Ultraviolet B inhibition of DNMT1 activity via AhR activation dependent SIRT1 suppression in CD4+ T cells from systemic lupus erythematosus patients.

BACKGROUND: Previous studies have reported that ultraviolet B (UVB) inhibits DNA methyltransferase1 (DNMT1) activity in CD4+ T cells from systemic lupus erythematosus (SLE) patients. Silent mating type information regulation 2 homolog 1 (SIRT1) is a type of Class III histone deacetylases (HDACs), and has been reported to play roles in the pathogenesis of different autoimmune diseases and can modulate DNMT1 activity. Moreover, aryl hydrocarbon receptor (AhR) has been reported to link UVB with SLE. However, the exact mechanisms by which DNMT1 activity is inhibited by UVB in lupus CD4+ T cells remain largely unknown. OBJECTIVE: To elucidate the exact mechanisms by which DNMT1 activity is inhibited by UVB in lupus CD4+ T cells. METHODS: Twenty-two newly diagnosed active SLE patients and 30 healthy controls were enrolled in the study. CD4+ T cells were isolated, cultured and treated. DNMT1 activity assay, quantitative real-time PCR (qRT-PCR), Western blotting, RNA interference using small interfering RNA and Chromatin Immunoprecipitation (ChIP) assay were employed. RESULTS: DNMT1 activity was inhibited in si-SIRT1-transfected CD4+ T cells, and increased by the established SIRT1 activator, SRT1720. Moreover, the mRNA and protein expression of SIRT1 were suppressed by UVB exposure in lupus CD4+ T cells. UVB-inhibited DNMT1 activity was reversed by SRT1720 in si-control-transfected lupus CD4+ T cells, but not in si-SIRT1-transfected lupus CD4 + T cells. Furthermore, AhR activation by VAF347 reduced the mRNA and protein expression of SIRT1. ChIP using an antibody against AhR in normal CD4+ T cells revealed a 16-fold stronger signal at the site about 1.6kb upstream from the translation start site of the SIRT1 promoter. Finally, UVB could activate AhR and inhibit the mRNA and protein expression of SIRT1. AhR knockdown abrogated the inhibition of UVB-mediated SIRT1 mRNA and protein expression and DNMT1 activity in lupus CD4+ T cells. CONCLUSION: UVB suppressed SIRT1 expression via activating AhR, and subsequently inhibited DNMT1 activity in CD4+ T cells from SLE patients.

Optical manipulation of the alpha subunits of heterotrimeric G proteins using photoswitchable dimerization systems.

Alpha subunits of heterotrimeric G proteins (Gα) are involved in a variety of cellular functions. Here we report an optogenetic strategy to spatially and temporally manipulate Gα in living cells. More specifically, we applied the blue light-induced dimerization system, known as the Magnet system, and an alternative red light-induced dimerization system consisting of Arabidopsis thaliana phytochrome B (PhyB) and phytochrome-interacting factor 6 (PIF6) to optically control the activation of two different classes of Gα (Gαq and Gαs). By utilizing this strategy, we demonstrate successful regulation of Ca2+ and cAMP using light in mammalian cells. The present strategy is generally applicable to different kinds of Gα and could contribute to expanding possibilities of spatiotemporal regulation of Gα in mammalian cells.

Effect of radiation on the Notch signaling pathway in osteoblasts.

Notch signaling has been shown to be important in osteoblast differentiation. Therapeutic radiation has been shown to alter the skeletal system, yet little information is available on the changes in Notch signaling in irradiated osteoblasts. The purpose of this study was to analyze the effect of radiation therapy with 2 and 4 Gy on Notch signaling in osteoblasts. In order to assess the radiation damage on osteoblast differentiation, total RNA and protein were collected three days after exposure to radiation. The effects of radiation on Notch signaling at the early and terminal stages of osteoblastic MC3T3-E1 cell differentiation was analyzed by qRT-PCR and western blot analysis. Our study applied a previously established method to induce MC3T3-E1 cell differentiation into osteoblasts and osteoblast precursors. Our results showed that the expression of Notch receptors (Notch1-4), ligands (Jagged1, Jagged2 and Delta1), target of Notch signaling (Hes1) and markers (ALP, M-CSF, RANKL and OPG) were altered following 2 and 4 Gy of irradiation. The present research did not indicate a strong relationship between Notch1 regulation and suppression of osteoblast differentiation. We found Hes1 may play a role in the radiation effect on osteoblast differentiation. Our results indicate that radiated osteoblast precursors and osteoblasts promoted osteoclast differentiation and proliferation.

Rapid blue-light-mediated induction of protein interactions in living cells.

Dimerizers allowing inducible control of protein-protein interactions are powerful tools for manipulating biological processes. Here we describe genetically encoded light-inducible protein-interaction modules based on Arabidopsis thaliana cryptochrome 2 and CIB1 that require no exogenous ligands and dimerize on blue-light exposure with subsecond time resolution and subcellular spatial resolution. We demonstrate the utility of this system by inducing protein translocation, transcription and Cre recombinase-mediated DNA recombination using light.

Phytochrome Interacting Factors: central players in phytochrome-mediated light signaling networks.

