Mechanical loading modulates phosphate related genes in rat bone

Ashwini Kumar Nepal; Hubertus W. van Essen; Christianne M. A. Reijnders; Paul Lips; Nathalie Bravenboer

doi:10.1371/journal.pone.0282678

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.

Citation: Nepal AK, van Essen HW, Reijnders CMA, Lips P, Bravenboer N (2023) Mechanical loading modulates phosphate related genes in rat bone. PLoS ONE 18(3): e0282678. https://doi.org/10.1371/journal.pone.0282678

Editor: Damian Christopher Genetos, University of California Davis, UNITED STATES

Received: August 2, 2022; Accepted: February 20, 2023; Published: March 7, 2023

Copyright: © 2023 Nepal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the manuscript and its Supporting Information files.

Funding: The author(s) received no specific funding for this work.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The ability of the skeleton to form bone after mechanical stimulation is a process that consists of mechanosensing, mechanotransduction and mechanoresponsiveness [1, 2]. Osteocytes function as mechanosensors via canalicular processes and communicating gap junctions in the early stage of bone remodelling [3]. Mechanotransduction involves biochemical signal molecules, which are produced after mechanical loading in a distinct temporal manner. Seconds after mechanical loading, the osteocytic production of factors such as NO and prostaglandins is induced [4]. Within two hours after a single bout of loading, Cfos mRNA expression is up regulated in the periosteum of the rat tibia [5]. Within a time frame of four hours after loading, osteocyte production of IGF-1 increased [6]. After several months, the cascade of biochemical signalling stimulates osteoblastic bone formation as a mechanoresponsive process that finally results in higher bone mass.

Bone mineralization is one of the various biological processes that govern phosphate homeostasis. Classic regulators of phosphate homeostasis are1,25-dihydroxyvitamin D3 (1,25(OH)₂D₃) and parathyroid hormone (PTH). In addition, it has been shown that fibroblast growth factor 23 (FGF23) is a regulator of phosphate homeostasis [7–9]. FGF23 is mainly produced by bone cells, such as osteocytes and osteoblasts [10] but recent studies have shown that Fgf23 expression was also present in bone marrow sinusoidal endothelial cells [11]. FGF23 can be detected in serum in an intact form but also the c-terminal and n-terminal fragments exist as a result of cleavage by furin proteases [12]. FGF23 predominantly exerts its effects in the kidney, where it inhibits the expression of 1-α-hydroxylase (encoded by the gene Cyp27b1) and decreases the production of 1,25(OH)₂D₃ [13]. Furthermore, FGF23 inhibits the expression of sodium/phosphate co-transporters (NPT2a and NPT2c) that concurrently results in increased phosphate excretion [14]. In the parathyroid gland, FGF23 inhibits PTH production [15, 16]. The production of FGF23 is stimulated by 1,25(OH)₂D₃ signalling through the vitamin D receptor (VDR) [13, 17] and by phosphate [18], but in addition appears to be influenced by local factors in bone such as matrix extracellular phosphoglycoprotein (MEPE), Dentin matrix protein-1 (DMP1) and phosphate regulating gene with homologies to endopeptidases on the X-chromosome (PHEX). DMP1, MEPE and PHEX all affect bone mineralization. Initially, DMP1 locally promotes deposition of hydroxyapatite [19, 20]. Cathepsin B is able to cleave a peptide (ASARM peptide) from MEPE, which is known to locally inhibit bone mineralization. Finally, PHEX protein prevents the formation of ASARM peptide [21].

Previous studies revealed that Mepe mRNA expression was up-regulated six hours after mechanical loading of the tibia [22] while Fgf23 gene expression was downregulated six hours after axial compression loading of the ulna [23]. Other investigators have agreed that mechanical loading of bone increased the expression of Mepe [24, 25] and Dmp1 [26]. These findings suggest that bone mineralization can be regulated by mechanical loading, possibly to obtain newly formed bone with optimal quality. However it is not clear whether the immediate effect of mechanical loading on these locally produced factors results in systemic changes in phosphate homeostasis. Therefore we investigated the effect of four-point bending of the rat tibia on gene expression in bone of the phosphate homeostasis related genes Mepe, Dmp1, Phex, Fgf23, on the protein expression of FGF23 in bone tissue and on the FGF23 concentration in serum. We also analyzed the gene expression of vitamin D related genes Vdr and Cyp27b1after mechanical loading. We hypothesised that mechanical loading of bone affects expression of Fgf23 which results in a change in serum concentration of FGF23.

Materials and methods

Experimental animals

Two experiments were performed to detect changes in mRNA and protein expression after mechanical loading. The animal experiments were in accordance with the governmental guidelines for care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee (IACUC) of the VU University Medical Center, Amsterdam, The Netherlands (KE 02–03).

Experiment A.

The first experiment aimed to detect the effect of mechanical loading on the mRNA expression of selected genes involved in phosphate homeostasis. Sixty 12-week-old female Wistar rats (Harlan, Horst, The Netherlands), weighing 226 ± 8 grams (mean ± SD) were randomly assigned to one of six groups (n = 10). The rats in the control group did not receive mechanical loading. The rats in the five load groups received a single bout of mechanical loading and were sacrificed 4, 5, 6, 7 or 8 hours after loading. From all rats, serum was obtained from blood at sacrifice. Moreover, tibiae were dissected and cleaned of soft tissue and periosteum. The proximal and distal ends of the tibia were removed, after which bone marrow was flushed out of the remaining diaphysis with RNAse free water. Lastly, the diaphyses were frozen in liquid nitrogen and stored at -80°C until RNA extraction.

Experiment B.

In the second experiment, we tested whether changes in gene expression would be detectable at the protein level. Thirty-two 12 weeks-old female Wistar rats, weighing 218 ± 15 grams (mean ± SD) were randomly assigned to a single load group of 14 rats, a repeated-load group of 9 rats and a control group of 9 rats. The rats in the load group received a single bout of mechanical loading and were sacrificed 6 hours after loading. The rats in the repeated-load group underwent a single bout of mechanical loading, five days a week, for two weeks. Six hours after the final loading bout, the rats were sacrificed. The rats in the control group received no mechanical loading. The tibiae of all rats in the repeated-load group and five rats in the single load group were dissected, cleaned of soft tissue and prepared for immunohistochemistry. From all rats serum was obtained by cardiac puncture at sacrifice.

