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Squamous Cell Carcinomas and Increased Apoptosis in Skin with Inhibited Rel/Nuclear Factor-B Signaling¹

Department of Bioscience at Novum, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden [M. v. H., R. T.]; Department of Clinical Pathology and Cytology, F49, Huddinge University Hospital, S-141 86 Huddinge, Sweden [B. L. R.]; and Department of Medical Nutrition, Huddinge University Hospital, NOVUM, S-141 86 Huddinge, Sweden [L. Ä-R.]

	ABSTRACT

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The Rel/nuclear factor-B (Rel/NF-B) transcription factors have been implicated previously in control of apoptosis, cell proliferation, and oncogenesis. Here we show that selective inhibition of Rel/NF-B signaling in murine skin, by targeted overexpression of a super-repressor form of IB-, results in an increased basal frequency of apoptotic cells and the spontaneous development of squamous cell carcinomas. Presence of hyperplasia and hair follicle degeneration demonstrate an important role for Rel/NF-B signaling in normal epidermal development and homeostasis. Transgenic skin, in addition, showed an enhanced sensitivity to UV-induced apoptosis. These data suggest an involvement of the Rel/NF-B signaling pathway in apoptosis and cancer development of the skin.

	Introduction

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The Rel/NF-B³ transcription factors serve an important regulatory role in 2inflammation and the immune response as well as the cellular stress response and have been implicated in control of cell proliferation and oncogenesis (1 , 2) . In nonstimulated cells, Rel/NF-B proteins are retained in the cytoplasm by an inhibitory protein, IB- (1) . Upon stimulation, IB- is rapidly phosphorylated and degraded, allowing Rel/NF-B to translocate to the nucleus. Stimuli activating Rel/NF-B include cytokines, phorbol esters, and bacterial products (1) , and in the skin, we have shown previously that activation of Rel/NF-B by UVB in keratinocytes is mediated by nonligand-dependent activation of tumor necrosis factor receptor 1 (3) . Recent studies show that activation of Rel/NF-B suppresses apoptosis induced by TNF- or the chemotherapeutic agent daunorubicin (4) , suggesting that the Rel/NF-B signaling pathway may also play an important role in regulating UV-induced cutaneous apoptosis. Supporting a role for Rel/NF-B signaling in skin physiology, previous studies generating mice lacking the IB- gene demonstrated severe dermatitis (5) .

In this study, we have examined the role of IB--dependent signaling in mouse skin. The results demonstrate that selective inhibition of Rel/NF-B signaling in the skin leads to a disturbed epidermal homeostasis and hair follicle development, an increased frequency of apoptotic keratinocytes, and the spontaneous development of squamous cell carcinomas.

	Materials and Methods

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Generation of Transgenic Mice.
The 1.05 IB- cDNA was subcloned by blunt-end ligation into the SnaBI restriction site of the p5'BK5 expression vector (6) , which was a kind gift from J. Jorcano (CIEMAT, Madrid, Spain). Serines 32 and 36 in the IB- cDNA were mutated to glycine and alanine, respectively. The transgene was excised from the expression plasmid with SalI and NotI and microinjected into the pronuclei of fertilized FVB/N or [CBA x C57Bl/6] F₂ oocytes. Transgene integration was confirmed by PCR-analysis of genomic DNA extracted from tail biopsies.

RNA Analysis.
Total RNA was prepared from mouse skin by a standard guanidinium isothiocyanate procedure. RT-PCR analysis was performed using the Access RT-PCR kit (Promega) according to the manufacturer’s instructions with the following modifications: a denaturation step was added (94°C for 2 min) before addition of the AMV reverse transcriptase. The Tfl polymerase was added to the reaction after inactivation of the AMV reverse transcriptase. Primer RT-pcrtg1 (5'-TGCTCTTTCTCTCCAGCACCTCGGATC-3') was derived from the 5' untranslated region of the keratin 5 gene and extends into the ß-globin sequence of the expression vector. Primer RT-pcrtg2 (5'-ATTGCCAAGTGCAGGAACGAGTCTCCG-3') was derived from the IB- cDNA sequence. The primers span over the ß-globin intron in the expression vector, and amplification of mRNA yields a 438-bp product. Amplification of contaminating genomic DNA would yield a 1011-bp product. Amplification of endogenous ß-actin mRNA using primers actin1 (5'-CATCGTGGGCCGCTCTAGGCACCAAG-3') and actin2 (5'-GCACAGCTTCTCTTTGATGTCACGCAC-3') served as an internal control and yielded a 553-bp product. PCR was performed by 37 cycles of amplification (denaturation at 94°C for 1 min, annealing and extension at 72°C for 4 min with a 5-s extension per cycle), with a final extension at 72°C for 10 min. The upstream primer actin1 was added after 10 cycles to keep the amplification reaction of the ß-actin mRNA in the exponential phase.

