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Genes to Cells (2007) 12, 197-207. doi:10.1111/j.1365-2443.2007.01043.x
© 2007 Blackwell Publishing or its licensors
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Suppression of keratinocyte stratification by a dominant negative JunB mutant without blocking cell proliferation

Dai Ikebe, Bei Wang, Hiroyuki Suzuki and Mitsuyasu Kato*

Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Keratinocytes make a stratified epidermoid structure when cultured at an air-liquid interface. The three-dimensional (3D) culture of keratinocytes has been successfully used for more than 25 years, but it is still unclear why keratinocytes stratify in response to air exposure. AP-1 proteins are ubiquitous transcription factors that regulate many biological processes, including cell proliferation, differentiation and apoptosis. We established HaCaT-JunB{Delta}N, a human keratinocyte cell line that expressed a mutant JunB with a dominant negative effect on AP-1 activity. Stratification of HaCaT-JunB{Delta}N cells was markedly suppressed in a 3D culture condition, in which HaCaT cells stratified similarly to stratified squamous epithelia. However, HaCaT-JunB{Delta}N cells had proliferation activities that were closely equivalent to those of HaCaT cells, under both two-dimensional (2D) and 3D culture conditions. To screen for the candidate gene responsible for the different stratification ability, we examined the gene expression profile of HaCaT cells before and after air exposure. Several genes with an antioxidative function, such as aldo-keto reductase and selenoprotein P were highly expressed after air exposure in HaCaT cells but not in HaCaT-JunB{Delta}N cells. Our findings indicate the presence of a novel role of AP-1 activity when HaCaT cells make a stratified epidermoid structure under 3D culture conditions.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
How can cells make a specific multicellular tissue and behave in such a condition? This is a major subject of study in current molecular and cell biology. Cultured epithelial cells have been shown to make three-dimensional (3D) tissues such as acini, follicles, tubules and stratified squamous epithelia under specific culture conditions (Regnier et al. 1990; Bissell & Labarge 2005; Debnath & Brugge 2005; Karihaloo et al. 2005). These cells reveal patterns of gene expression and other biological activities that more closely reflect what happens in living organism rather than the usual two-dimensional (2D) cell cultures (Abbott 2003; Mehul et al. 2004). For example, antibodies against ß1-integrin caused breast cancer cells to lose abnormal tissue structure and growth patterns in 3D but this never occurred in 2D (Weaver et al. 1997). After these works, the usefulness and necessity of 3D culture study were widely accepted and began to be used in many epithelial cell systems (Abbott 2003).

Epidermal keratinocytes were the first epithelial cells successfully maintained and propagated in serial cultivation (Rheinwald & Green 1975). These cells were further shown to make a stratified epidermoid structure and to differentiate terminally when cultured at an air-liquid interface on a contracted collagen lattice containing dermal fibroblasts (Bell et al. 1981). This organoid 3D culture has been used for the study of keratinocyte biology and pathology for more than 25 years and it has been clinically applied (Boyce & Hansbrough 1988; Alonso & Fuchs 2003). Through the combination of genetic engineering with cell biological studies, the mechanisms underlying the development and differentiation of the epidermis are becoming increasingly clear (Fuchs & Raghavan 2002; Watt 2002). However, the reasons why an air-liquid interface culture initiates the behavior of keratinocytes to closely resemble that of the parent tissue, has yet to be revealed.

Oxidative stress disturbs the cellular redox status, inducing oxidative damage to cellular macromolecules (DNA, lipid and protein), and alters the gene expression profile, most likely primarily by post-transcriptional modification of redox-sensitive transcription factors. Activator Protein-1 (AP-1) is a redox-sensitive, early responsive, transcription factor composed of the Jun and Fos families of nuclear proteins (Angel et al. 2001; Sakon et al. 2003). In response to oxidative stress, AP-1 proteins regulate the transcription of genes associated with antioxidant defense, cell cycle control and apoptosis (Shaulian & Karin 2002; Eferl & Wagner 2003; Yan & Hales 2005). The AP-1 component was originally found as a viral oncogene and a target of potent skin tumor promoters such as TPA (Chinenov & Kerppola 2001; Eferl & Wagner 2003). In vivo experiments demonstrated that c-Jun is a key transcriptional regulator of EGFR and HB-EGF, and is required for the development of a skin tumor (Young et al. 1999; Zenz et al. 2003). Furthermore, the over-expression of JunB enhanced the malignant phenotype of keratinocytes in vitro (Robinson et al. 2001). These previous works revealed essential roles of AP-1 activity in the regulation of keratinocyte growth and tumor formation both in vivo and in vitro.

