Sequence and functional characterization of hypoxia inducible factors, HIF1α, HIF2αa, and HIF3α, from the estuarine fish, Fundulus heteroclitus

Author Posting. © The Author(s), 2016. This is the author's version of the work. It is posted here under a nonexclusive, irrevocable, paid-up, worldwide license granted to WHOI. It is made available for personal use, not for redistribution. The definitive version was published in American Journal of Physiology-Regulatory Integrative and Comparative Physiology, 312 (2017): R412-R425, doi:10.1152/ajpregu.00402.2016.The hypoxia inducible factor (HIF) family of transcription factors plays central roles in the development, physiology, pathology, and environmental adaptation of animals. Because many aquatic habitats are characterized by episodes of low dissolved oxygen, fish represent ideal models to study the roles of HIF in the response to aquatic hypoxia. The estuarine fish Fundulus heteroclitus occurs in habitats prone to hypoxia, it responds to low oxygen via behavioral, physiological, and molecular changes, and one member of the HIF family, HIF2α, has been previously described. Herein, cDNA sequencing, phylogenetic analyses, and genomic approaches were used to determine other members of the HIFα family from F. heteroclitus and their relationships to HIFα subunits from other vertebrates. In vitro and cellular approaches demonstrated that full-length forms of HIF1α, 2α, and 3α independently formed complexes with the β subunit (ARNT) to bind to hypoxia response elements and activate reporter gene expression. Quantitative PCR showed that HIFα mRNA abundance varied among organs of normoxic fish in an isoform-specific fashion. Analysis of the F. heteroclitus genome revealed a locus encoding a second HIF2α, HIF2αb, a predicted protein lacking oxygen sensing and transactivation domains. Finally, sequence analyses demonstrated polymorphism in the coding sequence of each F. heteroclitus HIFα subunit, suggesting that genetic variation in these transcription factors may play a role in the variation in hypoxia responses among individuals or populations.This research was supported in part by the National Science Foundation (IBN-0236494 and DEB-1120263) and by National Institute of Environmental Health Sciences (NIEHS) grant P42ES007381 (Superfund Basic Research Program at Boston University). Data interpretation was aided by reference to a preliminary draft of the F. heteroclitus genome sequence, which was supported by funding from the National Science Foundation (collaborative research grants DEB-1120512, DEB-1265282, DEB-1120013, DEB-1120263, DEB-1120333, DEB-1120398)

A functional genomic and proteomic perspective of sea urchin calcium signaling and egg activation

AbstractThe sea urchin egg has a rich history of contributions to our understanding of fundamental questions of egg activation at fertilization. Within seconds of sperm–egg interaction, calcium is released from the egg endoplasmic reticulum, launching the zygote into the mitotic cell cycle and the developmental program. The sequence of the Strongylocentrotus purpuratus genome offers unique opportunities to apply functional genomic and proteomic approaches to investigate the repertoire and regulation of Ca2+ signaling and homeostasis modules present in the egg and zygote. The sea urchin “calcium toolkit” as predicted by the genome is described. Emphasis is on the Ca2+ signaling modules operating during egg activation, but the Ca2+ signaling repertoire has ramifications for later developmental events and adult physiology as well. Presented here are the mechanisms that control the initial release of Ca2+ at fertilization and additional signaling components predicted by the genome and found to be expressed and operating in eggs at fertilization. The initial release of Ca2+ serves to coordinate egg activation, which is largely a phenomenon of post-translational modifications, especially dynamic protein phosphorylation. Functional proteomics can now be used to identify the phosphoproteome in general and specific kinase targets in particular. This approach is described along with findings to date. Key outstanding questions regarding the activation of the developmental program are framed in the context of what has been learned from the genome and how this knowledge can be applied to functional studies

The sea urchin kinome: A first look

AbstractThis paper reports a preliminary in silico analysis of the sea urchin kinome. The predicted protein kinases in the sea urchin genome were identified, annotated and classified, according to both function and kinase domain taxonomy. The results show that the sea urchin kinome, consisting of 353 protein kinases, is closer to the Drosophila kinome (239) than the human kinome (518) with respect to total kinase number. However, the diversity of sea urchin kinases is surprisingly similar to humans, since the urchin kinome is missing only 4 of 186 human subfamilies, while Drosophila lacks 24. Thus, the sea urchin kinome combines the simplicity of a non-duplicated genome with the diversity of function and signaling previously considered to be vertebrate-specific. More than half of the sea urchin kinases are involved with signal transduction, and approximately 88% of the signaling kinases are expressed in the developing embryo. These results support the strength of this nonchordate deuterostome as a pivotal developmental and evolutionary model organism