Histology and Immunohistochemistry.
Skin biopsies were fixed overnight in 4% paraformaldehyde and paraffin embedded. Sections were stained with H&E. For immunohistochemistry, sections were deparaffinized in xylene, passed through a graded alcohol series, and then incubated in 0.3% H₂O₂ in methanol to block endogenous peroxidase activity. After rinsing in H₂O, sections were microwaved for 5 min (IB-) or 10 min (p65, TNF-) in 10 mM sodium citrate buffer (pH 6.0). Sections were washed in PBS and blocked in 10% goat serum (p65, IB-) or 5% rabbit serum (TNF-) in PBS containing 0.1% Triton X-100 (p65) or 0.1% saponin (TNF-, IB-). Sections were incubated with primary antibody in PBS containing 5% goat serum or 2% rabbit serum and 0.1% detergent. After washing in PBS, sections were incubated with biotinylated secondary antibody in PBS containing 5 or 2% serum and 0.1% detergent, washed in PBS, and incubated with Vectastain Elite ABC reagent (Vector Laboratories). The sections were washed in PBS and visualized using diaminobenzidine (Sigma Chemical Co.). Sections were counterstained with hematoxylin. Primary antibodies used were: rabbit polyclonal anti-p65 (Santa Cruz Biotechnology) 1:300, rabbit polyclonal anti-IB- (Santa Cruz Biotechnology) 1:200, and rat monoclonal anti-TNF- (PharMingen) 1:25. Secondary antibodies were biotinylated goat anti-rabbit (Vector Laboratories) 1:350 and biotinylated rabbit anti-rat (Vector Laboratories) 1:200. As negative controls, all experiments were also performed in the presence of equal concentrations of normal rabbit IgG or rat IgG1 isotype control or by preincubating with control peptide.

Detection of Apoptotic Cells.
Skin biopsies were fixed in 4% parafor-maldehyde and paraffin embedded. Sections were stained with H&E. Apoptotic keratinocytes were counted using the following criteria: dark pyknotic nuclei, cytoplasmic eosinophilia, vacuolization of the cytoplasm, and absence of cellular contacts (7) . For the UV experiments, mouse back skin was shaved and depilated. Twenty-four h later, mice were irradiated with UVB under anesthesia to prevent movement, using a bank of four TL12 (Philips) bulbs with a UVB emission of 16 W/m². UV irradiation was measured using a DRC-100 x radiometer (Spectroline).

	Results

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Expression of Signal-resistant IB- in Mouse Skin.
To study the importance of IB--dependent signaling in the skin, we selectively blocked Rel/NF-B activation by targeted expression of a super-repressor form of IB- in the epidermis of transgenic mice.

For this purpose, we inserted the IB- cDNA into an expression vector containing the bovine keratin 5 gene promoter, which directs expression to the basal layer of interfollicular epidermis and the outer root sheath of the hair follicles (Fig. 1A ; Ref. 8 ).

