. Glass Protein concentrations in the supernatant were determined rings were placed on the gels to allow keratinocyte seeding. using the BCA Protein Assay Reagent (Pierce, Rockford, IL, The gels were equilibrated for 24 h in FAD medium with 10% USA). FCS, 10À10 M cholera toxin, 0.4 mg hydrocortisone, and 50 mg L- ascorbic acid (Sigma) per ml. Cocultures were obtained by Western blot analysis seeding 1 Â 106 keratinocytes inside the glass rings (3 Â 105 cells Proteins were separated by SDS polyacrylamide gel electro- per cm2). After submersed incubation overnight, the glass rings phoresis and transferred to a nitrocellulose membrane. Anti- were removed and the medium level was lowered to the base of body incubations were performed in 3% non-fat dry milk in the dermal equivalent, thereby exposing the culture surface to TBS-T (10 mM Tris-HCl pH7.4, 150 mM NaCl, 0.1% Tween the air-medium interface. Cultivation was continued for 2 or 4 20). The following antibodies were used: a rabbit polyclonal weeks in FAD medium with 10% FCS, 10À10 M cholera toxin, antibody against Id1 (Santa Cruz Biotechnology, Santa Cruz, 0.4 mg hydrocortisone, and 50 mg L-ascorbic acid/ml with CA, USA), a rabbit polyclonal antibody against the HA medium change every other day. epitope (Santa Cruz), and a mouse monoclonal antibody against b-actin (Sigma). Detection was performed with the Hybridization of cDNA arrays enhanced chemoluminescence detection system (ECL; Amer- Polyadenylated RNA was prepared from total cellular RNA sham). using the mRNA Separator Kit (Clontech Laboratories, Palo Alto, CA, USA) following the instructions of the manu- þ Immunofluorescence and histological analysis facturer. The poly(A) -mRNA was isolated by affinity chro- Tail skin from BALB/c and CD1 mice and wounds from matography using oligo-d(T)-cellulose columns. The Atlas BALB/c mice were frozen in tissue freezing medium (Jung, human cDNA expression array I (Clontech) was hybridized Nussloch, Germany). Acetone-fixed frozen sections (6 mm) with radioactively labeled cDNA from quiescent and activin were incubated overnight at 41C with rabbit polyclonal A-treated HaCaT cells according to the manufacturer’s antibodies directed against Id1, Id2, or Id3 (Santa Cruz) instructions. diluted in PBS containing 12% BSA, 0.1% NP-40, and 0.1% Tween 20. After three washes with PBS/0.1% Tween 20, Generation of HaCaT cells stably expressing Id1-HA sections were incubated for 1 h with an anti-rabbit-Cy3 The full-length human id1 cDNA was amplified from HaCaT secondary antibody (Jackson ImmunoResearch Laboratories, cDNA using RT–PCR. In an additional PCR step, an in-frame West Grove, PA, USA), washed again and mounted with HA epitope was inserted in front of the stop codon of the id1 Mowiol (Hoechst, Frankfurt, Germany). Organotypic cultures cDNA. The construct was subcloned into the expression vector were fixed at 41C in acidic ethanol and embedded in paraffin. pIRES/neo (Clontech). For stable transfection, HaCaT cells Alternatively, they were frozen in tissue-freezing medium were seeded at 50–70% confluence and transfected using without prior fixation. Paraffin sections (7 mm) were de-waxed LipofectAMINE (Invitrogen, Basel, Switzerland) according to and incubated overnight at 41C with a mouse monoclonal the manufacturer’s instructions. Cells stably transfected with antibody directed against keratin 10 (Dako), or rabbit the pIRES/neo expression vector were selected by cultivation polyclonal antibodies directed against keratin 14, filaggrin, in growth medium containing 400 mg/l G418 (Invitrogen). loricrin (all from BabCO, Richmond, CA, USA), and the HA- epitope (Santa Cruz). Frozen sections were fixed with ice-cold RNA isolation and RPA acetone or 4% paraformaldehyde and incubated with the Id1 Isolation of total cellular RNA was performed as described by antibody described above, a rat monoclonal antibody directed Chomczynski and Sacchi (1987). RPAs were carried out against a6-integrin (BD Bioscience PharMingen, San Diego, according to Werner et al. (1993). The following cDNA CA, USA), or rabbit polyclonal antibodies directed against the templates were amplified by RT–PCR using RNA from mouse g2-chain of laminin-5 (kindly provided by Dr Takako Sasaki, wounds or HaCaT keratinocytes: a human id1 cDNA Martinsried, Germany) or cleaved caspase-3 (Cell Signaling corresponding to nucleotides (nt) 121–365 of the published Technology, Beverly, MA, USA). As secondary antibodies, sequence (Hara et al., 1994), a human id2 cDNA (nt 96–308 of anti-rabbit-Cy3 (red), anti-rat-Cy3, and anti-mouse-Cy2 the cDNA; Hara et al., 1994), a human id3 cDNA (nt 738–936 (green) (Jackson ImmunoResearch) were used. Sections were of the cDNA; Deed et al., 1994), a mouse id1 cDNA counterstained with DAPI (1 mg/ml) and photographed using a (nt 251–528 of the cDNA; NM_010495), a mouse id2 cDNA Zeiss Axioplan fluorescence microscope. For histological (nt 1119–1349 of the cDNA; AF077860), and a mouse id3 analysis, paraffin-embedded OTCs were sectioned, dewaxed cDNA (nt 57–255 of the cDNA; NM_010813). These cDNAs and stained with hematoxylin/eosin according to standard were cloned into the transcription vector pBluescript KSII( þ ) procedures. (Stratagene, La Jolla, CA, USA). As a loading control, either 1 mg of the RNA samples was loaded on a 1% agarose gel prior BrdU incorporation studies to hybridization and stained with ethidium bromide or the 1.2 Â 105 HaCaT cells were seeded into 3.5 cm dishes. After RNA was hybridized with a probe for murine glyceraldehyde- 24 h of cultivation, cells were treated with 18 or 90 ng/ml 3-phosphate dehydrogenase (GAPDH) (nt 566–685 of the activin A or 1 ng/ml TGF-b1 for 24 h and incubated with cDNA; NM_008048.1) or with a probe for human GAPDH BrdU (Sigma, 100 mM) for 2 h before fixation with 4% PFA. (nt 580–695 of the cDNA; BC_001601). Autoradiograms were To permeabilize the cell membrane and to denature the DNA,