Mechanical loading

One bout of mechanical loading consisted of bending the right tibia of each rat externally in a four-point bending device for 300 cycles with a frequency of 2 Hz and a peak load of 60 N in the single load groups or 50 N in the repeated-load group [6, 27]. Loading was applied under general anaesthesia (2%-2.5% isoflurane/N₂O/O₂) and the rats received local subcutaneous analgesia (0.1 mg/kg Temgesic) prior to loading. The left tibia served as a non-loaded contra-lateral control. At the indicated time points the rats were sacrificed with CO₂ gas. A cardiac puncture was performed to collect terminal blood samples, serum was obtained and stored at –20°C.

RNA extraction and RT-qPCR

RNA isolation from the tibiae from experiment A was performed by pulverizing the diaphysis with a freezer mill in liquid nitrogen (Spex Certiprep 6750 FreezerMill, Spex Certiprep, Metuchen, NJ, USA). RNA was extracted from bone powder with Trizol for 1 hour at 37°C and re-extracted once with phenol and once with chloroform. Next, the samples were extracted again with Trizol in accordance with the manufacturer’s instructions (Invitrogen, Waltham, MA, USA). RNA extracts were incubated with DNAse to eliminate DNA contamination and stored at –80°C. The absorption at 260 and 280 nm was measured to determine the amount of RNA. A total of 100 ng of total RNA was transcribed into cDNA in a 20 μl mixture containing M-MLV Reverse Transcriptase (Promega, Fitchburg, WI, USA). All RNA samples were assayed in triplicate. Quantitative PCR was performed in a BioRad iCycler Real-time PCR system with three μl of each cDNA sample, 300 nM of each primer and SYBR Green Supermix (BioRad, Hercules, CA, USA) in a total volume of 25 μl. Table 1 lists the primer details of the genes of interest, which were Fgf23, Mepe, Dmp1, Phex, Cyp27b1 and Vdr and the housekeeping genes, which were hypoxanthine guanine phosphoribosyl transferase (Hprt) and porphobilinogen deaminase (Pbgd). After the PCR run a melting curve was conducted from 50°C to 95°C to check the specificity of the reactions. Mean Ct-values of each RNA sample assayed in triplicate were used. Gene expression for the genes of interest was normalized for the housekeeping genes ΔCt = (Ctgene of interest−Ct_{housekeeping gene)} and expressed as fold difference from the average of the housekeeping genes (2^-ΔCt) [28].

Download:

Table 1. Primer sequences for genes used in qPCR analyses.

https://doi.org/10.1371/journal.pone.0282678.t001

Tissue preparation and immunohistochemistry for FGF23

After dissection, the condyles at the proximal end of the tibiae were sawn off with a diamond saw and the distal end was cut off with scissors. The tibiae were immediately fixed for 24 hours at 4°C in 4% paraformaldehyde in phosphate buffered saline (PBS). After fixation the tibiae were decalcified in 10% EDTA and 0.5% paraformaldehyde in PBS at 4°C for 4 weeks. Proper decalcification was verified by x-ray photography. The tibiae were then dehydrated and embedded in paraffin and cut into 5 μm thick sections. Sections were rehydrated and endogenous peroxidase was quenched with 3% H₂O₂ in 40% methanol/PBS. Antigen retrieval was performed through incubation with 0.5% trypsin + 0.1% CaCl2 for 15 minutes at 37°C. After the blocking of the non-specific binding sites with blocking solution (Invitrogen, Waltham, MA, USA) for 1 hour, the sections were incubated overnight at 4°C with 1/1000 anti-intact FGF23 antibody (Santa Cruz Biotechnology Inc, Dallas, TX, USA). The sections were incubated with 1/100 biotinylated second antibody (Dako, Agilent Technologies, Inc., Santa Clara, CA, USA). Further enhancement was performed with the Tyramide Signal Amplification kit (Invitrogen, Waltham, MA, USA) and color development was performed with AEC (Zymed technologies, Waltham, MA, USA). The sections were counterstained with Mayer’s hematoxylin. Quantitative evaluation of the specific staining for FGF23 was performed with NIS Elements digital imaging software (Nikon, Tokyo, Japan). Digital images were made from the medial cortex of two sections of each tibia at 10x magnification. The area of positively-stained osteocytes, the area of positively-stained capillairy bloodvessels and the total bone area was measured. The percentage of positively-stained bone area and the percentage of positively stained vessel area were calculated. Data from the two sections of each tibia were averaged.

Serum analysis

In sera of all rats from the two experiments, intact FGF23 concentration was measured by ELISA (Kainos Laboratories, Tokyo, Japan) as described in Heijboer et al [29]. Serum inorganic phosphorus and calcium were analyzed using a Modular P800 automatic analyzer (Roche, Mannheim, Germany).

Statistical analysis

For statistical analysis of qPCR expression, data were log transformed to obtain a normal distribution. Differences in expression between the loaded tibiae and the non-loaded tibiae in all groups were tested with one-way analysis of variance (ANOVA) and Dunnet’s post-hoc test was used to test for significant differences between the time groups and the control group. A non-parametric paired t-test (Wilcoxon) was used to test for significant differences in immunohistochemical staining. Statistical analysis of the serum data was performed with one-way ANOVA. Results were considered significantly different at p < 0.05. Graphpad 4.0 (Graphpad Software, San Diego, CA, USA) was used for statistical analysis.

