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Mechanical loading modulates phosphate related genes in rat bone.

ABSTRACT: Mechanical loading determines bone mass and bone structure, which involves many biochemical signal molecules. Of these molecules, Mepe and Fgf23 are involved in bone mineralization and phosphate homeostasis. Thus, we aimed to explore whether mechanical loading of bone affects factors of phosphate homeostasis. We studied the effect of mechanical loading of bone on the expression of Fgf23, Mepe, Dmp1, Phex, Cyp27b1, and Vdr. Twelve-week old female rats received a 4-point bending load on the right tibia, whereas control rats were not loaded. RT-qPCR was performed on tibia mRNA at 4, 5, 6, 7 or 8 hours after mechanical loading for detection of Mepe, Dmp1, Fgf23, Phex, Cyp27b1, and Vdr. Immunohistochemistry was performed to visualise FGF23 protein in tibiae. Serum FGF23, phosphate and calcium levels were measured in all rats. Four-point bending resulted in a reduction of tibia Fgf23 gene expression by 64% (p = 0.002) and a reduction of serum FGF23 by 30% (p<0.001), six hours after loading. Eight hours after loading, Dmp1 and Mepe gene expression increased by 151% (p = 0.007) and 100% (p = 0.007). Mechanical loading did not change Phex, Cyp27b1, and Vdr gene expression at any time-point. We conclude that mechanical loading appears to provoke both a paracrine as well as an endocrine response in bone by modulating factors that regulate bone mineralization and phosphate homeostasis.

SUBMITTER: Nepal AK

PROVIDER: S-EPMC9990935 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Mechanical loading modulates phosphate related genes in rat bone.

Nepal Ashwini Kumar AK van Essen Hubertus W HW Reijnders Christianne M A CMA Lips Paul P Bravenboer Nathalie N

PloS one 20230307 3

Mechanical loading determines bone mass and bone structure, which involves many biochemical signal molecules. Of these molecules, Mepe and Fgf23 are involved in bone mineralization and phosphate homeostasis. Thus, we aimed to explore whether mechanical loading of bone affects factors of phosphate homeostasis. We studied the effect of mechanical loading of bone on the expression of Fgf23, Mepe, Dmp1, Phex, Cyp27b1, and Vdr. Twelve-week old female rats received a 4-point bending load on the right ...[more]

PMID: 36881582

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Project description:It is estimated that more than 90% of human genes express multiple mRNA transcripts due to alternative splicing. Consequently, the proteins produced by different splice variants will likely have different functions and expression levels. Several genes with splice variants are known in bone, with functions that affect osteoblast function and bone formation. The primary goal of this study was to evaluate the extent of alternative splicing in a bone subjected to mechanical loading and subsequent bone formation. We used the rat forelimb loading model, in which the right forelimb was loaded axially for 3 min, while the left forearm served as a non-loaded control. Animals were subjected to loading sessions every day, with 24 h between sessions. Ulnae were sampled at 11 time points, from 4 h to 32days after beginning loading. RNA was isolated and mRNA abundance was measured at each time point using Affymetrix exon arrays (GeneChip® Rat Exon 1.0 ST Arrays). An ANOVA model was used to identify potential alternatively spliced genes across the time course, and five alternatively spliced genes were validated with qPCR: Akap12, Fn1, Pcolce, Sfrp4, and Tpm1. The number of alternatively spliced genes varied with time, ranging from a low of 68 at 12h to a high of 992 at 16d. We identified genes across the time course that encoded proteins with known functions in bone formation, including collagens, matrix proteins, and components of the Wnt/?-catenin and TGF-? signaling pathways. We also identified alternatively spliced genes encoding cytokines, ion channels, muscle-related genes, and solute carriers that do not have a known function in bone formation and represent potentially novel findings. In addition, a functional characterization was performed to categorize the global functions of the alternatively spliced genes in our data set. In conclusion, mechanical loading induces alternative splicing in bone, which may play an important role in the response of bone to mechanical loading.

Project description:The advent of high-throughput measurements of gene expression and bioinformatics analysis methods offers new ways to study gene expression patterns. The primary goal of this study was to determine the time sequence for gene expression in a bone subjected to mechanical loading, during key periods of the bone formation process, including expression of matrix-related genes, the appearance of active osteoblasts, and bone desensitization. A standard model for bone loading was employed in which the right forelimb was loaded axially for three minutes per day, while the left forearm served as a non-loaded, contralateral control. We evaluated loading-induced gene expression over a time course of 4 hours to 32 days after the first loading session. Six distinct, time-dependent patterns of gene expression were identified over the time course and categorized into three primary clusters: genes upregulated early in the time course, genes upregulated during matrix formation, and genes downregulated during matrix formation. Genes were then grouped based on function and/or signaling pathways. Many gene groups known to be important in loading-induced bone formation were identified within the clusters, including AP-1-related genes in the early response cluster, matrix-related genes in the upregulated gene clusters, and Wnt/?-catenin signaling pathway inhibitors in the downregulated gene clusters. Several novel gene groups were identified as well, including chemokine-related genes which were upregulated early but downregulated later in the time course, solute carrier genes which were both up- and downregulated, and muscle-related genes which were primarily downregulated. Time Course with 11 time points, each plus & minus mechanical stimulation with 5 replicates per experimental group (except 12d group which has 4 replicates). Daily mechanical loading was applied to the forearm (24 hours between loading sessions), and ulnae were sampled at indicated time points (4h, 12h, 1d, 2d, 4d, 6d, 8d, 12d, 16d, 24d, or 32d).

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Mechanical loading modulates phosphate related genes in rat bone.

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Mechanical loading modulates phosphate related genes in rat bone.

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