In the present paper, we have examined the role of AP-1 activity in a human keratinocyte cell line HaCaT, when a stratified epidermoid structure was formed in an air-liquid interface culture. The blocking of AP-1 activity by a dominant negative JunB mutant did not alter the cell proliferation activity of HaCaT cells but still suppressed the formation of a multi-layered epidermoid structure in a 3D culture. Furthermore, the activity was specific to the JunB mutant, while the dominant negative c-Jun mutant did not have such activity.

These results suggested that the AP-1 activity, which is blocked by the JunB mutant but not by the c-Jun mutant, was therefore required for the formation of a multi-layered epidermoid structure due to some as yet unknown mechanisms other than cell growth regulation.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
HaCaT cells in a 3D culture at an air-liquid interface

HaCaT cells created a multi-layered tissue similar to stratified squamous epithelia when cultured at an air-liquid interface as described in the Experimental procedures section (Fig. 1). HaCaT is an immortalized human keratinocyte cell line carrying several chromosomal abnormalities but it never piled up without exposure to air, even on the collagen gel containing human dermal fibroblasts (Fig. 1B, day 0). After exposure to air, three or four layers of stratification were achieved within 1 day and flattened post-mitotic layers became obvious within 3 days. Therefore, HaCaT cells retained the property to start proliferation for stratification in response to air exposure and to stop growing in the upper layers as primary keratinocytes do (Fig. 1B, days 3 and 7).


Figure 1
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  		Figure 1  3D culture of HaCaT cells. (A) Schematic representation of a 3D culture. HaCaT cells were plated onto the gels and cultured under the fluid surface for 7 days. The surface of gels with HaCaT cells was then exposed to air (day 0) and cultured for further 7 days at an air-liquid interface. (B) At the indicated time points, cells on the gel discs were fixed and the vertical sections were stained with hematoxylin and eosin.

 
JunB{Delta}N and c-Jun{Delta}N have dominant negative effects on pAP-1-luc activity

To investigate the function of the AP-1 transcription factor, we generated deletion mutants of JunB and c-Jun, designated as JunB{Delta}N and c-Jun{Delta}N, respectively, which lacked an N-terminal transcriptional activator domain (1–147) (Fig. 2A). JunB, JunB{Delta}N or c-Jun{Delta}N were expressed transiently in HaCaT cells together with c-Jun or c-Fos, as indicated in Fig. 2B. The effects on AP-1 activities were measured using pAP1-luc as a reporter. Both JunB{Delta}N and c-Jun{Delta}N suppressed not only internal AP-1 activity in HaCaT cells but also the AP-1 activity induced by exogenously expressed c-Jun or c-Fos. Between JunB{Delta}N and c-Jun{Delta}N, JunB{Delta}N gave a stronger dominant negative effect than c-Jun{Delta}N regarding the AP-1 activity in this reporter assay. JunB weakly enhanced the endogenous AP-1 activity of HaCaT cells but suppressed the high AP-1 activities in the presence of exogenous c-Jun or c-Fos (Fig. 2B).


Figure 2
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  		Figure 2  JunB{Delta}N and c-Jun{Delta}N have dominant negative effects on pAP-1-luc activity. (A) Schematic representation of the AP-1 family and deletion mutants used in this study. (B) JunB{Delta}N and c-Jun{Delta}N suppress pAP-1-luc activity in HaCaT cells. pAP-1-luc was transfected into HaCaT cells, together with the expression constructs encoding c-Jun, c-Fos, JunB, JunB{Delta}N and c-Jun{Delta}N, as indicated. The luciferase activities were normalized to the ß-galactosidase activities in order to correct any possible variation of the transfection efficiencies. All values represent the mean ± SD (n = 3).