Results

Gene expression

Experiment A evaluated the effect of mechanical loading on gene expression (Fig 1A–1F) shows the normalized mRNA expression of, Mepe, Dmp1, Fgf23, Phex, Cyp27b1 and Vdr in the loaded and non-loaded tibiae. To avoid the possibility that systemic loading effects on the left non-loaded tibia would interfere with the analysis, the normalized expression of the right-loaded tibiae in the time groups was compared with the normalized expression in the right-control tibiae. The expression of Mepe and Dmp1 was increased at 5, 6 and 8 hours after mechanical loading, which was at a maximum of 103% for Mepe (p = 0.007) and 151% for Dmp1 (p = 0.007) eight hours after loading (Fig 1A and 1B). Fgf23 gene expression was decreased at 4, 6 and 7 hours after mechanical loading and the maximum reduction was 64% (p = 0.002), 7 hours after loading (Fig 1C). The expression of the other genes, Phex, Vdr and Cyp27b1 was not different in the right-loaded tibiae of the time groups compared to the expression in the right control tibia (Fig 1D–1F). No difference was observed in gene expression for any of the tested genes when the left tibiae from the loaded and control groups were compared.

Download:

Fig 1.

(A-F): Box whisker plots of gene expression of Mepe, Dmp1, Fgf23, Phex, Cyp27b1 and Vdr in the contra-lateral control (left) and loaded tibiae (right). Rats underwent a single bout of mechanical loading of the right tibia and were sacrificed after the indicated time. Rats in the control group underwent no mechanical loading. The box represents median, 25th and 75th percentiles, and the whiskers represent highest and lowest gene expression values. For statistical analysis the data were log transformed. *p<0.05 versus right tibia in the control group. **p<0.01 versus right tibia in the control group.

https://doi.org/10.1371/journal.pone.0282678.g001

Immunohistochemistry

To test whether the effect on gene expression was also detectable at the protein level, FGF23 protein was quantified in bone tissue by immunohistochemistry. FGF23 positive staining was observed in the osteocytes of the cortex and in the capillary blood vessels within the cortical bone. Weak staining of osteoblasts on the endocortical side of the cortex was detected. Osteocytes in trabecular bone only sporadically showed positive staining. Measurement of the area of FGF23 positive staining in osteocytes in the cortical bone of the tibiae did not reveal significant differences between the loaded tibiae and the contra-lateral control tibiae after single loading or after repeated loading. Likewise there was no significant difference in FGF23 positive staining of the capillary blood vessels between the loaded tibiae and the contralateral control tibiae. (Fig 2A–2E).

Download:

Fig 2.

(A-E): Quantitative analysis of immunohistochemistry. Rats received a single bout of mechanical loading of the right tibia (A and B) or repeated loading of the right tibia (C and D). Paraffin sections of the tibiae were cut and stained for FGF23. The FGF23 positive area in as a percentage of the total bone area was measured in osteocytes (A and C) and in capillary blood vessels (B and D). (E) Representative image of FGF23 positive staining in osteocytes (arrowheads) and in capillary blood vessels (arrows).

https://doi.org/10.1371/journal.pone.0282678.g002

Serum analysis

To further investigate the effect of mechanical loading on protein expression of FGF23, serum concentrations of FGF23, phosphate and calcium were measured in the control rats, the single-loaded rats and the repeated-loaded rats. Six hours after a single bout of mechanical loading, serum FGF23 concentration reduced significantly in loaded rats, compared to control rats (110 ± 7 pg/mL vs 157 ± 9 pg/mL: p<0.001) (Fig 3A). Serum FGF23 concentration did not change in repeated-loaded rats, compared to control rats (153 ± 10 pg/mL). Mechanical loading did not change the serum concentrations of inorganic phosphate (Fig 3B) and calcium (data not shown).

Download:

Fig 3.

(A and B): Serum concentration of intact FGF23 (A) and inorganic phosphate (B). Rats in the control group (n = 19) received no mechanical loading. Rats in the 1x Load group (n = 24) received a single bout of mechanical loading of the right tibia and were sacrificed 6 hours later. Rats in the 10x Load group (n = 9) received a single bout of mechanical loading of the right tibia every workday for two weeks and were sacrificed 6 hours after the last bout of mechanical loading. Blood samples were collected at sacrifice and FGF23 and inorganic phosphate were measured in serum. Values are means ± SD. ^**p<0.001.

https://doi.org/10.1371/journal.pone.0282678.g003

Discussion

Our results demonstrated that mechanical loading increased expression of Mepe and Dmp1, and decreased Fgf23, whereas mechanical loading showed no effects on expression of Phex, Vdr and Cyp27b1. FGF23 protein expression in bone and in capillary blood vessels measured by immunohistochemistry was not affected six hours after loading nor after repeated loading for 10 days. Loading decreased FGF23 in serum at 6 hours after loading, however, this decrease did not persist after repeated loading for 10 days. Loading did not affect serum levels of phosphate and calcium.

The decreased expression of Fgf23 confirms earlier results in a rat axial ulna loading study where a decrease of Fgf23 expression 6 hours after a single bout of loading was found [23]. There are only a few studies that investigated the effect of mechanical loading on the expression of Fgf23 and phosphate homeostasis. These studies have performed controlled mechanical loading in the form of axial compression of the rat ulna [30] or axial compression of the mouse tibia [31]. They reported that gene expression of Fgf23 decreased after several days of repeated mechanical loading. A different mouse training model was performed by Gardinier et al [32]. Here the mice exercised 30 minutes every day in a treadmill. Fgf23 gene expression was increased after 6 days of training, but not after 2 or 4 days of training.

We observed a strong staining for FGF23 in osteocytes in the cortex of the tibia by immunohistochemistry. However, strong staining was also observed in capillary blood vessels within the cortical bone. This could represent FGF23 being secreted by osteocytes into the circulation, but it could also confirm reports showing FGF23 expression in endothelial cells of small blood vessels [11]. We did not find an effect of mechanical loading on the protein expression in cortical bone, both in osteocytes and in capillary blood vessels. The discrepancy between mRNA and protein results could be due to methodological limitations. Immunohistochemistry is at best a semi-quantitative method. Automatically-quantified positive area was used to measure bone FGF23 protein expression. Other methods, such as counting positive cells or measuring the color density, might be more accurate but are technically still unfeasible. Moreover, immunohistochemical staining for FGF23 exhibited large variation, which suggests that a larger number of animals might be needed to detect local bone changes in FGF23 protein.

