Pro-Apoptotic and Anti-Invasive Properties Underscore the Tumor-Suppressing Impact of Myoglobin on a Subset of Human Breast Cancer Cells.

The expression of myoglobin (MB), well known as the oxygen storage and transport protein of myocytes, is a novel hallmark of the luminal subtype in breast cancer patients and correlates with better prognosis. The mechanisms by which MB impacts mammary tumorigenesis are hitherto unclear. We aimed to unravel this role by using CRISPR/Cas9 technology to generate MB-deficient clones of MCF7 and SKBR3 breast cancer cell lines and subsequently characterize them by transcriptomics plus molecular and functional analyses. As main findings, loss of MB at normoxia upregulated the expression of cell cyclins and increased cell survival, while it prevented apoptosis in MCF7 cells. Additionally, MB-deficient cells were less sensitive to doxorubicin but not ionizing radiation. Under hypoxia, the loss of MB enhanced the partial epithelial to mesenchymal transition, thus, augmenting the migratory and invasive behavior of cells. Notably, in human invasive mammary ductal carcinoma tissues, MB and apoptotic marker levels were positively correlated. In addition, MB protein expression in invasive ductal carcinomas was associated with a positive prognostic value, independent of the known tumor suppressor p53. In conclusion, we provide multiple lines of evidence that endogenous MB in cancer cells by itself exerts novel tumor-suppressive roles through which it can reduce cancer malignancy.

Splitting of circulating red blood cells as an in vivo mechanism of erythrocyte maturation in developing zebrafish, chick and mouse embryos.

Nucleated circulating red blood cells (RBCs) of developing zebrafish, chick and mouse embryos can actively proliferate. While marrow- or organ-mediated erythropoiesis has been widely studied, transforming in vivo processes of circulating RBCs are under little scrutiny. We employed confocal, stereo- and electron microscopy to document the maturation of intravascular RBCs. In zebrafish embryos (32-72 h post-fertilization), RBC splitting in the caudal vein plexus follows a four-step program: (i) nuclear division with continued cytoplasmic connection between somata; (ii) dumbbell-shaped RBCs tangle at transluminal vascular pillars; (iii) elongation; and (iv) disruption of soma-to-soma connection. Dividing RBCs of chick embryos, however, retain the nucleus in one of their somata. Here, RBC splitting acts to pinch off portions of cytoplasm, organelles and ribosomes. Dumbbell-shaped primitive RBCs re-appeared as circulation constituents in mouse embryos. The splitting of circulating RBCs thus represents a biologically relevant mechanism of RBC division and maturation during early vertebrate ontogeny.

Extensive transcriptional complexity during hypoxia-regulated expression of the myoglobin gene in cancer.

Recently, the ectopic expression of myoglobin (MB) was reported in human epithelial cancer cell lines and breast tumor tissues, where MB expression increased with hypoxia. The better prognosis of MB-positive breast cancer patients suggested that the globin exerts a tumor-suppressive role, possibly by impairing mitochondrial activity in hypoxic breast carcinoma cells. To better understand MB gene regulation in cancer, we systematically investigated the architecture of the human MB gene, its transcripts and promoters. In silico analysis of transcriptome data from normal human tissues and cancer cell lines, followed by RACE-PCR verification, revealed seven novel exons in the MB gene region, most of which are untranslated exons located 5'-upstream of the coding DNA sequence (CDS). Sixteen novel alternatively spliced MB transcripts were detected, most of which predominantly occur in tumor tissue or cell lines. Quantitative RT-PCR analyses of MB expression in surgical breast cancer specimen confirmed the preferential usage of a hitherto unknown, tumor-associated MB promoter, which was functionally validated by luciferase reporter gene assays. In line with clinical observations of MB up-regulation in avascular breast tumors, the novel cancer-associated MB splice variants exhibited increased expression in tumor cells subjected to experimental hypoxia. The novel gene regulatory mechanisms unveiled in this study support the idea of a non-canonical role of MB during carcinogenesis.

