Robert C. Smart

CCAAT/enhancer binding protein-β is a mediator of keratinocyte survival and skin tumorigenesis involving oncogenic Ras signaling

The basic leucine zipper transcription factor CCAAT/enhancer binding protein-β (C/EBPβ) is expressed in many cell types, including keratinocytes. C/EBPβ activity can be increased by phosphorylation through pathways stimulated by oncogenic Ras, although the biological implications of Ras-C/EBPβ signaling are not currently understood. We report here that C/EBPβ-nullizygous mice are completely refractory to skin tumor development induced by a variety of carcinogens and carcinogenesis protocols, including 7,12-dimethylbenz[a]anthracene-initiation/12-O-tetradecanoylphorbol 13-acetate promotion, that produce tumors containing oncogenic Ras mutations. No significant differences in TPA-induced epidermal keratinocyte proliferation were observed in C/EBPβ-null versus wild-type mice. However, apoptosis was significantly elevated (17-fold) in the epidermal keratinocytes of 7,12-dimethylbenz[a]anthracene-treated C/EBPβ-null mice compared with wild-type mice. In v-Ha-ras transgenic mice, C/EBPβ deficiency also led to greatly reduced skin tumor multiplicity and size, providing additional evidence for a tumorigenesis pathway linking Ras and C/EBPβ. Oncogenic Ras potently stimulated C/EBPβ to activate a C/EBP-responsive promoter-reporter in keratinocytes and mutating an ERK1/2 phosphorylation site (T188) in C/EBPβ abolished this Ras effect. Finally, we observed that C/EBPβ participates in oncogenic Ras-induced transformation of NIH 3T3 cells. These findings indicate that C/EBPβ has a critical role in Ras-mediated tumorigenesis and cell survival and implicate C/EBPβ as a target for tumor inhibition.

C/EBPbeta modulates the early events of keratinocyte differentiation involving growth arrest and keratin 1 and keratin 10 expression.

ABSTRACT The epidermis is a stratified squamous epithelium composed primarily of keratinocytes that become postmitotic and undergo sequential changes in gene expression during terminal differentiation. The expression of the transcription factor CCAAT/enhancer binding protein β (C/EBPβ) within mouse epidermis and primary keratinocytes has recently been described; however, the function of C/EBPβ within the epidermal keratinocyte is unknown. We report here that transient transfection of mouse primary keratinocytes with a C/EBP-responsive promoter-reporter construct resulted in a sevenfold increase in luciferase activity when keratinocytes were switched to culture conditions that induce growth arrest and differentiation. Forced expression of C/EBPβ in BALB/MK2 keratinocytes inhibited growth, induced morphological changes consistent with a more differentiated phenotype, and upregulated two early markers of differentiation, keratin 1 (K1) and keratin 10 (K10) but had a minimal effect on the expression of late-stage markers, loricrin and involucrin. Analysis of the epidermis of C/EBPβ-deficient mice revealed a mild epidermal hyperplasia and decreased expression of K1 and K10 but not of involucrin and loricrin. C/EBPβ-deficient primary keratinocytes were partially resistant to calcium-induced growth arrest. Analysis of terminally differentiated spontaneously detached keratinocytes or those induced to differentiate by suspension culture revealed that C/EBPβ-deficient keratinocytes displayed striking decreases in K1 and K10, while expression of later-stage markers was only minimally altered. Our results demonstrate that C/EBPβ plays an important role in the early events of stratified squamous differentiation in keratinocytes involving growth arrest and K1 and K10 expression.

Tak1 is a Master Regulator of Epidermal Homeostasis Involving Skin Inflammation and Apoptosis

Transforming growth factor β-activated kinase 1 (TAK1) functions downstream of inflammatory cytokines to activate c-Jun N-terminal kinase (JNK) as well as NF-κB in several cell types. However, the functional role of TAK1 in an in vivo setting has not been determined. Here we have demonstrated that TAK1 is the major regulator of skin inflammation as well as keratinocyte death in vivo. Epidermal-specific deletion of TAK1 causes a severe inflammatory skin condition by postnatal day 6-8. The mutant skin also exhibits massive keratinocyte death. Analysis of keratinocytes isolated from the mutant skin revealed that TAK1 deficiency results in a striking increase in apoptosis in response to tumor necrosis factor (TNF). TAK1-deficient keratinocytes cannot activate NF-κB or JNK upon TNF treatment. These results suggest that TNF induces TAK1-deficient keratinocyte death because of the lack of NF-κB (and possibly JNK)-mediated cell survival signaling. Finally, we have shown that deletion of the TNF receptor can largely rescue keratinocyte death as well as inflammatory skin condition in epidermal-specific TAK1-deficient mice. Our results demonstrate that TAK1 is a master regulator of TNF signaling in skin and regulates skin inflammation and keratinocyte death.