A significant decrease of the FGF23 concentration in serum was observed six hours after mechanical loading but this decrease in serum FGF23 was not observed after 10 days of repeated loading. Our results suggest that the changes in mechanical loading of the skeleton quickly and substantially affected systemic FGF23. A comparably quick response in serum FGF23 has been shown after injection of mice with 1,25(OH)2D. This resulted in an 1.6-fold increase in serum FGF23 concentration within four hours [33]. Likewise, a single injection of mice with erythropoietin can also lead to a 2–4 times increase in serum intact FGF23 within 4–6 hours after injection [34]. Nevertheless, the systemic changes observed in serum FGF23 by loading of a single tibia only at 6 hours could be arbitrary and not an outcome of loading. Contrary to our expectations, it was observed that when bone was subjected to two weeks of daily mechanical loading, the serum FGF23 concentration was not different from controls, suggesting activation of a negative feedback mechanism to restore the serum FGF23 concentration to its original level after its initial decrease [14]. Lower levels of serum FGF23 would increase phosphate retention [9]. Nevertheless we did not find a change in serum phosphate concentration. A likely explanation would be that negative feedback mechanisms could be activated to maintain serum phosphate concentration within narrow boundaries by modulating bone metabolism, phosphate absorption in the gut and phosphate excretion by the kidney [35]. Hence, changes in phosphate metabolism through actions of FGF23 are possibly detectable more precisely in urinary phosphate excretion than in serum phosphate levels.

In the present study, mechanical loading increased the expression of Mepe and Dmp1 while the expression of Phex did not change. This confirms earlier findings that Mepe [22, 24, 25] and Dmp1 [20, 26] expression was increased after mechanical loading. Increased DMP1 production promotes bone mineralization. However, increased MEPE/PHEX ratio might lead to a higher formation of ASARM peptides and these are associated with inhibition of the mineralization process. DMP1 and PHEX are known to inhibit FGF23 production [36]. A single bout of mechanical loading decreased Fgf23 mRNA expression as early as 4 hours after mechanical loading while increases of Mepe and Dmp1 started at 5 hours after loading. This suggests that the change in Fgf23 expression was not the result of increases in MEPE or DMP1 expression.

One finding is that mechanical loading did not change the expression of Cyp27b1 and Vdr within the studied time frame of 8 hours. This is in line with previous results in which we did not find a differences in Cyp27b1 expression, 6 hours after in vivo axial mechanical loading of the ulna [23]. However, an in vitro study reported a 2-fold increase in Cyp27b1 and 0.6 fold decrease in Vdr, 3 hours after mechanical loading, applied as a pulsating fluid flow [37]. 1,25(OH)2D interacts with mechanical loading in vitro at the level of mechanotransduction, in which modulation of the mechanoresponse occurs via a mechanism which is independent of VDR genomic actions [38].

We demonstrated that a single bout of mechanical loading decreased gene expression of Fgf23 in the loaded bone and at the same time decreased serum levels of FGF23. However we could not detect changes in protein expression by immunohistochemistry in the loaded bone directly after loading. The most obvious reason is that changes in protein production in the osteocytes and endothelial cells will be detectable at a later time point, but it is also possible that downstream processing of mRNA is influenced by mechanical loading. We did see a decrease in FGF23 concentration is serum. The ELISA method we performed to measure FGF23 in serum detects only intact FGF23. Therefore the decrease that was detected six hours after loading could be caused by a genuine quick decrease in protein production by the osteocytes but also by increased cleavage of the intact protein. The responses to acute mechanical loading seem to have disappeared when loading was applied for 10 days in a row. This may be the result of a feedback mechanism, possibly involving 1,25(OH)2D [17].

A limitation of this study is that we only performed immunohistochemistry in the repeated-loading experiment and we did not have a group for RNA extraction from bone, therefore we could not test Fgf23 gene expression after ten bouts of mechanical loading. A second limitation of this study is that we did not measure phosphate concentration in urine. With the changes in FGF23 concentration one would expect changes in phosphate retention but we could not conclude this from the serum phosphate results. Urinary phosphate concentration would more likely reflect changes in phosphate homeostasis. In this study we showed changes in phosphate related gene expression for Mepe, Fgf23 and Dmp1 at different time points after loading and decreased serum FGF23, 6 hours after loading, these results do not show a direct effect on bone mineralization, as bone adaptation is expected after days or weeks and not within the timeframe of this study.

Taken together, this study shows that mechanical loading of bone quickly decreases Fgf23 gene expression and simultaneously decreases serum FGF23 concentration. Decreased FGF23 can lead to retention of phosphate and increased production of 1,25(OH)2D. This indicates that mechanical loading plays a role in the maintenance of serum phosphate concentration. We demonstrated that mechanical loading induces alterations in expression of Mepe and Dmp1, which suggests that there are local influences of mechanical loading on bone mineralization. In conclusion, mechanical loading appears to provoke both paracrine as well as endocrine response in bone by modulating factors which regulate bone mineralization and phosphate homeostasis.

Supporting information

S1 File. Result Immunhistochemistry.

Percentage of FGF23 positive area in bone and vessels after single load or repeated load.

https://doi.org/10.1371/journal.pone.0282678.s001

(XLSX)

S2 File. Result gene expression.

Normalized expression of measured genes in the left and right tibias at the timepoints.

https://doi.org/10.1371/journal.pone.0282678.s002

(XLSX)

S3 File. Result serum measurements.

Serum concentrations of FGF23, phosphate and calcium.

https://doi.org/10.1371/journal.pone.0282678.s003

(XLSX)

Acknowledgments

The authors thank Josien Dijkstra-Lagemaat for excellent technical assistance with the determination of FGF23 concentration in serum.

