Transcriptome and phenotypic analysis reveals Gata3-dependent signalling pathways in murine hair follicles

Abstract The transcription factor Gata3 is crucially involved in epidermis and hair follicle differentiation. Yet, little is known about how Gata3 co-ordinates stem cell lineage determination in skin, what pathways are involved and how Gata3 differentially regulates distinct cell populations within the hair follicle. Here, we describe a conditional Gata3-/- mouse (K14-Gata3-/-) in which Gata3 is specifically deleted in epidermis and hair follicles. K14-Gata3-/- mice show aberrant postnatal growth and development, delayed hair growth and maintenance, abnormal hair follicle organization and irregular pigmentation. After the first hair cycle, the germinative layer surrounding the dermal papilla was not restored; instead, proliferation was pronounced in basal epidermal cells. Transcriptome analysis of laser-dissected K14-Gata3-/- hair follicles revealed mitosis, epithelial differentiation and the Notch, Wnt and BMP signaling pathways to be significantly overrepresented. Elucidation of these pathways at the RNA and protein levels and physiologic endpoints suggests that Gata3 integrates diverse signaling networks to regulate the balance between hair follicle and epidermal cell fates

R-loops trigger the release of cytoplasmic ssDNAs leading to chronic inflammation upon DNA damage

How DNA damage leads to chronic inflammation and tissue degeneration with aging remains to be fully resolved. Here, we show that DNA damage leads to cellular senescence, fibrosis, loss-of-tissue architecture, and chronic pancreatitis in mice with an inborn defect in the excision repair cross complementation group 1 (Ercc1) gene. We find that DNA damage-driven R-loops causally contribute to the active release and buildup of single-stranded DNAs (ssDNAs) in the cytoplasm of cells triggering a viral-like immune response in progeroid and naturally aged pancreata. To reduce the proinflammatory load, we developed an extracellular vesicle (EV)-based strategy to deliver recombinant S1 or ribonuclease H nucleases in inflamed Ercc1(−/−) pancreatic cells. Treatment of Ercc1(−/−) animals with the EV-delivered nuclease cargo eliminates DNA damage-induced R-loops and cytoplasmic ssDNAs alleviating chronic inflammation. Thus, DNA damage-driven ssDNAs causally contribute to tissue degeneration, Ercc1(−/−) paving the way for novel rationalized intervention strategies against age-related chronic inflammation

XPF interacts with TOP2B for R-loop processing and DNA looping on actively transcribed genes

Co-transcriptional RNA-DNA hybrids can not only cause DNA damage threatening genome integrity but also regulate gene activity in a mechanism that remains unclear. Here, we show that the nucleotide excision repair factor XPF interacts with the insulator binding protein CTCF and the cohesin subunits SMC1A and SMC3, leading to R-loop-dependent DNA looping upon transcription activation. To facilitate R-loop processing, XPF interacts and recruits with TOP2B on active gene promoters, leading to double-strand break accumulation and the activation of a DNA damage response. Abrogation of TOP2B leads to the diminished recruitment of XPF, CTCF, and the cohesin subunits to promoters of actively transcribed genes and R-loops and the concurrent impairment of CTCF-mediated DNA looping. Together, our findings disclose an essential role for XPF with TOP2B and the CTCF/cohesin complex in R-loop processing for transcription activation with important ramifications for DNA repair-deficient syndromes associated with transcription-associated DNA damage

Tissue-infiltrating macrophages mediate an exosome-based metabolic reprogramming upon DNA damage

