Vitamin D inhibits Fas ligand-induced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways

doi:10.1016/j.bone.2004.03.005

Bone

Volume 35, Issue 1, July 2004, Pages 57-64

https://doi.org/10.1016/j.bone.2004.03.005 Get rights and content

Abstract

Apoptosis plays an important role in the regulation of bone turnover. Previously, we showed that 1,25(OH)₂D₃, the active form of vitamin D, may increase osteoblast survival by inhibiting apoptosis induced by serum deprivation. Human osteoblasts express the Fas receptor on their surface and its interaction with Fas ligand has been closely associated with human osteoblast apoptosis. To investigate the mechanism of 1,25(OH)₂D₃ inhibition of apoptosis in osteoblasts isolated from human calvaria, cells were exposed to Fas antibody. Visualization of apoptotic cells using annexin V revealed a significant decrease in apoptosis at 48 h in the presence of 1,25(OH)₂D₃ (14 ± 4%, P < 0.04) compared with non-treated cells (52 ± 4%). Furthermore, flow cytometric analysis of TUNEL-labeled osteoblasts showed a significant decrease in apoptotic cells in 1,25(OH)₂D₃-treated cultures (12 ± 2%) at 48 h compared with non-treated cultures (44 ± 3%, P < 0.04). Additionally, cells treated with 1,25(OH)₂D₃ survived longer as found by MTS analysis. To further explore the mechanism of 1,25(OH)₂D₃-mediated inhibition of apoptosis, we examined the changes in activation of death domain proteins, cleavage of caspases and mitochondrial regulators of apoptosis by Western blot analysis. A significant inhibition of caspase-8 cleavage and activity in 1,25(OH)₂D₃-treated cells was observed in conjunction with a decrease in the expression of the proapoptotic protein Bax with a significant increase in the expression of antiapoptotic protein Bcl-2. Furthermore, the levels of p21^Cip1/WAF1, which inhibits the cleavage of caspase-8, was found to be highly induced in 1,25(OH)₂D₃-treated cells. In summary, these results demonstrate that the anti-apoptotic effect of 1,25(OH)₂D₃ in human osteoblasts after the activation of Fas-ligand is mediated by the regulation of components of both the mitochondrial and Fas-related pathways.

Introduction

The skeleton is a dynamic tissue in which there are continuous interactions between bone-forming osteoblasts and bone-resorbing osteoclasts. Together, these cells form a basic multicellular unit (BMU) that continuously remodels the skeleton replacing old bone with new one [1]. This remodeling process can be affected by multiple factors including hormone deprivation and aging during which apoptosis, or programmed cell death, may contribute to the imbalance between the number of osteoclasts and osteoblasts available in the BMU [2], [3], [4].

Both osteoclasts and osteoblasts can undergo apoptosis [3], [4], [5], [6]. Under physiological conditions, osteoclasts undergo apoptosis and are quickly removed by local phagocytes once they have eroded bone to a specific distance from the central axis, in cortical bone, or to a particular depth from the surface in cancellous bone [3], [4], [6]. Osteoclast apoptosis can be induced in vitro and in vivo by administration of estrogens (E₂) [7] or bisphosphonates [8] which leads to a decrease in bone resorption.

Mature osteoblasts can alter their phenotype to become either lining cells or osteocytes. However 65% of the osteoblasts present in a remodeling site cannot be accounted for in either of these ways and are thought to die by apoptosis [8]. Recent evidence suggests that osteoblast apoptosis in vitro can be induced by serum deprivation [5], [9] or by the activation of CD95/Fas receptor by its ligand [10], [11]. The lack of growth factors, glucose, and amino acids during serum deprivation activates an apoptotic pathway shared by tumor necrosis factor (TNF) receptor, Fas/Apo1, and TRAIL-R/Apo2 pathway with subsequent ligand binding and the recruitment and activation of caspase-8 [12].

In addition, human osteoblasts express CD95/Fas, and both membrane-bound and soluble forms of the Fas-ligand (Fas-L) [13]. Osteoblasts undergo apoptosis after being exposed to antibodies to the CD95/Fas receptor which activate two specific adaptor molecules such as Fas-associated death domain (FADD) and TNFα death domain (TRADD) with the subsequent activation of the caspases cascade [12]. Furthermore, CD95/Fas is involved in the activation of the apoptotic pathway not only by directly inducing the cleavage of caspase-8 but also by increasing the ratio of Bax (pro-apoptotic factor) to Bcl-2 (anti-apoptotic factor) which favors the formation of Bax homodimers [14], [15] which activate the effector caspases. Additionally, TNF-α and interleukin 1α are also known to activate the CD95/Fas pathway in human osteoblasts leading to recruitment and activation of caspase-8 [11], [16].