IL6 secreted by Ewing sarcoma tumor microenvironment confers anti-apoptotic and cell-disseminating paracrine responses in Ewing sarcoma cells.

BACKGROUND: The prognosis of patients with Ewing sarcoma (ES) has improved over the course of the last decades. However, those patients suffering from metastatic and recurrent ES still have only poor chances of survival and require new therapeutic approaches. Interleukin-6 (IL6) is a pleiotropic cytokine expressed by immune cells and a great variety of cancer cells. It induces inflammatory responses, enhances proliferation and inhibits apoptosis in cancer cells, thereby promoting chemoresistance. METHODS: We investigated expression of IL6, its receptors and the IL6 signal transduction pathway in ES tumor samples and cell lines applying reverse transcriptase PCR, immunoblot and immunohistochemistry. The impact of IL6 on cell viability and apoptosis in ES cell lines was analyzed by MTT and propidium iodide staining, migration assessed by chorioallantoic membrane (CAM) assay. RESULTS: Immunohistochemistry proved IL6 expression in the stroma of ES tumor samples. IL6 receptor subunits IL6R and IL6ST were expressed on the surface of ES cells. Treatment of ES cells with rhIL6 resulted in phosphorylation of STAT3. rhIL6 protected ES cells from serum starvation-induced apoptosis and promoted migration. IL6 blood serum levels were elevated in a subgroup of ES patients with poor prognosis. CONCLUSIONS: These data suggest that IL6 contributes to ES tumor progression by increasing resistance to apoptosis in conditions of cellular stress, such as serum starvation, and by promotion of metastasis.

Lack of conventional oxygen-linked proton and anion binding sites does not impair allosteric regulation of oxygen binding in dwarf caiman hemoglobin.

In contrast to other vertebrate hemoglobins (Hbs) whose high intrinsic O2 affinities are reduced by red cell allosteric effectors (mainly protons, CO2, organic phosphates, and chloride ions), crocodilian Hbs exhibit low sensitivity to organic phosphates and high sensitivity to bicarbonate (HCO3(-)), which is believed to augment Hb-O2 unloading during diving and postprandial alkaline tides when blood HCO3(-) levels and metabolic rates increase. Examination of α- and ß-globin amino acid sequences of dwarf caiman (Paleosuchus palpebrosus) revealed a unique combination of substitutions at key effector binding sites compared with other vertebrate and crocodilian Hbs: ß82Lys→Gln, ß143His→Val, and ß146His→Tyr. These substitutions delete positive charges and, along with other distinctive changes in residue charge and polarity, may be expected to disrupt allosteric regulation of Hb-O2 affinity. Strikingly, however, P. palpebrosus Hb shows a strong Bohr effect, and marked deoxygenation-linked binding of organic phosphates (ATP and DPG) and CO2 as carbamate (contrasting with HCO3(-) binding in other crocodilians). Unlike other Hbs, it polymerizes to large complexes in the oxygenated state. The highly unusual properties of P. palpebrosus Hb align with a high content of His residues (potential sites for oxygenation-linked proton binding) and distinctive surface Cys residues that may form intermolecular disulfide bridges upon polymerization. On the basis of its singular properties, P. palpebrosus Hb provides a unique opportunity for studies on structure-function coupling and the evolution of compensatory mechanisms for maintaining tissue O2 delivery in Hbs that lack conventional effector-binding residues.

Myoglobin in Brown Adipose Tissue: A Multifaceted Player in Thermogenesis.

