Niklas Rye Jørgensen

The influence of smoking on vitamin D status and calcium metabolism

Objective: To assess the influence of smoking on serum parathyroid hormone (PTH), serum vitamin D metabolites, serum ionized calcium, serum phosphate, and biochemical markers of bone turnover in a cohort of 510 healthy Danish perimenopausal women.Design: A cross-sectional study.Setting: Copenhagen, Denmark.Subjects: Five-hundred-and-ten healthy women aged 45–58 y, included 3–24 months after last menstrual bleeding. None were using hormone replacement therapy.Methods: The women were grouped according to their current smoking status. The two groups were compared with regard to serum levels of 25-hydroxyvitamin D (25OHD) and 1,25-dihydroxyvitamin D (1,25-(OH)2D), intact PTH, ionized calcium and phosphate, osteocalcin, as well as urine pyridinolines. Bone mineral density (BMD) was measured with DEXA-scans. Multiple regression analyses were performed to detect the effect of potentially confounding lifestyle factors, such as calcium and vitamin D intakes, alcohol and coffee consumption, sunbathing, and physical exercise.Results: Fifty percent were current smokers. Smokers had significantly reduced levels of serum 25OHD (P=0.02), 1,25(OH)2D (P=0.001), and PTH (P<0.001). There was no difference in serum ionized calcium between smokers and non-smokers. We found a negative effect of smoking on serum osteocalcin (P=0.01), while urinary pyridinolines were similar in the two groups. The small differences in lifestyle between the two groups could not explain these findings. Smokers had small but significant reductions in bone mineral density.Conclusions: Smoking has a significant effect on calcium and vitamin D metabolism, which is not likely to be explained by other confounding lifestyle factors. The depression of the vitamin D-PTH system seen among smokers may represent another potential mechanism for the deleterious effects of smoking on the skeleton, and may contribute to the reported risk of osteoporosis among smokers.Sponsorship: Grants from the Karen Elise Jensens Foundation.

Dexamethasone, BMP-2, and 1,25-dihydroxyvitamin D enhance a more differentiated osteoblast phenotype: validation of an in vitro model for human bone marrow-derived primary osteoblasts.

In vitro models of bone cells are important for the study of bone biology, including the regulation of bone formation and resorption. In this study, we have validated an in vitro model of human osteoblastic cells obtained from bone marrow biopsies from healthy, young volunteers, aged 20-31 years. Osteoblast phenotypes were induced by either dexamethasone (Dex) or bone morphogenetic protein-2 (BMP-2). Bone marrow was obtained from biopsies at the posterior iliac spine. Cells were isolated by gradient centrifugation and grown to confluence. Cells were treated with 1 nM 1,25-dihydroxyvitamin D (vitamin D), 100 nM Dex, and/or 100 ng/ml BMP-2. The osteoblast phenotype was assessed as alkaline phosphatase (AP) activity/staining, production of osteocalcin and procollagen type 1 (P1NP), parathyroid hormone (PTH)-induced cyclic adenosine mono-phosphate (cAMP) production, and in vitro mineralization. AP activity was increased by Dex, but not by BMP-2 treatment. P1NP production was decreased after Dex treatment, while BMP-2 had no effect on P1NP levels. Osteocalcin production was low in cultures not stimulated with vitamin D. Dex or BMP-2 treatment alone did not affect the basic osteocalcin levels, but in combination with vitamin D, BMP-2 increased the osteocalcin production, while Dex treatment completely suppressed osteocalcin production. Further, PTH-induced cAMP production was greatly enhanced by Dex treatment, whereas BMP-2 did not affect cAMP production. Finally, in vitro mineralization was greatly enhanced in cultures enriched with either BMP-2 or Dex. Cell proliferation was only increased significantly by Dex treatment. In conclusion, the model described produces cells with an osteoblastic phenotype, and both Dex and BMP-2 can be used as osteoblast inducers. However, the two treatments produce osteoblastic cells with different phenotypic characteristics, and a selective activation of some of the most important genes and functions of the mature osteoblast can thus be performed in vitro.

Human osteoblastic cells propagate intercellular calcium signals by two different mechanisms.

Effective bone remodeling requires the coordination of bone matrix deposition by osteoblastic cells, which may occur via soluble mediators or via direct intercellular communication. We have previously identified two mechanisms by which rat osteoblastic cell lines coordinate calcium signaling among cells: autocrine activation of P2 (purinergic) receptors leading to release of intracellular calcium stores, and gap junction‐mediated communication resulting in influx of extracellular calcium. In the current work we asked whether human osteoblastic cells (HOB) were capable of mechanically induced intercellular calcium signaling, and if so, by which mechanisms. Upon mechanical stimulation, human osteoblasts propagated fast intercellular calcium waves, which required activation of P2 receptors and release of intracellular calcium stores but did not require calcium influx or gap junctional communication. After the fast intercellular calcium waves were blocked, we observed slower calcium waves that were dependent on gap junctional communication and influx of extracellular calcium. These results show that human osteoblastic cells can propagate calcium signals from cell to cell by two markedly different mechanisms and suggest that these two pathways may serve different purposes in coordinating osteoblast functions.