The action of the active metabolite of vitamin D3, 1,25 dihydroxyvitamin D3 (1,25(OH)₂D₃) on bone is primarily indirect through the intestinal absorption of calcium (Ca⁺) and phosphorus, which are necessary for mineralization of bone matrix and prevention of rickets and osteomalacia [17], [18], [19], [20]. In addition, 1,25(OH)₂D₃ binds to vitamin D receptors (VDR) in osteoblasts and induces expression of receptor activator of NF-κB ligand (RANK-L) [21], [22], which in turn mediates osteoclastogenesis and osteoclastic bone resorption [22]. Although in previous work, it was shown that 1,25(OH)₂D₃ rescued serum-deprived osteoblasts from apoptosis [5], the potential effect of 1,25(OH)₂D₃ on Fas-induced osteoblast apoptosis remains unknown. In this study, we assess the key elements of the CD95/Fas pathway that could be regulated by 1,25(OH)₂D₃ after the activation of Fas-ligand in human osteoblasts. Our data might offer a new approach to the inhibition of osteoblast apoptosis and therefore to the promotion of osteoblastic activity and bone formation.

Section snippets

Culture of human osteoblasts

Newborn human calvaria osteoblasts (N976) were generated as previously described [23]. Briefly, cells were obtained by collagenase digestion from the coronal sutures of two aborted normal 26-week-old fetuses in accordance with the French Ethical Committee recommendations. The cells were immortalized by transfection with the SV40 oncogene as described [24]. Previous analyses have shown that this immortalization does not change the properties of the cells [24], [25]. In fact, these cells express

Annexin V

FACS analysis of cells stained with annexin V and propidium iodide is shown in Table 1. The percentage of apoptotic osteoblasts in non-treated cells was 52 ± 4% after treatment with Fas-Ab, respectively. In contrast, the number of apoptotic cells was reduced to 14 ± 4% in cells treated with Fas-Ab in the presence of 10⁻⁸ M 1,25(OH)₂D₃ (P < 0.01). A slight number of serum supplemented non-induced N976 cells showed apoptotic changes in both annexin and TUNEL assays. No changes in the number of

Discussion

Investigation of the role of apoptosis in mediating a balance between bone deposition and bone resorption has gained momentum in recent years as a consequence of understanding the mechanism of action of antiresorptive agents such as E₂ and bisphosphonates [3] and anabolic agents such as parathyroid hormone (PTH) [27]. E₂ induces osteoclast apoptosis possibly through the induction or activation of transforming growth factor-β [6]. Similarly, bisphosphonates induce osteoclast apoptosis either by

Acknowledgements

This work was supported by grant MT-10839 from the Canadian Institutes of Health Research (CIHR), the Dairy Farmers of Canada, and the Helen McCall Hutchinson Foundation. Dr. Duque and Dr. El Abdaimi are supported by a fellowship from the Royal Victoria Hospital Research Institute and Dr. Duque holds a bursary from the Fonds de la Recherche du Santé du Québec. We thank Dr. Lise Binderup (LEO Pharmaceuticals, Ballerup, Denmark) for kindly providing us with 25-OHD and 1,25(OH)₂D₃.

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Vitamin D inhibits Fas ligand-induced apoptosis in human osteoblasts by regulating components of both the mitochondrial and Fas-related pathways

Abstract

Introduction

Section snippets

Culture of human osteoblasts

Annexin V

Discussion

Acknowledgements

Am. J. Med.

Biochem. Biophys. Res. Commun.

J. Lab. Clin. Med.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Arch. Oral Biol.

J. Biol. Chem.

J. Biol. Chem.

J. Lab. Clin. Med.

J. Invest. Dermatol.

J. Steroid Biochem. Mol. Biol.

Adv. Enzyme Regul.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

Discoveries, drugs and skeletal disorders

Nat. Rev., Drug Discov.

Age-related bone loss: old bone, new facts

Gerontology

Birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis

Endocr. Rev.

Apoptosis in bone physiology and disease

Mol. Pathol.

Estrogen promotes apoptosis of murine osteoclasts mediated by TGFß

Nat. Med.

Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids

J. Clin. Invest.