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Research ArticleToxicology

Chronic Ethanol Feeding in Mice Decreases Expression of Genes for Major Structural Bone Proteins in a Nox4-Independent Manner

Kim B. Pedersen, Michelle L. Osborn, Alex C. Robertson, Ashlee E. Williams, James Watt, Alexandra Denys, Katrin Schröder and Martin J. Ronis
Journal of Pharmacology and Experimental Therapeutics June 2020, 373 (3) 337-346; DOI: https://doi.org/10.1124/jpet.119.264374
Kim B. Pedersen
Department of Pharmacology & Experimental Therapeutics, Louisiana State Health Sciences Center (LSUHSC), New Orleans, Louisiana (K.B.P., A.C.R., A.E.W., J.W., A.D., M.J.R.); Comparative Biomedical Sciences, Louisiana State University (LSU) School of Veterinary Medicine, Baton Rouge, Louisiana (M.L.O.); and Institute of Physiology I, Goethe-University, Frankfurt, Germany (K.S.)
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Michelle L. Osborn
Department of Pharmacology & Experimental Therapeutics, Louisiana State Health Sciences Center (LSUHSC), New Orleans, Louisiana (K.B.P., A.C.R., A.E.W., J.W., A.D., M.J.R.); Comparative Biomedical Sciences, Louisiana State University (LSU) School of Veterinary Medicine, Baton Rouge, Louisiana (M.L.O.); and Institute of Physiology I, Goethe-University, Frankfurt, Germany (K.S.)
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Alex C. Robertson
Department of Pharmacology & Experimental Therapeutics, Louisiana State Health Sciences Center (LSUHSC), New Orleans, Louisiana (K.B.P., A.C.R., A.E.W., J.W., A.D., M.J.R.); Comparative Biomedical Sciences, Louisiana State University (LSU) School of Veterinary Medicine, Baton Rouge, Louisiana (M.L.O.); and Institute of Physiology I, Goethe-University, Frankfurt, Germany (K.S.)
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Ashlee E. Williams
Department of Pharmacology & Experimental Therapeutics, Louisiana State Health Sciences Center (LSUHSC), New Orleans, Louisiana (K.B.P., A.C.R., A.E.W., J.W., A.D., M.J.R.); Comparative Biomedical Sciences, Louisiana State University (LSU) School of Veterinary Medicine, Baton Rouge, Louisiana (M.L.O.); and Institute of Physiology I, Goethe-University, Frankfurt, Germany (K.S.)
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James Watt
Department of Pharmacology & Experimental Therapeutics, Louisiana State Health Sciences Center (LSUHSC), New Orleans, Louisiana (K.B.P., A.C.R., A.E.W., J.W., A.D., M.J.R.); Comparative Biomedical Sciences, Louisiana State University (LSU) School of Veterinary Medicine, Baton Rouge, Louisiana (M.L.O.); and Institute of Physiology I, Goethe-University, Frankfurt, Germany (K.S.)
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Alexandra Denys
Department of Pharmacology & Experimental Therapeutics, Louisiana State Health Sciences Center (LSUHSC), New Orleans, Louisiana (K.B.P., A.C.R., A.E.W., J.W., A.D., M.J.R.); Comparative Biomedical Sciences, Louisiana State University (LSU) School of Veterinary Medicine, Baton Rouge, Louisiana (M.L.O.); and Institute of Physiology I, Goethe-University, Frankfurt, Germany (K.S.)
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Katrin Schröder
Department of Pharmacology & Experimental Therapeutics, Louisiana State Health Sciences Center (LSUHSC), New Orleans, Louisiana (K.B.P., A.C.R., A.E.W., J.W., A.D., M.J.R.); Comparative Biomedical Sciences, Louisiana State University (LSU) School of Veterinary Medicine, Baton Rouge, Louisiana (M.L.O.); and Institute of Physiology I, Goethe-University, Frankfurt, Germany (K.S.)
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Martin J. Ronis
Department of Pharmacology & Experimental Therapeutics, Louisiana State Health Sciences Center (LSUHSC), New Orleans, Louisiana (K.B.P., A.C.R., A.E.W., J.W., A.D., M.J.R.); Comparative Biomedical Sciences, Louisiana State University (LSU) School of Veterinary Medicine, Baton Rouge, Louisiana (M.L.O.); and Institute of Physiology I, Goethe-University, Frankfurt, Germany (K.S.)
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  • Fig. 1.
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    Fig. 1.

