Background
Osteoarthritis (OA) is a debilitating degenerative disease of the joint affecting middle-aged and older people and the pathogenesis is considered multifactorial [
1]. Over the last decades, mounting evidence suggests that OA develops as a cause of inflammation in addition to the mechanical aspects [
2]. Vitamin D is a predecessor of the secosteroid hormone 1α,25(OH)
2D, which plays a pivotal role for appropriately regulated calcium required for a normal bone turnover. Of importance, 1α,25(OH)
2D has been suggested to dampen inflammation-driven diseases such as rheumatoid- and osteoarthritis [
3]. Exogenous sources of vitamin D
3 are mainly fatty fish and fortified food items, whereas the predominant source is the endogenous production from cholesterol in sun-exposed skin. After hydroxylation in the liver, the vitamin circulates as the prohormone 25(OH)D
3 bound to D-binding protein (90%) and albumin (10%). Likewise, the active hormone 1α,25(OH)
2D
3 is transported by these proteins, but circulates in a concentration < 0.1% of that of the prohormone. Less than 1% of both metabolites are unbound by carrier proteins. The hydroxylation of 25(OH)D
3 to 1α,25(OH)
2D
3 is facilitated by the enzyme 1α-hydroxylase, which is primarily located in the kidneys. However, there is compelling evidence of an extra-renal distribution of the enzyme, including local induction by the immune system [
4]. The effect of vitamin D in the extra-renal compartments are considered auto- or paracrine, and represent an entirely new paradigm of vitamin D actions compared to the endocrine action of renal vitamin D [
5]. The active hormone binds the vitamin D receptor (VDR), which is abundantly expressed in the intestine, kidneys, parathyroid and bone; while it is expressed in costal cartilage, its presence in articular cartilage is less settled [
6].
Several studies have suggested a link between vitamin D status and OA; some have found that individuals deficient in vitamin D have an increased risk of progressive knee-osteoarthritis, and there are reports of a pain reducing effect of oral supplementation [
7,
8]. Both 25(OH)D
3 and 1α,25(OH)
2D
3 have been detected in synovial fluid [
4,
9]. The presence of the receptor in human articular cartilage and chondrocytes has been previously investigated with divergent outcomes [
10,
11], and the biological effect of vitamin D on cartilage pathophysiology remains uncertain. In rat chondrosarcoma cells, 1α,25(OH)
2D
3 dose-dependently induced MMP13, a matrix metalloprotease which degrades the extracelluar matrix [
12]. 1α,25(OH)
2D
3 has also been associated with hypertrophy and mineralization of OA chondrocytes [
13].
The aim of this study was to investigate whether the previously described expression patterns of VDR in human articular cartilage and subcultured cells could be confirmed by PCR, Western blots and immunolabelling. It was also questioned whether receptor activation occurred upon ligand binding, and whether this could be recorded by biological readouts such as promoted chondrogenesis, cell proliferation or cartilage signature gene expression. Furthermore, in view of the previously reported presence of 1α,25(OH)
2D
3 in synovial fluid, it was addressed whether the sole source was the circulation or if the hormone could also be attributed to a local production by chondrocytes expressing 1α-hydroxylase. Previous experiments are described using cartilage tissues and chondrocytes expanded in adherent monolayer cultures. Since chondrocytes are known to dedifferentiate upon monolayer expansion, thus undermining the extrapolation of findings to cartilage conditions, we included assays utilizing freshly isolated chondrocytes in a differentiated stage, and 3D culture assays where the chondrocyte redifferentiate to a chondrocyte-like phenotype [
14].
Discussion
Osteoarthritis (OA) is a disease of the entire joint affecting cartilage, subchondral bone, the synovial membrane and ligaments. Clinically, the disease is characterized by joint space narrowing, osteophyte formation and sclerosis. At a microscopic level, OA is characterized by a loss of extracellular matrix, chondrocyte hypertrophy, cell proliferation and calcification [
29]. The role of Vitamin D in the OA context remains controversial. Synoviocytes secrete inflammatory mediators into the synovial fluid, and it appears that elevated levels of 1α,25(OH)
2D
3 may increase the OPG/RANKL ratio, along with a reduced IL-6 production that together contributes to a dampened inflammation [
30]. In contrast, it was reported that in human articular chondrocytes, 1α,25(OH)
2D
3 induced MMPs and calcification that are usually considered detrimental events [
11,
13]. The picture becomes more entangled after considering the studies that have investigated VDR expression in human cartilage. An early study from the 80’s claimed that VDR is absent in resident cartilage cells, but acquires VDR during ex-vivo cultivation, i.e. cells considered as dedifferentiated [
10]. A more recent report argues that VDR is inconsistently expressed in healthy human cartilage (donor dependent), but enhanced in OA cartilage [
11]. Because of the existing dubiety, we pursued studying VDR expression in cartilage tissue and cells by different means. Preceded by the measurement of 25(OH)D
3 in synovial fluid from patients with rheumatoid arthritis (mean 11,0 nM, results not shown), it was importunate to question whether the congeneric 1α,25(OH)
2D
3 hormone could be produced locally in the joint by chondrocytes. This would require the recognition that chondrocytes express 1α-hydroxylase.