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Transgene expression in mouse skin. A, schematic representation of the IB- expression vector. IB- cDNA containing S32G and S36A mutations was inserted downstream of the bovine keratin 5 (K5) promoter. A rabbit ß-globin intron and polyadenylation signal and a SV40 polyadenylation signal (pA) were included. B, mRNA expression from the transgene in mouse skin. RNA was isolated from the skin of adult (Lanes 1–4) or 3-day-old (Lanes 5–8) mice. The RNA was analyzed by RT-PCR using primers specific for transgenic mRNA that do not recognize endogenous IB- mRNA. In adult mice, expression is seen in founder Tg.1 (Lane 2), Tg.2 (Lane 3), and founder line Tg.3 (Lane 4) but not in control mice (Lane 1). In the 3-day-old mice, expression was observed in founder line Tg.3 (Lane 8) but not in mice from founder line Tg.4 (Lane 6), Tg.5 (Lane 7), or control mice (Lane 5). C, overexpression of IB- protein in the epidermis. Skin sections from transgenic founder Tg.2 (TG) and control littermate (WT) at 2 months of age were immunostained using an anti-IB- antibody. Overexpression of IB- is seen in epidermis and hair follicles of the transgenic skin. Expression is also observed suprabasally, which is in concordance with the degradation resistance of the mutant IB- protein. Bar, 50 µm.

Transgenic mice were generated by microinjection of the transgene into the pronuclei of fertilized FVB/N or [CBA x C57Bl/6] F₂ oocytes. A total of seven founder transgenic mice were identified by PCR analysis of tail DNA. Two founders did not express the transgene, and another two were probably genetic mosaics and did not transmit the transgene to subsequent generations.

Expression of IB- mRNA from the transgene as assessed by RT-PCR is shown in Fig. 1B , and expression in the skin is clearly seen in both adult and 3-day-old mice. Overexpression of IB- protein was localized to the epidermis and hair follicle keratinocytes of transgenic mice as demonstrated by immunohistochemistry (Fig. 1C) . One founder, designated Tg.1, showed expression of the transgene in the RT-PCR assays but was phenotypically normal. However, the RT-PCR experiments are not quantitative, and subsequent immunohistochemical analyses revealed a low expression level in the Tg.1 founder (data not shown).

Spontaneous Development of Squamous Cell Carcinomas in Transgenic Skin.
Two founders, designated Tg.2 and Tg.3, exhibited a severe phenotype, macroscopically observable already at birth, that was characterized by flaky skin, impaired hair growth, hair loss, and hyperkeratotic lesions on the scalp, back skin, and the tail. These lesions, which spread to the limbs and ventral skin, developed into crusted areas with markedly thickened, wrinkled skin. The animals were also considerably smaller than control littermates.

Histological examination of lesional back skin from founder Tg.2 (FVB/N background), at 2 months of age, surprisingly revealed dysplasia of varying degrees of the epidermis and hair follicle epithelium. Both epithelia demonstrated hyperplasia, and in the epidermis the disturbance of maturation was often combined with hypergranulosis and hyperorthokeratosis. Keratin-filled invaginations, degenerated hair follicles, and keratocysts were found as well as frankly invasive, well-differentiated squamous cell carcinoma (Fig. 2, A and B) . In 2addition, an inflammatory response was seen, sometimes pronounced. Mice were kept under barrier conditions and routinely screened for pathogens, and Gram’s staining for bacteria did not reveal any microorganisms.

View larger version (94K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Histological features of transgenic skin. A, in founder Tg.2, the hyperplastic epithelium of the epidermis and hair follicles created an irregular pattern of papillary projections and invaginations with prominent hyperkeratosis. B, the dysplastic properties of the epidermis and the infiltrative strands of the well-differentiated squamous cell carcinoma in the underlying dermis. C, founder Tg.3 demonstrated similar changes including prominent keratin-filled cysts in the deep part of the dermis. D, the epidermal dysplasia and progression to infiltrating squamous cell carcinoma equals that of founder Tg.2. The Tg.3 x FVB/N offspring produced squamous cell carcinomas that were, at least focally, moderately well differentiated with a more florid growth (E) and a more pronounced cellular atypia (F). Skin biopsies were fixed in 4% paraformaldehyde and paraffin embedded. Sections were stained with H&E. Bar, 50 µm.

Because the progression to malignancy seemed to be more rapid in the Tg.2 founder, offspring from the Tg.3 founder were crossed to the inbred FVB/N strain.