    Conditional knockdown of Nox4 expression in long bones. (A) Model for PrxCre-mediated excision of exons 1 and 2 of the Nox4 gene. Indicated are genotyping amplicons for distinguishing the various Nox4 alleles. (B–D) The concentrations of Nox4 mRNA from the knockout (K/O) region, total Nox4 mRNA, and Nox2 relative to β-actin mRNA were determined in the femoral shaft and femoral bone marrow from 13-week-old mice by qRT-PCR. There were 5–11 mice per group. (E and F) The concentration of Nox4 mRNA was determined in liver and kidney aliquots from 32-week-old female mice. There were four mice per group. *P < 0.05; ***P < 0.001 vs. the Nox4 fl/fl genotype. PCR, polymerase chain reaction.

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    Fig. 2.

    Sex and age affect trabecular bone of mice maintained on standard chow. Males and females of genotypes Nox4 fl/fl, PrxCre Nox4 fl/fl, and Nox4 0/0 at 13 and 32 weeks of age were analyzed. There were 5–12 mice per group. Three-way ANOVAs were conducted with sex, age, and genotype as the three factors. The panels indicate significant main effects and important interaction effects. (A–H) BMD, bone mineral density; Trab., trabecular; Vol., volume.

  • Fig. 3.
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    Fig. 3.

    Age, sex, and the Nox4 genotype affect cortical bone of mice maintained on standard chow. Males and females of genotypes Nox4 fl/fl, PrxCre Nox4 fl/fl, and Nox4 0/0 at 13 and 32 weeks of age were analyzed. There were 5–12 mice per group. Three-way ANOVAs were conducted with sex, age, and genotype as the three factors. The panels indicate significant main effects and important interaction effects. (A–E) BMD, bone mineral density;

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    Fig. 4.

    Chronic ethanol feeding for 3 months affects body composition and bone turnover. (A) The diagram illustrates the time course of the body weights for males and females fed either the ethanol diet or control diet for 3 months. (B–D) Body weight, relative liver weight, and the concentration of liver triglycerides were determined at the end of the chronic ethanol feeding experiment. (E–G) Serum concentrations of ALT, Osteocalcin and CTX were determined. Three-way ANOVAs were conducted with sex, diet, and genotype as the three factors. The panels indicate significant main effects. For ALT and CTX, a separate two-way ANOVA for just the data for females was calculated. EtOH, ethanol.

  • Fig. 5.
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    Fig. 5.

    Trabecular data for mice from the chronic ethanol feeding experiment. (A–H) Trabecular bone parameters for a region of the tibiae 0.4–0.7 mm distal to the proximal growth plate. Three-way ANOVAs were conducted with sex, diet, and genotype as the three factors. The panels indicate significant main effects. BMD, bone mineral density; EtOH, ethanol;

  • Fig. 6.
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    Fig. 6.

    Cortical data for mice from the chronic ethanol feeding experiment. (A–E) Cortical bone parameters for the tibiae. Three-way ANOVAs were conducted with sex, diet, and genotype as the three factors. The panels indicate significant main effects. BMD, bone mineral density; EtOH, ethanol.

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    Fig. 7.

