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Annals of the Rheumatic Diseases logoLink to Annals of the Rheumatic Diseases
. 1999 Jun;58(6):372–378. doi: 10.1136/ard.58.6.372

Autocrine control of vitamin D metabolism in synovial cells from arthritic patients

S Smith 1, M Hayes 1, P Selby 1, E Mawer 1
PMCID: PMC1752897  PMID: 10340962

Abstract

OBJECTIVE—This study was designed to investigate whether 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), produced by activated synovial fluid macrophages, promotes its own catabolism by upregulating vitamin D-24-hydroxylase (24-OHase) in synovial fibroblasts through a vitamin D receptor (VDR) mediated mechanism.
METHODS—Synovial macrophages and fibroblasts were derived from patients with rheumatoid arthritis. Expression of VDR and 24-OHase mRNAs was determined using in situ hybridisation. Vitamin D hydroxylase activity was determined by incubating cells with [3H]-25-(OH)D3, or [3H]-1,25-(OH)2D3, and metabolite synthesis quantified using high performance liquid chromatography.
RESULTS—1,25-(OH)2D3 increased expression of mRNA for both VDR and 24-OHase in fibroblasts by approximately threefold over 24 hours. 1,25-(OH)2D3 increased fibroblast 24-OHase activity, yielding 24-hydroxylated, and more polar, metabolites. In co-culture, fibroblasts were able to catabolise macrophage derived 1,25-(OH)2D3.
CONCLUSIONS—1,25-(OH)2D3 is produced by macrophages in vitro at biologically relevant concentrations and can increase its own catabolism by synovial fibroblasts; this effect is probably mediated via upregulation of both synovial fibroblast VDR and 24-OHase.



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Figure 1  .

Figure 1  

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Photomicrographs showing hybridisation of [35S] labelled riboprobe for 24-OHase and VDR mRNAs in F733 synovial fibroblasts. F733 cells were pre-treated with 5 × 10-8 M 1,25-(OH)2D3, or vehicle (0.125% v/v ethanol), fixed, then hybridised overnight with [35S] labelled riboprobe for 24-OHase or VDR mRNA. (A) Twenty four hour vehicle treated cells, 24-OHase probe; (B) 24 hour 1,25-(OH) 2D3 treated cells, 24-OHase probe; (C) RNase A treated control slide, 24-OHase probe; (D) 24 hour vehicle treated cells, VDR probe; (E) 24 hour 1,25-(OH)2D3 treated cells, VDR probe; (F) RNase A treated control slide, VDR probe. Bar = 50 µm, cells counterstained with eosin and Harris haematoxylin (Sigma).

Figure 2  .

Figure 2  

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HPLC profile of synovial fluid fibroblast (A) [3H]-25-(OH)D3 and (B) [3H]-1,25-(OH)2D3 metabolism. HPLC conditions are as described in Methods. The dotted traces show the positions of known tritiated forms of 25-(OH)D3 (peak A), 24,25-(OH) 2D3 (peak B), and 1,25-(OH) 2D3 (peak C). As no radioactive tracer was available, the expected position of a tritiated 1,24,25-(OH) 3D3 peak was calculated using an ultraviolet trace of unlabelled markers in conjunction with the radioactive trace above. The solid trace in figure 2 (A) shows the metabolism of substrate [3H]-25-(OH)D3 to its 24-hydroxylated form, [3H]-24,25-(OH)2D3, by synovial fluid fibroblasts pre-treated for 24 hours with 10-8 M 1,25-(OH) 2D3. The position of an unidentified product, which was more polar than [3H]-24,25-(OH) 2D3, is indicated by peak D. The solid trace in figure 2 (B) shows the metabolism of substrate [3H]-1,25-(OH)2D3 by synovial fluid fibroblasts pre-treated for 24 hours with 10-11 M 1,25-(OH)2D3. A metabolite with an elution time representing that of [3H]-1,24,25-(OH)3D3 is shown by peak E and the position of an unidentified product, which was more polar than [3H]-1,24,25-(OH)3D3, is indicated by peak F; dpm = disintegrations per minute.

Figure 3  .

Figure 3  

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Effects of 1,25-(OH)2D3 pre-treatment on the metabolism of [3H]-25-(OH)D3 and [3H]-1,25-(OH)2D3 by F733 synovial fibroblasts. Panels (A-C) are cells incubated with [3H]-25-(OH)D3 substrate, panels (D-F) are cells incubated with [3H]-1,25-(OH)2D3 substrate. (A) Synthesis of [3H]-24,25-(OH)2D3; (B) synthesis of an unidentified product (peak D in fig 2 (A)); (C) the radioactivity present in the aqueous phase; (D) synthesis of [3H]-1,24,25-(OH)3D3; (E) synthesis of an unidentified product (peak F in figure 2 (B)) and (F) radioactivity present in the aqueous phase. n=3 for each point. Results are shown as mean (SEM). *p<0.05, **p<0.01 compared with vehicle (0.25% ethanol) using ANOVA followed by Dunnett's multiple comparison test.

Selected References

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