Drosophila genome-wide obesity screen reveals hedgehog as a determinant of brown versus white adipose cell fate.

Over 1 billion people are estimated to be overweight, placing them at risk for diabetes, cardiovascular disease, and cancer. We performed a systems-level genetic dissection of adiposity regulation using genome-wide RNAi screening in adult Drosophila. As a follow-up, the resulting approximately 500 candidate obesity genes were functionally classified using muscle-, oenocyte-, fat-body-, and neuronal-specific knockdown in vivo and revealed hedgehog signaling as the top-scoring fat-body-specific pathway. To extrapolate these findings into mammals, we generated fat-specific hedgehog-activation mutant mice. Intriguingly, these mice displayed near total loss of white, but not brown, fat compartments. Mechanistically, activation of hedgehog signaling irreversibly blocked differentiation of white adipocytes through direct, coordinate modulation of early adipogenic factors. These findings identify a role for hedgehog signaling in white/brown adipocyte determination and link in vivo RNAi-based scanning of the Drosophila genome to regulation of adipocyte cell fate in mammals.

Organizer formation in Hydra is disrupted by thalidomide treatment.

Thalidomide is a drug that is well known for its teratogenic properties in humans. Surprisingly, thalidomide does not have teratogenic effects on mouse development. We investigated the effect of thalidomide on patterning in hydra, an early metazoan with a very simple axial symmetry. Hydra develops asexually via Wnt-dependent organizer formation, leading to the budding of a new organism. We observe both induction and inhibition of organizer formation depending on cellular context. Interestingly, thalidomide treatment altered budding and the developing organizer, but had little effect on the adult. Expression of Hybra1, a marker of the organizer increased upon thalidomide treatment. However when the organizer is induced by ectopic activation of Wnt signaling via GSK3 inhibition, thalidomide suppresses induction. We show that inhibition of Wnt signaling is not mediated by induction of the BMP pathway. We show that thalidomide activity on organizer formation in hydra depends on the activity of casein kinase1 and the abundance of ß-catenin. Finally, we find that interstitial cells, multipotent cells which give rise to nemoatocytes, neural, digestive and germline cells, are partially responsible for the inhibitory effect of thalidomide.

DJ-1, a novel regulator of the tumor suppressor PTEN.

The phosphatidylinositol 3' kinase (PI3'K) pathway, which regulates cell survival, is antagonized by the PTEN tumor suppressor. The regulation of PTEN is unclear. A genetic screen of Drosophila gain-of-function mutants identified DJ-1 as a suppressor of PTEN function. In mammalian cells, DJ-1 underexpression results in decreased phosphorylation of PKB/Akt, while DJ-1 overexpression leads to hyperphosphorylation of PKB/Akt and increased cell survival. In primary breast cancer samples, DJ-1 expression correlates negatively with PTEN immunoreactivity and positively with PKB/Akt hyperphosphorylation. In 19/23 primary non-small cell lung carcinoma samples, DJ-1 expression was increased compared to paired nonneoplastic lung tissue, and correlated positively with relapse incidence. DJ-1 is thus a key negative regulator of PTEN that may be a useful prognostic marker for cancer.

Correction: The Rho Guanine Nucleotide Exchange Factor DRhoGEF2 Is a Genetic Modifier of the PI3K Pathway in Drosophila.

[This corrects the article DOI: 10.1371/journal.pone.0152259.].

Neither ovariectomy nor progestin treatment prevents endometrial neoplasia in pten+/- mice.