To adapt to the surrounding environment, plants constantly monitor and respond to changes in the red and far-red regions of the light spectrum through the phytochrome family of photoreceptors. Extensive efforts using genetic, molecular and photobiological techniques have led to the identification of a group of basic helix-loop-helix transcription factors called the Phytochrome Interacting Factors, PIFs, which directly bind to the photoactivated phytochromes. Members of the PIF family have been shown to control light-regulated gene expression directly and indirectly. PIF1, PIF3, PIF4 and PIF5 are degraded in response to light signals, and physical interaction of PIF3 with phytochromes is necessary for the light-induced phosphorylation and degradation of PIF3. PIFs constitute an excellent model for the investigation of the biochemical mechanisms of signal transfer from photoactivated phytochromes and the light-regulation of gene expression that controls photomorphogenesis in plants.

Characterization of a set of phytochrome-interacting factor-like bHLH proteins in Oryza sativa.

The model dicotyledon Arabidopsis thaliana has a characteristic small sub-family of phytochrome-interacting bHLH (basic Helix-Loop-Helix) factors, which are collectively designated the PIL (or PIF) (PHYTOCHROME INTERACTING FACTOR-LIKE) family proteins. In this study, we identified and characterized a set of highly homologous members (designated OsPIL11 to OsPIL16) in the model monocotyledon rice (Oryza sativa). Some of them (OsPIL11, OsPIL12, and OsPIL13) showed the ability to interact with the putative OsPRR1 (PSEUDO-RESPONSE REGULATOR 1) clock component, as far as the results of yeast two-hybrid assays were concerned. It was found that the expression of OsPIL13 is under the control of circadian rhythms (clock), while the expression of OsPIL15 is negatively regulated by light upon the onset to light exposure of etiolated seedlings. When the rice genes (OsPIL11 to OsPIL15) were over-expressed in A. thaliana, the resulting transgenic seedlings displayed anomalous morphologies with very long hypocotyls during early photomorphogenesis. These results suggest the view that the identified OsPILs are functional counterparts (or orthologs) of AtPILs, which are known to play important roles in red light-mediated (phyA and/or phyB-dependent) signal transduction pathways at immediate positions downstream of the photoreceptor in A. thaliana.

The aryl hydrocarbon receptor and light.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that has been intensively studied with respect to the toxicity of xenobiotics. However, its function in response to light has never been summarized. Here, we provide an overview of AhR activation by light with a focus on the role of tryptophan in light-induced AhR activation. We discuss the involvement of the AhR in different biological rhythms and speculate on the possible role of the AhR in UV-induced responses in skin. Furthermore, this review points out future research needs in this field.

Light activates the degradation of PIL5 protein to promote seed germination through gibberellin in Arabidopsis.

Angiosperm seeds integrate various environmental signals, such as water availability and light conditions, to make a proper decision to germinate. Once the optimal conditions are sensed, gibberellin (GA) is synthesized, triggering germination. Among environmental signals, light conditions are perceived by phytochromes. However, it is not well understood how phytochromes regulate GA biosynthesis. Here we investigated whether phytochromes regulate GA biosynthesis through PIL5, a phytochrome-interacting bHLH protein, in Arabidopsis. We found that pil5 seed germination was inhibited by paclobutrazol, the ga1 mutation was epistatic to the pil5 mutation, and the inhibitory effect of PIL5 overexpression on seed germination could be rescued by exogenous GA, collectively indicating that PIL5 regulates seed germination negatively through GA. Expression analysis revealed that PIL5 repressed the expression of GA biosynthetic genes (GA3ox1 and GA3ox2), and activated the expression of a GA catabolic gene (GA2ox) in both PHYA- and PHYB-dependent germination assays. Consistent with these gene-expression patterns, the amount of bioactive GA was higher in the pil5 mutant and lower in the PIL5 overexpression line. Lastly, we showed that red and far-red light signals trigger PIL5 protein degradation through the 26S proteasome, thus releasing the inhibition of bioactive GA biosynthesis by PIL5. Taken together, our data indicate that phytochromes promote seed germination by degrading PIL5, which leads to increased GA biosynthesis and decreased GA degradation.

Ionizing radiation induces apoptosis and inhibits neuronal differentiation in rat neural stem cells via the c-Jun NH2-terminal kinase (JNK) pathway.

A substantial number of neural stem cells (NSCs) continue to proliferate and generate neurons in the central nervous system throughout life. Ionizing radiation, an important adjuvant therapy for glioma patients, may damage NSCs and cause neuronal deficits, such as cognitive dysfunction and memory impairment. However, the precise mechanism of radiation effects on death and differentiation of NSCs remains largely unknown. Here, we found that radiation induced apoptosis in NSCs via the mitochondrial pathway, upregulating the ratio of Bax to Bcl-2 and releasing cytochrome c into the cytoplasm. Radiation also inhibited neuronal differentiation of NSCs by 50%. Of the three stress-associated mitogen-activated protein kinases (MAPKs), only c-Jun NH(2)-terminal kinase (JNK) was activated in NSCs after radiation. Interestingly, JNK inhibition by the specific inhibitor SP600125 rescued NSCs from apoptosis and improved neuronal differentiation. Furthermore, we examined whether radiation directly inhibits neuronal differentiation or not. Radiation did not affect the promoter activity of NeuroD, a basic helix-loop-helix transcription factor that regulates the expression of neuronal differentiation markers. Radiation induced more apoptosis in NeuroD-positive cells than NeuroD-negative cells. We concluded that radiation activates JNK and induces apoptosis, especially in neural progenitor cells, resulting in the inhibition of neurogenesis. Our findings raise the possibility that JNK inhibition has therapeutic potential in protecting NSCs from the adverse effects of radiation.