Literature Review | June 2017

Select a topic from the list below:

• Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells
   
• Serotonin signaling through the 5-HT1B receptor and NADPH oxidase 1 in pulmonary arterial hypertension
   
• Blocking FSH induces thermogenic adipose tissue and reduces body fat
   
• Inhibition of hepatic lipogenesis in mice enhances liver tumorigenesis by increasing antioxidant defense and promoting cell survival

Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells.
Ganini D, Leinisch F, Kumar A, Jiang J, Tokar EJ, Malone CC, Petrovich RM, Mason RP. Redox Biol. 2017 Mar 10;12:462-468. doi: 10.1016/j.redox.2017.03.002.

Since their discovery, fluorescent proteins have been pivotal in our understanding of living systems and disease. The diverse applicability of these fluorescent molecules allows researchers to label proteins for subcellular distribution and protein degradation studies, monitor and characterize protein-protein interactions through FRET and fluorescence complementation, label organelles and populations of cells, interrogate promoter activity and monitor biological activity using genetically encoded sensors. While this list is not exhaustive the potential of fluorescent molecules is obvious. The idea that fluorescent chromophores of fluorescent molecules yield reactive oxygen species (ROS) during chromophore maturation through non-catalytic reactions is known and is thought to carry minimal cellular toxicity unless used as a photosensitizer in response to photo-irradiation. However, a recent paper by Ganini et al. published research in the journal Redox Biology shows compelling evidence that fluorescent molecules like enhanced GFP (eGFP) catalytically generate ROS in mammalian and non-mammalian cells in the presence of NADPH that supersedes basal levels produced by non-catalytic reactions. Notably, overexpression of eGFP in HeLa cells complemented with microarray studies showed that eGFP dramatically influenced expression of genes involved in the biological processes of inflammation, hedgehog signaling, pathogen response and hypoxia among others. Conclusively, this study points out a significant drawback of using fluorescent proteins like eGFP. Although this study used gene expression to measure the change in biological activity it is possible that other cellular processes, such as protein oxidation and signal transduction, are influenced by ROS produced by fluorescent proteins. Studies, both previous and future, using fluorescent proteins will need to be evaluated taking careful consideration that the data generated may be a consequence of the fluorescent molecules ability to produce ROS. (Prepared by Brandon Schickling, University of Iowa)

Serotonin signaling through the 5-HT_1B receptor and NADPH oxidase 1 in pulmonary arterial hypertension.
Katie Y Hood, Kirsty M Mair, Adam P Harvey, Augusto C Montezano, Rhian M Touyz, Margaret R MacLean. Arterioscler Thromb Vasc Biol 2017;37:00-00 DOI: 10.1161/ATVBAHA.116.308929.

Pulmonary arterial hypertension (PAH) is a rare, but terminal, disease involving high pulmonary vascular resistance, leading to pressure overload-induced right ventricular failure. Improvement of pulmonary vasodilation is the main treatment strategy currently employed, however, there is an unmet need for better therapeutics to prevent/reverse RV remodeling. PAH is characterized by redox – nitric oxide (NO) imbalance and several of the approved PAH drugs target NO signaling, such as phosphodiesterase inhibitors and soluble guanylate cyclase stimulators. It is now evident that NADPH oxidase (Nox) isoforms 1 and 4 are involved in PAH, therefore, selective targeting of these may be beneficial. The manuscript by Hood et al, published online in April, investigated how the potent pulmonary vasoconstrictor, serotonin, is involved in PAH. Using a combination of selective pharmacological inhibition and genetic ablation models they found that serotonin stimulates rapid Nox1-dependent superoxide generation in pulmonary arterial smooth muscle cells. Serotonin treatment resulted in increased Nrf2 activity in normal cells but this effect was absent in cells from PAH patients, resulting in reduced antioxidant capacity due to dysregulated Nrf2 activity. The combined effect was a lowering of catalase activity resulting in increased hydrogen peroxide and increased sulfonylation-induced deactivation of peroxiredoxin. This led to oxidation and carbonylation-dependent DNA damage, causing vascular smooth muscle cell proliferation, remodelling, and dysfunction, all hallmarks of PAH. As a proof of concept, serotonin transporter overexpressing mice had increased right ventricular systolic pressure (indicative of pulmonary pressure) and increased pulmonary oxidative stress (detected by 8-hydroxyguanosine) and these were reduced by a selective serotonin receptor antagonist. Nrf2 activation is currently undergoing clinical trial for PAH treatment and the novel pathway that has been identified in this manuscript, particularly pertaining to Nox1, may prove an additional beneficial target for PAH treatment. (Prepared by Kristen Bubb, University of Sydney, Australia)