Brown adipose tissue (BAT) plays an important role in energy homeostasis by generating heat from chemical energy via uncoupled oxidative phosphorylation. Besides its high mitochondrial content and its exclusive expression of the uncoupling protein 1, another key feature of BAT is the high expression of myoglobin (MB), a heme-containing protein that typically binds oxygen, thereby facilitating the diffusion of the gas from cell membranes to mitochondria of muscle cells. In addition, MB also modulates nitric oxide (NO•) pools and can bind C16 and C18 fatty acids, which indicates a role in lipid metabolism. Recent studies in humans and mice implicated MB present in BAT in the regulation of lipid droplet morphology and fatty acid shuttling and composition, as well as mitochondrial oxidative metabolism. These functions suggest that MB plays an essential role in BAT energy metabolism and thermogenesis. In this review, we will discuss in detail the possible physiological roles played by MB in BAT thermogenesis along with the potential underlying molecular mechanisms and focus on the question of how BAT-MB expression is regulated and, in turn, how this globin regulates mitochondrial, lipid, and NO• metabolism. Finally, we present potential MB-mediated approaches to augment energy metabolism, which ultimately could help tackle different metabolic disorders.

Endogenous myoglobin expression in mouse models of mammary carcinoma reduces hypoxia and metastasis in PyMT mice.

Myoglobin (MB) is expressed in different cancer types and may act as a tumor suppressor in breast cancer. The mechanisms by which basal MB expression level impacts murine mammary tumorigenesis are unclear. We investigated how MB expression in breast cancer influences proliferation, metastasis, tumor hypoxia, and chemotherapy treatment in vivo. We crossed PyMT and WapCreTrp53flox mammary cancer mouse models that differed in tumor grade/type and onset of mammary carcinoma with MB knockout mice. The loss of MB in WapCre;Trp53flox mice did not affect tumor development and progression. On the other hand, loss of MB decreased tumor growth and increased tissue hypoxia as well as the number of lung metastases in PyMT mice. Furthermore, Doxorubicin therapy prevented the stronger metastatic propensity of MB-deficient tumors in PyMT mice. This suggests that, although MB expression predicts improved prognosis in breast cancer patients, MB-deficient tumors may still respond well to first-line therapies. We propose that determining the expression level of MB in malignant breast cancer biopsies will improve tumor stratification, outcome prediction, and personalized therapy in cancer patients.

Endogenous myoglobin in breast cancer is hypoxia-inducible by alternative transcription and functions to impair mitochondrial activity: a role in tumor suppression?

Recently, immunohistochemical analysis of myoglobin (MB) in human breast cancer specimens has revealed a surprisingly widespread expression of MB in this nonmuscle context. The positive correlation with hypoxia-inducible factor 2α (HIF-2α) and carbonic anhydrase IX suggested that oxygen regulates myoglobin expression in breast carcinomas. Here, we report that MB mRNA and protein levels are robustly induced by prolonged hypoxia in breast cancer cell lines, in part via HIF-1/2-dependent transactivation. The hypoxia-induced MB mRNA originated from a novel alternative transcription start site 6 kb upstream of the ATG codon. MB regulation in normal and tumor tissue may thus be fundamentally different. Functionally, the knockdown of MB in MDA-MB468 breast cancer cells resulted in an unexpected increase of O(2) uptake and elevated activities of mitochondrial enzymes during hypoxia. Silencing of MB transcription attenuated proliferation rates and motility capacities of hypoxic cancer cells and, surprisingly, also fully oxygenated breast cancer cells. Endogenous MB in cancer cells is apparently involved in controlling oxidative cell energy metabolism, contrary to earlier findings on mouse heart, where the targeted disruption of the Mb gene did not effect myocardial energetics and O(2) consumption. This control function of MB seemingly impacts mitochondria and influences cell proliferation and motility, but it does so in ways not directly related to the facilitated diffusion or storage of O(2). Hypothetically, the mitochondrion-impairing role of MB in hypoxic cancer cells is part of a novel tumor-suppressive function.

Myoglobin regulates fatty acid trafficking and lipid metabolism in mammary epithelial cells.

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O2-dependent shuttle of oxidizable fatty acid substrates. MB's impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate. Thus, the novel functions of MB in breast epithelia described herein range from controlling fatty acid turnover and homeostasis to influencing inflammatory signalling cascade. Future work is needed to analyse to what extent these novel roles of MB also apply to myocytic cell physiology and malignant cell behaviour, respectively.

