ATF2, a paradigm of the multifaceted regulation of transcription factors in biology and disease.

Stringent transcriptional regulation is crucial for normal cellular biology and organismal development. Perturbations in the proper regulation of transcription factors can result in numerous pathologies, including cancer. Thus, understanding how transcription factors are regulated and how they are dysregulated in disease states is key to the therapeutic targeting of these factors and/or the pathways that they regulate. Activating transcription factor 2 (ATF2) has been studied in a number of developmental and pathological conditions. Recent findings have shed light on the transcriptional, post-transcriptional, and post-translational regulatory mechanisms that influence ATF2 function, and thus, the transcriptional programs coordinated by ATF2. Given our current knowledge of its multiple levels of regulation and function, ATF2 represents a paradigm for the mechanistic complexity that can regulate transcription factor function. Thus, increasing our understanding of the regulation and function of ATF2 will provide insights into fundamental regulatory mechanisms that influence how cells integrate extracellular and intracellular signals into a genomic response through transcription factors. Characterization of ATF2 dysfunction in the context of pathological conditions, particularly in cancer biology and response to therapy, will be important in understanding how pathways controlled by ATF2 or other transcription factors might be therapeutically exploited. In this review, we provide an overview of the currently known upstream regulators and downstream targets of ATF2.

Vaginal LPS changed gene transcriptional regulation response to ischemic reperfusion and increased vulnerability of fetal brain hemorrhage.

During pregnancy, both ischemic reperfusion and bacterial agent LPS are known risk factors for fetal brain damage. However, there is a lack of evidence to explain whether vaginal LPS affects the fetus response to ischemic reperfusion. Here we reported that there was more than 2 folds higher vulnerability of fetal brain hemorrhage response to ischemic reperfusion when mother mouse was treated with vaginal LPS. As our previously reported, ischemic reperfusion induces P53-dependent fetal brain damage was based on a molecular mechanism: the transcriptional pattern was changed from HIF-1alpha-dependent to P53-dependent immediately. In the present work, only with vaginal LPS precondition, phosphorylation of activated transcriptional factor (ATF) 2 at Thr71 appeared in response to ischemic reperfusion. Moreover, this phosphorylation was completely blocked by pre-treatment with a P53 inhibitor, pifithrin-α. We concluded that vaginal LPS precondition trigged the p53-dependent phosphorylation of ATF2 in response to ischemic reperfusion, which played an important role of increasing vulnerability to hemorrhage in fetus.

Role of the Exocyst Complex Component Sec6/8 in Genomic Stability.

The exocyst is a heterooctomeric complex well appreciated for its role in the dynamic assembly of specialized membrane domains. Accumulating evidence indicates that this macromolecular machine also serves as a physical platform that coordinates regulatory cascades supporting biological systems such as host defense signaling, cell fate, and energy homeostasis. The isolation of multiple components of the DNA damage response (DDR) as exocyst-interacting proteins, together with the identification of Sec8 as a suppressor of the p53 response, suggested functional interactions between the exocyst and the DDR. We found that exocyst perturbation resulted in resistance to ionizing radiation (IR) and accelerated resolution of DNA damage. This occurred at the expense of genomic integrity, as enhanced recombination frequencies correlated with the accumulation of aberrant chromatid exchanges. Sec8 perturbation resulted in the accumulation of ATF2 and RNF20 and the promiscuous accumulation of DDR-associated chromatin marks and Rad51 repairosomes. Thus, the exocyst supports DNA repair fidelity by limiting the formation of repair chromatin in the absence of DNA damage.

Defining the Biological Effectiveness of Components of High-LET Track Structure.

