International Journal of
Molecular Sciences Article
Vitamin D’s Effect on the Proliferation and Inflammation of Human Intervertebral Disc Cells in Relation to the Functional Vitamin D Receptor Gene FokI Polymorphism Paola De Luca 1 , Laura de Girolamo 1 ID , Carlotta Perucca Orfei 1 , Marco Viganò 1 Riccardo Cecchinato 2 , Marco Brayda-Bruno 3 and Alessandra Colombini 1, * ID 1
2 3
*
ID
,
Orthopaedic Biotechnology Lab, IRCCS Galeazzi Orthopaedic Institute, Via R. Galeazzi 4, 20161 Milan, Italy;
[email protected] (P.D.L.);
[email protected] (L.d.G.);
[email protected] (C.P.O.);
[email protected] (M.V.) GSpine4, IRCCS Galeazzi Orthopaedic Institute, Via R. Galeazzi 4, 20161 Milan, Italy;
[email protected] Scoliosis Unit, Department of Orthopedics and Traumatology-Spine Surgery III, IRCCS Galeazzi Orthopaedic Institute, Via R. Galeazzi 4, 20161 Milan, Italy;
[email protected] Correspondence:
[email protected]; Tel.: +39-026-621-4067
Received: 15 June 2018; Accepted: 6 July 2018; Published: 9 July 2018
Abstract: Vitamin D is known to have immunomodulatory effects, is involved in osteo-cartilaginous metabolism, and may have a role in human intervertebral disc pathophysiology. Although a link between vitamin D receptor (VDR) gene variants and disc degeneration-related pathologies has been observed, its functional contribution to pathologic processes has not been assessed yet. The aim of this study was to investigate the response of disc cells to vitamin D in terms of the regulation of proliferation, metabolism, and inflammatory processes, with a particular focus on the FokI VDR genotype. However, although it was found that vitamin D had a pro-apoptotic effect regardless of genotype, an up-regulation of IL-1Ra and downregulation of IL-6 was found to be evident only in Ff cells. Regarding the metabolic effects, in Ff cells, vitamin D promoted an upregulation of the aggrecan in inflammatory conditions but did not have an effect on the expression of collagen-related markers. Moreover, cells bearing the Ff genotype were the most responsive to vitamin D in the upregulation of catabolic markers. In addition, in contrast to the FF genotype, vitamin D downregulated the vitamin D-dependent signaling pathway in inflamed Ff cells, counteracting the inflammation-mediated catabolic effects. In conclusion, Ff cells were found to be more responsive to the anti-inflammatory and catabolic effects of vitamin D, which is likely to be related to matrix remodeling. Keywords: intervertebral disc; vitamin D; vitamin D receptor polymorphism; proliferation; inflammation
1. Introduction The involvement of the vitamin D endocrine system in the pathophysiology of the human intervertebral disc is still a topic of debate which is not fully explored. Few in-vitro studies have reported that vitamin D regulates proliferation, the expression of matrix genes, production of structural proteins, cytokines, and growth factors in cells obtained from the two main anatomical components of the disc, the nucleus pulposus (NP), and the annulus fibrosus (AF), and expressing the vitamin D receptor (VDR) [1,2]. The need to perform functional studies to analyze the effects of vitamin D on the fibro-cartilaginous disc and the osteo-cartilaginous endplate (CEP) resides in a number of evidences
Int. J. Mol. Sci. 2018, 19, 2002; doi:10.3390/ijms19072002
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW
2 of 13
The need to perform functional studies to analyze the effects of vitamin D on the fibro2 of 13 cartilaginous disc and the osteo-cartilaginous endplate (CEP) resides in a number of evidences describing the association between the four most studied, known genetic VDR variants (FokI, BsmI, TaqI and ApaI), and the between disc degeneration-related pathologies [3,4], VDR although inconsistent describing the association the four most studied, known genetic variants (FokI, BsmI, associations have and beenthe reported [5,6]. TaqI and ApaI), disc degeneration-related pathologies [3,4], although inconsistent associations Recently, a correlation have been reported [5,6]. between the aforementioned genetic variants and specific lumbar spine pathologies, sucha correlation as herniation, discopathy, and osteochondrosis, has beenand observed Some Recently, between the aforementioned genetic variants specific[7–10]. lumbar spine VDR alleles andsuch genotypes predisposed to lumbar pathologies have been observed identified[7–10]. in patients pathologies, as herniation, discopathy, andspine osteochondrosis, has been Some with a concomitant increase of type II collagen degradation products, which likely derives from the VDR alleles and genotypes predisposed to lumbar spine pathologies have been identified in patients degradation of the disc’s matrix [11,12]. However, there are still few findings thethe with a concomitant increase of type II collagen degradation products, which likelyconcerning derives from contribution of these variants to pathologic processes. The FokI polymorphism is particularly degradation of the disc’s matrix [11,12]. However, there are still few findings concerning the contribution interesting for its functional role—in fact, it isThe located the start codon the VDR and consists for of aits of these variants to pathologic processes. FokI in polymorphism is of particularly interesting C functional to T transition, determining the transcription a shorter, (F consists allele), or T (f role—in fact, it is located in the startof codon of theallele VDR C and of longer a C to Tallele transition, allele) polypeptide [13]. The shorter polypeptide couples more efficiently with the transcription factor determining the transcription of a shorter, allele C (F allele), or longer allele T (f allele) polypeptide [13]. II The B than the longer peptidescouples and leads to aefficiently higher transcriptional rate of vitamin shorter polypeptide more with the transcription factorD-dependent II B than thegenes longer [14,15]. peptides and leads to a higher transcriptional rate of vitamin D-dependent genes [14,15]. Moreover, given itsits involvement inin osteo-cartilaginous metabolism, vitamin DD might have a a Moreover, given involvement osteo-cartilaginous metabolism, vitamin might have crucial role inin the degenerative development ofof the disc and endplate [3]. crucial role the degenerative development the disc and endplate [3]. Finally, the immunomodulatory effects of vitamin D have been suggested [16], without albeit Finally, the immunomodulatory effects of vitamin D have been also also suggested [16], albeit without any clear explanation as to its possible involvement in the regulation of the inflammatory any clear explanation as to its possible involvement in the regulation of the inflammatory and catabolic and catabolic processes present in the discs degenerate processes present in the degenerate [17,18].discs [17,18]. Based on this background, the aim of this study was toto investigate thethe in-vitro regulation of of Based on this background, the aim of this study was investigate in-vitro regulation proliferation, inflammatoryprocesses processes of disc in response to vitamin D proliferation,metabolism, metabolism, and and inflammatory of disc cellscells in response to vitamin D treatment, treatment, with a focus on the functional FokI VDR genotype. This study attempts to clarify the with a focus on the functional FokI VDR genotype. This study attempts to clarify the functional functional meaning of the of association ofvariant this genetic with theto predisposition toofthe meaning of the association this genetic with thevariant predisposition the development disc development of disc degeneration-related pathologies. degeneration-related pathologies. Int. J. Mol. Sci. 2018, 19, 2002
Results 2. 2. Results 2.1. Anti-Proliferative Effect Vitamin Related Induction Apoptosis 2.1. Anti-Proliferative Effect of of Vitamin DD Is Is Related to to Induction of of Apoptosis Vitamin treatment caused a decrease the numberofofviable viablecells cells(−2.7%, (−2.7%, < 0.01). Vitamin DD treatment caused a decrease inin the number p
