Regulation of NR4A nuclear receptors by p38

In Drosophila, the melanization reaction is an important defense mechanism against injury and invasion of microorganisms. Drosophila tyrosine hydroxylase (TH, also known as Pale) and dopa decarboxylase (Ddc), key enzymes in the dopamine synthesis pathway, underlie the melanin synthesis by providing the melanin precursors dopa and dopamine, respectively. It has been shown that expression of Drosophila TH and Ddc is induced in various physiological and pathological conditions, including bacterial challenge; however, the mechanism involved has not been fully elucidated. Here, we show that ectopic activation of p38 MAPK induces TH and Ddc expression, leading to upregulation of melanization in the Drosophila cuticle. This p38-dependent melanization was attenuated by knockdown of TH and Ddc, as well as by that of Drosophila HR38, a member of the NR4A family of nuclear receptors. In mammalian cells, p38 phosphorylated mammalian NR4As and Drosophila HR38 and potentiated these NR4As to transactivate a promoter containing NR4A-binding elements, with this transactivation being, at least in part, dependent on the phosphorylation. This suggests an evolutionarily conserved role for p38 MAPKs in the regulation of NR4As. Thus, p38-regulated gene induction through NR4As appears to function in the dopamine synthesis pathway and may be involved in immune and stress responses

Prevention of Apoptosis by Mitochondrial Phosphatase PGAM5 in the Mushroom Body Is Crucial for Heat Shock Resistance in Drosophila melanogaster

The heat shock (HS) response is essential for survival of all organisms. Although the machinery of the HS response has been extensively investigated at the cellular level, it is poorly understood at the level of the organism. Here, we show the crucial role of the mushroom body (MB) in the HS response in Drosophila. Null mutants of the mitochondrial phosphatase Drosophila PGAM5 (dPGAM5) exhibited increased vulnerability to HS, which was reversed by MB-specific expression of the caspase inhibitor p35, and similar vulnerability was induced in wild-type flies by knockdown of MB dPGAM5. Elimination of the MB did not affect the HS response of wild-type flies, but did increase the resistance of dPGAM5-deficient flies to HS. Thus, the MB may possess an apoptosis-dependent toxic function, the suppression of which by dPGAM5 appears to be crucial for HS resistance

Proton beam therapy for the isolated recurrence of endometrial cancer in para-aortic lymph nodes : a case report

Background: Proton beam therapy penetrates tumor tissues with a highly concentrated dose. It is useful when normal structures are too proximate to the treatment target and, thus, may be damaged by surgery or conventional photon beam therapy. However, proton beam therapy has only been used to treat recurrent endometrial cancer in a few cases; therefore, its effectiveness remains unclear. Case presentation: We herein report a case of the isolated recurrence of endometrial cancer in the para-aortic lymph nodes in a 59-year-old postmenopausal woman that was completely eradicated by proton beam therapy. The patient was diagnosed with stage IIIC2 endometrial cancer and treated with 6 courses of doxorubicin (45 mg/m2) and cisplatin (50 mg/m2) in adjuvant chemotherapy. Fifteen months after the initial therapy, the isolated recurrence of endometrial cancer was detected in the para-aortic lymph nodes. The site of recurrence was just under the left renal artery. Due to the potential risks associated with left kidney resection due to the limited surgical space between the tumor and left renal artery, proton beam therapy was administered instead of surgery or conventional photon beam therapy. Following proton beam therapy, the complete resolution of the recurrent lesion was confirmed. No serious complications occurred during or after treatment. There have been no signs of recurrence more than 7 years after treatment. Conclusions: Proton beam therapy is a potentially effective modality for the treatment of recurrent endometrial cancer where the tumor site limits surgical interventions and the use of conventional photon beam therapy

Flies deficient in dPGAM5 are vulnerable to HS.

