J.P.T.M. van Leeuwen

1,25-Dihydroxyvitamin D3 modulates Th17 polarization and interleukin-22 expression by memory T cells from patients with early rheumatoid arthritis

OBJECTIVE To examine the immunologic mechanism by which 1,25-dihydroxyvitamin D(3) (1,25[OH](2)D(3)) may prevent corticosteroid-induced osteoporosis in patients with early rheumatoid arthritis (RA), with a focus on T cell biology. METHODS Peripheral blood mononuclear cells (PBMCs) and CD4+CD45RO+ (memory) and CD4+CD45RO- (non-memory) T cells separated by fluorescence-activated cell sorting (FACS) from treatment-naive patients with early RA were stimulated with anti-CD3/anti-CD28 in the absence or presence of various concentrations of 1,25(OH)(2)D(3), dexamethasone (DEX), and 1,25(OH)(2)D(3) and DEX combined. Levels of T cell cytokines were determined by enzyme-linked immunosorbent assay and flow cytometry. RESULTS The presence of 1,25(OH)(2)D(3) reduced interleukin-17A (IL-17A) and interferon-gamma levels and increased IL-4 levels in stimulated PBMCs from treatment-naive patients with early RA. In addition, 1,25(OH)(2)D(3) had favorable effects on tumor necrosis factor alpha (TNFalpha):IL-4 and IL-17A:IL-4 ratios and prevented the unfavorable effects of DEX on these ratios. Enhanced percentages of IL-17A- and IL-22-expressing CD4+ T cells and IL-17A-expressing memory T cells were observed in PBMCs from treatment-naive patients with early RA as compared with healthy controls. Of note, we found no difference in the percentage of CD45RO+ and CD45RO- cells between these 2 groups. Interestingly, 1,25(OH)(2)D(3), in contrast to DEX, directly modulated human Th17 polarization, accompanied by suppression of IL-17A, IL-17F, TNFalpha, and IL-22 production by memory T cells sorted by FACS from patients with early RA. CONCLUSION These data indicate that 1,25(OH)(2)D(3) may contribute its bone-sparing effects in RA patients taking corticosteroids by the modulation of Th17 polarization, inhibition of Th17 cytokines, and stimulation of IL-4.

Case-control analysis of bone resorption markers, disability, and hip fracture risk: the Rotterdam study

Several factors besides bone mineral mass have been related to the risk of hip fracture. Bone quality, the rate of bone loss, and non-skeletal factors have been identified as important.1 2 High rates of bone resorption may be associated with disruption of the trabecular network as well as with an increased rate of bone loss. Furthermore, immobility associated with disability induces bone resorption not followed by increased bone formation.3 Urinary pyridinium crosslinks are markers of bone resorption. We investigated whether these were associated with the risk of hip fracture and also whether such an association was attributable to disability. This nested case-control analysis was conducted as part of the Rotterdam study, a prospective cohort study of the incidence of and risk factors for chronic disabling diseases.4 Briefly, all 10275 residents of a district of Rotterdam aged 55 or over were invited to participate. The study consisted of an initial home interview followed by a series …

Evidence for auto/paracrine actions of vitamin D in bone: 1α-hydroxylase expression and activity in human bone cells