Lineage-specific patterns of functional diversification in the alpha- and beta-globin gene families of tetrapod vertebrates.

The alpha- and beta-globin gene families of jawed vertebrates have diversified with respect to both gene function and the developmental timing of gene expression. Phylogenetic reconstructions of globin gene family evolution have provided suggestive evidence that the developmental regulation of hemoglobin synthesis has evolved independently in multiple vertebrate lineages. For example, the embryonic beta-like globin genes of birds and placental mammals are not 1:1 orthologs. Despite the similarity in developmental expression profiles, the genes are independently derived from lineage-specific duplications of a beta-globin pro-ortholog. This suggests the possibility that other vertebrate taxa may also possess distinct repertoires of globin genes that were produced by repeated rounds of lineage-specific gene duplication and divergence. Until recently, investigations into this possibility have been hindered by the dearth of genomic sequence data from nonmammalian vertebrates. Here, we report new insights into globin gene family evolution that were provided by a phylogenetic analysis of vertebrate globins combined with a comparative genomic analysis of three key sauropsid taxa: a squamate reptile (anole lizard, Anolis carolinensis), a passeriform bird (zebra finch, Taeniopygia guttata), and a galliform bird (chicken, Gallus gallus). The main objectives of this study were 1) to characterize evolutionary changes in the size and membership composition of the alpha- and beta-globin gene families of tetrapod vertebrates and 2) to test whether functional diversification of the globin gene clusters occurred independently in different tetrapod lineages. Results of our comparative genomic analysis revealed several intriguing patterns of gene turnover in the globin gene clusters of different taxa. Lineage-specific differences in gene content were especially pronounced in the beta-globin gene family, as phylogenetic reconstructions revealed that amphibians, lepidosaurs (as represented by anole lizard), archosaurs (as represented by zebra finch and chicken), and mammals each possess a distinct independently derived repertoire of beta-like globin genes. In contrast to the ancient functional diversification of the alpha-globin gene cluster in the stem lineage of tetrapods, the physiological division of labor between early- and late-expressed genes in the beta-globin gene cluster appears to have evolved independently in several tetrapod lineages.

Targeting neovascularization and respiration of tumor grafts grown on chick embryo chorioallantoic membranes.

Since growing tumors stimulate angiogenesis, via vascular endothelial growth factor (VEGF), angiogenesis inhibitors (AIs, blockers of the VEGF signaling pathway) have been introduced to cancer therapy. However, AIs often yielded only modest and short-lived gains in cancer patients and more invasive tumor phenotypes in animal models. Combining anti-VEGF strategies with lactate uptake blockers may boost both efficacy and safety of AIs. We assessed this hypothesis by using the ex ovo chorioallantoic membrane (CAM) assay. We show that AI-based monotherapy (Avastin®, AVA) increases tumor hypoxia in human CAM cancer cell xenografts and cell spread in human as well as canine CAM cancer cell xenografts. In contrast, combining AVA treatment with lactate importer MCT1 inhibitors (α-cyano-4-hydroxycinnamic acid (CHC) or AZD3965 (AZD)) reduced both tumor growth and cell dissemination of human and canine explants. Moreover, combining AVA+AZD diminished blood perfusion and tumor hypoxia in human explants. Thus, the ex ovo CAM assay as an easy, fast and cheap experimental setup is useful for pre-clinical cancer research. Moreover, as an animal-free experimental setup the CAM assay can reduce the high number of laboratory animals used in pre-clinical cancer research.

Myoglobin, expressed in brown adipose tissue of mice, regulates the content and activity of mitochondria and lipid droplets.

