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Published in final edited form as: J Invest Dermatol. 2011 Feb 3;131(5):1149–1158. doi: 10.1038/jid.2011.1

c-Jun Promotes whereas JunB Inhibits Epidermal Neoplasia

Jane Yingai Jin 1, Hengning Ke 1, Russell P Hall 1, Jennifer Y Zhang 1,2
PMCID: PMC3108157  NIHMSID: NIHMS279073  PMID: 21289643

Abstract

Deregulation of the AP1 family gene regulators have been implicated in a wide range of diseases, including cancer. Here, we report that c-Jun was activated in human squamous cell carcinoma (SCC) and coexpression of c-Jun with oncogenic Ras was sufficient to transform primary human epidermal cells into malignancy in a regenerated human skin grafting model. In contrast, JunB was not induced in a majority of human SCC cells. Moreover, exogenous expression of JunB inhibited tumorigenesis driven by Ras or spontaneous human SCC cells. Conversely, the dominant negative JunB mutant (DNJunB) promoted tumorigenesis, which is in contrast to the tumor suppressor function of the corresponding c-Jun mutant. At the cellular level, JunB induced epidermal cell senescence and slowed cell growth in a cell-autonomous manner. Consistently, coexpression of JunB and Ras induced premature epidermal differentiation concomitant with upregulation of p16 and filaggrin and downregulation of cyclinD1 and CDK4. These findings indicate that JunB and c-Jun differentially regulate cell growth and differentiation and induce opposite effects on epidermal neoplasia.

Keywords: AP1, JunB, c-Jun and squamous cell carcinoma

INTRODUCTION

JunB and c-Jun, along with JunD and Fos group proteins (c-fos, FosB, Fra1 and Fra2), comprise the core members of the AP1 family of transcription factors. These proteins share common structural and functional motifs that include a transactivation(TA) domain for regulating target gene transcription, an amphipathic leucine-zipper (bZIP) domain for protein dimerization and a basic domain for DNA-binding (Angel et al., 2001; Eckert et al., 1997; Eferl and Wagner, 2003; Jochum et al., 2001). Recent mouse genetic studies have demonstrated that AP1 plays key roles in regulating a wide spectrum of biological processes, including embryonic development, tissue homeostasis, tumorigenesis and inflammation (Angel et al., 2001; Eferl and Wagner, 2003; Hess et al., 2004; Jochum et al., 2001). On the other hand, different AP1 proteins display structural and functional differences. For example, both JunB and c-Jun contain a JNK-docking site but only c-Jun has a typical JNK-phosphorylation site in the TA-domain. Thus, JunB is not activated by JNK in the same manner as c-Jun, and is often recognized as an inhibitor to target gene expression (Chiu et al., 1989; Deng and Karin, 1993). However, the latter notion has been recently challenged by the findings demonstrating that each AP1 subunit can either induce or suppress gene expression in a cell context- and target gene-dependent manner (Chinenov and Kerppola, 2001; Hess et al., 2004).

In skin, AP1 subunits are expressed in a species-specific pattern and are implicated in the specification of temporal and spatial patterns of gene expression during terminal differentiation of keratinocytes and the manifestation of inflammatory responses (Basset-Seguin et al., 1990; Briata et al., 1993; Mehic et al., 2005; Welter and Eckert, 1995). Of particular interest, mice with epidermal-specific deletion of JunB (JunBepi−/−) or both JunB and c-Jun (JunBepi−/−c-Junepi−/−) are born alive with no obvious skin defects; but within a few weeks after birth these mice develop bone and inflammatory skin abnormalities resembling arthritic and psoriatic responses in human (Meixner et al., 2008; Zenz et al., 2005). Moreover, JunBepi−/− but not c-Junepi−/− mice exhibit defects in hematopoiesis, kidney and lymph node organogenesis (Zenz et al., 2005). These findings underscore that JunB and c-Jun have overlapping yet differential roles in regulating mouse epidermal homeostasis and can act in the epidermis to control systemic well-being.