Blocking FSH induces thermogenic adipose tissue and reduces body fat.
Peng Liu et al., Nature 2017 Jun 1;546(7656):107-112. doi: 10.1038/nature22342. PMID:28538730.

Women are burdened not only by their pregnancies, nursing and nurturing babies, but, once the procreation period ends, they undergo menopause which causes loss of bone mass, an increase in visceral and subcutaneous fat. Such events coincide with the emergence in disrupted energy balance and reduced physical activity and greatly affect the quality of the life of women. These events are due to the failure in ovaries and in turn an increase in pituitary hormone follicle-stimulating hormone (Fsh). Fsh is one of the few evolutionary highly conserved hormones (also Acth, oxytocin, vasopression) in invertebrate and vertebrate that have multiple, still unrecognized functions; this opens the possibilities for much needed therapeutic interventions. A manuscript authored by Liu et al describes the study on polyclonal antibodies that target the β-subunit of Fsh. The antibody is raised against a 13-amino acid sequence of the β-subunit of Fsh. It inhibits ovariectomy-induced bone loss in mice, triggers beiging of white adipocytes ('brown-like' adipocytes, also known as beige cells) and reduced adiposity in ovariectomized rats. Moreover, authors show that reduction in adiposity is coupled with reduction in production of mitochondria-rich Ucp1-high thermogenic adipose tissue. Fsh reduces Ucp1 by reducing cAMP levels in both white and brown adipose tissues. Among targets that induce beige adipose tissue is the Arb3 pathway with numerous downstream molecules, such as CEBP and PRDM16; drugs that would modulate this pathway are not yet sufficiently developed to be tested in people. Authors then make monoclonal antibodies Hf2 to a corresponding human FSHβ, which binds both mouse and human FSHβ. Its injection into mice reduced subcutaneous and visceral fat and induced beiging. Such antibodies, in addition to treating postmenopausal osteoporosis and obesity, may also be beneficial for treatment of other diseases such as mitochondrial syndrome, cardiovascular disease, cancer, diabetes and polycystic ovarian syndrome. (Prepared by Ines Batinic-Haberle, Duke University)

Inhibition of hepatic lipogenesis in mice enhances liver tumorigenesis by increasing antioxidant defense and promoting cell survival.
Nelson, M. E., Lahiri, S., Chow, J. D., Byrne, F. L., Hargett, S. R., Breen, D. S., Olzomer, E. M., Wu, L. E., Cooney, G. J., Turner, N., James, D. E., Slack-Davis, J. K., Lackner, C., Caldwell, S. H., and Hoehn, K. L. (2017) Inhibition of hepatic lipogenesis enhances liver tumorigenesis by increasing antioxidant defense and promoting cell survival. Nat Commun 8, 14689).

Lipid metabolism has been closely associated with tumorigenesis and de novo lipogenesis is frequently upregulated in human hepatocellular carcinoma (HCC). Clinical evidence indicates that upregulation of lipogenesis is associated with poor prognosis. Thus blocking lipogenesis is hypothesized to prevent liver tumorigenesis. However, in a recent study, liver-specific knockout (KO) of acetyl-CoA carboxylase (ACC) genes (both ACC1 and 2) led to an unexpected twofold increase in tumor incidence and multiplicity compared to controls in a mouse model of diethylnitrosamine (DEN)-induced HCC. ACC KO mice had decreased lipogenesis without affecting other metabolic disorders such as obesity, hepatic steatosis, glucose intolerance, and hyperinsulinemia. Further metabolomic analysis demonstrated that ACC KO resulted in a marked increase in antioxidants including NADPH and reduced glutathione. This study suggests that lipogenesis is dispensable for liver tumorigenesis in mouse model of HCC. However, the discrepancy between the animal model and humans remains to be reconciled. (Summarized by Huiyong Yin, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China)