 
HaCaT cells stably expressing JunB{Delta}N (HaCaT-JunB{Delta}N) specifically decrease endogenous AP-1 activities, but the growth of the cells was not affected

We next established HaCaT cells that stably expressed JunB{Delta}N and c-Jun, respectively. Western blotting confirmed that HaCaT-JunB{Delta}N6 and HaCaT-JunB{Delta}N8 cells expressed a JunB{Delta}N protein, and HaCaT-c-Jun2 and HaCaT-c-Jun12 cell lines expressed exogenous c-Jun (Fig. 3A), respectively. Using these cell lines, their AP-1 activity and ARE (antioxidant response element) activity were measured by luciferase assays. The AP-1 activity of HaCaT-JunB{Delta}N6 and HaCaT-JunB{Delta}N8 cell lines was significantly suppressed, while the HaCaT-c-Jun2 and HaCaT-c-Jun12 cell lines had an increased AP-1 activity as expected (Fig. 3B). On the other hand, the JunB{Delta}N cell lines did not suppress the ARE-driven transcriptional activity, suggesting the specificity of the dominant negative effect of JunB{Delta}N on AP-1 activity (Fig. 3C). We then evaluated the effects of JunB{Delta}N on HaCaT cell growth. The growth curves are shown in Fig. 3D. In the presence of 5% FBS, no significant difference was observed to the growth curves of HaCaT cells by the expression of JunB{Delta}N (Fig. 3D).


Figure 3
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  		Figure 3  Establishment of HaCaT cells stably expressing c-Jun and JunB{Delta}N. (A) The expression of c-Jun in HaCaT-c-Jun2 and HaCaT-c-Jun12 cells, and JunB{Delta}N in HaCaT-JunB{Delta}N6 and HaCaT-JunB{Delta}N8 cells. Immunoblot analyses were performed using anti-FLAG and anti-ß-actin antibodies. (B, C) pAP-1-luc activity (B) or pNQO1-ARE-luc activity (C) in HaCaT-c-Jun- and HaCaT-JunB{Delta}N cells. The luciferase activities were normalized to ß-galactosidase activities to correct the possible variation of transfection efficiencies. All values represent the mean ± SD (n = 3). P values when compared with Mock-transfected HaCaT cells. *P > 0.05, **P < 0.05. (D) Growth curves of HaCaT-c-Jun, HaCaT-JunB{Delta}N and Mock-transfected HaCaT cells. All cells were seeded in 6 well plates on day 0 (3 x 104 cells/well). All values represent the mean (n = 3). The number of cells in each cell line showed no significant differences with the Mock-transfected HaCaT cells except for HaCaT-c-Jun2 cells on day 6.

 
HaCaT-JunB{Delta}N cells proliferate but never create a multi-layered structure in 3D culture

We next cultured HaCaT-JunB{Delta}N cell lines in the 3D culture method. After 7 days of an air-liquid interface culture, cells were fixed in formalin solution, embedded in paraffin and vertical sections were stained with hematoxylin and eosin (Fig. 4A). The mean thickness of the epidermoid layers created by the Mock transfected HaCaT cells was about 120 µm but that of HaCaT-JunB{Delta}N6 and HaCaT-JunB{Delta}N8 cells was 35 and 40 µm, respectively. Both of HaCaT-JunB{Delta}N6 and HaCaT-JunB{Delta}N8 cell lines could not create a multi-layered 3D structure and the thickness of epithelial layers remained at less than one third of the Mock transfectants (Fig. 4B). On the other hand, HaCaT-c-Jun cells showed almost the same degrees of stratification as the control cells did. We have already shown that HaCaT-JunB{Delta}N cells proliferate as well as HaCaT cells in a 2D culture condition (Fig. 3D). However, there is a possibility that HaCaT-JunB{Delta}N cells stop growing only in a 3D culture condition. Therefore, we examined whether the difference of thickness in the stratified epidermoid layers depended on altered cell proliferation activities. For this purpose, we performed immunohistochemical staining for Ki-67 on 3D cultured cells (Fig. 4C) and counted the numbers of Ki-67 positive cells (Fig. 4D). The number of Ki-67 positive cells on day 7 after air-exposure was not significantly different among the Mock transfected HaCaT cells, HaCaT-JunB{Delta}N cells and HaCaT-c-Jun cells.