AP1 has been generally characterized as a dominant promoter of skin cancer. AP1 inhibition through multiple genetic approaches, including epidermal expression of the dominant-negative mutant of c-Jun (DNc-Jun, also known as TAM67) in transgenic mice or genetic deletion of either c-Jun or c-fos, suppresses murine skin carcinogenesis induced by chemicals, UV radiation or viral oncogenes (Cooper et al., 2003; Dhar et al., 2004; Jochum et al., 2001; Saez et al., 1995; Thompson et al., 2002; Young et al., 2002; Zhang et al., 2007). In addition, expression of DNc-Jun inhibits tumorigenesis of murine squamous cell carcinoma (SCC) cell lines in nude mice (Domann et al., 1994). Conversely, AP1 activation by upstream MKK7/JNK signaling cascade is sufficient to couple with oncogenic Ras to induce human epidermal malignancy (Zhang et al., 2007). Likewise, overexpression of JunB enhances the malignant phenotype of transformed rat keratinocytes in vitro (Bernstein and Colburn, 1989). In addition, JunB mRNA is upregulated in advanced skin cancers induced by the DMBA/TPA carcinogenesis protocol (Schlingemann et al., 2003), though it is unclear whether the increased JunB expression is involved in the malignant progression or is a secondary response to tumor advancement. Taken together, these findings indicate that AP1 has a dominant role in epidermal tumorigenesis. On the other hand, JunB has been identified as a key regulator responsible for the resistance of JB6(−) mouse SCC cells to tumor promotion and the suppression of B9(SQ) mouse SCC cells to epithelial-to-mesenchymal transition (Finch et al., 2002; Hulboy et al., 2001), which pinpoints JunB as a tumor suppressor. These controversial findings imply that JunB functions in a species- or cell context-specific manner. Such possibility has been shown in the lymphoid system where JunB inhibits transformation of B-cells but not T-cells (Szremska et al., 2003). Thus, it is essential to examine the role of Jun proteins directly in human SCC.

In this study, we took advantage of using the human SCC model regenerated on immunodeficient mice and the spontaneous human SCC samples to determine how JunB and c-Jun were involved in regulating epidermal growth and neoplasia. We found that c-Jun activation was relevant to human SCC and was sufficient to couple with oncogenic Ras to transform normal epidermal cells into malignancy. In contrast, JunB inhibited epidermal tumorigenesis driven by defined genetic changes and spontaneous human SCC cells. Conversely, the dominant negative JunB mutant (DNJunB) promoted neoplasia. At the cellular level, JunB induced epidermal senescence and differentiation, which was accompanied with an upregulation of the cell cycle inhibitor p16 and the differentiation marker filaggrin and a downregulation of cell cycle promoter CDK4. Our findings indicate that JunB and c-Jun have opposite roles in human epidermal neoplasia and that their functional specificities are dependent on both N- and C-terminal domains.

RESULTS

JunB and c-Jun are differentially induced in human SCC

To determine the clinical relevance of JunB and c-Jun, we first examined their expression status in human SCC. By immunostaining, we found that both JunB and c-Jun were expressed in almost all layers of normal human epidermis and displayed both cytoplasmic and nuclei localization (Figure 1a–b), as described in previous studies (Mehic et al., 2005; Welter and Eckert, 1995). In SCC samples, c-Jun was detected primarily in the nuclei of the bulk of cancer cells. In contrast, JunB was present in the nuclei of a limited number of cells on the tumor tissues (Figure 1a–c). In agreement with these data, immunoblotting showed that c-Jun was highly activated in A431, a human SCC cell line, as indicated by the increased levels of phosphorylated c-Jun (p-c-Jun), as compared to normal human keratinocytes (Figure 1d). Similarly, p-c-Jun was increased in a majority of SCC samples as compared to the normal skin. In contrast, JunB and p-JunB were expressed at lower levels in A431 cells than they do in normal keratinocytes. In addition, despite the increased levels of p-JunB in SCC, the total levels of JunB were significantly reduced in 6 out of the 10 SCC tissues examined (Figure 1d). These data indicate that c-Jun is activated while JunB is reduced in human SCC, underscoring a clinical relevance of these proteins to human SCC.

Figure 1. c-Jun but not JunB is induced in human SCC.