In line with previous publications [
10,
31], VDR transcripts were detected in cartilage tissue and monolayers, while in our hands detecting VDR protein in native cartilage by immunohistochemistry and Western blot was less evident. The expected VDR band is hardly recognizable in Fig.
2e (cartilage D1, the D2 and D3 are judged negative). Of note, the β-actin protein band from cartilage samples was also weak even though equal amount of proteins were loaded in wells. This may reflect a very low concentration of cell associated proteins compared to ECM proteins in samples prepared by short enzymatic digestion. In Fig.
5a, corresponding to the Western blot of suspension cells phenotypically similar to native cells and devoid of matrix proteins, it is more evident that the VDR protein is present in the samples. No previous publication could be recovered for a comparison on the Western blot subject, and our judgment is that VDR is expressed by resident cells in cartilage, although at very low levels.
Through immunolabelling it has previously been demonstrated that chondrocytes express the VDR protein in OA cartilage and monolayer cells [
11]. The latter was confirmed here (Fig.
2c), though no signal was recorded by histological immunolabelling (Fig.
2a). It has been claimed that many VDR antibodies not only bind VDR, but also possess non-specific interactions with other unidentified proteins, determined by both immunoblotting and histochemistry [
15]. These authors recommended using the antibody applied in the present study for both purposes. The questioned utility of different antibodies could explain the current divergent findings. All in all, despite the failure to detect the VDR protein in cartilage tissue by immunohistochemistry, the results from protein and transcript detection in suspension cell cultures represent an evidence that differentiated chondrocytes express VDR, which is also in agreement with both the previous report and reports on enhanced expression in OA cartilage and rheumatoid lesions [
31]. In line with what we observed in tissue, a negative immunolabelling of redifferentiated cells, i.e. those in spheroids was recorded. Although VDR transcripts were detected, no or an insufficient amount of protein in spheroids enabled VDR detection.
After entering the cell, 1α,25(OH)
2D
3 binds the VDR, and the VDR-ligand complex translocates to the nucleus where it triggers a tissue-specific change in gene-transcription, resulting in altered growth, differentiation or functional activity [
32]. In adherent chondrocytes, receptor translocation is evident in Fig.
4 a-d where the immunostaining shifts from a mixed nuclear/cytoplasmic stain in untreated chondrocytes, to a predominant nuclear staining after the addition of 1α,25(OH)
2D
3. This provides evidence of the internalization of 1α,25(OH)
2D
3 and subsequent receptor engagement.
An objective in this study was to investigate a putative alteration of receptor or enzyme expression during an inflammatory condition arranged by treating cells with cytokine or hormone. Cells in both suspension and monolayers were subjected to either IL-1β or 1α,25(OH)
2D
3 treatment, and relative amounts of VDR protein were recorded by Western blot. In monolayer samples, the VDR was significantly upregulated upon treatment with IL-1β (Fig.
5b), while this effect could not be detected in a suspension culture condition (Fig.
5a). The upregulation detected in monolayer cultures is in agreement with previous publications reporting upregulated VDR expression during various inflammatory conditions and could be associated to the elevated receptor expression reported in OA cartilage samples [
31].
In osteoclasts, there has been observed an upregulation of VDR transcripts upon 1α,25(OH)
2D
3 stimulation at 10
−7 M, but not at a 10
−8 M level [
33]. In the present study, 10
−8 M was used to resemble the amounts found in the synovial fluid, yet it appeared insufficient to affect VDR expression at the protein level (Fig.
5). This indicates that a supplementary local hormone production is required [
34], or that osteoblasts and chondrocytes are unrelated cells on this subject.
It is claimed that in general 1α,25(OH)
2D
3 has an anabolic effect on tissues [
35]. In studies of proliferation, the mutual potency of 25(OH)D
3 and 1α,25(OH)
2D
3 was proven by the changes observed with each of the compounds. A similar pattern was seen in three donors, indicating enhanced proliferation after 25(OH)D
3 or 1α,25(OH)
2D
3 treatment (Fig.
5e). Chondrocyte proliferation is frequently observed in OA cartilage [
29], possibly as an attempt of cells to repair the damaged cartilage or to compensate the catabolic processes established in the joint.
