High-Throughput Gene and Protein Analysis Revealed the Response of Disc Cells to Vitamin D, Depending on the VDR FokI Variants.

Alessandra Colombini,Marco Brayda-Bruno,Michela Malacarne,Marco Viganò,Carlotta Perucca Orfei,Enrico Ragni,Domenico Coviello,Davide Cangelosi,Paola De Luca,Mauro Castagnetta,Laura De Girolamo

doi:10.3390/ijms22179603

Abstract

Vitamin D showed a protective effect on intervertebral disc degeneration (IDD) although conflicting evidence is reported. An explanation could be due to the presence of the FokI functional variant in the vitamin D receptor (VDR), observed as associated with spine pathologies. The present study was aimed at investigating—through high-throughput gene and protein analysis—the response of human disc cells to vitamin D, depending on the VDR FokI variants. The presence of FokI VDR polymorphism was determined in disc cells from patients with discopathy. 1,25(OH)2D3 was administered to the cells with or without interleukin 1 beta (IL-1β). Microarray, protein arrays, and multiplex protein analysis were performed. In both FokI genotypes (FF and Ff), vitamin D upregulated metabolic genes of collagen. In FF cells, the hormone promoted the matrix proteins synthesis and a downregulation of enzymes involved in matrix catabolism, whereas Ff cells behaved oppositely. In FF cells, inflammation seems to hamper the synthetic activity mediated by vitamin D. Angiogenic markers were upregulated in FF cells, along with hypertrophic markers, some of them upregulated also in Ff cells after vitamin D treatment. Higher inflammatory protein modulation after vitamin D treatment was observed in inflammatory condition. These findings would help to clarify the clinical potential of vitamin D supplementation in patients affected by IDD.

Highlights

Vitamin D supplementation has been proposed in several pathological contexts to improve skeletal and non-skeletal health, but a considerable body of clinical trials has failed to establish clear evidence of benefit [1].In general, to establish the clinical success of a hormonal supplementation, the mechanism of action of the hormone should be firstly in vitro defined and the safety and effectiveness of its administration should be validated in vivo.Considering the intervertebral disc degeneration (IDD)-related pathologies, few in vitro studies reported the presence of human cells expressing the vitamin D receptor (VDR) inside the disc, which are able to respond to the hormone by modulating proliferative and metabolic pathways [2,3]
The most relevant upregulated genes in the context of intervertebral disc (IVD) pathophysiology in homozygous FF disc cells were ADAMTS15, ACAN, BGN, KRT18, KRT19, SOX9, IGFBP2, IGFBP5, IL7 and TIMP3, COL11A2 along with COL10A1, COMP, CCR3, CCR5, and VEGFD (Figure 1 and Supplementary Table S1), that were upregulated in Ff disc cells
49 genes upregulated in FF disc cells were downregulated in Ff cells, among which ACVRIC, CCL13, CCR2, COL15A1, COL2A1, COL9A3, CXCR2, BMP7, DCN, FGF18, FRZB, INFG, IL10, IL17A, IL1R2, IL2, IL31RA, IL5, IHH, IDO2, NODAL, TGFB1, and TNMD, together with other 46 genes that were exclusively downregulated in Ff such as KRT14, TERT, and VEGFR (Figure 2 and Supplementary Table S1)

Summary

Introduction

Vitamin D supplementation has been proposed in several pathological contexts to improve skeletal and non-skeletal health, but a considerable body of clinical trials has failed to establish clear evidence of benefit [1].In general, to establish the clinical success of a hormonal supplementation, the mechanism of action of the hormone should be firstly in vitro defined and the safety and effectiveness of its administration should be validated in vivo.Considering the intervertebral disc degeneration (IDD)-related pathologies, few in vitro studies reported the presence of human cells expressing the vitamin D receptor (VDR) inside the disc, which are able to respond to the hormone by modulating proliferative and metabolic pathways [2,3]. The presence of the FokI variant (rs2228570) in the VDR, determines the functional modification of the encoded protein, with the shorter polypeptide (F allele) couples more efficiently with the transcription factor II B than the longer peptides (f allele) and leads to a higher transcriptional rate of vitamin D-dependent genes [6,7]. This could be responsible for differences in the response to the vitamin D supplementation and should be considered in view of a personalized medical treatment. FokI variants in the VDR were observed as associated with spine pathologies [8,9,10,11,12,13], but inconsistent associations have been reported [14]

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