Figure 1

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(a–b) Immunostaining of (a) c-Jun and (b) JunB in normal human skin (NS) and representative patient SCC samples. c-Jun and JunB [orange]; nuclei [blue, Hoechst 3342]; Scale bar=40 μm. (c) Percent of cells with positive nuclei staining for c-Jun and JunB in NS and SCC samples. Graph represent cell counts from 4–5 sections of NS or SCC + standard deviation (SD). (d) Immunoblotting for c-Jun, p-c-Jun, JunB, p-JunB and Actin with protein lysates isolated from human keratinocytes (KC), A431 SCC cells, NS or 10 SCC samples. Semi-quantitative densitometry was analyzed using Kodak imaging system and the number shown below each band represents the relative reading unit obtained by normalizing the reading of each band to that of actin of the same sample and then to the normalized number of keratinocyte or NS. Please note the samples shown in (a–b) and (d) were from different patients.

c-Jun promotes whereas JunB inhibits epidermal neoplasia

To determine whether Jun proteins have primary roles in the pathogenesis of epidermal neoplasia, we first tested their gain-of-function effects on the Ras-driven human SCC model (Dajee et al., 2003). To do this, primary human epidermal cells were isolated from normal human skin and transduced with retroviruses for expression of the oncogenic Ras along with JunB or c-Jun. These cells were then seeded onto pieces of devitalized and split-thickness human dermis for human skin regeneration on immunodeficient SCID mice. The skin grafts were collected at 6 weeks post-grafting and were confirmed to express the transduced genes by immunostaining (Figure S1a). Interestingly, the grafts expressing Ras and c-Jun developed features of epidermal malignancy with resemblance to human SCC. These tissues lacked a clear tissue boundary between dermal and epidermal compartments and were hyperproliferative as indicated by the disordered histological appearances and the abundance of Ki-67-positive cells (Figure 2a–b), respectively. In addition, they expressed high levels of cytokeratin 8 (Figure 2c), a simple epithelial marker detected in malignant SCC (Watanabe et al., 1995). Moreover, vimentin, a mesenchymal cell marker, was highly expressed in these tissues, whereas filaggrin, a late epidermal differentiation marker, was nearly absent in them (Figure 2d–e), which indicate that these tumors have undergone epithelial-mesenchymal transition but not differentiation. In contrast, the grafts expressing Ras and JunB appeared normal as judged by the delineated tissue boundary and the basal layer-confined cell proliferation, as well as the absence of vimentin and cytokeratin 8 and the presence of filaggrin (Figure 2a–e). Thus, c-Jun but not JunB is able to act in synergy with Ras to induce epidermal tumorigenesis.

Figure 2. JunB and c-Jun display opposite roles in Ras-driven epidermal neoplasia.

Figure 2

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(a) Histological appearances of 6 weeks old skin grafts regenerated on immunodeficient SCID mice. Primary human keratinocytes were transduced to express genes as indicated on the upright corner of each image and then used for skin grafting. [E= epidermal tissue, D=dermis, M=muscle, V=Blood vessel]. Grafts (n=4–6) displayed 100% phenotypic penetrance. (b–e) Immunofluorescent staining for Ki-67, cytokeratin 8 (K8), vimentin and filaggrin [orange]; nuclei [blue, Hoechst 3342], Scale bar=40 μm. (f) Soft agar colony formation. A431 cells transduced for expression of LacZ control, JunB-ER or c-Jun-ER were used for soft agar colony formation in the absence or presence of 100 nM 4-hydroxy-tamoxifen (4-OHT). The average numbers of colonies from triplicate wells + SD were presented.

To further test the differential effects of Jun proteins on SCC, we transfected A431 cells for expression of JunB-ER and c-Jun-ER. These fusion proteins have been previously validated to be functionally inducible by exogenous 4-hydroxytomoxifen (4-OHT) (Andrecht et al., 2002; Fialka et al., 1996). We then performed soft agar colony formation, an assay commonly used to assess anchorage-independent cell growth of cancer cells. We found that cells expressing JunB-ER developed a reduced number of soft agar colonies in response to 4-OHT-treatment. In contrast, cells expressing c-Jun-ER developed an increased number of colonies following 4-OHT induction (Figure 2f). These findings confirm that JunB and c-Jun have opposite effects on human SCC with JunB acting as an inhibitor for tumorigenesis.

