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.circadian clock | cryptochrome | sunbathing | tanning salons S kin cancer is the most common form of cancer in the United States. With over 1.3 million new cases each year, it constitutes nearly 40% of all diagnosed cancers (1). Moreover, because of changes in lifestyle and the environment, the incidence of skin cancer is steadily increasing (2). The main causative agent of skin cancer is the UV component of sunlight. UV radiation (UVR) produces two major lesions in DNA, the cyclobutane pyrimidine dimer (CPD) and the (6-4) photoproduct [(6-4) PP], both of which are mutagenic and carcinogenic in animal model systems and are thought to be the primary cause of skin cancer in humans (3-7).In mice and humans, nucleotide excision repair is the sole repair system for removing CPDs and (6-4) PPs from DNA. As a consequence, humans with hereditary mutations in excision repair genes suffer from xeroderma pigmentosum, a syndrome characterized by a nearly 5,000-fold increase in skin cancer in sunlight-exposed areas of the afflicted individuals (8). Excision repair involves photoproduct removal by dual incisions bracketing the lesion, removal of the damage in the form of a 24-to 32-nt-long oligomer, filling in the resulting single-stranded gap, and sealing by ligase (9). The dual incision is carried out by six excision repair factors: RPA, xeroderma pigmentosum group A (XPA), XPC, TFIIH, XPG, and XPF-ERCC1 (10). Recently, in a study that analyzed liver and brain tissues from mice, it was found that XPA, a critical protein involved in damage recognition and a rate-limiting factor in excision repair, is controlled by the core molecular circadian clock (11, 12). As a consequence, excision repair activity exhibited circadian rhythmicity in these organs, increasing during the day to reach a maximum at 4-6:00 PM and decreasing during the night to a minimum at 4-6:00 AM.Here we analyzed the expression pattern of XPA and excision repair activity in mouse skin. We found that protein and repair activity exhibit a circadian rhythm similar to that found in the liver and brain. To determine whether this rhythmicity affected UV-induced skin cancer development we exposed a...

The basic leucine zipper transcription factor CCAAT͞enhancer binding protein-␤ (C͞EBP␤) is expressed in many cell types, including keratinocytes. C͞EBP␤ activity can be increased by phosphorylation through pathways stimulated by oncogenic Ras, although the biological implications of Ras-C͞EBP␤ signaling are not currently understood. We report here that C͞EBP␤-nullizygous mice are completely refractory to skin tumor development induced by a variety of carcinogens and carcinogenesis protocols, including 7,12-dimethylbenz-[a]anthracene-initiation͞12-O-tetradecanoylphorbol 13-acetate promotion, that produce tumors containing oncogenic Ras mutations. No significant differences in TPA-induced epidermal keratinocyte proliferation were observed in C͞EBP␤-null versus wild-type mice. However, apoptosis was significantly elevated (17-fold) in the epidermal keratinocytes of 7,12-dimethylbenz[a]anthracene-treated C͞EBP␤-null mice compared with wild-type mice. In v-Ha-ras transgenic mice, C͞EBP␤ deficiency also led to greatly reduced skin tumor multiplicity and size, providing additional evidence for a tumorigenesis pathway linking Ras and C͞EBP␤. Oncogenic Ras potently stimulated C͞EBP␤ to activate a C͞EBP-responsive promoter-reporter in keratinocytes and mutating an ERK1͞2 phosphorylation site (T188) in C͞EBP␤ abolished this Ras effect. Finally, we observed that C͞EBP␤ participates in oncogenic Ras-induced transformation of NIH 3T3 cells. These findings indicate that C͞EBP␤ has a critical role in Ras-mediated tumorigenesis and cell survival and implicate C͞EBP␤ as a target for tumor inhibition.T he Ras family of GTP binding proteins function as intracellular mediators of extracellular signals to regulate cell proliferation, apoptosis, survival, senescence, and differentiation (1-5). Ras protooncogenes are frequently mutated in tumors, and Ϸ25% of human cancers contain transforming mutations in ras. Therefore, understanding oncogenic Ras-signaling pathways is critical for elucidating the mechanisms that underlie cellular transformation and for designing effective therapeutic strategies to prevent the development or block the growth of many classes of tumors. Ras has numerous effectors, and its pathways are multifaceted (3, 6, 7). Ras activation by growth factors or oncogenic mutations elicits activation of several transcription factors, which in turn regulate the expression of genes that control the cellular responses to Ras signaling, including oncogenesis. The transcription factors Ets, c-jun, c-myc, and NF-B are known to have roles in oncogenic ras-induced cellular transformation (8-11).The basic leucine zipper (bZIP) transcription factor CCAAT͞ enhancer binding protein-␤ (C͞EBP␤, also known as NF-IL6, IL-6DBP, LAP, CRP2, and NF-M) is expressed in a variety of cell types (12, 13) including keratinocytes (14,15), where it plays a role in squamous differentiation (16). C͞EBP␤ is also involved in regulating differentiation of specific mesenchymal, epithelial, and hematopoietic cell types (17-21). C͞EBP␤ activity can be activ...