References

1. Cowin SC, Moss-Salentijn L, Moss ML. Candidates for the mechanosensory system in bone. J Biomech Eng. 1991;113(2):191–7. pmid:1875693
- View Article
- PubMed/NCBI
- Google Scholar
2. Turner CH, Pavalko FM. Mechanotransduction and functional response of the skeleton to physical stress: the mechanisms and mechanics of bone adaptation. J Orthop Sci. 1998;3(6):346–55. pmid:9811988
- View Article
- PubMed/NCBI
- Google Scholar
3. Schaffler MB, Cheung WY, Majeska R, Kennedy O. Osteocytes: master orchestrators of bone. Calcif Tissue Int. 2014;94(1):5–24. pmid:24042263
- View Article
- PubMed/NCBI
- Google Scholar
4. Bakker AD, Soejima K, Klein-Nulend J, Burger EH. The production of nitric oxide and prostaglandin E(2) by primary bone cells is shear stress dependent. J Biomech. 2001;34(5):671–7. pmid:11311708
- View Article
- PubMed/NCBI
- Google Scholar
5. Raab-Cullen DM, Thiede MA, Petersen DN, Kimmel DB, Recker RR. Mechanical loading stimulates rapid changes in periosteal gene expression. Calcif Tissue Int. 1994;55(6):473–8. pmid:7895187
- View Article
- PubMed/NCBI
- Google Scholar
6. Reijnders CM, Bravenboer N, Tromp AM, Blankenstein MA, Lips P. Effect of mechanical loading on insulin-like growth factor-I gene expression in rat tibia. J Endocrinol. 2007;192(1):131–40. pmid:17210750
- View Article
- PubMed/NCBI
- Google Scholar
7. Liu S, Quarles LD. How fibroblast growth factor 23 works. J Am Soc Nephrol. 2007;18(6):1637–47. pmid:17494882
- View Article
- PubMed/NCBI
- Google Scholar
8. Nabeshima Y. The discovery of alpha-Klotho and FGF23 unveiled new insight into calcium and phosphate homeostasis. Cell Mol Life Sci. 2008;65(20):3218–30. pmid:18726073
- View Article
- PubMed/NCBI
- Google Scholar
9. Bergwitz C, Jüppner H. Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23. Annu Rev Med. 2010;61:91–104. pmid:20059333
- View Article
- PubMed/NCBI
- Google Scholar
10. Yoshiko Y, Wang H, Minamizaki T, Ijuin C, Yamamoto R, Suemune S, et al. Mineralized tissue cells are a principal source of FGF23. Bone. 2007;40(6):1565–73. pmid:17350357
- View Article
- PubMed/NCBI
- Google Scholar
11. Heil J, Olsavszky V, Busch K, Klapproth K, de la Torre C, Sticht C, et al. Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation. Nat Commun. 2021;12(1):6963. pmid:34845225
- View Article
- PubMed/NCBI
- Google Scholar
12. Mattoo RL. The Roles of Fibroblast Growth Factor (FGF)-23, alpha-Klotho and Furin Protease in Calcium and Phosphate Homeostasis: A Mini-Review. Indian J Clin Biochem. 2014;29(1):8–12.
- View Article
- Google Scholar
13. Lanske B, Densmore MJ, Erben RG. Vitamin D endocrine system and osteocytes. Bonekey Rep. 2014;3:494. pmid:24605211
- View Article
- PubMed/NCBI
- Google Scholar
14. Andrukhova O, Zeitz U, Goetz R, Mohammadi M, Lanske B, Erben RG. FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1/2-SGK1 signaling pathway. Bone. 2012;51(3):621–8. pmid:22647968
- View Article
- PubMed/NCBI
- Google Scholar
15. Ben-Dov IZ, Galitzer H, Lavi-Moshayoff V, Goetz R, Kuro-o M, Mohammadi M, et al. The parathyroid is a target organ for FGF23 in rats. J Clin Invest. 2007;117(12):4003–8. pmid:17992255
- View Article
- PubMed/NCBI
- Google Scholar
16. Shimada T, Hasegawa H, Yamazaki Y, Muto T, Hino R, Takeuchi Y, et al. FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res. 2004;19(3):429–35. pmid:15040831
- View Article
- PubMed/NCBI
- Google Scholar
17. Kolek OI, Hines ER, Jones MD, LeSueur LK, Lipko MA, Kiela PR, et al. 1alpha,25-Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: the final link in a renal-gastrointestinal-skeletal axis that controls phosphate transport. Am J Physiol Gastrointest Liver Physiol. 2005;289(6):G1036–42. pmid:16020653
- View Article
- PubMed/NCBI
- Google Scholar
18. Vervloet MG, van Ittersum FJ, Büttler RM, Heijboer AC, Blankenstein MA, ter Wee PM. Effects of dietary phosphate and calcium intake on fibroblast growth factor-23. Clin J Am Soc Nephrol. 2011;6(2):383–9. pmid:21030580
- View Article
- PubMed/NCBI
- Google Scholar
19. Huang B, Maciejewska I, Sun Y, Peng T, Qin D, Lu Y, et al. Identification of full-length dentin matrix protein 1 in dentin and bone. Calcif Tissue Int. 2008;82(5):401–10. pmid:18488132
- View Article
- PubMed/NCBI
- Google Scholar
20. Yang W, Lu Y, Kalajzic I, Guo D, Harris MA, Gluhak-Heinrich J, et al. Dentin matrix protein 1 gene cis-regulation: use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo. J Biol Chem. 2005;280(21):20680–90. pmid:15728181
- View Article
- PubMed/NCBI
- Google Scholar
21. Rowe PS. Regulation of bone-renal mineral and energy metabolism: the PHEX, FGF23, DMP1, MEPE ASARM pathway. Crit Rev Eukaryot Gene Expr. 2012;22(1):61–86. pmid:22339660
- View Article
- PubMed/NCBI
- Google Scholar
22. Reijnders CM, van Essen HW, van Rens BT, van Beek JH, Ylstra B, Blankenstein MA, et al. Increased expression of matrix extracellular phosphoglycoprotein (MEPE) in cortical bone of the rat tibia after mechanical loading: identification by oligonucleotide microarray. PLoS One. 2013;8(11):e79672. pmid:24255709
- View Article
- PubMed/NCBI
- Google Scholar
23. Nepal AK, van Essen HW, van der Veen AJ, van Wieringen WN, Stavenuiter AWD, Cayami FK, et al. Mechanical stress regulates bone regulatory gene expression independent of estrogen and vitamin D deficiency in rats. J Orthop Res. 2021;39(1):42–52. pmid:32530517
- View Article
- PubMed/NCBI
- Google Scholar
24. Kulkarni RN, Bakker AD, Everts V, Klein-Nulend J. Inhibition of osteoclastogenesis by mechanically loaded osteocytes: involvement of MEPE. Calcif Tissue Int. 2010;87(5):461–8. pmid:20725825
- View Article
- PubMed/NCBI
- Google Scholar
25. Gluhak-Heinrich J, Pavlin D, Yang W, MacDougall M, Harris SE. MEPE expression in osteocytes during orthodontic tooth movement. Arch Oral Biol. 2007;52(7):684–90. pmid:17270144
- View Article
- PubMed/NCBI
- Google Scholar