During space travel, astronauts are exposed to a wide array of high-linear energy transfer (LET) particles, with differing energies and resulting biological effects. Risk assessment of these exposures carries a large uncertainty predominantly due to the unique track structure of the particle's energy deposition. The complex damage elicited by high charge and energy (HZE) particles results from both lesions along the track core and from energetic electrons, δ rays, generated as a consequence of particle traversal. To better define how cells respond to this complex radiation exposure, a normal hTERT immortalized skin fibroblast cell line was exposed to a defined panel of particles carefully chosen to tease out track structure effects. Phosphorylation kinetics for several key double-strand break (DSB) response proteins (γ-H2AX, pATF2 and pSMC1) were defined after exposure to ten different high-LET radiation qualities and one low-LET radiation (X ray), at two doses (0.5-2 Gy) and time points (2 and 24 h). The results reveal that the lower energy particles (Fe 300, Si 93 and Ti 300 MeV/u), with a narrower track width and higher number and intensity of δ rays, cause the highest degree of persistent damage response. The persistent γ-H2AX signal at lower energies suggests that damage from these exposures are more difficult to resolve, likely due to the greater complexity of the associated DNA lesions. However, different kinetics were observed for the solely ATM-mediated phosphorylations (pATF2 and pSMC1), revealing a shallow induction at early times and a higher level of residual phosphorylation compared to γ-H2AX. The differing phospho-protein profiles exhibited, compared to γ-H2AX, suggests additional functions for these proteins within the cell. The strong correspondence between the predicted curves for energy deposition per nucleosome for each ion/energy combination and the persistent levels of γ-H2AX indicates that the nature of energy distribution defines residual levels of γ-H2AX, an indicator of unrepaired DSBs. Our results suggest that decreasing the energy of a particle results in more complex damage that may increase genomic instability and increase the risk of carcinogenesis.

ATF-2 immunoreactivity in post-mitotic and terminally differentiated human odontoblasts.

Activating transcription factor 2 (ATF-2/CRE-BP1; cAMP-responsive element binding protein 1) is a member of nuclear transcription factor activator protein-1 (AP-1) family. AP-1 regulates cellular processes including growth, proliferation, differentiation and apoptosis. However, biological relationship of cellular process to each member of the AP-1 family is not clear yet. The objective of the present study was to compare the ATF-2 immunoreactivity in the post-mitotic and terminally differentiated odontoblasts and in the pulpal fibroblasts which can be divided by mitosis when required. Fibroblasts at various stages of differentiation co-exist in the human dental pulp. ATF-2 was investigated immunohistochemically in 20 permanent human teeth. According to the findings obtained, the mean percentage of ATF-2 positive cells was 68.5 ± 19.2% in the odontoblasts and 22.8 ± 13.7% in the pulpal fibroblasts. The comparison of ATF-2 positivity revealed a statistically significant difference between odontoblasts and pulpal fibroblasts. These findings have suggested that ATF-2 is more associated with cell survival rather than cell proliferation, and revealed much of effectiveness in maintaining terminal differentiation than the various differentiation stages of the cells.

Inheritance of stress-induced, ATF-2-dependent epigenetic change.

Atf1, the fission yeast homolog of activation transcription factor-2 (ATF-2), contributes to heterochromatin formation. However, the role of ATF-2 in chromatin assembly in higher organisms remains unknown. This study reveals that Drosophila ATF-2 (dATF-2) is required for heterochromatin assembly, whereas the stress-induced phosphorylation of dATF-2, via Mekk1-p38, disrupts heterochromatin. The dATF-2 protein colocalized with HP1, not only on heterochromatin but also at specific loci in euchromatin. Heat shock or osmotic stress induced phosphorylation of dATF-2 and resulted in its release from heterochromatin. This heterochromatic disruption was an epigenetic event that was transmitted to the next generation in a non-Mendelian fashion. When embryos were exposed to heat stress over multiple generations, the defective chromatin state was maintained over multiple successive generations, though it gradually returned to the normal state. The results suggest a mechanism by which the effects of stress are inherited epigenetically via the regulation of a tight chromatin structure.

[Effects of antenatal administration of dexamethasone and betamethasone on signal transduction of bone morphogenetic protein in the fetal lungs of rats].

OBJECTIVE: To study the role of antenatal glucocorticoid (dexamethasone and betamethasone) on bone morphogenetic protein (BMP) signal transduction of the rat fetal lungs. METHODS: Fifteen pregnant rats were randomly divided into five groups: the rats treated with dexamethasone for 1 day (1D-DEX) or 3 days (3D-DEX), with betamethasone for 1 day (1D-BEX) or 3 days (3D-BEX) or with normal saline (control group), followed cesarean section on the 19th day of gestation. The mRNA levels of BMP4, BMPR-II, Smad1 and ATF-2 of fetal rat lungs were ascertained by reverse transcriptase polymerase chain reaction (RT-PCR). The expression of BMP4, BMPR-II, Smad1 and ATF-2 antigen expression in fetal lungs was assessed by immune histochemical staining. The expression of BMP4 and BMPR-II was determined by Western blot. RESULTS: The levels of BMP4, BMPR-II and Smad1 mRNA expression were up-regulated in the 1D-BEX, 3D-BEX and 3D-DEX groups compared with those in the control group (P<0.05). The immune histochemiscal analysis showed that the expression of BMP4, BMPR-II, Phospho-Smad1 (pSmad1) and ATF-2 in the 1D-BEX, 3D-BEX and 3D-DEX groups was significantly higher than that in the control group (P<0.01). The results of Western blot demonstrated that the expression of BMP4 and BMPR-II protein increased significantly in the 1D-BEX, 3D-BEX and 3D-DEX groups when compared with the control group (P<0.01). CONCLUSIONS: Betamethasone and dexamethasone may play important roles in the regulation of BMP signal transduction in the rat fetal lungs. Up-regulation of BMP4, BMPR-II and Smad1 might be one of crucial factors for the glucocorticoid-induced maturity of fetal lungs.