Figure 4
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  		Figure 4  Suppression of stratified tissue formation by JunB{Delta}N. (A) HaCaT-c-Jun, HaCaT-JunB{Delta}N and Mock-transfected HaCaT clones were cultured by a 3D culture method as described in the Experimental procedures section and Fig. 1A. Vertical sections of 3D cultured cells at day 7 after air exposure were stained with hematoxylin and eosin. (B) Thickness of stratified epidermoid layers. All values represent mean ± SD (n = 5). Both HaCaT-JunB{Delta}N clones did not make thick stratified layers. P values when compared with Mock-transfected HaCaT cells. *P > 0.05, **P < 0.05. (C) Distributions of Ki-67 positive proliferative cells in a 3D culture. (D) Numbers of Ki-67 positive cells (/200 µm in width) were counted. All values represent mean ± SD (n = 5). *P > 0.05, significant differences were not detected among examined cell lines.

 
Time course of the cell proliferation activities and stratification of HaCaT cells expressing c-Jun, JunB{Delta}N or c-Jun{Delta}N in a 3D culture

To study the reason why HaCaT-JunB{Delta}N cell lines could not make stratified layers, we examined the time course of both the stratification and cell proliferation activities of HaCaT cell lines. To perform this experiment, we added HaCaT-c-Jun{Delta}N cell lines that express comparative amounts of c-Jun{Delta}N with the expression levels of JunB{Delta}N in HaCaT-JunB{Delta}N cell lines (Fig. 5A). Representative pictures of 3D cultured cells were shown in Fig. 5B and the numbers of Ki-67 positive proliferative cells were counted (Fig. 5C). HaCaT-c-Jun cells and HaCaT-c-Jun{Delta}N cells had started stratification at day 1. No significant differences were observed in cell proliferation activities at day 1 by the expression of c-Jun, JunB{Delta}N or c-Jun{Delta}N, but only HaCaT-JunB{Delta}N cells could not start to make stratified layers. HaCaT-c-Jun cells gradually accumulated stratified cell layers until day 7. These cells had higher proliferative activities on day 3 than HaCaT-JunB{Delta}N and HaCaT-c-Jun{Delta}N cells. HaCaT-JunB{Delta}N cells had stable cell proliferation activities but never made stratified layers except for the thin accumulation of cornified layer-like substances. HaCaT-c-Jun{Delta}N cells had similar proliferative activities with HaCaT-JunB{Delta}N cells until day 3 but then the cell proliferation activities decreased along with the accumulation of the post-mitotic upper-layer cells. HaCaT-c-Jun{Delta}N cells clearly kept the stratification activities nevertheless the proliferation activities were still lower than for HaCaT-JunB{Delta}N cells.


Figure 5
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  		Figure 5  The time course of the stratification and cell proliferation activities of HaCaT-JunB{Delta}N, HaCaT-c-Jun and HaCaT-c-Jun{Delta}N cells. (A) The expression of c-Jun{Delta}N in HaCaT-c-Jun{Delta}N3 and HaCaT-c-Jun{Delta}N15 cells. Immunoblot analyses were performed using an anti-FLAG and anti-ß-actin antibodies. (B) HaCaT-JunB{Delta}N, HaCaT-c-Jun, and HaCaT-c-Jun{Delta}N clones were cultured by a 3D culture method and then were fixed on days 1, 3 and 7 after air exposure, as indicated. Vertical sections of the 3D cultured cells were stained with hematoxylin and eosin or anti-Ki-67 immunostaining. (C) The numbers of Ki-67 positive cells (/200 µm in width) were counted and compared with Mock transfected HaCaT cells. All values represent the mean (n = 5). **P < 0.05, in comparison to the Mock-transfected HaCaT cells.