<p>(<b>A</b>) <i>PGAM5¹</i> flies are vulnerable to HS. Survival curves of adult male control (<i>y¹w¹/Y</i>) and <i>PGAM5¹</i> flies (<i>y¹</i>, <i>PGAM5¹/Y</i>) subjected to oxidative stress (0.1% H₂O₂, <i>n</i> = 66; left), starvation (<i>n</i> = 180; middle), or HS (37°C, <i>n</i> = 190; right) are shown. Control vs. <i>PGAM5¹</i> in HS, <i>p</i><0.0001 by the log-rank test. (<b>B</b>) dPGAM5 and dPINK1 act independently in HS response. Survival curves of the indicated adult male flies (day 5–15) subjected to HS are shown (control, <i>n</i> = 120; <i>PGAM5¹</i>, <i>n</i> = 112; <i>PINK1^B9</i>, <i>n</i> = 105; <i>PINK1^B9, PGAM5¹</i>, <i>n</i> = 116). <i>PINK1^B9</i> vs <i>PGAM5¹, PINK1^B9</i>, p<0.0001 by the log-rank test. The genotypes are revertant <i>PINK1^REV/Y</i> (Control), <i>PGAM5¹/Y</i> (<i>PGAM5¹</i>), <i>PINK1^B9/Y</i> (<i>PINK1^B9</i>) and <i>PGAM5¹, PINK1^B9/Y</i> (<i>PGAM5¹, PINK1^B9</i>). (<b>C</b>) Efficiency of IR-mediated <i>dPGAM5</i> knockdown. Protein expression of dPGAM5 in <i>daughterless (da)>LacZ IR</i> flies (<i>da-GAL4/+; UAS-LacZ IR/+</i>) and <i>da>dPGAM5 IR</i> flies (<i>da-GAL4/+</i>; <i>UAS-dPGAM5 IR/+</i>) was detected by immunoblotting with dPGAM5 antibody. Actin was also detected as a loading control. (<b>D</b>) Knockdown of <i>dPGAM5</i> in the whole body induces vulnerability of flies to HS. Survival curves of adult male <i>da>LacZ IR</i> flies and <i>da>dPGAM5 IR</i> flies subjected to HS are shown (<i>n</i> = 110). <i>p</i><0.0001 by the log-rank test. (<b>E</b>) Transient knockdown of <i>dPGAM5</i> prior to HS is sufficient to induce vulnerability of flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown. IR RNA was induced two days prior to sustained HS by two cycles of HS pretreatment, each of which was composed of 37°C for 30 min, 25°C for 5 hr, 37°C for 30 min and 25°C for 18 hr. “Pre-HS +” and “pre-HS −” indicate flies expressing and not expressing IR RNA, respectively, when they were subjected to sustained HS. The genotypes are <i>hs-GAL4/UAS-LacZ-IR</i> (<i>hs>LacZ-IR</i>) and <i>hs-GAL4/UAS-dPGAM5-IR</i> (<i>hs>dPGAM5-IR</i>). <i>hs>LacZ-IR</i> (pre-HS +) flies (<i>n</i> = 79), <i>hs>dPGAM5-IR</i> (pre-HS +) flies (<i>n</i> = 68), <i>hs>LacZ-IR</i> (pre-HS −) flies (<i>n</i> = 50) and <i>hs>dPGAM5-IR</i> (pre-HS −) flies (<i>n</i> = 45) were examined. <i>hs>LacZ-IR</i> (pre-HS +) vs <i>hs>dPGAM5-IR</i> (pre-HS +), p<0.0001 by the log-rank test.</p

The MB gains a toxic function, rather than loses a protective function, in response to HS in the absence of dPGAM5.

<p>(<b>A</b>) Ablation of the MB. Ablation of the MB in adult flies pretreated with hydroxyurea (HU) at the larval stage was confirmed by MB-specific expression of mCD8::GFP. Scale bar = 50 µm. The genotype is <i>c739-GAL4/+; UAS-mCD8::GFP/+</i>. (<b>B</b>) Ablation of the MB attenuates the vulnerability of <i>PGAM5¹</i> flies to HS. Survival curves of adult male <i>PGAM5¹</i> flies (<i>y¹</i>, <i>PGAM5¹/Y</i>) untreated (−) or treated (+) with HU are shown (<i>n</i> = 110). <i>p</i><0.0001 by the log-rank test. (<b>C</b>) Ablation of the MB does not affect the response of control flies to HS. Survival curves of adult male control (<i>y¹w¹/Y</i>) flies untreated (−) or treated (+) with HU are shown (<i>n</i> = 110). (<b>D</b>) Suppression of neurotransmission does not decrease vulnerability of <i>PGAM5¹</i> flies to HS. Survival curves of the indicated adult male flies (day 9–15) subjected to HS are shown. <i>c739-GAL4</i> flies (<i>+/Y; c739-GAL4/+; Sb/+</i>, <i>n</i> = 91), <i>c739>shi^TS</i> flies (<i>+/Y; c739-GAL4/+; UAS- shi^TS/+</i>, <i>n</i> = 100), <i>PGAM5¹, c739-GAL4</i> flies (<i>PGAM5¹/Y; c739-GAL4/+; Sb/+</i>, <i>n</i> = 100) and <i>PGAM5¹, c739>shi^TS</i> flies (<i>PGAM5¹/Y; c739-GAL4/+; UAS-shi^TS/+</i>, <i>n</i> = 100) were examined.</p

Locomotor activity is reduced in dPGAM5-deficient flies.