The identification of novel physiological regulators that stimulate energy expenditure through brown adipose tissue (BAT) activity in substrate catalysis is of utmost importance to understand and treat metabolic diseases. Myoglobin (MB), known to store or transport oxygen in heart and skeletal muscles, has recently been found to bind fatty acids with physiological constants in its oxygenated form (i.e., MBO2). Here, we investigated the in vivo effect of MB expression on BAT activity. In particular, we studied mitochondrial function and lipid metabolism as essential determinants of energy expenditure in this tissue. We show in a MB-null (MBko) mouse model that MB expression in BAT impacts on the activity of brown adipocytes in a twofold manner: i) by elevating mitochondrial density plus maximal respiration capacity, and through that, by stimulating BAT oxidative metabolism along with the organelles` uncoupled respiration; and ii) by influencing the free fatty acids pool towards a palmitate-enriched composition and shifting the lipid droplet (LD) equilibrium towards higher counts of smaller droplets. These metabolic changes were accompanied by the up-regulated expression of thermogenesis markers UCP1, CIDEA, CIDEC, PGC1-α and PPAR-α in the BAT of MB wildtype (MBwt) mice. Along with the emergence of the "browning" BAT morphology, MBwt mice exhibited a leaner phenotype when compared to MBko littermates at 20 weeks of age. Our data shed novel insights into MB's role in linking oxygen and lipid-based thermogenic metabolism. The findings suggest potential new strategies of targeting the MB pathway to treat metabolic disorders related to diminishing energy expenditure.

Characterization of the first beta-class carbonic anhydrase from an arthropod (Drosophila melanogaster) and phylogenetic analysis of beta-class carbonic anhydrases in invertebrates.

BACKGROUND: The beta-carbonic anhydrase (CA, EC 4.2.1.1) enzymes have been reported in a variety of organisms, but their existence in animals has been unclear. The purpose of the present study was to perform extensive sequence analysis to show that the beta-CAs are present in invertebrates and to clone and characterize a member of this enzyme family from a representative model organism of the animal kingdom, e.g., Drosophila melanogaster. RESULTS: The novel beta-CA gene, here named DmBCA, was identified from FlyBase, and its orthologs were searched and reconstructed from sequence databases, confirming the presence of beta-CA sequences in 55 metazoan species. The corresponding recombinant enzyme was produced in Sf9 insect cells, purified, kinetically characterized, and its inhibition was investigated with a series of simple, inorganic anions. Holoenzyme molecular mass was defined by dynamic light scattering analysis and gel filtration, and the results suggested that the holoenzyme is a dimer. Double immunostaining confirmed predictions based on sequence analysis and localized DmBCA protein to mitochondria. The enzyme showed high CO2 hydratase activity, with a kcat of 9.5 x 105 s-1 and a kcat/KM of 1.1 x 108 M-1s-1. DmBCA was appreciably inhibited by the clinically-used sulfonamide acetazolamide, with an inhibition constant of 49 nM. It was moderately inhibited by halides, pseudohalides, hydrogen sulfide, bisulfite and sulfate (KI values of 0.67 - 1.36 mM) and more potently by sulfamide (KI of 0.15 mM). Bicarbonate, nitrate, nitrite and phenylarsonic/boronic acids were much weaker inhibitors (KIs of 26.9 - 43.7 mM). CONCLUSIONS: The Drosophila beta-CA represents a highly active mitochondrial enzyme that is a potential model enzyme for anti-parasitic drug development.

Taking advantage of tumor cell adaptations to hypoxia for developing new tumor markers and treatment strategies.