All transgenic mice from the F₁ and F₂ generations (n = 16) developed tumors behind the ears, on the back, on the hind legs, or at the base of the tail, usually within 3 months. Also, these lesions consisted of infiltrative squamous cell carcinoma, mainly well differentiated, but focally demonstrating a more pronounced cellular atypia and aggressive growth pattern corresponding to a moderately well-differentiated tumor (Fig. 2, E and F) . Increased susceptibility to malignant conversion has been shown previously to be a dominant trait in FVB/N mice (10) .

Expression of the transgene leads to a block of Rel/NF-B signaling.
The development of squamous cell carcinomas in two founders on different genetic backgrounds strongly indicates that the changes observed are the result of the expression of the IB- transgene leading to a block of Rel/NF-B signaling.

To further substantiate this conclusion, we examined the presence of such a block by performing nuclear translocation studies using an antibody directed against RelA/p65. Treatment of back skin with 10 nmol of the phorbol ester TPA resulted, as expected, in translocation of p65 to the nucleus within 2 h in normal mice, whereas in transgenic skin translocation of p65 was completely inhibited (Fig. 3A) .

View larger version (91K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. A, nuclear translocation of p65 is inhibited in transgenic skin. Nonlesional back skin of transgenic mice (TG) of the Tg.3 line or control littermates (WT) was treated with 10 nmol of TPA in acetone or acetone alone, as indicated. Sections were immunostained using an anti-p65 antibody. B, expression of TNF-. Back skin from control littermate (WT) or lesional skin from transgenic (TG) Tg.2 mice was sectioned and immunostained using a monoclonal anti-TNF- antibody. Bars, 50 µm.

In an attempt to investigate Rel/NF-B target gene expression and understand the appearance of an inflammatory response, we examined the expression of TNF-. Surprisingly, lesional transgenic skin showed increased expression of TNF- as assessed by immunohistochemistry (Fig. 3B) . This paradoxical result may be due to a secondary effect where infiltrating inflammatory cells induce the expression of TNF- in keratinocytes, perhaps through activation of transcription factors other than NF-B. Several regulatory elements have been identified in the TNF- promoter (12) .

The underlying cause for the inflammatory response may be the presence of degenerated hair follicles, as observed previously in mice with compromised epidermal growth factor receptor function (6) , or alternatively, release of cytokines from hyperplastic interfollicular keratinocytes. In this scenario, the increased expression of TNF- is a secondary response that may contribute to the severe phenotype observed in the transgenic mice, because overexpression of TNF- in the skin has been shown to result in severe dermatitis (13) .

Increased Frequency of Apoptotic Cells in Transgenic Skin.
It is now well established that Rel/NF-B signaling can elicit a protective effect against TNF- and chemotherapy-induced apoptosis in diverse cell types (2 , 4) . Histological examination of transgenic skin revealed multiple cases of basal and suprabasal keratinocytes undergoing apoptosis (Fig. 4A) .

View larger version (50K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Increased frequency of apoptotic cells in transgenic skin. A, squamous epithelium of a keratin-filled cyst in founder Tg.2 demonstrating multiple apoptotic cells, in basal and suprabasal position. Arrows, apoptotic cells. Sections were stained with H&E. Bar, 50 µm. B, increased UV-induced apoptosis in transgenic skin. Nonlesional back skin from transgenic mice (TG) of the Tg.3 line or control littermates (WT) was shaved, depilated, and irradiated with 500 or 1000 J/m2 UVB as indicated or was left unirradiated. Skin samples were collected at the indicated time points, and sections were analyzed for sunburn cell formation per cm of epidermal length (sbc/cm). The data presented are the average of at least two sections/mouse/time point and four mice/group. Bars, SE. Statistical analysis was performed using the Student’s t test. **, P < 0.01; ***, P < 0.001.

