MicroRNAs (miRNAs) have been recognized as significantly involved in prostate cancer (PCa). Since androgen receptor (AR) plays a central role in PCa carcinogenesis and progression, it is imperative ...to systematically elucidate the causal association between AR and miRNAs, focusing on the molecular mechanisms by which miRNAs mediate AR signalling. In this study, we performed a series of time-course microarrays to observe the dynamic genome-wide expressions of mRNAs and miRNAs in parallel in hormone-sensitive prostate cancer LNCaP cells stimulated by androgen. Accordingly, we introduced Response Score to identify AR target miRNAs, as well as Modulation Score to identify miRNA target mRNAs. Based on theoretical identification and experimental validation, novel mechanisms addressing cell viability in PCa were unravelled for 3 miRNAs newly recognized as AR targets. (1) miR-19a is directly up-regulated by AR, and represses SUZ12, RAB13, SC4MOL, PSAP and ABCA1, respectively. (2) miR-27a is directly up-regulated by AR, and represses ABCA1 and PDS5B. (3) miR-133b is directly up-regulated by AR, and represses CDC2L5, PTPRK, RB1CC1, and CPNE3, respectively. Moreover, we found miR-133b is essential to PCa cell survival. Our study gives certain clues on miRNAs mediated AR signalling to cell viability by influencing critical pathways, especially by breaking through androgen's growth restriction effect on normal prostate tissue.
Modifications at the N-terminal tails of nucleosomal histones are required for efficient transcription in vivo. We analyzed how H3 histone methylation and demethylation control expression of ...estrogen-responsive genes and show that a DNA-bound estrogen receptor directs transcription by participating in bending chromatin to contact the RNA polymerase II recruited to the promoter. This process is driven by receptor-targeted demethylation of H3 lysine 9 at both enhancer and promoter sites and is achieved by activation of resident LSD1 demethylase. Localized demethylation produces hydrogen peroxide, which modifies the surrounding DNA and recruits 8-oxoguanine-DNA glycosylase 1 and topoisomeraseIIβ, triggering chromatin and DNA conformational changes that are essential for estrogen-induced transcription. Our data show a strategy that uses controlled DNA damage and repair to guide productive transcription.
Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal illness whose pathogenesis remains poorly understood. Recent evidence suggests oxidative stress as a key player in the ...establishment/progression of lung fibrosis in animal models and possibly in human IPF. The aim of the present study was to characterize the cellular phenotype of fibroblasts derived from IPF patients and identify underlying molecular mechanisms.
We first analyzed the baseline differentiation features and growth ability of primary lung fibroblasts derived from 7 histology proven IPF patients and 4 control subjects at different culture passages. Then, we focused on the redox state and related molecular pathways of IPF fibroblasts and investigated the impact of oxidative stress in the establishment of the IPF phenotype. IPF fibroblasts were differentiated into alpha-smooth muscle actin (SMA)-positive myofibroblasts, displayed a pro-fibrotic phenotype as expressing type-I collagen, and proliferated lower than controls cells. The IPF phenotype was inducible upon oxidative stress in control cells and was sensitive to ROS scavenging. IPF fibroblasts also contained large excess of reactive oxygen species (ROS) due to the activation of an NADPH oxidase-like system, displayed higher levels of tyrosine phosphorylated proteins and were more resistant to oxidative-stress induced cell death. Interestingly, the IPF traits disappeared with time in culture, indicating a transient effect of the initial trigger.
Robust expression of α-SMA and type-I collagen, high and uniformly-distributed ROS levels, resistance to oxidative-stress induced cell death and constitutive activation of tyrosine kinase(s) signalling are distinctive features of the IPF phenotype. We suggest that this phenotype can be used as a model to identify the initial trigger of IPF.
Although chemotherapy (CHT) exposure is an established cause of telomere attrition, determinants of telomere length (TL) dynamics after chemotherapy are poorly defined. In this study, we analyzed ...granulocyte telomere dynamics in 34 adult lymphoma patients undergoing first‐line CHT. TL was measured by southern blot at each CHT cycle and after 1 year from CHT completion. Median age was 59 yrs (range 22–77). Median number of CHT cycles was 6 (range 3–6). The majority of patients (79%, n = 27) experienced TL shortening following CHT exposure. Mean telomere loss was 673 base pairs (bp) by cycle 6. Telomere shortening was an early event as 87% of the total telomere loss (mean 586 bp) occurred by the end of cycle 3, with no significant recovery after 1 year. A significant correlation was observed between baseline TL and total or fractional telomere loss (p < 0.001), with telomere shortening by cycle 3 observed predominantly in male patients with long telomeres at pre‐treatment evaluation. Stratifying the analysis by gender and age only young women (<51 years of age) did not show significant telomere shortening following chemotherapy exposure. These findings indicate that gender and baseline TL are major determinants of TL dynamics following chemotherapy exposure in lymphoma patients.