26. Gluhak-Heinrich J, Ye L, Bonewald LF, Feng JQ, MacDougall M, Harris SE, et al. Mechanical loading stimulates dentin matrix protein 1 (DMP1) expression in osteocytes in vivo. J Bone Miner Res. 2003;18(5):807–17. pmid:12733719
- View Article
- PubMed/NCBI
- Google Scholar
27. Forwood MR, Bennett MB, Blowers AR, Nadorfi RL. Modification of the in vivo four-point loading model for studying mechanically induced bone adaptation. Bone. 1998;23(3):307–10. pmid:9737355
- View Article
- PubMed/NCBI
- Google Scholar
28. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):Research0034. pmid:12184808
- View Article
- PubMed/NCBI
- Google Scholar
29. Heijboer AC, Levitus M, Vervloet MG, Lips P, ter Wee PM, Dijstelbloem HM, et al. Determination of fibroblast growth factor 23. Ann Clin Biochem. 2009;46(Pt 4):338–40. pmid:19564163
- View Article
- PubMed/NCBI
- Google Scholar
30. Mantila Roosa SM, Liu Y, Turner CH. Gene expression patterns in bone following mechanical loading. J Bone Miner Res. 2011;26(1):100–12. pmid:20658561
- View Article
- PubMed/NCBI
- Google Scholar
31. Chermside-Scabbo CJ, Harris TL, Brodt MD, Braenne I, Zhang B, Farber CR, et al. Old Mice Have Less Transcriptional Activation But Similar Periosteal Cell Proliferation Compared to Young-Adult Mice in Response to in vivo Mechanical Loading. J Bone Miner Res. 2020;35(9):1751–64. pmid:32311160
- View Article
- PubMed/NCBI
- Google Scholar
32. Gardinier JD, Al-Omaishi S, Morris MD, Kohn DH. PTH signaling mediates perilacunar remodeling during exercise. Matrix Biol. 2016;52–54:162–75. pmid:26924474
- View Article
- PubMed/NCBI
- Google Scholar
33. Liu S, Tang W, Zhou J, Stubbs JR, Luo Q, Pi M, et al. Fibroblast growth factor 23 is a counter-regulatory phosphaturic hormone for vitamin D. J Am Soc Nephrol. 2006;17(5):1305–15. pmid:16597685
- View Article
- PubMed/NCBI
- Google Scholar
34. Toro L, Barrientos V, Leon P, Rojas M, Gonzalez M, Gonzalez-Ibanez A, et al. Erythropoietin induces bone marrow and plasma fibroblast growth factor 23 during acute kidney injury. Kidney Int. 2018;93(5):1131–41.
- View Article
- Google Scholar
35. Rendenbach C, Yorgan TA, Heckt T, Otto B, Baldauf C, Jeschke A, et al. Effects of extracellular phosphate on gene expression in murine osteoblasts. Calcif Tissue Int. 2014;94(5):474–83. pmid:24366459
- View Article
- PubMed/NCBI
- Google Scholar
36. Martin A, Liu S, David V, Li H, Karydis A, Feng JQ, et al. Bone proteins PHEX and DMP1 regulate fibroblastic growth factor Fgf23 expression in osteocytes through a common pathway involving FGF receptor (FGFR) signaling. FASEB J. 2011;25(8):2551–62. pmid:21507898
- View Article
- PubMed/NCBI
- Google Scholar
37. van der Meijden K, Bakker AD, van Essen HW, Heijboer AC, Schulten EA, Lips P, et al. Mechanical loading and the synthesis of 1,25(OH)2D in primary human osteoblasts. J Steroid Biochem Mol Biol. 2016;156:32–9. pmid:26625962
- View Article
- PubMed/NCBI
- Google Scholar
38. Atkins GJ, Anderson PH, Findlay DM, Welldon KJ, Vincent C, Zannettino AC, et al. Metabolism of vitamin D3 in human osteoblasts: evidence for autocrine and paracrine activities of 1 alpha,25-dihydroxyvitamin D3. Bone. 2007;40(6):1517–28. pmid:17395559
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Cowin SC, Moss-Salentijn L, Moss ML. Candidates for the mechanosensory system in bone. J Biomech Eng. 1991;113(2):191–7. pmid:1875693
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Turner CH, Pavalko FM. Mechanotransduction and functional response of the skeleton to physical stress: the mechanisms and mechanics of bone adaptation. J Orthop Sci. 1998;3(6):346–55. pmid:9811988
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Schaffler MB, Cheung WY, Majeska R, Kennedy O. Osteocytes: master orchestrators of bone. Calcif Tissue Int. 2014;94(1):5–24. pmid:24042263
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Bakker AD, Soejima K, Klein-Nulend J, Burger EH. The production of nitric oxide and prostaglandin E(2) by primary bone cells is shear stress dependent. J Biomech. 2001;34(5):671–7. pmid:11311708
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Raab-Cullen DM, Thiede MA, Petersen DN, Kimmel DB, Recker RR. Mechanical loading stimulates rapid changes in periosteal gene expression. Calcif Tissue Int. 1994;55(6):473–8. pmid:7895187
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Reijnders CM, Bravenboer N, Tromp AM, Blankenstein MA, Lips P. Effect of mechanical loading on insulin-like growth factor-I gene expression in rat tibia. J Endocrinol. 2007;192(1):131–40. pmid:17210750
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Liu S, Quarles LD. How fibroblast growth factor 23 works. J Am Soc Nephrol. 2007;18(6):1637–47. pmid:17494882
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Nabeshima Y. The discovery of alpha-Klotho and FGF23 unveiled new insight into calcium and phosphate homeostasis. Cell Mol Life Sci. 2008;65(20):3218–30. pmid:18726073
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Bergwitz C, Jüppner H. Regulation of phosphate homeostasis by PTH, vitamin D, and FGF23. Annu Rev Med. 2010;61:91–104. pmid:20059333
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Yoshiko Y, Wang H, Minamizaki T, Ijuin C, Yamamoto R, Suemune S, et al. Mineralized tissue cells are a principal source of FGF23. Bone. 2007;40(6):1565–73. pmid:17350357
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Heil J, Olsavszky V, Busch K, Klapproth K, de la Torre C, Sticht C, et al. Bone marrow sinusoidal endothelium controls terminal erythroid differentiation and reticulocyte maturation. Nat Commun. 2021;12(1):6963. pmid:34845225
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Mattoo RL. The Roles of Fibroblast Growth Factor (FGF)-23, alpha-Klotho and Furin Protease in Calcium and Phosphate Homeostasis: A Mini-Review. Indian J Clin Biochem. 2014;29(1):8–12.
View Article
Google Scholar

