Positive regulation of Hedgehog signaling via phosphorylation of GLI2/GLI3 by DYRK2 kinase.

Hedgehog (Hh) signaling, an evolutionarily conserved pathway, plays an essential role in development and tumorigenesis, making it a promising drug target. Multiple negative regulators are known to govern Hh signaling; however, how activated Smoothened (SMO) participates in the activation of downstream GLI2 and GLI3 remains unclear. Herein, we identified the ciliary kinase DYRK2 as a positive regulator of the GLI2 and GLI3 transcription factors for Hh signaling. Transcriptome and interactome analyses demonstrated that DYRK2 phosphorylates GLI2 and GLI3 on evolutionarily conserved serine residues at the ciliary base, in response to activation of the Hh pathway. This phosphorylation induces the dissociation of GLI2/GLI3 from suppressor, SUFU, and their translocation into the nucleus. Loss of Dyrk2 in mice causes skeletal malformation, but neural tube development remains normal. Notably, DYRK2-mediated phosphorylation orchestrates limb development by controlling cell proliferation. Taken together, the ciliary kinase DYRK2 governs the activation of Hh signaling through the regulation of two processes: phosphorylation of GLI2 and GLI3 downstream of SMO and cilia formation. Thus, our findings of a unique regulatory mechanism of Hh signaling expand understanding of the control of Hh-associated diseases.

Pregnancy and Lactation Alter Vitamin A Metabolism and Kinetics in Rats under Vitamin A-Adequate Dietary Conditions.

BACKGROUND: Vitamin A (VA) plays critical roles in prenatal and postnatal development; however, limited information is available regarding maternal VA metabolism during pregnancy and lactation. OBJECTIVES: We investigated the impact of pregnancy and lactation on VA metabolism and kinetics in rats, hypothesizing that changes in physiological status would naturally perturb whole-body VA kinetics. METHODS: Eight-week old female rats (n = 10) fed an AIN-93G diet received an oral tracer dose of 3H-labeled retinol to initiate the kinetic study. On d 21 after dosing, six female rats were mated. Serial blood samples were collected from each female rat at selected times after dose administration until d 14 of lactation. Model-based compartmental analysis was applied to the plasma tracer data to develop VA kinetic models. RESULTS: Our compartmental model revealed that pregnancy resulted in a gradual increase in hepatic VA mobilization, presumably to support different stages of fetal development. Additionally, the model indicates that during lactation, VA derived from dietary intake was the primary source of VA delivered to the mammary gland for milk VA secretion. CONCLUSION: During pregnancy and lactation in rats with an adequate VA intake and previous VA storage, the internal redistribution of VA and increased uptake from diet supported the maintenance of VA homeostasis.

Investigation of the effects of urea cycle amino acids on the expression of ALB and CEBPB in the human hepatocellular carcinoma cell line FLC-4.

Cell lines are powerful tools for research into liver function at the molecular level. However, they are generally unsuitable for rigorously assessing the effects of amino acid composition, because many lines require serum-containing medium for their maintenance. Here, we aimed to investigate the effects of ornithine and arginine, which are included in the characteristic metabolic process in hepatocyte, on a human hepatoma-derived cell line (FLC-4) that can be cultured in serum-free medium. FLC-4 cells were cultured under the following three conditions: + ornithine/ - arginine, - ornithine/ - arginine, and -ornithine/ + arginine. Albumin expression evaluated by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay and showed no obvious differences based on the presence of ornithine or arginine. However, the mRNA levels of two liver-enriched transcription factors (CEBPB and HNF1A), which are involved in regulating albumin expression, were significantly higher in cells grown in medium-containing arginine than that in cells grown in ornithine-containing medium. Western blotting showed that the levels both activating and inhibitory C/EBPß isoforms were significantly increased in cells grown in arginine medium. Furthermore, we have found that depletion of both ornithine and arginine, the polyamine sources, in the medium did not cause polyamine deficiency. When ornithine and arginine were depleted, albumin production was significantly reduced at the mRNA level, CEBPB mRNA levels were increased, and the level of activating form of C/EBPß was increased. The results of this study suggest that in hepatocyte, these two amino acids might have different functions, and because of which they elicit disparate cellular responses.

Novel ubiquitin-independent nucleolar c-Myc degradation pathway mediated by antizyme 2.

The proto-oncogene c-Myc encodes a short-lived protein c-Myc that regulates various cellular processes including cell growth, differentiation and apoptosis. Degradation of c-Myc is catalyzed by the proteasome and requires phosphorylation of Thr-58 for ubiquitination by E3 ubiquitin ligase, Fbxw7/ FBW7. Here we show that a polyamine regulatory protein, antizyme 2 (AZ2), interacts with c-Myc in the nucleus and nucleolus, to accelerate proteasome-mediated c-Myc degradation without ubiquitination or Thr-58 phosphorylation. Polyamines, the inducer of AZ2, also destabilize c-Myc in an AZ2-dependent manner. Knockdown of AZ2 by small interfering RNA (siRNA) increases nucleolar c-Myc and also cellular pre-rRNA whose synthesis is promoted by c-Myc. AZ2-dependent c-Myc degradation likely operates under specific conditions such as glucose deprivation or hypoxia. These findings reveal the targeting mechanism for nucleolar ubiquitin-independent c-Myc degradation.

Polyamine regulating protein antizyme binds to ATP citrate lyase to accelerate acetyl-CoA production in cancer cells.

Antizyme (AZ) regulates cellular polyamines (i.e., putrescine, spermidine, and spermine) through binding to ornithine decarboxylase and subsequent ubiquitin-independent degradation of the enzyme protein by the 26S proteasome. Screening for AZ-binding proteins using a yeast two-hybrid system identified ATP citrate lyase (ACLY), a cytosolic enzyme which catalyzes the production of acetyl-CoA that is used for lipid anabolism or acetylation of cellular components. We confirmed that both AZ1 and AZ2 bind to ACLY and AZ colocalizes with ACLY to the cytoplasm. Unexpectedly, neither AZ1 nor AZ2 accelerated ACLY degradation. Additionally, purified AZ, particularly AZ1, increased the activity of purified ACLY in a dose-dependent manner in vitro, suggesting that AZ activates ACLY through protein-protein interaction. Polyamines themselves had no effect on the ACLY activity in vitro. Knockdown of AZ1 and/or AZ2 in human cancer cells significantly decreased the ACLY activity as well as cellular levels of acetyl-CoA and cholesterol. Our results are the first to show the crosstalk between polyamine and acetyl-CoA metabolism. We hypothesize that AZ may promote acetyl-CoA synthesis to downregulate spermidine and spermine through acetylation.