Moreover, from the spheroid model that investigates chondrogenic potential, a corresponding effect was observed resulting in a significant loss of matrix production (Fig.
6 a-d) during treatment with 25(OH)D
3 or 1α,25(OH)
2D
3, but only after applying the assay to chondrocytes that also were expanded in the presence of 25(OH)D
3 or 1α,25(OH)
2D
3. Spheroids prepared from chondrocytes propagated in standard growth medium were indifferent to the presence of 1α,25(OH)
2D
3 during 3D culture (data not shown), underpinning that chondrocytes in 3D cultures rapidly repress VDR expression.
The gene expression profile of cultured cells exposed to 25(OH)D
3 and 1α,25(OH)
2D
3 indicated an unfavourable effect on proteoglycan transcripts ACAN and VCAN, while the expression of VDR, COL1A1 and SOX9 was unchanged. This outcome is in accordance with the lower expression of proteoglycans observed by Alcian blue staining of treated spheroids. Interestingly, the CYP24A1, that was included as a positive control of 1α,25(OH)
2D
3 effects, showed increased expression also upon 25(OH)D
3 treatment (Fig.
6 f), supporting the notion that chondrocytes endogenously express 1α-hydroxylase.
Striking and novel findings in this study were the expression of transcripts for the enzyme 1α-hydroxylase and the presence of the encoded protein in human osteoarthritic articular cartilage, in suspension cells, in monolayers and 3D spheroid cultures (Fig.
1). The expression in all these conditions could indicate a constitutive expression pattern. No evidence for 1α-hydroxylase regulation by IL-1β or 1α,25(OH)
2D
3 in differentiated suspension cells or dedifferentiated monolayer cells was recorded, which is in line with previous studies on osteoblasts [
33]. Previously, the enzyme was detected in rat growth plate chondrocytes [
36], but to the best of our knowledge there are no reports on its presence in human articular cartilage or chondrocytes. Expression of 1α-hydroxylase in cartilage from healthy donors remains to be determined.
The level of 25(OH)D
3 and 1α,25(OH)
2D
3 in the synovial fluid of inflamed joints in patients with rheumatoid arthritis has been measured to 20 nM and 25 pM, respectively [
4]. After oral administration of vitamin D, the level of 1α,25(OH)
2D
3 in synovial fluid has been measured to up to 100 nM [
37]. Based on these measurements, and to meet the sensitivity of the assay, chondrocytes were challenged with 50, 250 and 500 nM 25(OH)D
3, resulting in a conversion to 50, 150 and 300 pM 1α,25(OH)
2D
3, respectively (Fig.
4e). Since cell-free controls were subtracted to account for spontaneous 1α-hydroxylation, the results imply that the chondrocyte exhibits 1α-hydroxylase activity that may contribute to the pool of 1α,25(OH)
2D
3 in synovial fluid, an action that has previously been attributed solely to macrophages in synovial fluid [
4,
38].
This study has provided strong evidence for 1α-hydroxylase being expressed in human articular chondrocytes, at least in OA-derived chondrocytes, whereas evidence for a VDR expression is weaker, except in culture-expanded cells. The activity of the 1α-hydroxylase was supported by the conversion of 25(OH)D
3 to 1α,25(OH)
2D
3 (Fig.
4e) and indirectly by the assays presented in Fig.
6 showing comparable outcomes from treatment with 25(OH)D
3 and 1α,25(OH)
2D
3. Hence, the co-expression of these proteins enables auto- and paracrine cell activity, exemplified here by impaired matrix production, augmented cell proliferation and shifted ACAN/VCAN expression after the application of either 1α,25(OH)
2D
3 or 25(OH)D
3 (Fig.
6). These changes in chondrocyte activity are indeed associated with OA progression [
39], however since the functional experiments were conducted on dedifferentiated cells, some caution is advised in extrapolating these results to in vivo conditions. On the other hand, as a result of the increased expression of VDR during inflammatory conditions reported by us and others [
31], some of the actions described in Fig.
6 may be part of the picture during OA and RA pathology.
The extent and types of effects 1α,25(OH)
2D
3 have on resident cells in healthy cartilage remains to be uncovered, yet a probable paracrine action affecting neighbouring cells and tissues is apparent. However, the numerous VDR responsive elements in DNA and VDR’s capability to additionally engage several intracellular signalling systems, such as protein kinase C and phosphatidyl-inositol-3′ kinase reviewed in [
40], vouch for a plethora of biological readouts beyond the scope of this study to be investigated. Thus, this study does not rule out any anti-inflammatory effects of vitamin D on the joint as a whole.