<p><i>PGAM5¹</i> flies are less active than control flies under both HS (37°C; <b>A</b>) and unstressed (25°C; <b>B</b>) conditions. Locomotor activity was monitored using the <i>Drosophila</i> activity monitor (DAM) system as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030265#s4" target="_blank">Materials and Methods</a>. Data are shown as the mean counts ± s.e.m of total flies examined for each genotype [Control, <i>n</i> = 77 and 79; <i>PGAM5¹</i>, n = 76 and 78; at 37°C and 25°C, respectively]. The genotypes are <i>y¹w¹/Y</i> (Control) and <i>PGAM5¹/Y</i> (<i>PGAM5¹</i>).</p

HS induces a rapid degeneration of the nuclei of the MB neurons in the absence of dPGAM5.

<p>The number of cell bodies of the KCs decreases in HS-treated <i>PGAM5¹</i> flies. Representative images of lobes (<b>A</b>–<b>D</b>) and calyxes (<b>E</b>–<b>H</b>) visualized by mCD8::GFP are shown. The genotypes are <i>c739-GAL4/+; UAS-mCD8::GFP/+</i> (Control) and <i>PGAM5¹</i>/<i>Y; c739</i>-<i>Gal4</i>/+; <i>UAS</i>-<i>mCD8::GFP</i>/+ (<i>PGAM5¹</i>). Scale bar (<b>A</b>–<b>H</b>) = 50 µm. Representative images of the KC nuclei visualized by Histone2B::ECFP (<b>I</b>–<b>L</b>; scale bar = 20 µm) and the total number of KC nuclei (<b>M</b>) are shown. The latter was calculated by summing up the number counted manually in every tenth section, and the results were shown as the mean cell number per fly ± s.e.m. [<i>n</i> = 6, Control HS (−) and (+) and <i>PGAM5¹</i> HS (−); <i>n</i> = 8, <i>PGAM5¹</i> HS (+)]. ** P<0.01, unpaired t-test (<b>M</b>). The genotypes are <i>c739-GAL4</i>/<i>UAS-Histone2B::ECFP</i> (Control) and <i>PGAM5¹</i>/<i>Y</i>; <i>c739-GAL4</i>/<i>UAS-Histone2B::ECFP</i> (<i>PGAM5¹</i>).</p

dPGAM5 exerts its protective effect against HS by preventing apoptosis in the MB.

<p>(<b>A–F</b>) TUNEL-positive KCs are detected in the MB of HS-treated <i>PGAM5¹</i> flies, but not in that of control flies. TUNEL staining of the MB of control flies (<b>A</b>) and <i>PGAM5¹</i> flies (<b>B</b>) treated with HS for 75 min is shown. Counter nuclear staining with Hoechst33258 (<b>C</b> and <b>D</b>) and merged images of TUNEL and Hoechst 33258 staining (<b>E</b> and <b>F</b>) are also shown. Scale bar (<b>A</b>–<b>F</b>) = 20 µm. The genotypes are <i>c739-GAL4/UAS-Histone2B::ECFP</i> (Control) and <i>PGAM5¹/Y; c739-GAL4/UAS-Histone2B::ECFP</i> (<i>PGAM5¹</i>). (<b>G</b>) Expression of p35 in the MB attenuates the vulnerability of <i>PGAM5¹</i> flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown (<i>n</i> = 105). <i>PGAM5¹</i> vs. <i>PGAM5¹; c739>p35</i>, <i>p</i><0.0001 by the log-rank test. The genotypes are <i>c739-GAL4/+</i> (Control), <i>PGAM5¹/Y; c739-GAL4/+</i> (<i>PGAM5¹</i>) and <i>PGAM5¹/Y; c739-GAL4/+; UAS-p35/+</i> (<i>PGAM5¹; c739>p35</i>). (<b>H</b>) Expression of p35 in the MB does not affect the response of control flies to HS. Survival curves of the indicated adult male flies subjected to HS are shown (<i>n</i> = 130). The genotypes are <i>c739-GAL4/+</i> (Control) and <i>c739-GAL4/+; UAS-p35/+</i> (Control; <i>c739>p35</i>).</p