Vitamin D is an important regulator of mineral homeostasis and bone metabolism. 1α‐Hy‐droxylation of 25‐(OH)D3 to form the bioactive vitamin D hormone, 1α,25‐(OH)2D3, is classically considered to take place in the kidney. However, 1α‐hydroxylase has been reported at extrarenal sites. Whether bone is a 1α,25‐(OH)2D3 synthesizing tissue is not univocal. The aim of this study was to investigate an autocrine/ paracrine function for 1α,25‐(OH)2D3 in bone. We show that 1α‐hydroxlase is expressed in human osteoblasts, as well as the vitamin D binding protein receptors megalin and cubilin. Functional analyses demonstrate that after incubation with the 1α‐hydoxylase substrate 25‐(OH)D3, the osteoblasts can produce sufficient 1α,25‐(OH)2D3 to modulate osteoblast activity, resulting in induced alkaline phosphatase (ALP) activity, osteocalcin (OC) and CYP24 mRNA expression, and mineralization. The classical renal regulators of 1α‐hydroxylase, parathyroid hormone, and ambient calcium do not regulate 1α‐hydroxylase in osteoblasts. In contrast, interleukin (IL)‐1β strongly induces 1α‐hydroxylase. Besides the bone‐forming cells, we demonstrate 1α‐hydroxylase activity in the bone resorbing cells, the osteoclasts. This is strongly dependent on osteoclast inducer RANKL. This study showing expression, activity, and functionality of 1α‐hydoxylase unequivocally demonstrates that vitamin D can act in an auto/paracrine manner in bone.—van Driel, M., Koedam, M., Buurman, C. J., Hewison, M., Chiba, H., Uitterlinden, A. G., Pols, H. A. P., van Leeuwen, J. P. T. M. Evidence for auto/paracrine actions of vitamin D in bone: 1α‐hydroxylase expression and activity in human bone cells. FASEB J. 20, E1811–E1819 (2006)

The Effect of Vitamin D Supplementation on the Bone Mineral Density of the Femoral Neck Is Associated with Vitamin D Receptor Genotype

Recent studies suggest that variations of the vitamin D receptor (VDR) gene are related to bone mineral density (BMD). In this study, we examined the effect of vitamin D3 supplementation on BMD at the femoral neck in relation to VDR genotype. We analyzed 81 women, age 70 years and over, who participated in a placebo‐controlled clinical trial on the effect of vitamin D3 supplementation (400 IU daily for at least 2 years) on BMD and fracture incidence. VDR genotype was based on the presence (b) or absence (B) of the BsmI restriction site. Mean BMD of the right and left femoral neck was measured at baseline and after 1 and 2 years. Dietary calcium, body mass index, and years since menopause were assessed at baseline while biochemical markers were measured at baseline and after 1 year. There was no difference among the BB, Bb, and bb genotype for baseline measurements of BMD at the femoral neck (mean and SD, g/cm2: 0.70 (0.10), 0.71 (0.12), and 0.69 (0.10), respectively), nor for any of the biochemical indices. The mean increase of BMD in the vitamin D group relative to the placebo group, expressed as percentage of baseline BMD, was significantly higher (p = 0.03) in the BB (ΔBMD: 4.4%, p = 0.04) and Bb genotype (ΔBMD: 4.2%, p = 0.007) compared with the bb genotype (ΔBMD: −0.3%, p = 0.61). No significant changes were found for any of the other measured parameters. The VDR genotype‐dependent effect of vitamin D supplementation in these elderly subjects suggest a functional involvement of VDR gene variants in determining BMD.

Animal models for osteoarthritis: the effect of ovariectomy and estrogen treatment – a systematic approach

OBJECTIVE The prevalence of osteoarthritis (OA) increases dramatically in women after the age of 50. Animal models are used to study the effects of hormone depletion [by ovariectomy (OVX)] and estrogen treatment on OA. This review summarizes these animal studies, in order to get a better insight in the role of hormones on OA. METHOD The literature was systematically reviewed until May 2007. The results were divided into two parts: the effect of OVX on cartilage, and the effect of estrogen treatment on cartilage. Only studies with an appropriate control group (e.g., sham-operated) were included. RESULTS AND DISCUSSION Eleven out of 16 animal studies showed that OVX resulted in cartilage damage. When only studies using sexually mature animals were included, we saw that 11 out of 14 studies showed a detrimental effect, indicating considerable evidence for a relation between cartilage degeneration and OVX in mature animals. The effect of estrogen treatment was inconclusive with only 11 out of 22 animal studies reporting a beneficial effect on cartilage, whereas all six studies administering selective estrogen receptor modulators (SERMs) after OVX described protective effects. The discrepancy between the studies may be caused by the large variation in experimental set-up. We suggested a list of quality criteria for animal models since standardisation of design and outcome parameters of animal experiments may help to compare different studies and to gain better insight in the role of hormones in the osteoarthritic process.