Because UV radiation induces apoptosis in keratinocytes (15) and is a known activator of Rel/NF-B (1 , 3) , we wanted to investigate if inhibition of this part of the UV-response influences the formation of apoptotic cells. To this end, transgenic and control mice were irradiated with 500 or 1000 J/m² UVB. Skin samples were taken at different time points and stained with H&E, and apoptotic cells were counted. Apoptotic keratinocytes, known as sunburn cells, are characterized by dark pyknotic nuclei, cytoplasmic eosinophilia, vacuolization of the cytoplasm, and absence of cellular contacts (15) . Skin from transgenic mice showed an increase of 80% in the frequency of apoptotic cells in the epidermis 12 h after UV radiation compared to wild-type littermates (Fig. 4b) , indicating that activation of Rel/NF-B signaling by UV radiation serves a protective function with regard to programmed cell death in intact skin.

	Discussion

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The most striking and unexpected result in our study is the rapid and spontaneous development of malignant squamous cell carcinomas in transgenic mice expressing a skin-targeted IB- super-repressor. Involvement of NF-B/IB proteins in oncogenesis has been implicated in several cases (reviewed in Ref. 16 ). Previous studies in cell culture using NIH 3T3 cells have shown that expression of IB- antisense RNA can result in malignant transformation (17) . In a recent study, Mayo et al. (2) , also using NIH 3T3 cells, showed that NF-B activation by oncogenic ras is required to suppress apoptosis and suggested that such activation is an important part of ras-mediated oncogenesis. However, fibroblast cell lines established from IB- -/- mice are not transformed, indicating that loss of IB- function is not sufficient to transform primary cells (16) . Also, NF-B is activated by the anticancer agent taxol and several other antineoplastic agents, including vinblastine and daunomycin, suggesting a potential protective role (18) . We think that the cell type-specific properties of Rel/NF-B signaling in keratinocytes, i.e., negative regulation of cell growth (Ref. 11 and this study) and ras-independent regulation of Rel/NF-B activity (19) , are the most likely reasons behind these apparent discrepancies.

The growth-inhibiting role of NF-B in epidermis could be explained by an effect on cell cycle regulators. Previous studies have shown that expression of c-Rel in HeLa cells led to growth arrest at the G₁-S-phase transition associated with p21^WAF1 induction (20) .

Because genomic instability is known to be a hallmark of multistage carcinogenesis (21) , it is also an interesting possibility that Rel/NF-B signaling, at least in epithelial skin cells, is involved in maintaining genomic stability.

The disturbed regulation of NF-B-dependent genes in the transgenic skin could also lead to an impaired immune response. Wrighton et al. (22) showed previously that adenovirus-mediated expression of IB- resulted in inhibited expression of VCAM-1, IL-1, IL-6, and IL-8 and fully supressed leukocyte adhesion in endothelial cells.

The loss of growth control combined with an impaired immune response could hypothetically lead to expansion of aberrant keratinocyte clones, which are not recognized by the immune system, resulting in the observed tumorigenesis. These possibilities can now be experimentally investigated in the IB- transgenic model.

Our data demonstrate that selective inhibition of Rel/NF-B signaling in epithelial skin cells disrupts normal epidermal homeostasis and hair follicle development, increases the number of keratinocytes undergoing apoptosis, and strongly predisposes to malignant skin tumor formation. The potential for such effects has important implications when considering pharmacological inhibition of Rel/NF-B signaling as a treatment modality to facilitate apoptosis or inhibit inflammatory responses.

	ACKNOWLEDGMENTS

 
We thank R. Taub for the IB- cDNA clone, J. Jorcano for the K5 expression vector, and U. Brockstedt for help with the TdT-mediated dUTP-X nick end labeling assay.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by grants from the Swedish Cancer Fund and from the Karolinska Institute.

² To whom requests for reprints should be addressed, at Department of Biosciences at Novum, Karolinska Institute, NOVUM, S-141 57 Huddinge, Sweden. Phone: 46-8-6089152; Fax: 46-8-6081501; E-mail: Rune.Toftgard{at}cnt.ki.se

³ The abbreviations used are: Rel/NF-B, Rel/nuclear factor-B; TNF, tumor necrosis factor; RT-PCR, reverse transcription-PCR; TPA, 12-O-tetradecanoylphorbol-13-acetate.

Received 2/26/99. Accepted 6/ 1/99.

	REFERENCES

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