Marjolein van Driel

24,25-Dihydroxyvitamin D3 and bone metabolism

The 1alpha-hydroxylated metabolite of 25-hydroxyvitamin D(3), 1,25-dihydroxyvitamin D(3), is the biologically most active metabolite of vitamin D. The 24-hydroxylated metabolites were generally considered as degradation products of a catabolic pathway finally leading to excretion of calcitroic acid. Studies with analogues fluorinated at the C-24 position did not indicate a physiological function for 24R,25(OH)(2)D(3). Nevertheless throughout the years various studies showed biologic effects of other metabolites than 1alpha,25(OH)(2)D(3). In particular the metabolite 24R,25(OH)(2)D(3) has been functionally analyzed, e.g. with respect to a role in normal chicken egg hatchability and effects on chondrocytes in the resting zone of cartilage. Numerous studies have shown the presence of the vitamin D receptor in bone cells and effects of 1alpha,25(OH)(2)D(3) on bone and bone cells. Also for 24R,25(OH)(2)D(3) studies have been performed focusing on effects on bone and bone cells. The purpose of this review is to summarize the data regarding 24R,25(OH)(2)D(3) and bone and to evaluate its role in bone biology.

Vitamin D endocrine system and osteoblasts

The interaction between vitamin D and osteoblasts is complex. In the current review we will give an overview of the current knowledge of the vitamin D endocrine system in osteoblasts. The presence of the vitamin D receptor in osteoblasts enables direct effects of 1α,25dihydroxyvitamin D3 (1α,25D3) on osteoblasts, but the magnitude of the effects is subject to the presence of many other factors. Vitamin D affects osteoblast proliferation, as well as differentiation and mineralization, but these effects vary with the timing of treatment, dosage and origin of the osteoblasts. Vitamin D effects on differentiation and mineralization are mostly stimulatory in human and rat osteoblasts, and inhibitory in murine osteoblasts. Several genes and mechanisms are studied to explain the effects of 1α,25D3 on osteoblast differentiation and bone formation. Besides the classical VDR, osteoblasts also express a membrane-localized receptor, and in vitro studies have shown that osteoblasts are capable of the synthesis of 1α,25D3.

Proteomic signatures of extracellular vesicles secreted by nonmineralizing and mineralizing human osteoblasts and stimulation of tumor cell growth

Beyond forming bone, osteoblasts play pivotal roles in various biologic processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. We focused on the proteomic characterization of nonmineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV‐HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic, and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast‐specific EV proteins. Label‐free quantification demonstrated mineralization stage‐dependent 5‐fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV‐regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to 2‐fold increase in PC3 cell growth compared to cell‐free culture medium‐derived vesicle controls. Our findings elucidate the mineralization stage‐specific protein content of osteoblast‐secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention.—Morhayim, J., van de Peppel, J., Demmers, J. A. A., Kocer, G., Nigg, A. L., van Driel, M., Chiba, H., van Leeuwen, J. P., Proteomic signatures of extracellular vesicles secreted by nonmineralizing and mineralizing human osteoblasts and stimulation of tumor cell growth. FASEB J. 29, 274–285 (2015). www.fasebj.org

Basic Techniques in Human Mesenchymal Stem Cell Cultures: Differentiation into Osteogenic and Adipogenic Lineages, Genetic Perturbations, and Phenotypic Analyses

This unit describes basic techniques in human mesenchymal stem cell (hMSC) cultures. It includes protocols for the differentiation of hMSCs into osteogenic and adipogenic lineages, genetic perturbations, and phenotypic analyses. hMSCs can be differentiated with dexamethasone and β-glycerophosphate into mineralizing osteoblasts within 2 to 3 weeks, or with dexamethasone, indomethacin, and 3-isobutyl-1-methylxanthine into lipid vesicle-containing adipocytes within 1 to 2 weeks. Phenotypic changes during those highly dynamic differentiation processes can be detected by biochemical and histological assays and gene expression analyses of differentiation markers. In addition, this unit describes an electroporation method that allows the transient genetic perturbation of hMSCs.

Primary human osteoblasts in response to 25-hydroxyvitamin D3, 1,25-dihydroxyvitamin D3 and 24R,25-dihydroxyvitamin D3.

The most biologically active metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has well known direct effects on osteoblast growth and differentiation in vitro. The precursor 25-hydroxyvitamin D3 (25(OH)D3) can affect osteoblast function via conversion to 1,25(OH)2D3, however, it is largely unknown whether 25(OH)D3 can affect primary osteoblast function on its own. Furthermore, 25(OH)D3 is not only converted to 1,25(OH)2D3, but also to 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) which may have bioactivity as well. Therefore we used a primary human osteoblast model to examine whether 25(OH)D3 itself can affect osteoblast function using CYP27B1 silencing and to investigate whether 24R,25(OH)2D3 can affect osteoblast function. We showed that primary human osteoblasts responded to both 25(OH)D3 and 1,25(OH)2D3 by reducing their proliferation and enhancing their differentiation by the increase of alkaline phosphatase, osteocalcin and osteopontin expression. Osteoblasts expressed CYP27B1 and CYP24 and synthesized 1,25(OH)2D3 and 24R,25(OH)2D3 dose-dependently. Silencing of CYP27B1 resulted in a decline of 1,25(OH)2D3 synthesis, but we observed no significant differences in mRNA levels of differentiation markers in CYP27B1-silenced cells compared to control cells after treatment with 25(OH)D3. We demonstrated that 24R,25(OH)2D3 increased mRNA levels of alkaline phosphatase, osteocalcin and osteopontin. In addition, 24R,25(OH)2D3 strongly increased CYP24 mRNA. In conclusion, the vitamin D metabolites 25(OH)D3, 1,25(OH)2D3 and 24R,25(OH)2D3 can affect osteoblast differentiation directly or indirectly. We showed that primary human osteoblasts not only respond to 1,25(OH)2D3, but also to 24R,25(OH)2D3 by enhancing osteoblast differentiation. This suggests that 25(OH)D3 can affect osteoblast differentiation via conversion to the active metabolite 1,25(OH)2D3, but also via conversion to 24R,25(OH)2D3. Whether 25(OH)D3 has direct actions on osteoblast function needs further investigation.