Systemic sclerosis (scleroderma) is characterized by immunologic abnormalities, injury of endothelial cells, and tissue fibrosis. Abnormal oxidative stress has been documented in scleroderma and ...linked to fibroblast activation. Since platelet-derived growth factor (PDGF) stimulates the production of reactive oxygen species (ROS) and since IgG from patients with scleroderma reacts with human fibroblasts, we tested the hypothesis that patients with scleroderma have serum autoantibodies that stimulate the PDGF receptor (PDGFR), activating collagen-gene expression.
We analyzed serum from 46 patients with scleroderma and 75 controls, including patients with other autoimmune diseases, for stimulatory autoantibodies to PDGFR by measuring the production of ROS produced by the incubation of purified IgG with mouse-embryo fibroblasts carrying inactive copies of PDGFR alpha or beta chains or the same cells expressing PDGFR alpha or beta. Generation of ROS was assayed with and without specific PDGFR inhibitors. Antibodies were characterized by immunoprecipitation, immunoblotting, and absorption experiments.
Stimulatory antibodies to the PDGFR were found in all the patients with scleroderma. The antibodies recognized native PDGFR, inducing tyrosine phosphorylation and ROS accumulation. Autoantibody activity was abolished by preincubation with cells expressing the PDGFR alpha chain or with recombinant PDGFR or by PDGFR tyrosine kinase inhibitors. Stimulatory PDGFR antibodies selectively induced the Ha-Ras-ERK1/2 and ROS cascades and stimulated type I collagen-gene expression and myofibroblast phenotype conversion in normal human primary fibroblasts.
Stimulatory autoantibodies against PDGFR appear to be a specific hallmark of scleroderma. Their biologic activity on fibroblasts strongly suggests that they have a causal role in the pathogenesis of the disease.
A higher incidence of cancer in scleroderma patients compared with the general population has been suggested by several observational studies, reporting, however, different estimates. Therefore, we ...aimed to perform a systematic review and meta-analysis to definitely assess this association.
We searched MEDLINE and Embase for all original articles of observational studies on cancer incidence in scleroderma patients without language restriction published up to December 2011. Two independent authors reviewed all titles/abstracts and retrieved detailed full-text of potentially relevant articles to identify studies according to predefined selection criteria. Summary estimates were derived using random-effects model and reported as relative risk (RR). Publication bias was evaluated by trim and fill analysis.
From articles initially identified, 16 original studies, involving more than 7000 patients, were included in the present review. Compared with the general population, the summary RR to develop all invasive cancers in scleroderma patients was 1.75 (95% CI 1.41, 2.18). The results for selected cancer sites indicated a strong association with lung cancer (RR 4.35; 95% CI 2.08, 9.09), and a significant increased risk also for haematological neoplasms (RR 2.24; 95% CI 1.53, 3.29). The relation with breast cancer, suggested in some previous epidemiological studies, was not confirmed (RR 1.05; 95% CI 0.86, 1.29).
The present meta-analysis, the first on scleroderma and cancer risk, provides definite estimates on the association between scleroderma and cancer.
Objective
To describe a skin–SCID mouse chimeric model of systemic sclerosis (SSc; scleroderma) fibrosis based on engraftment of ex vivo–bioengineered skin using skin cells derived either from ...scleroderma patients or from healthy donors.
Methods
Three‐dimensional bioengineered skin containing human keratinocytes and fibroblasts isolated from skin biopsy specimens from healthy donors or SSc patients was generated ex vivo and then grafted onto the backs of SCID mice. The features of the skin grafts were analyzed by immunohistochemistry, and the functional profile of the graft fibroblasts was defined before and after treatment with IgG from healthy controls or SSc patients. Two procedures were used to investigate the involvement of platelet‐derived growth factor receptor (PDGFR): 1) nilotinib, a tyrosine kinase inhibitor, was administered to mice before injection of IgG from SSc patient sera (SSc IgG) into the grafts, and 2) human anti‐PDGFR monoclonal antibodies were injected into the grafts.
Results
Depending on the type of bioengineered skin grafted, the regenerated human skin exhibited either the typical scleroderma phenotype or the healthy human skin architecture. Treatment of animals carrying healthy donor skin grafts with SSc IgG resulted in the appearance of a bona fide scleroderma phenotype, as confirmed by increased collagen deposition and fibroblast activation markers. Results of the experiments involving administration of nilotinib or monoclonal antibodies confirmed the involvement of PDGFR.
Conclusion
Our results provide the first in vivo demonstration of the fibrotic properties of anti‐PDGFR agonistic antibodies. This bioengineered skin–humanized mouse model can be used to test in vivo the progression of the disease and to monitor response to antifibrotic drugs.