JunB suppression of tumorigenesis requires its TA-domain

Previous studies have shown that JunB and c-Jun inhibits and promotes mouse embryonic cell proliferation, respectively. Their distinctive effects on cell growth have been attributed to the differences in their TA-domains such that c-Jun displays a higher transcriptional activity than JunB (Chiu et al., 1989; Shaulian and Karin, 2001). Like DNc-Jun which lacks a TA-domain and functions a dominant negative c-Jun mutant (Brown et al., 1993), DNJunB, the corresponding JunB mutant, induced a dominant negative effect when tested by AP1-luciferase gene reporter assay (Ikebe et al., 2007) (Figure 3a). We predicted that, if the functional divergence between JunB and c-Jun was caused by the differences in their TA-domains, DNJunB and DNc-Jun would induce similar effects on epidermal cell growth and neoplasia. DNc-Jun has been previously shown to inhibit tumorigenesis in both mouse and human skin tumor models (Cooper et al., 2003; Dhar et al., 2004; Domann et al., 1994; Jochum et al., 2001; Thompson et al., 2002; Young et al., 2002; Zhang et al., 2007). To determine DNJunB effects on human SCC, we used a previously established human SCC model, in which coexpression of Ras and MKK7, an upstream activator of AP1, transforms normal human epidermal cells into malignancy (Zhang et al., 2007). We transduced primary human keratinocytes for expression of Ras and MKK7 along with JunB or DNJunB and then used these cells for skin regeneration on immunodeficient mice. Expression of the transduced genes, including Ras, MKK7, JunB and DNJunB, were confirmed by immunostaining of the regenerated skin tissues (Figure S1b). In line with the inhibitory effect of JunB on A431 cells, the grafts expressing Ras, MKK7 and JunB appeared normal with an intact tissue boundary and basal cell-confined cell proliferation (Figure 3b–c). They displayed negative detection of cytokeratin 8 and vimetin and positive detection of filaggrin (Figure 3d–f). In contrast, the grafts expressing Ras, MKK7 and DNJunB developed malignant features similar to those observed in tissues expressing Ras and c-Jun. These features include tissue invasion, abnormal cell proliferation and epithelial-mesenchymal transition, as indicated by the appearance of disrupted tissue boundaries, the high numbers of Ki-67-positive cells and the abundant expression of cytokeratin 8 and vimentin but not filaggrin (Figure 3b–f). These data indicate that DNJunB does not inhibit tumorigenesis driven by Ras and MKK7, which lead us to predict that DNJunB might be sufficient by itself to promote Ras-driven tumorigenesis. Consistent with this idea, the grafts expressing Ras and DNJunB displayed invasive tumor growth in a manner similar to those expressing Ras, MKK7 and DNJunB (Figure 3b–f). Furthermore, both DNJunB and c-Jun are sufficient to support subcutaneous tumor growth when coupled with Ras (Figure S2). These data indicate that, contrary to DNc-Jun, DNJunB promotes Ras-driven tumorigenesis. Thus, the N-terminal TA-domain of JunB is required for its inhibitory effect on epidermal tumorigenesis while its C-terminus is actively involved in promoting tumorigenesis.

Figure 3. DNJunB promotes Ras-driven epidermal neoplasia.

Figure 3

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(a) AP1-gene reporter assay. 293T cells were co-transfected with an AP1-luciferase gene reporter construct along with expression constructs encoding LacZ, c-Fos, DNJunB or DNc-Jun. Graph represents means of relative luciferase reading units (RLU) + SD. (b) Histological appearances of skin grafts regenerated on SCID mice with primary human keratinocytes which have been transduced to express genes as indicated on the upper left corner of each image. [E= epidermal tissue, D=dermis, M=muscle, V=Blood vessel]. Grafts (n=4–6) displayed 100% phenotypic penetrance. (c–f) Immunofluorescent staining for Ki-67, cytokeratin 8, vimentin and filaggrin [orange]; Nuclei [blue, Hoechst 3342]; Scale bar=40 μm.

