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Chronic Ethanol Feeding in Mice Decreases Expression of Genes for Major Structural Bone Proteins in a
Kim B Pedersen1, Michelle L Osborn1, Alex C Robertson1
1Department 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.).
Abstract:
Bone loss in response to alcohol intake has previously been hypothesized to be mediated by excessive production of reactive oxygen species via NADPH oxidase (Nox) enzymes. Nox4 is one of several Nox enzymes expressed in bone. We investigated the role of Nox4 in the chondro-osteoblastic lineage of the long bones in mice during normal chow feeding and during chronic ethanol feeding for 90 days. We generated mice with a genotype (PrxCre +/- Nox4 fl/fl) allowing conditional knockout of Nox4 in the limb bud mesenchyme. Adult mice had 95% knockdown of Nox4 expression in the femoral shafts. For mice on regular chow, only whole-body Nox4 knockout mice had clearly increased cortical thickness and bone mineral density in the tibiae. When chronically fed a liquid diet with and without ethanol, conditional Nox4 knockout mice had slightly reduced dimensions of the cortical and trabecular regions of the tibiae (P < 0.1). The ethanol diet caused a significant reduction in cortical bone area and cortical thickness relative to a control diet without ethanol (P < 0.05). The ethanol diet further reduced gene expression of Frizzled related protein (Frzb), myosin heavy chain 3, and several genes encoding collagen and other major structural bone proteins (P < 0.05), whereas the Nox4 genotype had no effects on these genes. In conclusion, Nox4 expression from both mesenchymal and nonmesenchymal cell lineages appears to exert subtle effects on bone. However, chronic ethanol feeding reduces cortical bone mass and cortical gene expression of major structural bone proteins in a Nox4-independent manner. SIGNIFICANCE STATEMENT: Excessive alcohol intake contributes to osteopenia and osteoporosis, with oxidative stress caused by the activity of NADPH oxidases hypothesized to be a mediator. We tested the role of NADPH oxidase (Nox) 4 in osteoblast precursors in the long bones of mice with a conditional Nox4 knockout model. We found that Nox4 exerted effects independent of alcohol intake, and ethanol effects on bone were Nox4-independent.
Insights
This study found that while Nox4 has minor effects on bone, chronic alcohol consumption significantly reduces bone mass and structural protein gene expression independently of Nox4. Alcohol
Area of Science:
- Bone Biology
- Oxidative Stress
- Alcohol-Induced Bone Loss
Background:
- Alcohol intake is linked to bone loss, potentially via reactive oxygen species from NADPH oxidase (Nox) enzymes.
- Nox4 is a key Nox enzyme expressed in bone cells.
Purpose of the Study:
- To investigate the role of Nox4 in the chondro-osteoblastic lineage during normal and chronic ethanol feeding in mice.
- To determine if Nox4 mediates alcohol-induced bone loss.
Main Methods:
- Generated conditional Nox4 knockout mice (PrxCre +/- Nox4 fl/fl) for targeted Nox4 reduction in limb bud mesenchyme.
- Administered chronic ethanol feeding for 90 days.
- Assessed bone parameters including cortical thickness, bone mineral density, and gene expression.
Main Results:
- Whole-body Nox4 knockout increased tibial cortical thickness and bone mineral density in mice on regular chow.
- Conditional Nox4 knockout mice showed slightly reduced bone dimensions on ethanol diet.
- Ethanol diet significantly reduced cortical bone area, thickness, and expression of structural bone protein genes (e.g., collagen, Frzb).
- Nox4 genotype did not affect these ethanol-induced changes.
Conclusions:
- Nox4 has subtle effects on bone, independent of alcohol intake.
- Chronic ethanol feeding reduces bone mass and structural protein gene expression in a Nox4-independent manner.
- Oxidative stress from Nox enzymes may not be the primary mediator of alcohol-induced bone loss in this model.
