Dan F. Spandau

Both Transcriptional Regulation and Translational Control of ATF4 Are Central to the Integrated Stress Response

In response to different environmental stresses, phosphorylation of eIF2 (eIF2∼P) represses global translation coincident with preferential translation of ATF4. ATF4 is a transcriptional activator of the integrated stress response, a program of gene expression involved in metabolism, nutrient uptake, anti-oxidation, and the activation of additional transcription factors, such as CHOP/GADD153, that can induce apoptosis. Although eIF2-P elicits translational control in response to many different stress arrangements, there are selected stresses, such as exposure to UV irradiation, that do not increase ATF4 expression despite robust eIF2∼P. In this study we addressed the underlying mechanism for variable expression of ATF4 in response to eIF2∼P during different stress conditions and the biological significance of omission of enhanced ATF4 function. We show that in addition to translational control, ATF4 expression is subject to transcriptional regulation. Stress conditions such as endoplasmic reticulum stress induce both transcription and translation of ATF4, which together enhance expression of ATF4 and its target genes in response to eIF2∼P. By contrast, UV irradiation represses ATF4 transcription, which diminishes ATF4 mRNA available for translation during eIF2∼P. eIF2∼P enhances cell survival in response to UV irradiation. However, forced expression of ATF4 and its target gene CHOP leads to increased sensitivity to UV irradiation. This combination of transcriptional regulation and translational control allows the eIF2 kinase pathway to selectively repress or activate key regulatory genes subject to preferential translation, providing the integrated stress response versatility to direct the transcriptome that is essential for maintaining the balance between stress remediation and apoptosis.

Activation of the insulin-like growth factor-1 receptor promotes the survival of human keratinocytes following ultraviolet B irradiation

The ultraviolet B (UVB) component of sunlight causes non‐melanoma skin cancers due to the damage it inflicts on genomic DNA. The response of epidermal keratinocytes to sunlight depends on the dose of UVB received and the severity of the damage to the DNA. Mild DNA damage typically induces DNA‐repair pathways and cell survival, while severe DNA damage provokes apoptosis. Primary human keratinocytes grown in serum‐free media respond in a similar manner to UVB irradiation. However, we observed that keratinocytes are exquisitely more susceptible to UVB‐induced apoptosis if the growth medium is depleted of exogenous growth factors. Therefore, an exogenous growth factor could provide protection from UVB‐induced apoptosis. We found that the only growth factor that provided protection from UVB‐induced apoptosis was insulin and that the protective effect elicited by insulin was not due to binding the insulin receptor but, rather, to activation of the insulin‐like growth factor‐1 (IGF‐1) receptor. Additionally, activation of the IGF‐1 receptor in combination with UVB irradiation induced keratinocytes to become post‐mitotic. This survival function of the IGF‐1 receptor in response to UVB irradiation was influenced by activation of phosphatidylinositol‐3 kinase and MAP kinase. Prior to UVB irradiation, insulin or IGF‐1 had little to no effect on cell growth or viability. Therefore, activation of the IGF‐1 receptor in conjunction with UVB irradiation promotes keratinocyte survival at the expense of cell proliferation. Int. J. Cancer 80:431–438, 1999.

Ultraviolet B-radiation dose influences the induction of apoptosis and p53 in human keratinocytes

p53 is a tumor suppressor gene that has been implicated in a number of important cellular processes, including DNA repair and apoptosis. Genomic damage in human keratinocytes caused by ultraviolet B (UVB) irradiation has been shown to induce both apoptosis and p53 expression. We have previously observed that p53 expression in cultured normal human keratinocytes is predominantly perinuclear; however, exposure of cells to UVB radiation induces a major shift of p53 expression to the nucleus. Using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling, internucleosomal DNA ladders and flow cytometry, we correlated observed changes in p53 expression with the induction of apoptosis at low, intermediate and high doses of UVB radiation. High doses of UVB radiation induced cells to undergo apoptosis, whereas UVB radiation at low doses did not induce apoptosis but appeared to stimulate repair of the DNA damage induced by UVB radiation. Intermediate doses of UVB radiation induced a heterogeneous population of cells to undergo either DNA repair or apoptosis. The level of UVB radiation dose also influenced the induced cellular localization of p53. These observed differences in p53 cellular localization correlated with the induction of DNA repair or apoptosis. In cells undergoing apoptosis, p53 protein was found within the blebs of the degenerating nuclei. Our data give support to increasing evidence that p53 may play a role in both the repair of UV-radiation-induced DNA damage and the induction of apoptosis, and may function as a central control checkpoint in response to UVB-radiation-induced DNA damage.