Transforming growth factor ␤-activated kinase 1 (TAK1) functions downstream of inflammatory cytokines to activate c-Jun N-terminal kinase (JNK) as well as NF-B 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-B or JNK upon TNF treatment. These results suggest that TNF induces TAK1-deficient keratinocyte death because of the lack of NF-B (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. TAK12 (transforming growth factor ␤-activated kinase 1) is a member of the mitogen-activated protein kinase kinase kinase family and is activated by inflammatory cytokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) and Toll-like receptor ligands (1, 2). In IL-1, TNF, and Toll-like receptor ligand signaling pathways, TAK1 has been shown to be an essential signaling intermediate that functions upstream of IB kinase (IKK)-NF-B and c-Jun N-terminal kinase (JNK) in B cells and some culture cells (3-5). However, the role of TAK1 has not been established in an in vivo context due to embryonic death of TAK1 germ line knock out (3, 4).Skin homeostasis is maintained through a well balanced interplay of cytokines and growth factors (6). Several cytokines, including TNF, activate JNK and NF-B pathways (7, 8) that play critical roles in epidermal homeostasis involving skin inflammation and cancer development (9 -16). Inactivation of IKK␤ or IKK␥, which blocks the so-called canonical NF-B pathway, produces a severe inflammatory skin condition. Furthermore, NF-B hypofunction is implicated in epidermal squamous cell carcinoma. In contrast, activation of JNK pathway is involved in epidermal hyperplasia and subsequent cancer development (14, 15). Roles of IKK-NF-B and JNK in skin have been demonstrated by using genetic and pharmacological inhibitory approaches (12,14,15). However, the upstream regulators of NF-B and JNK pathways in skin have not yet been determined. In this study, we generated and characterized mice with epidermal-specific deletion of TAK1. We found that TAK1 is an essential intermediate in TNF signaling to activate both IKK and JNK in keratinocytes. TAK1 deficiency causes severe dysregulation of skin homeostasis. Our results suggest that the dysregulation in TA...

The hair follicle is a cyclic, self renewing epidermal structure which is thought to be controlled by signals from the dermal papilla, a specialized cluster of mesenchymal cells within the dermis. Topical treatments with 17-8-estradiol to the clipped dorsal skin of mice arrested hair follicles in telogen and produced a profound and prolonged inhibition of hair growth while treatment with the biologically inactive stereoisomer, 17-a-estradiol, did not inhibit hair growth. Topical treatments with ICI 182,780, a pure estrogen receptor antagonist, caused the hair follicles to exit telogen and enter anagen, thereby initiating hair growth. Immunohistochemical staining for the estrogen receptor in skin revealed intense and specific staining of the nuclei of the cells of the dermal papilla. The expression of the estrogen receptor in the dermal papilla was hair cycle-dependent with the highest levels of expression associated with the telogen follicle. 17-8-Estradiol-treated epidermis demonstrated a similar number of 5-bromo-2'-deoxyuridine (BrdUrd) S-phase cells as the control epidermis above telogen follicles; however, the number of BrdUrd S-phase basal cells in the control epidermis varied according to the phase ofthe cycle ofthe underlying hair follicles and ranged from 2.6% above telogen follicles to 7.0% above early anagen follicles. These findings indicate an estrogen receptor pathway within the dermal papilla regulates the telogen-anagen follicle transition and suggest that diffusible factors associated with the anagen follicle influence cell proliferation in the epidermis.The hair follicle cycle is characterized by a period of follicle growth (anagen), followed by period of degeneration and rearrangement (catagen), and finally by a resting period (telogen) (1, 2). The hair follicle is a self-renewing system and is therefore governed by a slow cycling stem cell (1-5). Recently, the bulge activation hypothesis has been proposed which states that the follicular stem cell resides in the bulge area of the permanent portion of the hair follicle and that this stem cell is stimulated during early-anagen to divide and produce transient amplifying stem cells (4). It appears that a small group of highly specialized mesenchymal cells referred to as the dermal papilla provides the signal that initiates anagen and instructs the bulge follicular stem cell to divide (3,4,(6)(7)(8) (3, 4, 6). The matrix cells have a finite life span and are thought to terminally differentiate as the follicle enters catagen. After degeneration of the lower follicle, the follicle enters telogen and remains in telogen until the dermal papilla signals the bulge stem cells to divide and the hair follicle cycle begins again. While the dermal papilla appears to be critical in the regulation of hair follicle cycle (8, 9) the actual signals that initiate and terminate the cycles of hair growth as well as those that induce the differentiation of the matrix cells remain poorly understood.Developmental as well as recent transgenic studies in mice hav...