Cancer cells in hypoxic areas of solid tumors are to a large extent protected against the action of radiation as well as many chemotherapeutic drugs. There are, however, two different aspects of the problem caused by tumor hypoxia when cancer therapy is concerned: One is due to the chemical reactions that molecular oxygen enters into therapeutically targeted cells. This results in a direct chemical protection against therapy by the hypoxic microenvironment, which has little to do with cellular biological regulatory processes. This part of the protective effect of hypoxia has been known for more than half a century and has been studied extensively. However, in recent years there has been more focus on the other aspect of hypoxia, namely the effect of this microenvironmental condition on selecting cells with certain genetic prerequisites that are negative with respect to patient prognosis. There are adaptive mechanisms, where hypoxia induces regulatory cascades in cells resulting in a changed metabolism or changes in extracellular signaling. These processes may lead to changes in cellular intrinsic sensitivity to treatment irrespective of oxygenation and, furthermore, may also have consequences for tissue organization. Thus, the adaptive mechanisms induced by hypoxia itself may have a selective effect on cells, with a fine-tuned protection against damage and stress of many kinds. It therefore could be that the adaptive mechanisms may take advantage of for new tumor labeling/imaging and treatment strategies. One of the Achilles' heels of hypoxia research has always been the exact measurements of tissue oxygenation as well as the control of oxygenation in biological tumor models. Thus, development of technology that can ease this control is vital in order to study mechanisms and perform drug development under relevant conditions. An integrated EU Framework project 2004-2009, termed EUROXY, demonstrates several pathways involved in transcription and translation control of the hypoxic cell phenotype and evidence of cross-talk with responses to pH and redox changes. The carbonic anhydrase isoenzyme CA IX was selected for further studies due to its expression on the surface of many types of hypoxic tumors. The effort has led to marketable culture flasks with sensors and incubation equipment, and the synthesis of new drug candidates against new molecular targets. New labeling/imaging methods for cancer diagnosing and imaging of hypoxic cancer tissue are now being tested in xenograft models and are also in early clinical testing, while new potential anti-cancer drugs are undergoing tests using xenografted tumor cancers. The present article describes the above results in individual consortium partner presentations.

Regulating cellular oxygen sensing by hydroxylation.

Oxygen homeostasis under conditions of limited O2 supply requires hypoxia-dependent gene regulation. The transcription factor complex hypoxia-inducible factor 1 (HIF-1) has been recognized as the master regulator that mediates the adaptational genetic response to ensure restoration of energy supply. This review will focus on the recent advances in understanding the hypoxia-induced cellular response with particular respect to cellular O2 sensing for adequate control of HIF-1 activation.

A genetically encoded biosensor for visualising hypoxia responses in vivo.

Cells experience different oxygen concentrations depending on location, organismal developmental stage, and physiological or pathological conditions. Responses to reduced oxygen levels (hypoxia) rely on the conserved hypoxia-inducible factor 1 (HIF-1). Understanding the developmental and tissue-specific responses to changing oxygen levels has been limited by the lack of adequate tools for monitoring HIF-1 in vivo. To visualise and analyse HIF-1 dynamics in Drosophila, we used a hypoxia biosensor consisting of GFP fused to the oxygen-dependent degradation domain (ODD) of the HIF-1 homologue Sima. GFP-ODD responds to changing oxygen levels and to genetic manipulations of the hypoxia pathway, reflecting oxygen-dependent regulation of HIF-1 at the single-cell level. Ratiometric imaging of GFP-ODD and a red-fluorescent reference protein reveals tissue-specific differences in the cellular hypoxic status at ambient normoxia. Strikingly, cells in the larval brain show distinct hypoxic states that correlate with the distribution and relative densities of respiratory tubes. We present a set of genetic and image analysis tools that enable new approaches to map hypoxic microenvironments, to probe effects of perturbations on hypoxic signalling, and to identify new regulators of the hypoxia response.

Linker chains of the gigantic hemoglobin of the earthworm Lumbricus terrestris: primary structures of linkers L2, L3, and L4 and analysis of the connectivity of the disulfide bonds in linker L1.