Control of skin cancer by the circadian rhythm

Skin cancer is the most common form of cancer in the United States. The main cause of this cancer is DNA damage induced by the UV component of sunlight. In humans and mice, UV damage is removed by the nucleotide excision repair system. Here, we report that a rate-limiting subunit of excision repair, the xeroderma pigmentosum group A (XPA) protein, and the excision repair rate exhibit daily rhythmicity in mouse skin, with a minimum in the morning and a maximum in the afternoon/evening. In parallel with the rhythmicity of repair rate, we find that mice exposed to UV radiation (UVR) at 4:00 AM display a decreased latency and about a fivefold increased multiplicity of skin cancer (invasive squamous cell carcinoma) than mice exposed to UVR at 4:00 PM. We conclude that time of day of exposure to UVR is a contributing factor to its carcinogenicity in mice, and possibly in humans.

Correlation between expression of peroxisome proliferator-activated receptor β and squamous differentiation in epidermal and tracheobronchial epithelial cells

Previously, several members of the nuclear receptor superfamily have been implicated in the regulation of epidermal differentiation. In this study, we analyze the expression of members of the PPAR nuclear receptor subfamily in relation to the process of squamous differentiation in normal human epidermal keratinocytes (NHEK), human tracheobronchial epithelial (HBE) cells and the epidermis in vivo. Our results demonstrate that induction of differentiation in NHEK by either treatment with the phorbol ester phorbol 12-myristate-13-acetate (PMA), suspension culture or confluence greatly enhances the expression of PPARbeta mRNA. Likewise, topical treatment of mouse skin with PMA results in increased PPARbeta mRNA expression in the epidermis. In addition, the induction of squamous differentiation in HBE cells was also associated with an upregulation of PPARbeta mRNA expression. Finally, in situ hybridization analysis localized PPARbeta mRNA to the suprabasal layers of normal human skin. Our results demonstrate that the expression of PPARbeta is associated with squamous differentiation suggesting a regulatory role for this receptor in the control of specific genes during this differentiation process.

Cell Cycle-Dependent Phosphorylation of C/EBPβ Mediates Oncogenic Cooperativity between C/EBPβ and H-RasV12

ABSTRACT CCAAT/enhancer binding protein β (C/EBPβ) is a widely expressed transcription factor whose activity is regulated by oncogenic Ha-RasV12 signaling. C/EBPβ is essential for the development of mouse skin tumors containing Ras mutations and can cooperate with RasV12 to transform NIH 3T3 cells. Here we have investigated Ras-induced phosphorylation of C/EBPβ in fibroblasts and report a novel proline-directed phosphoacceptor site at Ser64 within the transactivation domain. Ser64 phosphorylation was induced by activated Ras and Raf but was not blocked by chemical inhibitors of MEK1/2, phosphatidylinositol 3-kinase, JNK, or p38 mitogen-activated protein kinases. Ser64 was efficiently phosphorylated in vitro by the cyclin-dependent kinases Cdk2 and Cdc2. Thr189, previously identified as an ERK1/2 phosphorylation site that regulates C/EBPβ activity, was also a substrate for Cdk phosphorylation. Ser64 and Thr189 phosphorylation was low in serum-starved (G0) cells but was strongly increased in mid-G1 cells and in cells arrested in S or M phase. In addition, phosphorylation on both sites was blocked by treating cells with the Cdk inhibitor roscovitine. In contrast to wild-type C/EBPβ, which enhances transformation of NIH 3T3 cells, mutants bearing alanine substitutions at Ser64 and/or Thr189 inhibited RasV12-induced focus formation. Our findings support a role for C/EBPβ as a nuclear effector of Ras signaling and transformation, and they indicate that cell cycle-dependent phosphorylation of C/EBPβ on Ser64 and Thr189 is required to promote Ras-induced transformation of NIH 3T3 cells.