Oncogene Id proteins in epidermal homeostasis D Rotzer et al 2080 cells were treated with 2 M HCl/0.1% Triton X-100 for 30 min. diameter, were made on either side of the dorsal midline as Subsequently, 0.1 M Na-borate (pH 8.5) was added for 5 min. described previously (Werner et al., 1994). Wounds were left Immunoﬂuorescence staining of BrdU-positive cells was uncovered and harvested at different time points after injury. performed by overnight incubation at 41C with an FITC- For RPA, the complete wounds including 2 mm of the conjugated monoclonal antibody directed against BrdU epithelial margins were excised and immediately frozen in (Roche, Rotkreuz, Switzerland). Cells were counterstained liquid nitrogen until used for RNA isolation. Nonwounded with propidium iodide (2 mg/ml), mounted, and photographed back skin served as a control. For immunoﬂuorescence using a Zeiss AxioCam HRc camera. For prolifera- analyses, the complete wounds were isolated, bisected, and tion analysis, the ratio of total cell number to the number frozen in tissue-freezing medium. All experiments with mice of BrdU-positive cells was determined by counting cells were performed with permission from the local veterinary using Openlab software (GraphPad Software Inc, San Diego, authorities of Zurich, Switzerland. USA).

Animals Acknowledgements Transgenic activin bA-overexpressing CD1 mice were described previously (Munz et al., 1999). BALB/c mice were We thank Christiane Born-Berclaz and Iris Nord for excellent obtained from RCC (Fu¨ llinsdorf, Switzerland). Mice were technical assistance and Dr Takako Sasaki, Max-Planck-Institute housed and fed according to federal guidelines. of Biochemistry, Martinsried, Germany, for kindly providing the laminin-5 antibody. This work was supported by a grant from Wounding and preparation of wound tissue the Swiss National Science Foundation (31-61358.00) to SW and Mice were anesthetized by intraperitoneal injection of keta- a grant from the EC/Swiss Ministry for Education and Research mine/xylazine. Two full-thickness excisional wounds, 5 mm in (LSHG-CT-2003-503447, WOUND) to SW and PB.