    RNA-Seq analysis of femoral shafts of mice from the chronic ethanol feeding experiment. RNA-Seq was conducted on femoral shaft RNA isolated from male mice. A two-way ANOVA was conducted for each gene with diet and genotype as the two factors. The test probabilities were not adjusted for the multiplicity of genes. Volcano plots depicting the test probability for each gene against the fold change in gene expression are shown for the main effect of diet (A), the main effect of genotype (B), and the interaction effect between diet and genotype (C). Points above the red horizontal dashed line represent genes with test probabilities P < 0.05. Red points indicate genes with at least a 2-fold change in gene expression and a test probability P < 0.05. (D) Among the 100 genes with the highest expression levels in Nox4 fl/fl mice on the control diet, the change in gene expression caused by ethanol is depicted for the 16 genes that show a significant (P < 0.05) main diet effect. EtOH, ethanol.

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    Fig. 8.

    qRT-PCR gene expression analysis replicates findings from RNA-Seq. Gene expression was determined for RNA isolated from the femur shafts of mice from the chronic ethanol feeding experiments by qRT-PCR. The expression levels were normalized to the concentration of total RNA. The expression levels were determined for total Nox4 mRNA (A), Frzb (B), Myh3 (C), Col2a1 (D), osteocalcin (E), RANKL (F), Nox2 (G), GAPDH (H), and Cyp2s1 (I). 3-way ANOVAs were conducted with sex, diet, and genotype as the three factors. The panels indicate significant main effects. EtOH, ethanol.

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    TABLE 1

    qRT-PCR primers

    GeneGene SymbolForward PrimerReverse Primer
    Nox4 knockout regionNox4acc tct gcc tgc tca ttt ggcct agg ccc aac att tgg tga
    Total Nox4Nox4gga tca cag aag gtc cct agcgtt gag ggc att cac caa gtg
    Nox2Cybbggg atg aat ctc agg cca atgcc gtc cat aca gag tct tc
    β-ActinActbaga tga ccc aga tca tgt ttg agacca gag gca tac agg gac agc
    GAPDHGapdhcat ctt cca gga gcg aga cccct tca agt ggg ccc cg
    RANKLTnfsf11cag cat cgc tct gtt cct gtactg cgt ttt cat gga gtc tca
    Cyp2S1Cyp2s1ttg gca tcc gtt tgc cct atgag aac atc tcg tag gcc tgg
    OsteocalcinBglapagc ctt cat gtc caa gca gga ggac tga ggc tcc aag gta gcg
    Col2a1Col2a1ggc cag gat gcc cga aaa ttacgc acc ctt ttc tcc ctt gt
    FrzbFrzbaca act atg tca tcc ggg cttga cgg tgt ccc ttg gaa tg

Additional Files

  • Figures
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  • Data Supplement

    • Supplemental Figures -

      1. (A)Nox4 and Cyp2s1 mRNA expression were compared in two Nox4 knockout models.

      2. (A)Western blot for detection of nitrotyrosinewith 0 –50 ng of nitrotyrosinebovine serum albumin ...

    • Supplemental Table -

      mRNAseq data set.

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Journal of Pharmacology and Experimental Therapeutics: 373 (3)
Journal of Pharmacology and Experimental Therapeutics
Vol. 373, Issue 3
1 Jun 2020
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Research ArticleToxicology

Independent Effects of Nox4 and Ethanol on Bone Biology

Kim B. Pedersen, Michelle L. Osborn, Alex C. Robertson, Ashlee E. Williams, James Watt, Alexandra Denys, Katrin Schröder and Martin J. Ronis
Journal of Pharmacology and Experimental Therapeutics June 1, 2020, 373 (3) 337-346; DOI: https://doi.org/10.1124/jpet.119.264374

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Research ArticleToxicology

Independent Effects of Nox4 and Ethanol on Bone Biology

Kim B. Pedersen, Michelle L. Osborn, Alex C. Robertson, Ashlee E. Williams, James Watt, Alexandra Denys, Katrin Schröder and Martin J. Ronis
Journal of Pharmacology and Experimental Therapeutics June 1, 2020, 373 (3) 337-346; DOI: https://doi.org/10.1124/jpet.119.264374
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