JunB induces cell growth inhibition

Cancer is a result of abnormal activities of multiple oncogenes and/or tumor suppressors. Of particular relevance, activation of oncogenic Ras alone induces epidermal cell growth arrest and senescence (Dajee et al., 2003). Therefore, additional molecular changes, such as JNK activation or NF-κB blockade, are required to overcome Ras-induced cell growth arrest and senescence; and as a result, JNK activation or NF-κB blockade is necessary to act in concert with Ras to transform normal epidermal cells into malignancy (Dajee et al., 2003; Ke et al., 2010; Zhang et al., 2007). The inability of JunB in promoting Ras-driven tumorigenesis led us to predict that JunB is not able to surmount Ras-induced cell growth arrest. Consistent with this idea, keratinocytes expressing JunB grew slower than GFP-control cells when cultured either in separate dishes or in co-cultures (Figure 4a), which indicates that JunB inhibits epidermal cell growth in a cell-autonomous fashion. Similarly, JunB-ER induced a cell growth inhibition in a 4-OHT dose-response manner (Figure 4b), further confirming that the inhibitory effect on cell growth is a result of JunB function. In addition, induction of JunB-ER with 4-OHT increased cell senescence as indicated by the increased number of cells positive in β-galactosidase (Figure 4c–d). Further cell cycle analysis by flow cytometry showed that keratinocytes transduced to express JunB were less efficient in entering S phase than control cells (Figure 4e). Consistent with these in vitro data, the core G1-S phase cell cycle promoters, including cyclinD1 and CDK4, were downregulated in tissues expressing JunB and Ras either with or without MKK7, whereas the cell cycle inhibitor p16 was upregulated in these tissues. On the contrary, cyclinD1 and CDK4 were increased and p16 was decreased in the tumor tissues expressing Ras and c-Jun or DNJunB (Figure 4f). These data indicate that JunB inhibits human epidermal cell growth in a cell-autonomous fashion and is unable to overcome Ras-induced cell growth inhibition.

Figure 4. JunB inhibits human epidermal cell growth and increases cell senescence.

Figure 4

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(a) Cell growth analysis. Primary human keratinocytes were transduced with retroviruses for expression of GFP or JunB and then plated in triplicates in separate or coculture dishes. GFP-positive and GFP-negative cells were trypsinized and counted under microscope 72 hours later. (b) Cell growth analysis. Keratinocytes transduced to express LacZ or JunB-ER were plated in triplicates in the presence of 4-OHT in a dose-response manner and then collected for cell counting 72 hours later. (c) Cell senescence analysis. Mock trasnduced control (Con) cells and cells expressing JunB-ER were treated with 100 nM 4-OHT for 48 hours and then processed for β-galactosidase staining (β-Gal, blue); Scale bar=100 μm. Inducible expression of JunB-ER was confirmed by immunoblotting with an antibody against ERα with Actin for control. (d) Quantitative index of β-Gal positive cells. (e) Cell cycle distribution analysis by flow cytometry of keratinocytes transduced to express GFP or JunB. (f) Immunofluorescent staining of the regenerated skin grafts with antibodies against cyclinD1, CDK4 and p16 [orange]; nuclei [blue, Hoechst 3342]; Scale bar=40 μm. Graphs in (a, b, d and e) represent the mean values + SD.

DISSCUSSION

Cancer is a result of genetic changes leading to gain-of-function of classical oncogenes and/or loss-of-function of tumor suppressors. However, a single genetic change is usually not sufficient to induce tumor growth, which is exemplified by the fact that oncogenic Ras induces epidermal cell senescence (Dajee et al., 2003). Abnormal induction or suppression of other key molecules due to genetic, epigenetic or environmental perturbations is therefore essential in the malignant transform of cells harboring one or more oncogenic mutations. Here, we showed that c-Jun was able to act in synergy with oncogenic Ras to transform normal epidermal cells into malignancy. In contrast, JunB not only failed to couple with Ras but also inhibited tumorigenesis induced by coexpression of Ras and MKK7. Most interestingly, the JunB mutant missing the TA-domain is a potent promoter of epidermal malignancy, which is in sharp contrast to the tumor inhibitory effect of the corresponding c-Jun mutant. Our findings indicate that the functional differences between JunB and c-Jun are not simply determined by the differential transactivation potential of their N-terminal TA-domains, as indicated by previous studies (Chiu et al., 1989; Shaulian and Karin, 2001). Their c-terminal domains are also functionally distinct, which is consistent with the biochemical data demonstrating that these domains are involved in governing the specificity of target gene recognition and transcriptional regulation (Deng and Karin, 1993).