OBJECTIVE: Hormonal therapy for type I (endometrioid) endometrial carcinoma is employed as both a conservative treatment option and for advanced or recurrent disease, but outcome is often poor. Our objective was to test whether ovariectomy, or ovariectomy followed by progestin treatment prevents the development of endometrial lesions in the pten+/- mouse model of endometrial cancer. METHODS: pten+/- mice underwent ovariectomy or sham surgery at 6 or 12 weeks of age. Groups of mice were sacrificed at 24, 30 or 40 weeks. Different cohorts of pten+/- mice were ovariectomized at 6 or 12 weeks of age, followed by medroxyprogesterone acetate (MPA) treatment at low or high-dose (25 or 200 mg total dose, respectively) over 21 days, beginning at 30 weeks of age. Uteri from all mice were examined by routine histology and immunohistochemistry. RESULTS: Without MPA treatment, 16 of 18 ovariectomized animals developed endometrial neoplasms (atypical hyperplasia or adenocarcinoma), as did all 9 sham surgery mice. Immunophenotypes for all tumors were consistent with activation of the phosphoinositidyl-3-kinase (PI3K) pathway, showing staining for phosphorylated PKB/Akt, phosphorylated S6 ribosomal protein and phosphorylated GSK3alpha/beta. All 10 mice treated with either low or high-dose MPA developed endometrial tumors, again with persistent activation of the PI3K signaling pathway. CONCLUSIONS: Development of endometrial neoplasms and constitutive activation of the PI3K pathway in pten+/- mice is not affected by hormonal ablation or by progestin treatment. Loss of PTEN expression is common during human endometrial cancer development, and this may render lesions resistant to the effects of hormonal manipulation leading to treatment failure.

Glycogen synthase kinase 3, circadian rhythms, and bipolar disorder: a molecular link in the therapeutic action of lithium.

BACKGROUND: Bipolar disorder (BPD) is a widespread condition characterized by recurring states of mania and depression. Lithium, a direct inhibitor of glycogen synthase kinase 3 (GSK3) activity, and a mainstay in BPD therapeutics, has been proposed to target GSK3 as a mechanism of mood stabilization. In addition to mood imbalances, patients with BPD often suffer from circadian disturbances. GSK3, an essential kinase with widespread roles in development, cell survival, and metabolism has been demonstrated to be an essential component of the Drosophila circadian clock. We sought to investigate the role of GSK3 in the mammalian clock mechanism, as a possible mediator of lithium's therapeutic effects. METHODS: GSK3 activity was decreased in mouse embryonic fibroblasts (MEFs) genetically and pharmacologically, and changes in the cyclical expression of core clock genes--mPer2 in particular--were examined. RESULTS: We demonstrate that genetic depletion of GSK3 in synchronized oscillating MEFs results in a significant delay in the periodicity of the endogenous clock mechanism, particularly in the cycling period of mPer2. Furthermore, we demonstrate that pharmacological inhibition of GSK3 activity by kenpaullone, a known antagonist of GSK3 activity, as well as by lithium, a direct inhibitor of GSK3 and the most common treatment for BPD, induces a phase delay in mPer2 transcription that resembles the effect observed with GSK3 knockdown. CONCLUSION: These results confirm GSK3 as a plausible target of lithium action in BPD therapeutics, and suggest the circadian clock mechanism as a significant modulator of lithium's clinical benefits.

The Rho Guanine Nucleotide Exchange Factor DRhoGEF2 Is a Genetic Modifier of the PI3K Pathway in Drosophila.

The insulin/IGF-1 signaling pathway mediates various physiological processes associated with human health. Components of this pathway are highly conserved throughout eukaryotic evolution. In Drosophila, the PTEN ortholog and its mammalian counterpart downregulate insulin/IGF signaling by antagonizing the PI3-kinase function. From a dominant loss-of-function genetic screen, we discovered that mutations of a Dbl-family member, the guanine nucleotide exchange factor DRhoGEF2 (DRhoGEF22(l)04291), suppressed the PTEN-overexpression eye phenotype. dAkt/dPKB phosphorylation, a measure of PI3K signaling pathway activation, increased in the eye discs from the heterozygous DRhoGEF2 wandering third instar larvae. Overexpression of DRhoGEF2, and it's functional mammalian ortholog PDZ-RhoGEF (ArhGEF11), at various stages of eye development, resulted in both dPKB/Akt-dependent and -independent phenotypes, reflecting the complexity in the crosstalk between PI3K and Rho signaling in Drosophila.