 
Stratification of JunB- or c-Jun knock down HaCaT cells in a 3D culture

We further examined the stratification activities of HaCaT cells after knocking down the endogenous JunB or c-Jun by RNA interference technique. Decreases of either JunB or c-Jun were confirmed by Western blotting (Fig. 6A). Both HaCaT-sh(JunB) cells and HaCaT-sh(c-Jun) cells showed a marginally decreased number of stratified layers in a 3D culture at day 7 (Fig. 6B). Especially, HaCaT-sh(JunB) cells showed a dose-dependent collapse of the post-mitotic upper-layers. However, the inhibition of the stratification of these cells was not as complete as that observed in HaCaT-JunB{Delta}N cells.


Figure 6
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  		Figure 6  Stratified tissue formation of JunB or c-Jun knock down HaCaT cells in 3D culture. (A) JunB or c-Jun knock down HaCaT cells were established as indicated in the Experimental procedures section. The expression of JunB in HaCaT-sh(JunB)4 and HaCaT-sh(JunB)8 cells, and c-Jun in HaCaT-sh(c-Jun)9 and HaCaT-sh(c-Jun)17 cells were examined by immunoblot analyses using an anti-JunB, anti-c-Jun, and anti-ß-actin antibodies, as indicated. (B) HaCaT-sh(JunB)4, HaCaT-sh(JunB)8, HaCaT-sh(c-Jun)9 and HaCaT-sh(c-Jun)17 cells were cultured by a 3D culture method and compared with Mock-transfected HaCaT-sh(Luc) (Control) and HaCaT-JunB{Delta}N8 cells at day 7 after air exposure.

 
Genes activated by a 3D culture

To screen the genome-wide alterations of transcriptional activities induced by the air exposure, we investigated the gene expression profile of HaCaT cells using CodeLink human genome arrays (Amersham). After a 24 h air-liquid interface culture, the expression of 112 genes more than doubled and the expression of 140 other genes decreased down to less than half of their expression levels before air exposure. We verified the results of the gene array experiments by using a semi-quantitative RT-PCR to confirm the alteration of the gene expression levels. The HaCaT-JunB{Delta}N cell line was also used to examine the dependency of the alteration in the gene expression on the AP-1 activity. Among the 28 known increased genes listed by the microarray experiment, aldo-keto reductase family 1 member B10, selenoprotein P and superoxide dismutase 1 (SOD1) were confirmed to increase when exposed to air and the responses were completely absent in the HaCaT-JunB{Delta}N cell line, suggesting that the enhanced expression of these genes was mediated by AP-1 activity. Other AP-1 target genes implicated in the response to air exposure includes fatty acid binding proteins-4/5 and chemokine CXCL14 (Fig. 7).


Figure 7
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  		Figure 7  The genes activated by a 3D culture depending on AP-1 activity. The mock-transfected HaCaT and HaCaT-JunB{Delta}N cells were cultured on collagen gels with or without air exposure for 24 h. The genes, expression levels of those are enhanced after exposure to air, were listed in an oligonucleotide microarray analysis using total RNA extracted from Mock-transfected HaCaT cells and the expression levels of selected genes were confirmed by semi-quantitative RT-PCR. The AP-1 dependency of the gene expression was examined by using HaCaT-JunB{Delta}N cells.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
A 3D culture of keratinocytes has been successfully used for more than 25 years and is well known as the organotypic culture, air-liquid interface culture and raft culture (Bell et al. 1981; Regnier et al. 1990). However, it is not elucidated why an exposure to air induces a multi-layered epidermoid tissue formation. We used human HaCaT cells to analyze this mechanism. HaCaT cells never pile up without an exposure to air, even on the collagen gel containing human dermal fibroblasts, and the cells retained the property to start proliferation for stratification in response to air exposure and stop growing in the upper layers as normal keratinocytes do (Fig. 1). The advantage of using HaCaT cells depends on their immortality. Because of this, we could make stable transfectants that altered the activity of a specific molecule by using HaCaT cells as a recipient. We have examined the role of AP-1 activity in this study. For this purpose, we modified AP-1 activity through the stable expression of JunB{Delta}N, c-Jun{Delta}N or c-Jun. HaCaT cells with depleted endogenous JunB or c-Jun were also made by RNA interference technique. The different members of the Jun and Fos families exhibit significant differences in DNA-binding and transcriptional activation, suggesting specific functions in gene regulation for individual AP-1 dimers (Shaulian & Karin 2002). We therefore tried to make HaCaT-c-Fos, HaCaT-Fra1 and HaCaT-JunB cells but positive clones were never obtained after more than a hundred colonies of screening. By using a 3D culture of HaCaT-JunB{Delta}N, HaCaT-c-Jun{Delta}N and HaCaT-c-Jun cells, we found that HaCaT cells with a suppressed AP-1 activity by JunB{Delta}N could not make a stratified epidermoid structure, although they did proliferate as well as wild-type HaCaT cells in the 2D culture and as a comparative level with wild-type HaCaT cells in a 3D culture condition (Figs 3–5). Chang et al. (2004) has previously shown that E2F/DP-1 activity is essential for the proliferation of keratinocytes and epidermal stratification. Cell proliferation is essential for the multi-layered tissue formation. In addition to this, our findings indicated for the first time that cell proliferation is not sufficient for stratification alone.