DNA damage and metabolic disorders are intimately linked with premature disease onset but the underlying mechanisms remain poorly understood. Here, we show that persistent DNA damage accumulation in tissue-infiltrating macrophages carrying an ERCC1-XPF DNA repair defect (Er1(F/-)) triggers Golgi dispersal, dilation of endoplasmic reticulum, autophagy and exosome biogenesis leading to the secretion of extracellular vesicles (EVs) in vivo and ex vivo. Macrophage-derived EVs accumulate in Er1(F/-) animal sera and are secreted in macrophage media after DNA damage. The Er1(F/-) EV cargo is taken up by recipient cells leading to an increase in insulin-independent glucose transporter levels, enhanced cellular glucose uptake, higher cellular oxygen consumption rate and greater tolerance to glucose challenge in mice. We find that high glucose in EV-targeted cells triggers pro-inflammatory stimuli via mTOR activation. This, in turn, establishes chronic inflammation and tissue pathology in mice with important ramifications for DNA repair-deficient, progeroid syndromes and aging

Tissue-specific suppression of thyroid hormone signaling in various mouse models of aging

DNA damage contributes to the process of aging, as underscored by premature aging syndromes caused by defective DNA repair. Thyroid state changes during aging, but underlying mechanisms remain elusive. Since thyroid hormone (TH) is a key regulator of metabolism, changes in TH signaling have widespread effects. Here, we reveal a significant common transcriptomic signature in livers from hypothyroid mice, DNA repair-deficient mice with severe (Csbm/m/Xpa-/-) or intermediate (Ercc1-/Δ-7) progeria and naturally aged mice. A strong induction of TH-inactivating deiodinase D3 and decrease of TH-activating D1 activities are observed in Csbm/m/Xpa-/- livers. Similar findings are noticed in Ercc1-/Δ-7, in naturally aged animals and in wild-type mice exposed to a chronic subtoxic dose of DNAdamaging agents. In contrast, TH signaling in muscle, heart and brain appears unaltered. These data show a strong suppression of TH signaling in specific peripheral organs in premature and normal aging, probably lowering metabolism, while other tissues appear to preserve metabolism. D3-mediated TH inactivation is unexpected, given its expression mainly in fetal tissues. Our studies highlight the importance of DNA damage as the underlying mechanism of changes in thyroid state. Tissue-specific regulation of deiodinase activities, ensuring diminished TH signaling, may contribute importantly to the protective metabolic response in aging

Photolyases: Capturing the light to battle skin cancer

Photolyases comprise efficient enzymes to remove the major UV-induced DNA lesions, cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). While photolyases are present in all three kingdoms of life (i.e., bacteria, prokaryotes and eukaryotes), placental mammals appear to have lost these enzymes when they diverted from marsupials during evolution. Consequently, man and mice have to rely solely on the more complex and, for these lesions, less efficient nucleotide excision repair (NER) system. To assess the relative contribution of CPDs and 6-4PPs to the cytotoxic and genotoxic effects of the UV component of sunlight, we have recently generated a comprehensive set of transgenic mice expressing CPD and/or 6-4PP photolyases. Here, we discuss the use of photolyase transgenic mice as effective tools to study the adverse effects of UV irradiation

Elevated serum levels of the apoptosis related molecules TNF-α, Fas/Apo-1 and Bcl-2 in oral lichen planus

BACKGROUND: The serum circulatory levels of apoptosis related molecules measured in patients with oral lichen planus (OLP) and healthy individuals in order to investigate possible alterations associated with the clinical forms of OLP. METHODS: Serum levels of tumor necrosis factor (TNF)-α, soluble Fas (sFas) and Bcl-2 studied by enzyme-linked immunosorbent assay in whole blood samples in 13 OLP reticular, 13 OLP atrophic-erosive form patients and 26 healthy subjects. RESULTS: Significantly elevated levels of TNF-α and sFas detected in OLP patients as compared with controls. Serum concentrations of Bcl-2 although increased in 17/26 patients, they were not statistically significant. Reticular OLP exhibited slightly elevated TNF-α and significantly elevated Bcl-2 serum levels, compared with erosive OLP. CONCLUSIONS: These data suggest that a putative dysfunction in the Fas/FasL mediated apoptosis might be involved in the OLP pathogenesis. A downregulation of Bcl-2 serum levels in the atrophic-erosive OLP may be associated with promotion of the disease activity

