Impact of Nox5 Polymorphisms on Basal and Stimulus-Dependent ROS Generation

<div><p>Nox5 is an EF-hand containing, calcium-dependent isoform of the NADPH oxidase family of reactive oxygen species (ROS) generating enzymes. Altered expression and activity of Nox5 has been reported in cardiovascular diseases and cancers but the absence of Nox5 in rodents has precluded a greater understanding of its physiological and pathophysiological roles. Multiple polymorphisms have been identified within the coding sequence of human Nox5, but whether this translates into altered enzyme function is unknown. Herein, we have generated 15 novel mutants of Nox5β to evaluate the effect of exonic SNPs on basal and stimulated enzyme activity. Compared to the WT enzyme, ROS production was unchanged or slightly modified in the majority of mutants, but significantly decreased in 7. Focusing on M77K, Nox5 activity was dramatically reduced in unstimulated cells and following challenge with both calcium- and phosphorylation-dependent stimuli despite equivalent levels of expression. The M77K mutation did not influence the Nox5 phosphorylation or the ability to bind Hsp90, but in cell-free assays with excess co-factors and calcium, ROS production was dramatically reduced. A more conservative substitution M77V arising from another SNP yielded a different profile of enzyme activity and suggests a critical role of M77 in calcium-dependent ROS production. Two C-terminal mutants, R530H and G542R, were observed that had little to no activity and relatively high minor allele frequency (MAF). In conclusion, we have identified 7 missense SNPs in Nox5 that result in little or no enzyme activity. Whether humans with dysfunctional Nox5 variants have altered physiology or disease remains to be determined.</p></div

The M77K mutation decreases the calcium-induced activation of Nox5.

<p>(<b>A</b>) Stimulated superoxide release was measured over time from COS-7 cells expressing HA-Nox5 or the mutant M77K following addition with ionomycin (1 µM). Results show the maximum level of superoxide produced and are presented as mean ± SEM (n = 4–6), *P<0.05, versus HA-Nox5. (<b>B</b>) The activity of Nox5 in cell free extracts was determined in the absence or in the presence of free calcium (mean ± SEM n = 4). *<i>P</i><0.05, <i>versus</i> HA-Nox5.</p

Demographics of SNP induced mutations in Nox5.

<p>ASW: Americans of African Ancestry in SW USA.</p><p>CEU: Utah Residence (CEPH) with Western and Northern European Ancestry.</p><p>CHB: Han Chineses in Beijing, China.</p><p>CHS: Southern Han Chinese.</p><p>CLM: Colombians from Medelin, Colombia.</p><p>FIN: Finnish in Finland.</p><p>GBR: British in England and Scotland.</p><p>IBS: Iberian population in Spain.</p><p>JPT: Japanese in Tokyo, Japan.</p><p>LWK: Luhya in Webuye, Kenya.</p><p>MXL: Mexican Ancestry from Los Angeles USA.</p><p>PUR: Puerto Ricans from Puerto Rico.</p><p>TSI: Toscani in Italia.</p><p>YRI: Yoruba in Ibadan, Nigera.</p

Stimulated superoxide production from Nox5 mutants.

<p>Stimulated superoxide production was monitored in COS-7 cells transfected with HA-Nox5 or mutants via L-012 chemiluminescence (A–O). Superoxide release was monitored over time from cells stimulated with ionomycin (1 µM) or PMA (100 nM). Maximal superoxide produced is presented as mean ± SEM (n = 4–6), *<i>P</i><0.05, <i>versus</i> HA-Nox5.</p

Basal superoxide production from Nox5 mutants.

<p>Superoxide production was monitored using L-012 in COS-7 cells transfected with HA-Nox5 or mutants based on SNPs listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100102#pone-0100102-t002" target="_blank"><b>Table 2</b></a> (A–O). Upper panel shows unstimulated or basal superoxide release from HA-Nox5 or the mutants. Western blots in the lower panels reveal full length protein expression of transgenes versus the loading control, GAPDH. Results are presented as mean ± SEM (n = 4–6), *<i>P</i><0.05, <i>versus</i> HA-Nox5.</p

The M77K mutant does not influence Nox5 phosphorylation.

<p>(<b>A</b>) Stimulated superoxide production was measured over time from COS-7 cells expressing HA-Nox5 or the mutant M77K following the addition of PMA (100 nM). Results show the maximum level of superoxide produced and are presented as mean ± SEM (n = 4–6), *P<0.05, versus HA-Nox5. (<b>B</b>) COS-7 cells expressing HA-Nox5 or the mutant M77K with vehicle or PMA (100 nM) after 2 h serum starvation and the phosphorylation of Nox5 at Ser490, Thr494 and Ser498 were determined by Western blot using phosphorylation state specific antibodies relative to total Nox5. Quantitation and representative blots from 3 independent experiments are shown. *<i>P</i><0.05, <i>versus</i> Basal WT-Nox5.</p

Sequences of Nox5 mutagenic primers.

<p>Sequences of Nox5 mutagenic primers.</p

Nonsense and missense single nucleotide polymorphisms within the gene coding region of Nox5. Position numbers refer to AF325189.1

<p>*EA: European Ameriacan.</p><p>*AA: African American.</p><p>*Reference species: <i>Homo sapiens, Bos taurus, Canis lupus familiaris, Oryctolagus cuniculus, Equus caballus, Macaca mulatta, Gorilla gorilla, Ovis aries, Felis catus and Sus scrofa.</i></p

Additional file 1 of Dense phenotyping from electronic health records enables machine learning-based prediction of preterm birth

Additional file 1. Supplementary figures, tables, and methods

Prevention and management of osteoporosis and osteoporotic fractures in persons with a spinal cord injury or disorder: A systematic scoping review

<p>Objectives: The primary objective was to review the literature regarding methodologies to assess fracture risk, to prevent and treat osteoporosis and to manage osteoporotic fractures in SCI/D.</p> <p>Study Design: Scoping review.</p> <p>Settings/Participants: Human adult subjects with a SCI/D.</p> <p>Outcome measures: Strategies to identify persons with SCI/D at risk for osteoporotic fractures, nonpharmacological and pharmacological therapies for osteoporosis and management of appendicular fractures.</p> <p>Results: 226 articles were included in the scoping review. Risk of osteoporotic fractures in SCI is predicted by a combination of DXA-defined low BMD plus clinical and demographic characteristics. Screening for secondary causes of osteoporosis, in particular hyperparathyroidism, hyperthyroidism, vitamin D insufficiency and hypogonadism, should be considered. Current antiresorptive therapies for treatment of osteoporosis have limited efficacy. Use of surgery to treat fractures has increased and outcomes are good and comparable to conservative treatment in most cases. A common adverse event following fracture was delayed healing.</p> <p>Conclusions: Most of the research in this area is limited by small sample sizes, weak study designs, and significant variation in populations studied. Future research needs to address cohort definition and study design issues.</p