Melanoma prognostic model using tissue microarrays and genetic algorithms.

PURPOSE: As a result of the questionable risk-to-benefit ratio of adjuvant therapies, stage II melanoma is currently managed by observation because available clinicopathologic parameters cannot identify the 20% to 60% of such patients likely to develop metastatic disease. Here, we propose a multimarker molecular prognostic assay that can help triage patients at increased risk of recurrence. METHODS: Protein expression for 38 candidates relevant to melanoma oncogenesis was evaluated using the automated quantitative analysis (AQUA) method for immunofluorescence-based immunohistochemistry in formalin-fixed, paraffin-embedded specimens from a cohort of 192 primary melanomas collected during 1959 to 1994. The prognostic assay was built using a genetic algorithm and validated on an independent cohort of 246 serial primary melanomas collected from 1997 to 2004. RESULTS: Multiple iterations of the genetic algorithm yielded a consistent five-marker solution. A favorable prognosis was predicted by ATF2 ln(non-nuclear/nuclear AQUA score ratio) of more than -0.052, p21(WAF1) nuclear compartment AQUA score of more than 12.98, p16(INK4A) ln(non-nuclear/nuclear AQUA score ratio) of < or = -0.083, beta-catenin total AQUA score of more than 38.68, and fibronectin total AQUA score of < or = 57.93. Primary tumors that met at least four of these five conditions were considered a low-risk group, and those that met three or fewer conditions formed a high-risk group (log-rank P < .0001). Multivariable proportional hazards analysis adjusting for clinicopathologic parameters shows that the high-risk group has significantly reduced survival on both the discovery (hazard ratio = 2.84; 95% CI, 1.46 to 5.49; P = .002) and validation (hazard ratio = 2.72; 95% CI, 1.12 to 6.58; P = .027) cohorts. CONCLUSION: This multimarker prognostic assay, an independent determinant of melanoma survival, might be beneficial in improving the selection of stage II patients for adjuvant therapy.

Spatiotemporal expression of transcriptional regulators in concert with the maternal-to-embryonic transition during bovine in vitro embryogenesis.

Cleavage-stage bovine embryos are transcriptionally quiescent until they reach the 8- to 16-cell stage, and thus rely on the reserves provided by the stored maternal mRNAs and proteins found in the oocytes to achieve their first cell divisions. The objective of this study was to characterize the expression and localization of the transcriptional and translational regulators, Y box binding protein 2 (YBX2), TATA box-binding protein (TBP), and activating transcription factor 2 (ATF2), during bovine early embryo development. Germinal vesicle (GV)- and metaphase II (MII)-stage oocytes, as well as 2-, 4-, 8-, 16-cell-stage embryos, morula, and blastocysts, produced in vitro were analyzed for temporal and spatial protein expression. Using Q-PCR, ATF2 mRNA expression was shown to remain constant from the GV-stage oocyte to the four-cell embryo, and then decreased through to the blastocyst stage. By contrast, the protein levels of ATF2 remained constant throughout embryo development and were found in both the cytoplasm and the nucleus. Both TBP and YBX2 showed opposite protein expression patterns, as YBX2 protein levels decreased throughout development, while TBP levels increased through to the blastocyst stage. Immunolocalization studies revealed that TBP protein was localized in the nucleus of 8- to 16-cell-stage embryos, whereas the translational regulator YBX2 was exclusively cytoplasmic and disappeared from the 16-cell stage onward. This study shows that YBX2, TBP, and ATF2 are differentially regulated through embryo development, and provides insight into the molecular events occurring during the activation of the bovine genome during embryo development in vitro.

Suppressor role of activating transcription factor 2 (ATF2) in skin cancer.