Single nucleotide polymorphisms in the P2X7 gene are associated to fracture risk and to effect of estrogen treatment

Objectives The purinergic P2RX7 receptor (P2RX7) has been shown to play a role in the regulation of osteoblast and osteoclast activity. The aim of this study was to determine the presence of polymorphisms in exon 13 of the P2X7 gene and the association with osteoclast apoptosis in vitro and bone status in vivo. Methods A total of 1764 postmenopausal women were genotyped for three single nucleotide polymorphisms detected after sequencing of exon 13 of P2X7. Bone markers, bone mineral density of the hip and lumbar spine were determined at baseline and after 10 years, and vertebral fracture incidence after 10 years. In-vitro ATP-induced caspase-1 determinations were performed on osteoclasts from the different genotypes. Results Three polymorphisms were detected (Gln460Arg, Glu496Ala, and Ile568Asn). None of the polymorphisms was related to bone mineral density or changes in bone mineral density over 10 years in hormone replacement therapy naïve women. The Ile568Asn polymorphism was however, associated with effect of hormone replacement therapy. Furthermore, the 10-year fracture incidence was significantly associated with both the Glu496Ala and the Ile568Asn. The Glu496Ala polymorphism was closely related to ATP-induced osteoclast apoptosis in vitro, as osteoclasts from individuals homozygous for the C allele had significantly decreased apoptotic activity. Conclusion The P2X7 Glu496Ala and the Ile568Asn single nucleotide polymorphisms are associated with 10-year fracture risk in postmenopausal women and response to hormone replacement therapy treatment. Further, the Glu496Ala polymorphism is strongly influencing osteoclast apoptosis in vitro, which could contribute to increased fracture risk.

Parathyroid hormone and bone healing.

Fracture healing is a complex process, and a significant number of fractures are complicated by impaired healing and non-union. Impaired healing is prevalent in certain risk groups, such as the elderly, osteoporotics, people with malnutrition, and women after menopause. Currently, no pharmacological treatments are available. There is therefore an unmet need for medications that can stimulate bone healing. Parathyroid hormone (PTH) is the first bone anabolic drug approved for the treatment of osteoporosis, and intriguingly a number of animal studies suggest that PTH could be beneficial in the treatment of fractures and could thus be a potentially new treatment option for induction of fracture healing in humans. Furthermore, fractures in animals with experimental conditions of impaired healing such as aging, estrogen withdrawal, and malnutrition can heal in an expedited manner after PTH treatment. Interestingly, fractures occurring at both cancellous and cortical sites can be treated successfully, indicating that both osteoporotic and nonosteoporotic fractures can be the target of PTH-induced healing. Finally, the data suggest that PTH partly prevents the delay in fracture healing caused by aging. Recently, the first randomized, controlled clinical trial investigating the effect of PTH on fracture healing was published, indicating a possible clinical benefit of PTH treatment in inducing fracture healing. The aim of this article is therefore to review the evidence for the potential of PTH in bone healing, including the underlying mechanisms for this, and to provide recommendations for the clinical testing and use of PTH in the treatment of impaired fracture healing in humans.

Sunbed Radiation Provokes Cutaneous Vitamin D Synthesis in Humans—A Randomized Controlled Trial

We wanted to investigate whether the use of sunbeds with sunlamps emitting mainly UVA and only 0.5% or 1.4% UVB will increase the level of serum 25‐hydroxyvitamin D (25(OH)D). In a randomized, controlled, open study on healthy, Caucasian females (>50 years) sunbed radiation was given as follows: four 6‐min sunbed sessions (days 0, 2, 4 and 7) and four 12‐min sunbed sessions (days 9, 11, 14 and 16 ) with sunlamps emitting 0.5% UVB (n = 20) or with sunlamps emitting 1.4% UVB (n = 15). The controls (n = 21) had no intervention. Serum levels of 25(OH)D were measured on days 0, 9 and 18 in all three groups. The average increase in serum 25(OH)D from day 0 to day 9 was 12 nmol L−1 (SD 11 nmol L−1, P = 0.0002) in the 0.5% UVB group and 27 nmol L−1 (SD 9 nmol L−1, P < 0.0001) in the 1.4% UVB group. From day 9 to day 18 a further but not significant increase in serum 25(OH)D of 3 nmol L−1 (SD 9 nmol L−1, P = 0.2) in the 0.5% UVB group and 0.6 nmol L−1 (SD 18 nmol L−1, P = 0.9) in the 1.4% UVB group was seen. No significant changes were found in the control group. Increasing with UVB dose and exposure time, 37–64% of the sunbed sessions resulted in side effects such as erythema or polymorphic light eruption. The results showed that sunbeds emitting 0.5% and 1.4% UVB increased 25(OH)D serum levels. The increases were dose dependent but reached a plateau after few sessions. Sunbed use as vitamin D source is, however, not generally recommendable due to the well‐known carcinogenicity and high frequency of acute side effects.