c-Jun was shown to be primarily a positive regulator of cell proliferation. c-Jun seems to be essential for the development of skin tumors (Young et al. 1999; Zenz et al. 2003). c-Jun-deficient fibroblasts have a marked proliferation defect and liver regeneration is severely impaired in c-Jun-deficient liver cells (Schreiber et al. 1999; Wisdom et al. 1999; Behrens et al. 2002). On the other hand, JunB suppresses proliferaion of keratinocytes and combined depletion of c-Jun and JunB from mouse epidermis caused psoriasis-like proliferative skin disease (Zenz & Wagner 2006). All of these works elucidated significant roles of c-Jun and JunB in the regulation of cell proliferation. In addition to this, JunB and JunD but not c-Jun has been reported to activate involucrin promoter together with Fra-1, which was activated during the late stages of terminal differentiation of keratinocytes (Welter et al. 1995). An increased AP-1 activity, which is activated by an air-liquid interface culture, induces stratification of differentiated keratinocytes as well as cell proliferation. JunB{Delta}N but not c-Jun{Delta}N is suggested to block the gene expression that is required for the stratification of differentiated keratinocytes independently of the regulation of cell proliferation. Therefore, the downstream targets of AP-1 that is essential for stratification is the next area of interest. Early in vitro studies indicated that increased AP-1 activities could lead to apoptosis in specific cell types (Shaulian & Karin 2002). In contrast, c-Jun is required for the survival of fetal hepatocytes, which undergo apoptosis in c-Jun-deficient mouse embryos (Eferl et al. 1999). Therefore, the effects of the AP-1 activity on cell death may be bi-directional depending on the cellular context. By oligonucleotide microarray experiments and RT-PCR experiments, the aldo-keto reductase family1 member B10, selenoprotein P and SOD1 were confirmed to increase by exposure to air and these responses were completely avoided in the HaCaT-JunB{Delta}N cell line. Selenoprotein P and SOD1 have been reported to be the direct targets of the AP-1 activity (Kim et al. 1994; Dreher et al. 1997). There have been no reports about the aldo-keto reductase family1 member B10 but this gene has two AP1 binding elements in a 5' enhancer region of the gene. These molecules have antioxidative activities (Nishinaka & Yabe-Nishimura 2001; Steinbrenner et al. 2006). The increased anti-oxidative activities by these AP-1 target genes may possibly block early cell death and the formation of multi-layered epidermoid structures. c-Jun{Delta}N affected cell proliferation but it did not block the stratification of HaCaT cells (Fig. 6). The suppression of the AP-1 activity by the dominant negative c-Jun mutant (TAM-67) was reported not to affect the normal architecture and thickness of the mouse epidermis (Young et al. 1999; Thompson et al. 2002). These results suggest to us that differential effects of JunB{Delta}N and c-Jun{Delta}N on the air exposure-induced alteration of gene expression profiles might help to identify the AP-1 components and the target genes required for the stratification of keratinocytes. We examined the oligonucleotide microarray data while paying a special interest to such cell adhesion molecules as integrins, desmocollin and desmoplakin, as well as AP-1-regulated apoptosis-related genes (Jamora & Fuchs 2002; Shaulian & Karin 2002; Watt 2002). However, the expression levels of any of these genes were not altered by a 3D culture of HaCaT cells (data not shown).