Role of glycogen synthase kinase-3 in cancer: regulation by Wnts and other signaling pathways.

Although glycogen synthase kinase-3 (GSK-3) is but one of more than a thousand distinct serine/threonine kinases present in the mammalian genome, this enzyme has attracted attention for its role in a diverse range of cellular processes and its positioning at a nexus of several signaling pathways that are important in cancer and other human diseases. The association of GSK-3 with widely different functions, from glycogen metabolism to fruit fly segmentation and slime mold differentiation, was initially perplexing. However, as the context of the biological processes involving this enzyme has been clarified, unifying themes have emerged that begin to explain its pleiotropic nature. Unlike most protein kinases involved in signaling, GSK-3 is active in unstimulated, resting cells. Its activity is inactivated during cellular responses and its substrates therefore tend to be dephosphorylated. As more of these targets have been identified and the effects of their modification by GSK-3 determined, most have been found to be functionally inhibited by GSK-3. Hence, this kinase appears to act as a general repressor, keeping its targets switched off or inaccessible under resting conditions. The rarity of this form of regulation is perhaps related to the diversity of its targets. Over the past decade, the importance of GSK-3 has been established by three significant properties: its remarkable evolutionary conservation, allowing analysis in genetically tractable organisms; its involvement in the Wnt/wingless signaling pathway; and its inhibition by agonists of the prosurvival phosphatidylinositol 3' kinase (P13'K) pathway. This review covers recent advances in understanding the physiological roles of this enzyme, particularly in the context of cancer.

Chk2 regulates irradiation-induced, p53-mediated apoptosis in Drosophila.

The tumor suppressor function of p53 has been attributed to its ability to regulate apoptosis and the cell cycle. In mammals, DNA damage, aberrant growth signals, chemotherapeutic agents, and UV irradiation activate p53, a process that is regulated by several posttranslational modifications. In Drosophila melanogaster, however, the regulation modes of p53 are still unknown. Overexpression of D. melanogaster p53 (Dmp53) in the eye induced apoptosis, resulting in a small eye phenotype. This phenotype was markedly enhanced by coexpression with D. melanogaster Chk2 (DmChk2) and was almost fully rescued by coexpression with a dominant-negative (DN), kinase-dead form of DmChk2. DN DmChk2 also inhibited Dmp53-mediated apoptosis in response to DNA damage, whereas overexpression of Grapes (Grp), the Drosophila Chk1-homolog, and its DN mutant had no effect on Dmp53-induced phenotypes. DmChk2 also activated the Dmp53 transactivation activity in cultured cells. Mutagenesis of Dmp53 amino terminal Ser residues revealed that Ser-4 is critical for its responsiveness toward DmChk2. DmChk2 activates the apoptotic activity of Dmp53 and Ser-4 is required for this effect. Contrary to results in mammals, Grapes, the Drosophila Chk1-homolog, is not involved in regulating Dmp53. Chk2 may be the ancestral regulator of p53 function.

Angiotensin-converting enzyme 2 is an essential regulator of heart function.

Cardiovascular diseases are predicted to be the most common cause of death worldwide by 2020. Here we show that angiotensin-converting enzyme 2 (ace2) maps to a defined quantitative trait locus (QTL) on the X chromosome in three different rat models of hypertension. In all hypertensive rat strains, ACE2 messenger RNA and protein expression were markedly reduced, suggesting that ace2 is a candidate gene for this QTL. Targeted disruption of ACE2 in mice results in a severe cardiac contractility defect, increased angiotensin II levels, and upregulation of hypoxia-induced genes in the heart. Genetic ablation of ACE on an ACE2 mutant background completely rescues the cardiac phenotype. But disruption of ACER, a Drosophila ACE2 homologue, results in a severe defect of heart morphogenesis. These genetic data for ACE2 show that it is an essential regulator of heart function in vivo.