Osteoporosis in chronic obstructive pulmonary disease patients

Purpose of review The purpose of this review is to examine the state of knowledge and clinical practice in the association of chronic obstructive pulmonary disease to osteoporosis and fracture incidence. Recent findings There is a clear association between chronic obstructive pulmonary disease and excessive bone loss/risk of fractures. Little is known about the pathophysiological processes involved in the bone loss, but recent reports point to a continuous systemic inflammatory state in patients with chronic obstructive pulmonary disease. This inflammation involves the release of inflammatory cytokines such as tumour necrosis factor-α and interleukin-1. During the course of chronic obstructive pulmonary disease, a protein catabolic process takes place, including increased production of catalytic enzymes (matrix metalloproteinases etc.), which together with the inflammatory cytokines induces bone resorption. Summary Patients with chronic obstructive pulmonary disease are at increased risk of osteoporosis and fractures. Risk factors such as smoking, advanced age, physical inactivity, malnutrition, and low weight may be responsible, but a number of pathophysiological explanations including the presence of a chronic inflammatory state with increased levels of proinflammatory cytokines and protein catalytic enzymes may also be involved. The use of oral glucocorticoids is also a significant risk factor. Increased awareness is highly warranted to diagnose osteoporosis at an early stage, and professionals should be aware of the risk of osteoporosis in this patient population.

Activation of L-type calcium channels is required for gap junction-mediated intercellular calcium signaling in osteoblastic cells

The propagation of mechanically induced intercellular calcium waves (ICW) among osteoblastic cells occurs both by activation of P2Y (purinergic) receptors by extracellular nucleotides, resulting in “fast” ICW, and by gap junctional communication in cells that express connexin43 (Cx43), resulting in “slow” ICW. Human osteoblastic cells transmit intercellular calcium signals by both of these mechanisms. In the current studies we have examined the mechanism of slow gap junction-dependent ICW in osteoblastic cells. In ROS rat osteoblastic cells, gap junction-dependent ICW were inhibited by removal of extracellular calcium, plasma membrane depolarization by high extracellular potassium, and the L-type voltage-operated calcium channel inhibitor, nifedipine. In contrast, all these treatments enhanced the spread of P2 receptor-mediated ICW in UMR rat osteoblastic cells. Using UMR cells transfected to express Cx43 (UMR/Cx43) we confirmed that nifedipine sensitivity of ICW required Cx43 expression. In human osteoblastic cells, gap junction-dependent ICW also required activation of L-type calcium channels and influx of extracellular calcium.

Relationships between bone mineral density, serum vitamin D metabolites and calcium:phosphorus intake in healthy perimenopausal women

Abstract. Brot C, Jørgensen N, Madsen OR, Jensen LB, Sørensen OH (Copenhagen Municipal Hospital, Copenhagen, Denmark). Relationships between bone mineral density, serum vitamin D metabolites and calcium:phosphorus intake in healthy perimenopausal women. J Intern Med 1999; 245: 509–516.

Mechanisms for the bone anabolic effect of parathyroid hormone treatment in humans.

Abstract Intermittent low-dose treatment with parathyroid hormone (PTH) analogues has become widely used in the treatment of severe osteoporosis. During normal physiological conditions, PTH stimulates both bone formation and resorption, and in patients with primary hyperparathyroidism, bone loss is frequent. However, development of the biochemical measurement of PTH in the 1980s led us to understand the regulation of PTH secretion and calcium metabolism which subsequently paved the way for the use of PTH as an anabolic treatment of osteoporosis as, when given intermittently, it has strong anabolic effects in bone. This could not have taken place without the basic understanding achieved by the biochemical measurements of PTH. The stimulatory effects of PTH on bone formation have been explained by the so-called ‘anabolic window’, which means that during PTH treatment, bone formation is in excess over bone resorption during the first 6–18 months. This is due to the following: (1) PTH up-regulates c-fos expression in bone cells, (2) IGF is essential for PTHs anabolic effect, (3) bone lining cells are driven to differentiate into osteoblasts, (4) mesenchymal stem cells adhesion to bone surface is enhanced, (5) PTH has a direct antiapoptotic effect on osteoblasts and (6) when PTH interferes with remodelling, the osteoblasts over-compensate, and (7) PTH also decreases sclerostin levels, thereby removing inhibition of Wnt signalling which is required for PTHs anabolic actions. Thus, the net formative effect of PTH given in intermittent treatment emerges through a complex network of pathways. In summary, the effects of PTH on bone turnover are dependent on the mode and dose of administration and studies investigating the mechanisms underlying this effect are reviewed in this article.