Anastrozole versus tamoxifen for the prevention of locoregional and contralateral breast cancer in postmenopausal women with locally excised ductal carcinoma in situ (IBIS-II DCIS): a double-blind, randomised controlled trial

Background Third-generation aromatase inhibitors are more effective than tamoxifen for preventing recurrence in postmenopausal women with hormone-receptor-positive invasive breast cancer. However, it is not known whether anastrozole is more effective than tamoxifen for women with hormone-receptor-positive ductal carcinoma in situ (DCIS). Here, we compare the efficacy of anastrozole with that of tamoxifen in postmenopausal women with hormone-receptor-positive DCIS. Methods In a double-blind, multicentre, randomised placebo-controlled trial, we recruited women who had been diagnosed with locally excised, hormone-receptor-positive DCIS. Eligible women were randomly assigned in a 1:1 ratio by central computer allocation to receive 1 mg oral anastrozole or 20 mg oral tamoxifen every day for 5 years. Randomisation was stratified by major centre or hub and was done in blocks (six, eight, or ten). All trial personnel, participants, and clinicians were masked to treatment allocation and only the trial statistician had access to treatment allocation. The primary endpoint was all recurrence, including recurrent DCIS and new contralateral tumours. All analyses were done on a modified intention-to-treat basis (in all women who were randomised and did not revoke consent for their data to be included) and proportional hazard models were used to compute hazard ratios and corresponding confidence intervals. This trial is registered at the ISRCTN registry, number ISRCTN37546358. Results Between March 3, 2003, and Feb 8, 2012, we enrolled 2980 postmenopausal women from 236 centres in 14 countries and randomly assigned them to receive anastrozole (1449 analysed) or tamoxifen (1489 analysed). Median follow-up was 7·2 years (IQR 5·6–8·9), and 144 breast cancer recurrences were recorded. We noted no statistically significant difference in overall recurrence (67 recurrences for anastrozole vs 77 for tamoxifen; HR 0·89 [95% CI 0·64–1·23]). The non-inferiority of anastrozole was established (upper 95% CI <1·25), but its superiority to tamoxifen was not (p=0·49). A total of 69 deaths were recorded (33 for anastrozole vs 36 for tamoxifen; HR 0·93 [95% CI 0·58–1·50], p=0·78), and no specific cause was more common in one group than the other. The number of women reporting any adverse event was similar between anastrozole (1323 women, 91%) and tamoxifen (1379 women, 93%); the side-effect profiles of the two drugs differed, with more fractures, musculoskeletal events, hypercholesterolaemia, and strokes with anastrozole and more muscle spasm, gynaecological cancers and symptoms, vasomotor symptoms, and deep vein thromboses with tamoxifen. Conclusions No clear efficacy differences were seen between the two treatments. Anastrozole offers another treatment option for postmenopausal women with hormone-receptor-positive DCIS, which may be be more appropriate for some women with contraindications for tamoxifen. Longer follow-up will be necessary to fully evaluate treatment differences

Spatial Restriction of PDK1 Activation Cascades by Anchoring to mAKAPα

The muscle A-kinase anchoring protein (mAKAP) tethers cAMP-dependent enzymes to perinuclear membranes of cardiomyocytes. We now demonstrate that two alternatively spliced forms of mAKAP are expressed: mAKAPα and mAKAPβ. The longer form, mAKAPα, is preferentially expressed in the brain. mAKAPβ is a shorter form of the anchoring protein that lacks the first 244 amino acids and is preferentially expressed in the heart. The unique amino terminus of mAKAPα can spatially restrict the activity of 3-phosphoinositide-dependent kinase-1 (PDK1). Biochemical and genetic analyses demonstrate that simultaneous recruitment of PDK1 and ERK onto mAKAPα facilitates activation and release of the downstream target p90RSK. The assembly of tissue-specific signaling complexes provides an efficient mechanism to integrate and relay lipid-mediated and mitogenic activated signals to the nucleus