Activating transcription factor 2 (ATF2) regulates transcription in response to stress and growth factor stimuli. Here, we use a mouse model in which ATF2 was selectively deleted in keratinocytes. Crossing the conditionally expressed ATF2 mutant with K14-Cre mice (K14.ATF2(f/f)) resulted in selective expression of mutant ATF2 within the basal layer of the epidermis. When subjected to a two-stage skin carcinogenesis protocol [7,12-dimethylbenz[a]anthracene/phorbol 12-tetradecanoate 13-acetate (DMBA/TPA)], K14.ATF2(f/f) mice showed significant increases in both the incidence and prevalence of papilloma development compared with the WT ATF2 mice. Consistent with these findings, keratinocytes of K14.ATF2(f/f) mice exhibit greater anchorage-independent growth compared with ATF2 WT keratinocytes. Papillomas of K14.ATF2(f/f) mice exhibit reduced expression of presenilin1, which is associated with enhanced beta-catenin and cyclin D1, and reduced Notch1 expression. Significantly, a reduction of nuclear ATF2 and increased beta-catenin expression were seen in samples of squamous and basal cell carcinoma, as opposed to normal skin. Our data reveal that loss of ATF2 transcriptional activity serves to promote skin tumor formation, thereby indicating a suppressor activity of ATF2 in skin tumor formation.

Renal p38 MAP kinase activity in experimental diabetes.

Renal cell activity of p38 mitogen-activated protein kinase (p38) is increased in the diabetic milieu. p38 mediates signals relevant for the development of diabetic nephropathy (DN). However, renal p38 in Type 1 diabetes in vivo, particularly in conditions reflecting the differences in metabolic control, and its activity in advanced stages of DN, has received less attention. We examined the p38 pathway in renal cortex of rats with streptozotocin diabetes (4 weeks) with poor (DS), moderate (DM), and intensive (DII) metabolic control, achieved by varying doses of insulin therapy. Renal p38 was also studied in 12-month diabetic rats with established nephropathy (DM12) and compared with age-matched controls. p38 activity (in vitro kinase assay and expression of phosphorylated (active) p38 (P-p38)) was increased in DM and DS rats, as compared with non-diabetic controls, and attenuated by intensive insulin treatment. In all groups, P-p38 was predominantly localized in macula densa cells. Diabetic rats also demonstrated P-p38 immunoreactivity in the distal tubule and glomeruli. Enhanced p38 activity in DS and DM rats was not associated with increases in expression of active mitogen-activated protein kinase 3/6, an activator of p38, but paralleled with increased expression of scaffolding protein transforming growth factor-beta-activated protein kinase 1-binding protein 1. Expression of mitogen-activated protein phosphatase-1 (MKP-1), one of the phosphatases involved in inactivation of mitogen-activated protein kinase signaling, was increased in all diabetic groups, irrespective of metabolic control. Renal p38 activation was also detectable in D12 rats with established albuminuria and glomerulosclerosis. In summary, renal cortical p38 activity was increased in diabetic rats at early and advanced stages of nephropathy, as compared with non-diabetic animals, and attenuated by improved metabolic control. p38 activation in diabetes is likely to occur via multiple pathways and cannot be explained by downregulation of MKP-1.

Reduced levels of ATF-2 predispose mice to mammary tumors.

Transcription factor ATF-2 is a nuclear target of stress-activated protein kinases, such as p38, which are activated by various extracellular stresses, including UV light. Here, we show that ATF-2 plays a critical role in hypoxia- and high-cell-density-induced apoptosis and the development of mammary tumors. Compared to wild-type cells, Atf-2(-/-) mouse embryonic fibroblasts (MEFs) were more resistant to hypoxia- and anisomycin-induced apoptosis but remained equally susceptible to other stresses, including UV. Atf-2(-/-) and Atf-2(+/-) MEFs could not express a group of genes, such as Gadd45alpha, whose overexpression can induce apoptosis, in response to hypoxia. Atf-2(-/-) MEFs also had a higher saturation density than wild-type cells and expressed lower levels of Maspin, the breast cancer tumor suppressor, which is also known to enhance cellular sensitivity to apoptotic stimuli. Atf-2(-/-) MEFs underwent a lower degree of apoptosis at high cell density than wild-type cells. Atf-2(+/-) mice were highly prone to mammary tumors that expressed reduced levels of Gadd45alpha and Maspin. The ATF-2 mRNA levels in human breast cancers were lower than those in normal breast tissue. Thus, ATF-2 acts as a tumor susceptibility gene of mammary tumors, at least partly, by activating a group of target genes, including Maspin and Gadd45alpha.