Evidence that both 1α,25‐dihydroxyvitamin D3 and 24‐hydroxylated D3 enhance human osteoblast differentiation and mineralization

Vitamin D plays a major role in the regulation of mineral homeostasis and affects bone metabolism. So far, detailed knowledge on the vitamin D endocrine system in human bone cells is limited. Here we investigated the direct effects of 1α,25‐(OH)2D3 on osteoblast differentiation and mineralization. Also, we studied the impact of 24‐hydroxylation, generally considered as the first step in the degradation pathway of vitamin D, as well as the role of the nuclear and presumed membrane vitamin D receptor (VDR). For this we used a human osteoblast cell line (SV‐HFO) that has the potency to differentiate during culture forming a mineralized extracellular matrix in a 3‐week period. Transcriptional analyses demonstrated that both 1α,25‐(OH)2D3 and the 24‐hydroxylated metabolites 24R,25‐(OH)2D3 and 1α,24R,25‐(OH)3D3 induced gene transcription. All metabolites dose‐dependently increased alkaline phosphatase (ALP) activity and osteocalcin (OC) production (protein and RNA), and directly enhanced mineralization. 1α,24R,25‐(OH)3D3 stimulated ALP activity and OC production most potently, while for mineralization it was equipotent to 1α,25‐(OH)2D3. The nuclear VDR antagonist ZK159222 almost completely blocked the effects of all metabolites. Interestingly, 1β,25‐(OH)2D3, an inhibitor of membrane effects of 1α,25‐(OH)2D3 in the intestine, induced gene transcription and increased ALP activity, OC expression and mineralization. In conclusion, not only 1α,25‐(OH)2D3, but also the presumed 24‐hydroxylated “degradation” products stimulate differentiation of human osteoblasts. 1α,25‐(OH)2D3 as well as the 24‐hydroxylated metabolites directly enhance mineralization, with the nuclear VDR playing a central role. The intestinal antagonist 1β,25‐(OH)2D3 acts in bone as an agonist and directly stimulates mineralization in a nuclear VDR‐dependent way. J. Cell. Biochem. 99: 922–935, 2006.

The essential role of glucocorticoids for proper human osteoblast differentiation and matrix mineralization

Glucocorticoids (GCs) exert profound effects on bone and are essential for human osteoblast differentiation. However, GCs are still interpreted as negative regulators of bone formation, mainly caused by the detrimental effects on bone after clinical use of GCs. In this paper we emphasize the importance of GCs for proper human osteoblast differentiation and matrix mineralization. We show that human osteoblast differentiation needs to be triggered by GCs in a specific time-window during the early stages of development. Exposure to GCs in the beginning of osteoblast development induces a dose dependent increase in alkaline phosphatase activity and matrix mineralization. GC-induced differentiation stimulated expression of genes involved in bone formation and suppressed genes that negatively regulate bone formation and mineralization. Furthermore we highlight the importance of local cortisol activation in osteoblasts by expression of 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1).

Evidence for Involvement of 17β-Estradiol in Intestinal Calcium Absorption Independent of 1,25-Dihydroxyvitamin D3 Level in the Rat