The extracellular hemoglobin (Hb) of the earthworm, Lumbricus terrestris, has four major kinds of globin chains: a, b, c, and d, present in equimolar proportions, and additional non-heme, non-globin scaffolding chains called linkers that are required for the calcium-dependent assembly of the full-sized molecule. The amino acid sequences of all four of the globin chains and one of the linkers (L1) have previously been determined. The amino acid sequences via cDNA of each of the three remaining linkers, L2, L3, and L4, have been determined so that the sequences of all constituent polypeptides of the hemoglobin are now known. Each linker has a highly conserved cysteine-rich segment of approximately 40 residues that is homologous with the seven ligand-binding repeats of the human low-density lipoprotein receptor (LDLR). Analysis of linker L1 shows that the connectivity of the three disulfide bonds is exactly the same as in the LDLR ligand-binding repeats. The presence of a calcium-binding site comprising one glutamyl and three aspartyl residues in both the LDLR repeats and in the linkers supports the suggestion that calcium is required for the folding and disulfide connectivity of the linkers as in the LDLR repeats. Linker L2 is markedly heterogeneous and contains unusual glycine-rich sequences near the NH2-terminus and a polar zipper-like sequence with imperfect repeats of Asp-Asp-His at the carboxyl terminus. Similar Asp-Asp-His repeats have been found in a protein homologous to superoxide dismutase in the hemolymph of certain mussels. These repeats may function as metal-binding sites.

A candidate juvenoid hormone receptor cis-element in the Daphnia magna hb2 hemoglobin gene promoter.

Hemoglobin levels are significantly elevated in the crustacean Daphnia magna by juvenoid hormones. The present study was undertaken to identify the specific globin (hb) genes that are induced by juvenoids and to identify putative juvenoid response elements (JREs) that may mediate this induction. Gene product of globin 2 (hb2), but not globin 1 and globin 3, was robustly elevated following juvenoid treatment of daphnids. A candidate JRE, located in the promoter of hb2, bound activated factor(s) in response to juvenoid treatment of daphnids. This hormone-induced protein:JRE interaction was robust when daphnids were reared at high oxygen tension but was inhibited when daphnids were reared under low pO2, implying that hypoxia might act to disrupt juvenoid-mediated endocrine signaling. The candidate JRE consists of a steroid/retinoid-response element-like core adjacent to a 5' AT-rich extension and thus bears resemblance to response elements that bind monomeric nuclear receptors. The induction of hb2 mRNA levels by juvenoid treatment occurred rapidly (within 4 h of exposure) and was not attenuated by treatment of daphnids with cycloheximide. In contrast, cycloheximide treatment did block hormone-mediated elevations in hemoglobin protein levels. Thus, induction of hb2 by juvenoids was not dependent upon the synthesis of secondary transcription factors that bound the JRE but was likely due to activation of the gene directly by the juvenoid-receptor complex. Affinity pull-down experiments with nuclear proteins extracted from juvenoid-treated daphnids using the JRE as bait yielded a 52kDa candidate for a monomeric nuclear receptor in D. magna that may mediate the regulatory activity of juvenoids.

Sensing and responding to hypoxia via HIF in model invertebrates.

This past decade has brought considerable progress towards elucidating the molecular mechanisms of oxygen sensing pathways by which mammalian cells are able to detect and adjust, or succumb, to hypoxia. In contrast, far less is known about the protein and DNA constituents that endow many invertebrate species to withstand and recover from even more severe and prolonged O2 limitations. In spite of these differences in hypoxia tolerance, inadequacy in oxygen supply is, from mammals to insects to nematodes, signaled onto the DNA level predominantly by hypoxia-inducible factors (HIFs). Across the animal kingdom, HIF accumulates in hypoxic, but not normoxic, cells and functions in a remarkably conserved pathway. Using crustacean (Daphnia magna) and insect (Drosophila melanogaster) models, work by us and others has implicated HIF in restoring O2 delivery via stimulated hemoglobin synthesis (Daphnia) or tracheal remodeling (Drosophila). HIF is essential for these arthropods to adapt and survive during moderate O2 limitations. A similar life-preserving role for HIF-signaling in hypoxic, but not anoxic, environments had previously been established for another stress-tolerant invertebrate model, the nematode Caenorhabditis elegans. Exploring regulations of oxygen-dependent Daphnia and Drosophila genes in cell culture and in vivo have furthermore aided in uncovering novel HIF-targeting mechanisms that might operate to fine-tune the activity of this transcription factor under steadily hypoxic, rather than changing, oxygen tensions. We conclude our review with yet another addition to the growing list of HIF's many functions: the control of cellular growth during fly development.