References

Bamberger C, Scha¨ rer A, Antsiferova M, Tychsen B, Pankow Maas-Szabowski N, Starker A, Fusenig NE. (2003). J Cell Sci S, Mu¨ ller M et al. (2005). Am J Pathol 167: 733–747. 116: 2937–2948. Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H. Martin P. (1997). Science 276: 75–81. (1990). Cell 61: 49–59. Massague J. (1990). Annu Rev Cell Biol 6: 597–641. Bjorntorp E, Parsa R, Thornemo M, Wennberg AM, Lindahl Mathews LS, Vale WW. (1993). Receptor 3: 173–181. A. (2003). Acta Derm Venerol 83: 403–409. Munz B, Smola H, Engelhardt F, Bleuel K, Brauchle M, Lein I Boukamp P, Petrussevska RT, Breitkreutz D, Hornung J, et al. (1999). EMBO J 18: 5205–5215. Markham A, Fusenig NE. (1988). J Cell Biol 106: Nakamura T, Takio K, Eto Y, Shibai H, Titani K, Sugino H. 761–771. (1990). Science 247: 836–838. Chambers RC, Leoni P, Kaminski N, Laurent GJ, Heller RA. Norton JD, Deed RW, Craggs G, Sablitzky F. (1998). Trends (2003). Am J Pathol 162: 533–546. Cell Biol 8: 58–65. Chaturvedi V, Bonish B, Bacon P, Qin JZ, Denning MF, Ohga E, Matsuse T, Teramoto S, Katayama H, Nagase T, Foreman K et al. (2003). Exp Dermatol 12: 255–260. Fukuchi Y et al. (1996). Biochem Biophys Res Commun 228: Chomczynski P, Sacchi N. (1987). Anal Biochem 162: 391–396. 156–159. Ruzinova MB, Benezra R. (2003). Trends Cell Biol 13: Cruise BA, Xu P, Hall AK. (2004). Dev Biol 271: 1–10. 410–418. Deed RW, Hirose T, Mitchell EL, Santibanez-Koref MF, Ryle CM, Breitkreutz D, Stark HJ, Leigh IM, Steinert PM, Norton JD. (1994). Gene 151: 309–314. Roop D et al. (1989). Differentiation 40: 42–54. Frank S, Madlener M, Werner S. (1996). J Biol Chem 271: Schaefer BM, Koch J, Wirzbach A, Kramer MD. (2001). Exp 10188–10193. Cell Res 266: 250–259. Hara E, Yamaguchi T, Nojima H, Ide T, Campisi J, Okayama Schoop VM, Mirancea N, Fusenig NE. (1999). J Invest H et al. (1994). J Biol Chem 269: 2139–2145. Dermatol 112: 343–353. Hu¨ bner G, Hu Q, Smola H, Werner S. (1996). Dev Biol 173: Seishima M, Nojiri M, Esaki C, Yoneda K, Eto Y, Kitajima 490–498. Y. (1999). J Invest Dermatol 112: 432–436. Kang Y, Chen CR, Massague J. (2003). Mol Cell 11: 915–926. Shimizu A, Kato M, Nakao A, Imamura T, ten Dijke P, Korchynskyi O, ten Dijke P. (2002). J Biol Chem 277: 4883– Heldin CH et al. (1998). Genes Cells 3: 125–134. 4891. Shipley GD, Pittelkow MR, Wille Jr JJ, Scott RE, Moses HL. Kowanetz M, Valcourt U, Bergstrom R, Heldin CH, (1986). Cancer Res 46: 2068–2071. Moustakas A. (2004). Mol Cell Biol 24: 4241–4254. Siegel PM, Shu W, Massague J. (2003). J Biol Chem 278: Kretschmer A, Moepert K, Dames S, Sternberger M, 35444–35450. Kaufmann J, Klippel A. (2003). Oncogene 22: 6748–6763. Sikder HA, Devlin MK, Dunlap S, Ryu B, Alani RM. (2003). Kurisaki K, Kurisaki A, Valcourt U, Terentiev AA, Pardali K, Cancer Cell 3: 525–530. Ten Dijke P et al. (2003). Mol Cell Biol 23: 4494–4510. Singer AJ, Clark RA. (1999). N Engl J Med 341: 738–746. Langlands K, Down GA, Kealey T. (2000). Cancer Res 60: Smola H, Stark HJ, Thiekotter G, Mirancea N, Krieg T, 5929–5933. Fusenig NE. (1998). Exp Cell Res 239: 399–410. Lasorella A, Uo T, Iavarone A. (2001). Oncogene 20: 8326– Stark HJ, Baur M, Breitkreutz D, Mirancea N, Fusenig NE. 8333. (1999). J Invest Dermatol 112: 681–691. Lin CQ, Parrinello S, Campisi J, Desprez PY. (1999). Endocr Tsuchida K, Nakatani M, Yamakawa N, Hashimoto O, Relat Cancer 6: 49–50. Hasegawa Y, Sugino H. (2004). Mol Cell Endocrinol 220: 59–65. Ling MT, Wang X, Tsao SW, Wong YC. (2002). Biochim Wankell M, Kaesler S, Zhang YQ, Florence C, Werner S, Biophys Acta 1570: 145–152. Duan R. (2001a). J Endocrinol 171: 385–395.

Oncogene Id proteins in epidermal homeostasis D Rotzer et al 2081 Wankell M, Munz B, Hubner G, Hans W, Wolf E, Goppelt A Werner S, Weinberg W, Liao X, Peters KG, Blessing M, et al. (2001b). EMBO J 20: 5361–5372. Yuspa SH et al. (1993). EMBO J 12: 2635–2643. Watt FM. (2002). EMBO J 21: 3919–3926. Yamashita S, Maeshima A, Kojima I, Nojima Y. (2004). JAm Werner S, Beer HD, Mauch C, Luscher B, Werner S. (2001). Soc Nephrol 15: 91–101. Oncogene 20: 7494–7504. Zhang L, Deng M, Parthasarathy R, Wang L, Werner S, Smola H, Liao X, Longaker MT, Krieg T, Mongan M, Molkentin JD et al. (2005). Mol Cell Biol 25: Hofschneider PH et al. (1994). Science 266: 819–822. 60–65.

Oncogene