Expression of the Platelet-activating Factor Receptor Results in Enhanced Ultraviolet B Radiation-induced Apoptosis in a Human Epidermal Cell Line

Recent studies have demonstrated that ultraviolet B radiation (UVB) damages human keratinocytes in part by inducing oxidative stress and cytokine production. Severe UVB damage to the keratinocyte can also result in apoptosis or programmed cell death. Although the lipid mediator platelet-activating factor (PAF) is synthesized in response to epidermal cell damage and epidermal cells express PAF receptors, it is not known whether PAF is involved in UVB-induced epidermal cell apoptosis. These studies examined the role of the PAF system in UVB-induced epidermal cell apoptosis using a novel model system created by retroviral-mediated transduction of the PAF receptor-negative human epidermal cell line KB with the human PAF receptor (PAF-R). Expression of the PAF-R in KB cells did not affect base-line growth or apoptosis, yet resulted in a decrease in the lag time between treatment of the cells and the induction of apoptosis following irradiation with 400 J/m2 UVB. This effect was inhibited by pretreatment with the PAF-R antagonists WEB 2086 and A-85783, confirming involvement of the PAF-R in this process. At lower doses (100–200 J/m2) of UVB, only KB cells that expressed the PAF-R became apoptotic. Treatment of PAF-R-expressing KB clones with the metabolically stable PAF-R agonist 1-hexadexyl-2-N-methylcarbamoyl-3-glycerophosphocholine (CPAF) alone did not induce apoptosis but augmented the degree of apoptosis observed if CPAF was used in combination with lower doses (200 J/m2) of UVB irradiation. Interestingly, UVB irradiation was found to stimulate PAF synthesis only in PAF-R-expressing KB cell clones. The antioxidants N-acetyl cysteine, 1,1,3,3-tetramethyl-2-thiourea, and vitamin E inhibited both UVB-induced PAF biosynthesis as well as the augmentation of UVB-induced apoptosis in PAF-R-expressing KB clones, suggesting the possibility that UVB stimulates the production of oxidized lipid species with PAF-R agonistic activity in this model system. Thus, these studies indicate that a component of UVB-induced epidermal cell cytotoxicity can be modulated by PAF-R activation through the production of PAF and PAF-like species.

The IGF-1/IGF-1R signaling axis in the skin: a new role for the dermis in aging-associated skin cancer.

The appropriate response of human keratinocytes to ultraviolet-B (UVB) is dependent on the activation status of the insulin-like growth factor 1 (IGF-1) receptor. Keratinocytes grown in conditions in which the IGF-1 receptor is inactive inappropriately replicate in the presence of UVB-induced DNA damage. In human skin, epidermal keratinocytes do not express IGF-1, and hence the IGF-1 receptor on keratinocytes is activated by IGF-1 secreted from dermal fibroblasts. We now show that the IGF-1 produced by human fibroblasts is essential for the appropriate UVB response of keratinocytes. Furthermore, the expression of IGF-1 is silenced in senescent fibroblasts in vitro. Using quantitative reverse transcriptase–PCR and immunohistochemisty, we can show that IGF-1 expression is also silenced in geriatric dermis in vivo. The diminished IGF-1 expression in geriatric skin correlates with an inappropriate UVB response in geriatric volunteers. Finally, the appropriate UVB response is restored in geriatric skin in vivo through pretreatment with exogenous IGF-1. These studies provide further evidence for a role of the IGF-1 receptor (IGF-1R) in suppressing UVB-induced carcinogenesis, suggest that fibroblasts have a critical role in maintaining appropriate activation of the keratinocyte IGF-1R, and imply that reduced expression of IGF-1 in geriatric skin could be an important component in the development of aging-related non-melanoma skin cancer.

UVB-induced Senescence in Human Keratinocytes Requires a Functional Insulin-like Growth Factor-1 Receptor and p53

To cope with the frequent exposure to carcinogenic UV B (UVB) wavelengths found in sunlight, keratinocytes have acquired extensive protective measures to handle UVB-induced DNA damage. Recent in vitro and epidemiological data suggest one these protective mechanisms is dependent on the functional status of the insulin-like growth factor-1 receptor (IGF-1R) signaling network in keratinocytes. During the normal UVB response, ligand-activated IGF-1Rs protect keratinocytes from UVB-induced apoptosis; however, as a consequence, these keratinocytes fail to proliferate. This adaptive response of keratinocytes to UVB exposure maintains the protective barrier function of the epidermis while ensuring that UVB-damaged keratinocytes do not replicate DNA mutations. In contrast, when keratinocytes are exposed to UVB in the absence of IGF-1R activation, the keratinocytes are more sensitive to UVB-induced apoptosis, but the surviving keratinocytes retain the capacity to proliferate. This aberrant UVB response represents flawed protection from UVB damage potentially resulting in the malignant transformation of keratinocytes. Using normal human keratinocytes grown in vitro, we have demonstrated that activation of the IGF-1R promotes the premature senescence of UVB-irradiated keratinocytes through increased generation of reactive oxygen species (ROS) and by maintaining the expression of the cyclin-dependent kinase inhibitor p21(CDKN1A). Furthermore, IGF-1R-dependent UVB-induced premature senescence required the phosphorylation of p53 serine 46. These data suggest one mechanism of keratinocyte resistance to UVB-induced carcinogenesis involves the induction of IGF-1R-dependent premature senescence.