The sex steroid 17β‐estradiol (17β‐E2) has a broad range of actions, including effects on calcium and bone metabolism. This study with 3‐month‐old Brown Norway rats was designed to investigate the role of 17β‐E2 in the regulation of calcium homeostasis. Rats were divided in four groups, sham‐operated, ovariectomized (OVX), and OVX supplemented with either a 0.025‐mg or 0.05‐mg 17β‐E2 pellet implanted subcutaneously. After 4 weeks, in none of the groups was serum calcium, phosphate, or parathyroid hormone altered compared with the sham group, while only in the OVX rats was a significant reduction in urinary calcium found. Bone mineral density and osteocalcin were modified, as can be expected after OVX and 17β‐E2 supplementation. OVX resulted in a nonsignificant increase in serum 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3). Supplementation with either one of the 17β‐E2 dosages resulted in an 80% reduction of 1,25(OH)2D3 and only a 20% reduction in 25‐hydroxyvitamin D3 levels. OVX, as well as supplementation with 17β‐E2, did not affect serum levels of vitamin D binding protein. As a consequence, the estimated free 1,25(OH)2D3 levels were also significantly decreased in the 17β‐E2‐supplemented group compared with the sham and OVX groups. Next, the consequences for intestinal calcium absorption were analyzed by the in situ intestinal loop technique. Although the 1,25(OH)2D3 serum level was increased, OVX resulted in a significant decrease in intestinal calcium absorption in the duodenum. Despite the strongly reduced 1,25(OH)2D3 levels (18.1 ± 2.1 and 16.4 ± 2.2 pmol/l compared with 143.5 ± 29 pmol/l for the OVX group), the OVX‐induced decrease in calcium absorption could partially be restored by supplementation with either 0.025 mg or 0.05 mg of 17β‐E2. None of the treatments resulted in a significant change in calcium handling in the jejunum, although the trends were similar as those observed in the duodenum. 17β‐E2 did not change the VDR levels in both the intestine and the kidney. In conclusion, the present study demonstrates that 17β‐E2 is positively involved in intestinal calcium absorption, and the data strengthen the assertion that 17β‐E2 exerts this effect independent of 1,25(OH)2D3. In general, 17β‐E2 not only affects bone turnover but also calcium homeostasis via an effect on intestinal calcium absorption.

Vitamin D: A modulator of cell proliferation and differentiation

1,25-Dihydroxyvitamin D3, [1,25(OH)2D3], the biologically most active metabolite of vitamin D3, is involved in the regulation of calcium homeostasis and bone metabolism. Recently, receptors for 1,25(OH)2D3 have also been shown in cells and tissues not directly related to calcium homeostasis. Experimental data obtained with leukaemic and cancer cell lines, both in vitro and in vivo, showed the effects of 1,25(OH)2D3 on cell differentiation and proliferation. However, high doses of the sterol have to be used to observe these effects. Additional studies are needed to establish whether 1,25(OH)2D3 or suitable analogues have a therapeutic potential in malignant diseases without unacceptable toxicity like the development of hypercalcemia.

ADAMTS5−/− mice have less subchondral bone changes after induction of osteoarthritis through surgical instability: implications for a link between cartilage and subchondral bone changes

OBJECTIVE Osteoarthritis (OA) is characterized by damaged articular cartilage and changes in subchondral bone. Previous work demonstrated aggrecanase-2 deficient (ADAMTS5-/-) mice to be protected from cartilage damage induced by joint instability. This study analyzed whether this protective effect on cartilage is also reflected in the subchondral bone structure. METHODS Right knee joints from 10-week old male wild type (WT) and ADAMTS5-/- mice received transection of the medial meniscotibial ligament to induce OA, whereas left knees were left unoperated. After 8 weeks knee joints were scanned by micro-CT. The proximal tibia was selected for further analysis. Histology was performed to evaluate cartilage damage and osteoclast presence. RESULTS ADAMTS5-/- joints had a significantly thinner subchondral plate and less epiphyseal trabecular bone compared to WT joints. Histology confirmed previous findings that ADAMTS5-/- mice have significantly less cartilage damage than WT in the instability-induced OA model. Although the subchondral bone plate became significantly thicker at the medial tibial plateau in operated joints of both genotypes, the percentage increase was significantly smaller in ADAMTS5-/- mice (WT: 20.7+/-4.7%, ADAMTS5-/-: 8.3+/-1.2% compared to the left unoperated control joint). In ADAMTS5-/- animals a significant decrease was found in both Oc.N./BS and Oc.S./BS. Finally, in WT but not in ADAMTS5-/- mice a significant correlation was found between medial subchondral bone plate thickness and cartilage damage at the medial tibial plateau. CONCLUSION ADAMTS5-/- joints that were protected from cartilage damage showed minor changes in the subchondral bone structure, in contrast to WT mice where substantial changes were found. This finding suggests links between the process of cartilage damage and subchondral bone changes in instability-induced OA.