C/EBPα Is a DNA Damage-Inducible p53-Regulated Mediator of the G1 Checkpoint in Keratinocytes

ABSTRACT The basic leucine zipper transcription factor, CCAAT/enhancer binding protein α (C/EBPα), is abundantly expressed in keratinocytes of the skin; however, its function in skin is poorly characterized. UVB radiation is responsible for the majority of human skin cancers. In response to UVB-induced DNA damage, keratinocytes activate cell cycle checkpoints that arrest cell cycle progression and prevent replication of damaged DNA, allowing time for DNA repair. We report here that UVB radiation is a potent inducer of C/EBPα in human and mouse keratinocytes, as well as in mouse skin in vivo. UVB irradiation of keratinocytes resulted in the transcriptional up-regulation of C/EBPα mRNA, producing a >70-fold increase in C/EBPα protein levels. N-Methyl-N′-nitro-N-nitrosoguanidine, etoposide, and bleomycin also induced C/EBPα. UVB-induced C/EBPα was accompanied by an increase in p53 protein and caffeine, an inhibitor of ataxia-telangiectasia-mutated kinase, and ataxia-telangiectasia-mutated and Rad3-related kinase inhibited UVB-induced increases in both C/EBPα and p53. UVB irradiation of p53-null or mutant p53-containing keratinocytes failed to induce C/EBPα. UVB irradiation of C/EBPα knockdown keratinocytes displayed a greatly diminished DNA damage G1 checkpoint, and this was associated with increased sensitivity to UVB-induced apoptosis. Our results uncover a novel role for C/EBPα as a p53-regulated DNA damage-inducible gene that has a critical function in the DNA damage G1 checkpoint response in keratinocytes.

PTEN Positively Regulates UVB-Induced DNA Damage Repair

Nonmelanoma skin cancer is the most common cancer in the United States, where DNA-damaging ultraviolet B (UVB) radiation from the sun remains the major environmental risk factor. However, the critical genetic targets of UVB radiation are undefined. Here we show that attenuating PTEN in epidermal keratinocytes is a predisposing factor for UVB-induced skin carcinogenesis in mice. In skin papilloma and squamous cell carcinoma (SCC), levels of PTEN were reduced compared with skin lacking these lesions. Likewise, there was a reduction in PTEN levels in human premalignant actinic keratosis and malignant SCCs, supporting a key role for PTEN in human skin cancer formation and progression. PTEN downregulation impaired the capacity of global genomic nucleotide excision repair (GG-NER), a critical mechanism for removing UVB-induced mutagenic DNA lesions. In contrast to the response to ionizing radiation, PTEN downregulation prolonged UVB-induced growth arrest and increased the activation of the Chk1 DNA damage pathway in an AKT-independent manner, likely due to reduced DNA repair. PTEN loss also suppressed expression of the key GG-NER protein xeroderma pigmentosum C (XPC) through the AKT/p38 signaling axis. Reconstitution of XPC levels in PTEN-inhibited cells restored GG-NER capacity. Taken together, our findings define PTEN as an essential genomic gatekeeper in the skin through its ability to positively regulate XPC-dependent GG-NER following DNA damage.

Conditional ablation of C/EBPβ demonstrates its keratinocyte-specific requirement for cell survival and mouse skin tumorigenesis

The CCAAT/enhancer binding protein β (C/EBPβ) is implicated in the regulation of many different molecular and physiological processes. Mice with a germline deletion of C/EBPβ (C/EBPβ−/−) display phenotypes in a multitude of cell types and organ systems, including skin where C/EBPβ−/− mice exhibit increased apoptosis in epidermal keratinocytes in response to carcinogen treatment and are completely resistant to carcinogen-induced skin tumorigenesis. To determine the contribution of systemic versus cell autonomous functions of C/EBPβ to specific phenotypes, mice with a conditional ‘floxed’ C/EBPβ null allele were generated. Epidermal-specific deletion of C/EBPβ was achieved by Cre recombinase expression from a keratin 5 (K5) promoter. Similar to C/EBPβ−/− mice, K5-Cre;C/EBPβfl/fl mice were completely refractory to 7,12 dimethylbenz[a]anthracene (DMBA)-induced skin tumorigenesis and these mice displayed increased DMBA-induced apoptosis in epidermal keratinocytes compared to wild-type mice. In contrast, mice lacking the related gene, C/EBPδ, were not resistant to DMBA-induced skin tumorigenesis, indicating a unique role of C/EBPβ in skin tumor development. Our findings demonstrate that C/EBPβ exerts an essential, keratinocyte-intrinsic role in cell survival in response to carcinogen treatment and the elimination of C/EBPβ in keratinocytes is sufficient to confer complete resistance of the skin to chemical carcinogenesis.

Molecular and biochemical toxicology

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