Cytokine serum levels in patients with chronic HCV infection

The pathogenic role of immune-mediated mechanisms in chronic hepatitis C virus (HCV) infection has not yet been elucidated. In this study, we report different cytokine expression profiles from hemodialysis (HD) and non-HD HCV (+) patients. IL-1β, IL-2, IL-4, IL-6, TNF-α, and TGF-β1 serum levels, and liver biochemical parameters were determined in 85 individuals (41 HD patients and 44 non-HD patients). Screening for HCV RNA and anti-HCV antibodies was performed using qualitative and quantitative reverse transcription polymerase chain reaction (RT-PCR), and standardized enzyme-linked immunosorbent assay (ELISA) and recombinant immunoblot assay (RIBA) methods, respectively. IL-4 and IL-1β demonstrated decreased serum levels in non-HD HCV carriers compared with healthy controls. Both T helper (Th) 1 and Th2 lymphocytes were highly associated with chronic HCV infection, as indicated by the increased IL-2, IL-4, and IL-6 cytokine circulating levels in all chronic active hepatitis (CAH) patients examined. An enhanced Th2 response (IL-4 and IL-6) coupled with increased TNF-α and IL-1β serum levels was reported in HD HCV (-) patients. In conclusion, our data show that a virus-induced Th2 and IL-1β immunosuppression is an early event in HCV-related chronicity. Long-term HD specifically exerts a chronic effect on IL-6, IL-1β, and TNF-α serum circulating levels. Irrespective of the HD status, HCV viremia, and liver biochemistry parameters, both Th1 and Th2 responses are highly associated with chronic HCV infection. © 2002 Wiley-Liss, Inc

Association of allelic loss at the FHIT locus and p53 alterations with tumour kinetics and chromosomal instability in non-small cell lung carcinomas (NSCLCs)

The FHIT gene, located at the FRA3B fragile site of chromosome 3p14.2, encodes a 16.8 kD homologue of the yeast enzyme diadenosine tetraphosphate (Ap4A) hydrolase. Frequent allelic losses at this region in various malignancies, including non-small cell lung carcinomas (NSCLCs), imply that FHIT may represent a tumour suppressor gene (TSG). Increasing evidence suggests that multiple TSG impairment has a synergistic effect on tumour growth. The present study of 67 NSCLCs investigated the allelic imbalance (AIm) within the FHIT locus and its relationship with p53 abnormalities, kinetic parameters [proliferative activity or proliferation index (PI) and apoptotic index (AI)], and ploidy status of the carcinomas. Allelic imbalance at FHIT was observed in 35 out of 55 informative (heterozygous: H) cases (64%). Similar frequencies of loss of heterozygosity (LOH) were noticed among squamous cell lung carcinomas and adenocarcinomas. The high percentage of AIm in stage I tumours (71%) is indicative of its relatively early involvement in NSCL carcinogenesis. No association was found between LOH at FHIT, kinetic parameters, and ploidy status of the tumours. Concurrent loss at FHIT and p53 overexpression [FHIT(LOH)/p53(P)] was the most frequent pattern and was observed in 39% of the informative cases. The latter pattern was not associated with smoking, supporting the hypothesis that in patients with a history of tobacco exposure, FHIT allelic loss may not be a consequence of p53 checkpoint defects, but the outcome of tobacco-induced mutagenesis. Statistically significant differences in the presence of FHIT(LOH)/p53(P) and FHIT(LOH)/p53(N) patterns were noted at the proliferative and apoptotic level, whereas ploidy was similar amongst all groups, implying that wild-type (wt) p53 may play a safeguard role against altered FHIT function. However, the possibility of a masking effect from wt p53 cannot be excluded, since the FHIT(LOH)/p53(P) profile demonstrated a higher growth index (GI = PI/AI mean value ratio) than FHIT(H)/p53(P) (32 vs. 8), although this was not significant. Further studies are needed in order to elucidate the role of FHIT and its relationships with other cell-cycle regulatory molecules involved in NSCL carcinogenesis. Copyright © 2000 John Wiley & Sons, Ltd