The functional differences between JunB and c-Jun are multifaceted and are in part manifested in their effects on cell growth and differentiation. JunB induced epidermal growth inhibition, yet cells expressing both JunB and Ras were able to generate live but non-invasive skin grafts. This is surprising because Ras inhibited epidermal cell growth and differentiation; and consequently cells transduced to express Ras alone were not able to produce live skin grafts (Dajee et al., 2002; Lazarov et al., 2002). Presumably, JunB overcomes Ras-inhibition of differentiation and thereby prevented skin graft failure. Interestingly, we observed that c-Jun did not increase cell proliferation of normal epidermal cells (data not shown). Yet, coexpression of c-Jun with Ras induced invasive epidermal tumor growth accompanied with the deficiency of terminal tissue differentiation and the occurrence of epithelial-mesenchymal transition as evidenced by the negative and positive detection of filaggrin and vimentin, respectively. The invasive phenotype induced by c-Jun and Ras is consistent with a previous study demonstrating that c-Jun promoted mouse SCC growth through induction of matrix metalloproteinase-2 and -9 expression (Zhang et al., 2006). In contrast to normal keratinocytes, human SCC cells are responsive to c-Jun induction by showing an increase in soft agar colony formation. These findings indicate that c-Jun acts synergistically with other oncogenes, such as Ras, to induce invasive epidermal growth.

JunB is able to substitute c-Jun during embryonic development (Passegue et al., 2002), indicating that Jun proteins have redundant roles in embryogenesis. Similarly, our findings implicate that JunB and c-Jun are both involved in epidermal stratification, which is in agreement with a recent report demonstrating that mice with expression of the dominant negative c-Jun mutant in the superbasal layer of epidermis had defects in epidermal differentiation and were resistance to tumor induction (Rorke et al., 2010). However, the molecular mechanisms governing the functional antagonism or synergism between JunB and c-Jun are yet to be defined. On this regard, JunB appears to inhibit c-Jun activation, as indicated by the cytoplasmic localization of c-Jun in tissues expressing Ras and JunB (Figure S1a–b) and its nuclei presence in the tumor tissues expressing Ras and DNJunB (Figure S1b). Of further interest, Ras-inhibition of epidermal stratification is dependent on the PI3K signaling pathway (Dajee et al., 2002). It is not clear whether Jun proteins cross-talk with the PI3K pathway to overcome the differentiation defect.

Consistent with the findings obtained with the experimental models of human SCC, JunB is reduced at the protein level in some primary human SCC samples. In contrast, c-Jun is highly expressed and activated in SCC, which is consistent with our previous data demonstrating that JNK activation is increased in a majority of human SCC (Ke et al., 2010; Zhang et al., 2007). Thus, JunB loss-of-function and c-Jun gain-of-function are both clinically relevant to human SCC. However, it is worth noting that both c-Jun and JunB genes were shown to be downregulated in well-differentiated locally invasive SCC at the mRNA levels (Haider et al., 2006). The downregulation of JunB mRNA is consistent with the reduced JunB activity as detected by immunostaining. The discrepancies between the mRNA and protein levels of c-Jun suggest that c-Jun is subject to regulation at a posttranslational level in human SCC. Of further interest, JunB-phosphorylation has been linked to both transcriptional activation and protein degradation (Bakiri et al., 2000; Li et al., 1999). The functional relevance of the positive detection of pJunB in SCC has yet to be investigated. Overall, findings to date establish a dominant role for MKK7/JNK/c-Jun signaling axis and a tumor suppressor role for JunB in human SCC. The findings of this study underscore the importance of distinguishing different AP1 subunits in future therapeutic designs.

MATERIAL AND METHODS

Cell culture, gene transfer and gene reporter assay

cDNAs encoding c-Jun, JunB and the N-terminal deletion mutant of JunB were subcloned into the LZRS retroviral vector (Ikebe et al., 2007; Treier et al., 1995). Viral production and infection of human keratinocytes were performed as described (Robbins et al., 2001). Primary human keratinocytes were isolated from neonatal foreskin and cultured in serum free keratinocyte media (KSFM) (Invitrogen Corporation, Carlsbad, CA). One day before viral infection, early passage (passages1–2) keratinocytes were plated at 5×105 and 3×104 cells per 10 cm or 35 mm dishes, respectively. Protein expression was confirmed by immunoblotting with protein lysates collected two days post gene-transduction. Dural-luciferase gene reporter assays were performed in 293T cells as described (Zhang et al., 2005). Relative luciferase unit was obtained by normalizing the firefly luciferase reading unit to that of the internal renilla-luciferase control and then to the normalized numbers of LacZ control cells.