It is also interesting to note how exposure to air increased the AP-1 activity. Reactive oxygen species (ROS) play an essential role in mediating long-lasting JNK activation. Therefore, oxidative stress that is induced by air exposure is suspected to activate AP-1 activity. We attempted to examine if ASK kinase, which is directly activated by ROS, is involved in this pathway but could not get any definitive results so far (data not shown).

In recent years, 3D culture has been used for the study of multicellular tissue structure formation and JNK signaling has been shown to be essential for the acinar formation of mammary epithelial cells (Murtagh et al. 2004). However, it is not elucidated which AP-1 component is essential also in this case. Knockout of a gene in the AP-1 family frequently resulted in embryonic lethality. Conditional knockout experiments started to clarify the role of each AP-1 component in recent years, but we do not have any study reporting a stratification deficient phenotype in keratinocyte-specific knockouts of the AP-1 family transcription factors. Multiple conditional knockout such as Fra-1 and JunB or JunD and JunB may be required to elucidate the roles of the AP-1 family on the multi-layered epidermoid tissue formation.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cells

HaCaT cells were obtained from N.E. Fusenig and maintained in MCDB153 medium (Sigma) supplemented with 0.1 mM calcium chloride, 10 ng/mL epidermal growth factor (Sigma), 1% of a penicillin–streptomycin solution (Gibco) and 5% dialyzed fetal bovine serum (FBS) as previously described (Kato et al. 1995).

DNA constructs

c-Fos, JunB, JunB{Delta}N, c-Jun and c-Jun{Delta}N cDNAs were obtained by reverse transcription and PCR using the total RNA extracted from HaCaT cells as a template, and then cloned into the pcDNA3-FLAG vector. JunB{Delta}N and c-Jun{Delta}N lack codons 1–147, including the transcriptional activation domain of the proteins. All DNA sequences were confirmed before use. pAP1-luc was purchased from STRATAGENE and pNQO1-ARE reporter plasmid was provided by M. Yamamoto (Kobayashi et al. 2006).

RNAi

RNAi-mediated knockdown of c-Jun or JunB was performed using the pSUPER RNAi system (Oligoengine). The 19-bp targeting sequence was 5'-AGATGGAAACGACCTTCTA-3' for c-Jun, 5'-GACCAAGAGCGCATCAAAG-3' for JunB and 5'-CGTACGCGGAATACTTCGA-3' for luciferase. Stable HaCaT clones trasfected with pSUPER.puro-sh(JunB), pSUPER.puro-sh(c-Jun) and pSUPER.puro-sh(Luc) (Control) were selected and maintained in the presence of 1 µg/mL of puromycin (Sigma).

DNA transfection and luciferase assay

Cells were transfected using the FuGENE6 transfection reagent (Roche Diagnostics) following the manufacturer's recommendations. Stable HaCaT clones transfected with pcDNA3-FLAG-c-Jun, pcDNA3-FLAG-JunB{Delta}N, pcDNA3-FLAG-c-Jun{Delta}N and pcDNA3 (Mock) were selected and maintained in the presence of 300 µg/mL of G418 (Gibco). AP-1 activity in these cells was determined by a luciferase reporter assay system (Promega) using a luminometer (AutoLumat LB953, EG & G Berthold). Luciferase activities were normalized to ß-galactosidase activity, which was induced by the co-transfected CH110 (Amersham).