[46] View Article

[47] Google Scholar

[ref13] 13. Lanske B, Densmore MJ, Erben RG. Vitamin D endocrine system and osteocytes. Bonekey Rep. 2014;3:494. pmid:24605211
View Article
PubMed/NCBI
Google Scholar

[49] View Article

[50] PubMed/NCBI

[51] Google Scholar

[ref14] 14. Andrukhova O, Zeitz U, Goetz R, Mohammadi M, Lanske B, Erben RG. FGF23 acts directly on renal proximal tubules to induce phosphaturia through activation of the ERK1/2-SGK1 signaling pathway. Bone. 2012;51(3):621–8. pmid:22647968
View Article
PubMed/NCBI
Google Scholar

[53] View Article

[54] PubMed/NCBI

[55] Google Scholar

[ref15] 15. Ben-Dov IZ, Galitzer H, Lavi-Moshayoff V, Goetz R, Kuro-o M, Mohammadi M, et al. The parathyroid is a target organ for FGF23 in rats. J Clin Invest. 2007;117(12):4003–8. pmid:17992255
View Article
PubMed/NCBI
Google Scholar

[57] View Article

[58] PubMed/NCBI

[59] Google Scholar

[ref16] 16. Shimada T, Hasegawa H, Yamazaki Y, Muto T, Hino R, Takeuchi Y, et al. FGF-23 is a potent regulator of vitamin D metabolism and phosphate homeostasis. J Bone Miner Res. 2004;19(3):429–35. pmid:15040831
View Article
PubMed/NCBI
Google Scholar

[61] View Article

[62] PubMed/NCBI

[63] Google Scholar

[ref17] 17. Kolek OI, Hines ER, Jones MD, LeSueur LK, Lipko MA, Kiela PR, et al. 1alpha,25-Dihydroxyvitamin D3 upregulates FGF23 gene expression in bone: the final link in a renal-gastrointestinal-skeletal axis that controls phosphate transport. Am J Physiol Gastrointest Liver Physiol. 2005;289(6):G1036–42. pmid:16020653
View Article
PubMed/NCBI
Google Scholar

[65] View Article

[66] PubMed/NCBI

[67] Google Scholar

[ref18] 18. Vervloet MG, van Ittersum FJ, Büttler RM, Heijboer AC, Blankenstein MA, ter Wee PM. Effects of dietary phosphate and calcium intake on fibroblast growth factor-23. Clin J Am Soc Nephrol. 2011;6(2):383–9. pmid:21030580
View Article
PubMed/NCBI
Google Scholar

[69] View Article

[70] PubMed/NCBI

[71] Google Scholar

[ref19] 19. Huang B, Maciejewska I, Sun Y, Peng T, Qin D, Lu Y, et al. Identification of full-length dentin matrix protein 1 in dentin and bone. Calcif Tissue Int. 2008;82(5):401–10. pmid:18488132
View Article
PubMed/NCBI
Google Scholar

[73] View Article

[74] PubMed/NCBI

[75] Google Scholar

[ref20] 20. Yang W, Lu Y, Kalajzic I, Guo D, Harris MA, Gluhak-Heinrich J, et al. Dentin matrix protein 1 gene cis-regulation: use in osteocytes to characterize local responses to mechanical loading in vitro and in vivo. J Biol Chem. 2005;280(21):20680–90. pmid:15728181
View Article
PubMed/NCBI
Google Scholar

[77] View Article

[78] PubMed/NCBI

[79] Google Scholar

[ref21] 21. Rowe PS. Regulation of bone-renal mineral and energy metabolism: the PHEX, FGF23, DMP1, MEPE ASARM pathway. Crit Rev Eukaryot Gene Expr. 2012;22(1):61–86. pmid:22339660
View Article
PubMed/NCBI
Google Scholar

[81] View Article

[82] PubMed/NCBI

[83] Google Scholar

[ref22] 22. Reijnders CM, van Essen HW, van Rens BT, van Beek JH, Ylstra B, Blankenstein MA, et al. Increased expression of matrix extracellular phosphoglycoprotein (MEPE) in cortical bone of the rat tibia after mechanical loading: identification by oligonucleotide microarray. PLoS One. 2013;8(11):e79672. pmid:24255709
View Article
PubMed/NCBI
Google Scholar

[85] View Article

[86] PubMed/NCBI

[87] Google Scholar

[ref23] 23. Nepal AK, van Essen HW, van der Veen AJ, van Wieringen WN, Stavenuiter AWD, Cayami FK, et al. Mechanical stress regulates bone regulatory gene expression independent of estrogen and vitamin D deficiency in rats. J Orthop Res. 2021;39(1):42–52. pmid:32530517
View Article
PubMed/NCBI
Google Scholar