Cancer and bone: a complex complex.

Primary and secondary bone cancers are rare events. However, once settled, a complex process is started involving an extensive amount of factors and interactions. The bone micro-environment is a preferential site for (metastatic) tumor cells to enter, stay, colonize and expand. The fact that the tumor cells affect the complete bone environment involving many cell types and regulatory pathways to stimulate their own growth and escape from therapy is devastating for the patient. Many efforts have been made to get more insight into the mechanisms underlying the communication between bone cells and cancer cells and progress is made in therapeutic interventions. This review will discuss the biological mechanisms of primary bone malignancies (osteosarcoma, Ewings sarcoma, chondrosarcoma, multiple myeloma) and secondary bone malignancies (bone metastases) and therapeutic interventions.

Vitamin D: Cancer and Differentiation

Publisher Summary This chapter provides an overview of the history and the current state of knowledge regarding the role of vitamin D in tumor cell growth regulation, treatment of cancer and development of potent synthetic vitamin D analogs. It addresses the recent developments in studies of vitamin D and cancer, regulation of tumor cells, possible mechanisms, and clinical applications. Based on this understanding, it indicates that the data obtained so far, on the distribution of the VDR in a broad range of tumors and the inhibition of cancer cell growth, angiogenesis, metastasis, inflammation, and PTHrP synthesis as well as the stimulation of differentiation and apoptosis by 1,25(OH)2D3, all hold promise for the development of treatment strategies based on vitamin D3 use in a wide range of cancers. Combination of vitamin D3 with other antitumor drugs, hormones, or growth factors is an important additional therapeutic option. The clinical application is enhanced by the development of 1,25(OH)2D3 analogs with potent growth-inhibitory actions and reduced hypercalcemic activity. Nevertheless it is crucial for the coming years to deliver strong clinical trials to support the potential of vitamin D in cancer treatment uncovered by investigation of cultured cells, animal models, and epidemiological studies.

Vitamin D endocrinology of bone mineralization

Bone is a dynamic tissue that is strongly influenced by endocrine factors to restore the balance between bone resorption and bone formation. Bone formation involves the mineralization of the extracellular matrix formed by osteoblasts. In this process the role of vitamin D (1α,25(OH)2D3) is both direct and indirect. The direct effects are enabled via the Vitamin D Receptor (VDR); the outcome is dependent on the presence of other factors as well as origin of the osteoblasts, treatment procedures and species differences. Vitamin D stimulates mineralization of human osteoblasts but is often found inhibitory for mineralization of murine osteoblasts. In this review we will overview the current knowledge of the role of the vitamin D endocrine system in controlling the mineralization process in bone.

The role of IL-23 receptor signaling in inflammation-mediated erosive autoimmune arthritis and bone remodeling

The IL‐23/Th17 axis has been implicated in the development of autoimmune diseases, such as rheumatoid arthritis (RA) and psoriatic arthritis (PsA). RA and PsA are heterogeneous diseases with substantial burden on patients. Increasing evidence suggests that the IL‐23 signaling pathway may be involved in the development of autoimmunity and erosive joint damage. IL‐23 can act either directly or indirectly on bone forming osteoblasts as well as on bone resorbing osteoclasts. As IL‐23 regulates the activity of cells of the bone, it is conceivable that in addition to inflammation‐mediated joint erosion, IL‐23 may play a role in physiological bone remodeling. In this review, we focus on the role of IL‐23 in autoimmune arthritis in patients and murine models, and provide an overview of IL‐23 producing and responding cells in autoimmune arthritic joints. In addition, we discuss the role of IL‐23 on bone forming osteoblasts and bone resorbing osteoclasts regarding inflammation‐mediated joint damage and bone remodeling. At last, we briefly discuss the clinical implications of targeting this pathway for joint damage and systemic bone loss in autoimmune arthritis.

Control of Osteoblast Function and Bone Extracellular Matrix Mineralization by Vitamin D

Vitamin D is the major regulator of calcium homeostasis and protects the organism from calcium deficiency via effects on the intestine, kidney, parathyroid gland, and bone. Disturbances in the vitamin D endocrine system, for example, vitamin D-dependent rickets type I and type II, result in profound effects on the mineralization of bone. Also, recent studies with vitamin D receptor (VDR) knockout mice show effects on bone. It is questioned whether vitamin D has a direct effect on bone formation and mineralization. In rickets and in particular vitamin D receptor knockout mice, calcium supplementation restores bone mineralization. However, the vitamin D receptor (see Vitamin D Receptor section) is present in osteoblasts, and vitamin D affects the expression of various genes in osteoblasts (see Introduction: Osteoblasts and Effects of Vitamin D on Osteoblast Function and Mineralization sections). Vitamin D regulates the expression of genes and osteoblast activity not in an independent manner but often in interaction with other hormones and/or growth factors (see section titled Interaction of Vitamin D with Other Factors).