3D culture

Human dermal fibroblasts were mixed into a neutralized type I collagen gel (Cellmatrix type I-A, Nitta Gelatin Inc.) following the manufacturer's recommendations. The mixed solution was placed into 6 well plates (3 mL/well) and was hardened for 30 min in a CO2 incubator at 37 °C in humidified air. HaCaT cells were then dispensed onto each gel at a density of 2 x 106 cells in 3 mL of complete 3D culture medium and were incubated overnight. The complete 3D culture medium consisted of a 1 : 1 mixture of MCDB153 and DMEM, supplemented with 10% FBS, 100 µg/mL bovine pituitary extract (KOHJINBIO), 10 ng/mL EGF, 0.1 mM ethanolamine, 60 µM putrescine, 100 U/mL penicillin and 100 µg/mL streptomycin. The following day, the hardened gels were detached from the plates and incubation was continued for 1 week until the size of the contracted gel stabilized. Cell strainers (Becton Dickinson) were placed upside-down into a fresh 6 well plate, after removal of the strainer handles using sterilized scissors, and the 3D culture medium was poured into the wells until the nylon mesh of the strainers was covered. The contracted-gel discs were then placed on the mesh so that the HaCaT cells laid on the top of gel discs and the fluid level was adjusted to just below the upper edge of the gel. It was important that the gel was steeped sufficiently in the fluid, while at the same time its surface was exposed to air. Throughout the experiment, half of culture fluid was renewed every other day. After 1 week of an air-liquid interface culture, the gel discs were fixed in a phosphate-buffered formalin solution, embedded in paraffin and vertical sections were stained with hematoxylin and eosin.

Immunohistochemical staining

Sections of 3D cultured cells were pre-treated with an autoclave in 10 mM citrate buffer (pH 6.0) before immunostaining with anti-Ki-67 (Dako). All following procedures were carried out according to a standard protocol using the EnVision kit (Dako) and then were counterstained lightly with hematoxylin.

Immunoblotting

Cells were solubilized in a buffer containing 20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 1.5% Trasylol and 1 mM phenylmethylsulfonyl fluoride (PMSF). After clearing with centrifugation, the total cell lysates were subjected to SDS-PAGE. The proteins were electrotransferred to Hybond-C Extra (Amersham) and subjected to immunoblotting. Anti-FLAG (M5, Sigma), anti-ß-actin (Sigma), anti-JunB (C-11, Santa Cruz) and anti-c-Jun (Cell Signaling) were used as the primary antibody for immunoblotting. Reacted antibodies were detected using an enhanced chemiluminescence detection system (Pierce).

Cell growth assay

Cells were seeded in 6 well plates at a density of 3 x 104 cells per well on day 0. Cell numbers were counted at the indicated time points using a hematocytometer.

Oligonucleotide microarray analysis

We carried out oligonucleotide microarray analysis using the CodeLink system (Amersham). HaCaT cells in the 3D culture system were cultured with or without exposure to air for 24 h. The total RNA was prepared from HaCaT cell layers using the RNA easy mini Kit (Qiagen). Biotinylated cRNA was synthesized following the manufacturer's protocols and hybridized to a Human Whole Genome Bioarray. The array slides were treated with streptavidin-Cy5 and analyzed with arrayWoRxe (Applied Precision).

Reverse transcription (RT)-PCR

Reverse transcription was performed with the First-strand cDNA synthesis kit (TaKaRa) using the total RNA obtained from 3D cultured HaCaT cells as a template, and PCR was carried out using the primers listed as follows:

Aldo-keto reductase family1, member B10, forward primer: 5'-GTCACCCATACCTCACACAG-3', reverse primer: 5'-AGCCATGCTTTTCTGTGATA-3'; Selenoprotein P, forward primer: 5'-AGAGATCAAGATCCAATGCT-3', reverse primer: 5'-CATCTTTGAGAGTCGTGAGA-3'; FABP4, forward primer: 5'-TAGGTACCTGGAAACTTGTC-3', reverse primer: 5'-ATATATCCCACAGAATGTTG-3'; FABP5, forward primer: 5'-AGGCTTTGATGAATACATGA-3', reverse primer: 5'-ATTGTTCATGACACACTCCA-3'; Chemokine ligand 14(CXCL14), forward primer: 5'-GACGGGTCCAAATGCAAGTG-3', reverse primer: 5'-CAGGCGTTGTACCACTTGAT-3'; ß-actin, forward primer: 5'-CAAGAGATGGCCACGGCTGCT-3', reverse primer: 5'-TCCTTCTGCATCCTGTCGGCA-3'.


   Acknowledgements
 
We are very grateful to Drs NE Fusenig and M Yamamoto for the HaCaT cells and pNQO1-ARE-luc reporter, respectively. This work was supported by the Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.


   Footnotes
 
Communicated by: Kohei Miyazono

* Correspondence: E-mail: mit-kato{at}md.tsukuba.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
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Received: 28 July 2006
Accepted: 2 November 2006





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