Systemic sclerosis (SSc) is an autoimmune disease characterized by extensive visceral organ and skin fibrosis. SSc patients have increased production of autoreactive antibodies and Wnt signaling ...activity. We found that expression of the gene encoding Wnt inhibitor factor 1 (WIF-1) was decreased in fibroblasts from SSc patient biopsies. WIF-1 deficiency in SSc patient cells correlated with increased abundance of the Wnt effector β-catenin and the production of collagen. Knocking down WIF-1 in normal fibroblasts increased Wnt signaling and collagen production. WIF-1 loss and DNA damage were induced in normal fibroblasts by either SSc patient immunoglobulins or oxidative DNA-damaging agents, such as ultraviolet light, hydrogen peroxide, or bleomycin. The DNA damage checkpoint kinase ataxia telangiectasia mutated (ATM) mediated WIF-1 silencing through the phosphorylation of the transcription factor c-Jun, which in turn activated the expression of the gene encoding activating transcription factor 3 (ATF3). ATF3 and c-Jun were recruited together with histone deacetylase 3 (HDAC3) to the WIF-1 promoter and inhibited WIF-1 expression. Preventing the accumulation of reactive oxygen species or inhibiting the activation of ATM, c-Jun, or HDACs restored WIF-1 expression in cultured SSc patient cells. Trichostatin A, an HDAC inhibitor, prevented WIF-1 loss, β-catenin induction, and collagen accumulation in an experimental fibrosis model. Our findings suggest that oxidative DNA damage induced by SSc autoreactive antibodies enables Wnt activation that contributes to fibrosis.
Objective
Reactive oxygen species (ROS) contribute to the pathogenesis of fibrosis in systemic sclerosis (SSc; scleroderma), and NADPH oxidase (NOX) is an important source of ROS. Since the role of ...single NOX isoforms has not been previously investigated in SSc, this study was undertaken to assess the expression of NOX in SSc fibroblasts compared to normal healthy cells and to analyze their role in cell activation.
Methods
Expression of NOX isoforms in dermal fibroblasts from patients with SSc and healthy control subjects was analyzed by real‐time polymerase chain reaction, immunoblotting, and immunofluorescence. NOX isoforms were silenced using small interfering RNA. Production of ROS was measured by fluorometry and confocal microscopy.
Results
Scleroderma fibroblasts showed up‐regulation of NOX‐2 and NOX‐4 protein and messenger RNA (mRNA) expression. Treatment of the cells with diphenyleneiodonium, a nonselective inhibitor of flavin‐containing enzymes, and silencing of NOX2 and NOX4 decreased the production of ROS as well as the expression of type I collagen and α‐smooth muscle actin in SSc fibroblasts. ROS generated by NOX‐2 and NOX‐4 were involved in DNA damage and activation of a DNA repair checkpoint. Incubation of healthy control fibroblasts with platelet‐derived growth factor (PDGF) or with IgG isolated from SSc patient serum enhanced the expression of NOX2 and NOX4 mRNA, via ROS, in a time‐dependent manner. Treatment with actinomycin D, a transcription inhibitor, reversed the effects of PDGF stimulation but not the effects of SSc IgG.
Conclusion
Both NOX2 and NOX4 generate ROS in SSc fibroblasts and play a critical role in cell activation and DNA damage. Expression of NOX‐2 and NOX‐4 in SSc fibroblasts is maintained by a ROS‐mediated loop.
Extensive chronic graft-versus-host disease (ecGVHD) is characterized by fibrosis similar to that of patients with systemic sclerosis (scleroderma). Since stimulatory autoantibodies against the ...platelet-derived growth factor (PDGF) receptor (PDGFR) have been found in patients with scleroderma and are responsible for the activation of skin fibroblasts, we tested the hypothesis that these autoantibodies are also present in patients affected by ecGVHD. Serum from 39 patients subjected to allogeneic stem cell transplantation for hematologic malignancies (22 with ecGVHD and 17 without cGVHD) and 20 healthy controls was assayed for the presence of stimulatory autoantibodies to the PDGFR by incubating purified IgG with mouse-embryo fibroblasts lacking PDGFR α or β chains or with the same cells expressing PDGFR α. Stimulatory antibodies to the PDGFR were found selectively in all patients with ecGVHD but in none of the patients without cGVHD. Higher levels were detected in patients with generalized skin involvement and/or lung fibrosis. Antibodies recognized native PDGFR, induced tyrosine phosphorylation, accumulation of reactive oxygen species (ROS), and stimulated type 1 collagen gene expression through the Ha-Ras-ERK1/2-ROS signaling pathway. The biologic activity of these autoantibodies suggests a role in the development of fibrosis and argues for a common pathogenetic trait in ecGVDH and scleroderma phenotypes.
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