Decreased incidence of nonmelanoma skin cancer in patients with type 2 diabetes mellitus using insulin: a pilot study.

Background  In order to prevent the propagation of genetic mutations, human keratinocytes irradiated with ultraviolet (UV) B light in vitro undergo premature stress‐induced senescence or apoptosis. This response to UVB irradiation is dependent on the functional activation of the insulin‐like growth factor‐1 receptor (IGF‐1R). Based on this in vitro functional data, we hypothesized that the increased serum levels of insulin in patients with type 2 diabetes may activate the IGF‐1R in skin and lead to a decreased frequency of skin cancer in these patients.

Loss of the Nf1 Tumor Suppressor Gene Decreases Fas Antigen Expression in Myeloid Cells

Genetic loss of surface Fas antigen expression leads to reduced apoptosis of myeloid and lymphoid progenitor cells, and a propensity to develop autoimmunity and myeloid leukemia in mouse models. Oncogenic p21(ras) decreases surface Fas antigen expression and renders fibroblasts resistant to Fas mediated apoptosis. Neurofibromin, which is encoded by NF1, is a GTPase activating protein that negatively regulates p21(ras) activity. NF1 loss leads to deregulation of p21(ras)-effector pathways, which control myeloid cell survival. Heterozygous inactivation of Nf1 increases mast cell numbers in Nf1 +/- mice, and enhances mast cell survival in response to c-kit ligand (kit-L). Here, we show that Nf1-deficient mast cells have reduced surface Fas antigen expression in response to kit-L and are resistant to Fas ligand-mediated apoptosis. Using genetic intercrosses between Nf1 +/- and class I (A)-PI-3K-deficient mice, we demonstrate that hyperactivation of the p21(ras)-class I(A) PI-3K pathway is the mechanism for this phenotype. Finally, we demonstrate that mast cells from both Fas antigen-deficient mice and Nf1 +/- mice are resistant to apoptosis following kit-L withdrawal in vivo. Thus, therapies designed to decrease p21(ras) activity and up-regulate Fas antigen expression may limit the pathological accumulation of myeloid cells in disease states where p21(ras) is hyperactivated.

UVB activation of NF-κB in normal human keratinocytes occurs via a unique mechanism

The transcription factor nuclear factor-κB (NF-κB) is comprised of a family of proteins that are implicated in a wide variety of cellular functions, including the control of cell proliferation, cell survival, and cellular differentiation. Although NF-κB is activated in response to inflammatory signals or cellular stress, in the skin NF-κB is also implicated to play a role in normal epidermal homeostasis. Often the cellular consequences of NF-κB activation are dependent on the specific triggering stimuli. Thus, we have compared the activation mechanism and the function of NF-κB following two common stimuli of normal human keratinocytes, inflammatory mediators (tumor necrosis factor alpha (TNFα)), and cellular stress (ultraviolet light B (UVB) irradiation). These experiments indicate that although both TNFα and UVB stimulate NF-κB DNA-binding activity in normal human keratinocytes, the mechanisms of NF-κB activation by each stimulus is different. In contrast to the NF-κB response following TNFα, activation of NF-κB by UVB is independent of IκBα degradation. Analyses of NF-κB-dependent gene expression following TNFα or UVB treatment demonstrate that each of these stimulatory signals results in a specific subset of genes that are activated or repressed. These studies provide further evidence of the stimuli and cell-type specific nature of NF-κB function.

Pten Loss Induces Autocrine FGF Signaling to Promote Skin Tumorigenesis

Inactivation of the Pten tumor suppressor negatively regulates the PI3K-mTOR pathway. In a model of cutaneous squamous cell carcinoma (SCC), we demonstrate that deletion of Pten strongly elevates Fgf10 protein levels without increasing Fgf10 transcription in vitro and in vivo. The translational activation of Fgf10 by Pten deletion is reversed by genetic disruption of the mTORC1 complex, which also prevents skin tumorigenesis in Pten mutants. We further show that ectopic expression of Fgf10 causes skin papillomas, whereas Pten deletion-induced skin tumors are inhibited by epidermal deletion of Fgfr2. Collectively, our data identify autocrine activation of FGF signaling as an essential mechanism in promoting Pten-deficient skin tumors.