Cell growth, flow cytometry and soft agar colony formation

For growth analysis, keratinocytes were infected as described above and trypsinized for cell counting three days post-infection. For coculture cell growth analysis, keratinocytes trasnduced to express GFP or JunB were trypsinized one day post-infection and equal numbers of GFP- and JunB-expressing cells were mixed for subculture. Cells were then trypsinized 72 hours later and counted under immunofluorescent microscope for GFP-positive and -negative cells. For cell cycle analysis, trypsinized cells were fixed in cold 70% ethanol for 24 hours and treated with 100 μg/ml DNase-free ribonuclease (Sigma-Aldrich, St. Louis, MO) at room temperature for 10 minutes. Cells were then stained with propidium iodide (50 μg/ml in PBS, Sigma) right before flow cytometry analysis (Duke Flow Cytometry Core Facility). A431 cells (ATCC) were cultured in 10% FBS/DMEM, and transfected with the pUbi-JunB-ER or pMV7c-Jun-ER fusion constructs using GenJet™ Plus reagent (SignaGen Laboratories, Ijamsville, MD) (Andrecht et al., 2002; Fialka et al., 1996). Cells were then used for growth and soft agar colony formation analysis as described (Ke et al., 2010).

Animal studies

Animal studies were conducted in accordance with protocols approved by the Duke Animal Care and Use Committee. CB.17.SCID immunodeficient mice were purchased from National Cancer Center, and human skin regeneration was performed as described (Dajee et al., 2003; Zhang et al., 2007). Briefly, primary human keratinocytes were transduced with retrovirus for expression of the interested genes and seeded onto 1 cm square devitalized human cadaver dermis (National Disease Research Institute). When cells reached confluence, the dermis was lifted from tissue culture plate and grafted onto the back of mice. Regenerated human tissue samples were analyzed at 6 weeks post-grafting. Data shown represent two sets of independently grafted animals with 3 animals per group. Phenotypic penetrance was observed in all animals with successful grafts.

β-galactosidase staining

Primary human keratinocytes were mock transduced or transduced with retroviruses encoding JunB-ER. One day post-transfection, cells were treated with 100 nM 4-OHT for 48 hours and then fixed in methanol followed by staining for the senescence-associated acidic β-Galactosidase (Cell Signaling Technology, Danvers, MA). Blue cells and non-blue cells were counted on photographed microscopic images (n=10) taken under Olympia BX41 microscopic imaging system.

Immunoblotting and immunohistology

Antibodies against p-c-Jun, c-Jun, JunB, p-JunB, Actin, CDK4, filaggrin and ERα were purchased from (Santa CruZ Biotechnology, Santa Cruz, CA), and the antibody against p16 was from (Genescript, Piscataway, NJ). For immunoblotting, protein lysates (20 μg each samples) isolated from surgically discarded human skin, SCC samples or cultured cells were separated on 12% SDS-PAGE gel. For hematoxylin and eosin (H&E) staining, tissue samples were fixed in 10% formalin and then embedded in paraffin blocks for tissue sectioning and staining in Duke Pathology Laboratory. For immunofluorescent staining, frozen tissue sections were fixed with 100% methanol, incubated with primary antibodies against Ki-67 (Labvision, Fremont, CA), CDK4, filaggrin or p16 and then detected with a secondary antibody conjugated to Alexa688 (Invitrogen). Sections were counterstained with 1μg/ml Hoechst. Pictures were taken under Olympia B×41 microscopic imaging system.

Supplementary Material

Supplemental data

Acknowledgments

This work was supported by K01AR051470 and R01AR057746 from NIH/NIAMS and a research grant from Skin Cancer Foundation to Jennifer Y. Zhang. We thank Dr. Mitsuyasu Kato (University of Tsukuba) for the JunB and Dirk Bohmann (University of Rochester Medical Center) for the c-Jun expression constructs, as well as Dr. Marina Schorpp-Kistner (German Cancer Research Center) and Dr. Meinrad Busslinger (Research Institute of Molecular Pathology, Austria) for the JunB-ER and c-Jun-ER fusion constructs, respectively. We also thank Benjamin Leshin and Michael Benson for their technical assistance in plasmid preparation.

Footnotes

CONFLICT OF INTEREST

The authors state no conflict of interest.

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