[89] View Article

[90] PubMed/NCBI

[91] Google Scholar

[ref24] 24. Kulkarni RN, Bakker AD, Everts V, Klein-Nulend J. Inhibition of osteoclastogenesis by mechanically loaded osteocytes: involvement of MEPE. Calcif Tissue Int. 2010;87(5):461–8. pmid:20725825
View Article
PubMed/NCBI
Google Scholar

[93] View Article

[94] PubMed/NCBI

[95] Google Scholar

[ref25] 25. Gluhak-Heinrich J, Pavlin D, Yang W, MacDougall M, Harris SE. MEPE expression in osteocytes during orthodontic tooth movement. Arch Oral Biol. 2007;52(7):684–90. pmid:17270144
View Article
PubMed/NCBI
Google Scholar

[97] View Article

[98] PubMed/NCBI

[99] Google Scholar

[ref26] 26. Gluhak-Heinrich J, Ye L, Bonewald LF, Feng JQ, MacDougall M, Harris SE, et al. Mechanical loading stimulates dentin matrix protein 1 (DMP1) expression in osteocytes in vivo. J Bone Miner Res. 2003;18(5):807–17. pmid:12733719
View Article
PubMed/NCBI
Google Scholar

[101] View Article

[102] PubMed/NCBI

[103] Google Scholar

[ref27] 27. Forwood MR, Bennett MB, Blowers AR, Nadorfi RL. Modification of the in vivo four-point loading model for studying mechanically induced bone adaptation. Bone. 1998;23(3):307–10. pmid:9737355
View Article
PubMed/NCBI
Google Scholar

[105] View Article

[106] PubMed/NCBI

[107] Google Scholar

[ref28] 28. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002;3(7):Research0034. pmid:12184808
View Article
PubMed/NCBI
Google Scholar

[109] View Article

[110] PubMed/NCBI

[111] Google Scholar

[ref29] 29. Heijboer AC, Levitus M, Vervloet MG, Lips P, ter Wee PM, Dijstelbloem HM, et al. Determination of fibroblast growth factor 23. Ann Clin Biochem. 2009;46(Pt 4):338–40. pmid:19564163
View Article
PubMed/NCBI
Google Scholar

[113] View Article

[114] PubMed/NCBI

[115] Google Scholar

[ref30] 30. Mantila Roosa SM, Liu Y, Turner CH. Gene expression patterns in bone following mechanical loading. J Bone Miner Res. 2011;26(1):100–12. pmid:20658561
View Article
PubMed/NCBI
Google Scholar

[117] View Article

[118] PubMed/NCBI

[119] Google Scholar

[ref31] 31. Chermside-Scabbo CJ, Harris TL, Brodt MD, Braenne I, Zhang B, Farber CR, et al. Old Mice Have Less Transcriptional Activation But Similar Periosteal Cell Proliferation Compared to Young-Adult Mice in Response to in vivo Mechanical Loading. J Bone Miner Res. 2020;35(9):1751–64. pmid:32311160
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref32] 32. Gardinier JD, Al-Omaishi S, Morris MD, Kohn DH. PTH signaling mediates perilacunar remodeling during exercise. Matrix Biol. 2016;52–54:162–75. pmid:26924474
View Article
PubMed/NCBI
Google Scholar

[125] View Article

[126] PubMed/NCBI

[127] Google Scholar

[ref33] 33. Liu S, Tang W, Zhou J, Stubbs JR, Luo Q, Pi M, et al. Fibroblast growth factor 23 is a counter-regulatory phosphaturic hormone for vitamin D. J Am Soc Nephrol. 2006;17(5):1305–15. pmid:16597685
View Article
PubMed/NCBI
Google Scholar

[129] View Article

[130] PubMed/NCBI

[131] Google Scholar

[ref34] 34. Toro L, Barrientos V, Leon P, Rojas M, Gonzalez M, Gonzalez-Ibanez A, et al. Erythropoietin induces bone marrow and plasma fibroblast growth factor 23 during acute kidney injury. Kidney Int. 2018;93(5):1131–41.
View Article
Google Scholar

[133] View Article

[134] Google Scholar

[ref35] 35. Rendenbach C, Yorgan TA, Heckt T, Otto B, Baldauf C, Jeschke A, et al. Effects of extracellular phosphate on gene expression in murine osteoblasts. Calcif Tissue Int. 2014;94(5):474–83. pmid:24366459
View Article
PubMed/NCBI
Google Scholar

[136] View Article

[137] PubMed/NCBI

[138] Google Scholar

[ref36] 36. Martin A, Liu S, David V, Li H, Karydis A, Feng JQ, et al. Bone proteins PHEX and DMP1 regulate fibroblastic growth factor Fgf23 expression in osteocytes through a common pathway involving FGF receptor (FGFR) signaling. FASEB J. 2011;25(8):2551–62. pmid:21507898
View Article
PubMed/NCBI
Google Scholar

[140] View Article

[141] PubMed/NCBI

[142] Google Scholar

[ref37] 37. van der Meijden K, Bakker AD, van Essen HW, Heijboer AC, Schulten EA, Lips P, et al. Mechanical loading and the synthesis of 1,25(OH)2D in primary human osteoblasts. J Steroid Biochem Mol Biol. 2016;156:32–9. pmid:26625962
View Article
PubMed/NCBI
Google Scholar

[144] View Article

[145] PubMed/NCBI

[146] Google Scholar

[ref38] 38. Atkins GJ, Anderson PH, Findlay DM, Welldon KJ, Vincent C, Zannettino AC, et al. Metabolism of vitamin D3 in human osteoblasts: evidence for autocrine and paracrine activities of 1 alpha,25-dihydroxyvitamin D3. Bone. 2007;40(6):1517–28. pmid:17395559
View Article
PubMed/NCBI
Google Scholar

[148] View Article

[149] PubMed/NCBI

[150] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Experimental animals

Experiment A.

Experiment B.

Mechanical loading

RNA extraction and RT-qPCR

Tissue preparation and immunohistochemistry for FGF23

Serum analysis

Statistical analysis

Results

Gene expression

Immunohistochemistry

Serum analysis

Discussion

Supporting information

S1 File. Result Immunhistochemistry.

S2 File. Result gene expression.

S3 File. Result serum measurements.

Acknowledgments

References