Elsevier

Bone

Volume 33, Issue 5, November 2003, Pages 760-770
Bone

Original article
Expression and function of TNF-family proteins and receptors in human osteoblasts☆

https://doi.org/10.1016/j.bone.2003.07.006Get rights and content

Abstract

We studied how tumor necrosis-factor (TNF)-family proteins interact with osteoblasts to resolve several controversial points. We measured expression of TNFs, TNF-receptors, and nonsignaling (decoy) TNF receptors in human osteoblasts derived from mesenchymal stem cells and in MG63 human osteosarcoma cells using unamplified mRNA screening, with secondary Western or PCR analysis where indicated, and studied the effects of TNFs on osteoblasts in cell culture. Expression of TNFs and receptors was similar in MG63 cells and osteoblasts. TNF-R1 (p55), TRAIL receptor 1 and 2 (DR4 and 5), and Fas were expressed; RANK was undetectable. TNF-family ligands RANKL, TRAIL, and TNFα were expressed, but mRNAs were typically at low levels relative to receptors, suggesting that osteoblastic TNF signals, including RANKL, require specific stimuli. Flow cytometry of MG63 cells confirmed TNFα receptors and identified subpopulations with high surface-bound TNFα. Decoy receptors expressed included a novel soluble form of TNFRSF25 (formerly DR3 or Apo3), implicated in rheumatoid-arthritis linkage studies, as well as osteoprotegerin, a well-characterized osteoblast protein that binds TRAIL and RANKL, and DcR2, which binds TRAIL. Osteoblast apoptosis was studied using terminal deoxynucleotidyl transferase labeling and annexin V binding. MG63 cells were resistant to apoptosis by exogenous TNFα except when grown in media promoting osteoblast-like growth or matrix nodules. However, in media supporting osteoblast-like phenotype, apoptosis was induced by anti-Fas or TNF, in contrast to other studies with human osteoblasts. TRAIL caused cell retraction, supporting functional TRAIL response in cell differentiation, but did not cause apoptosis. We conclude that human osteoblasts have functional receptors for FasL, TNFα, TRAIL, but not RANKL, and that osteoblasts are protected by multiple nonsignaling TNF receptors against destruction by TNF-family proteins under conditions favoring cell growth.

Introduction

Despite many studies on TNF-family ligands (TNFs) and receptors in bone, it is unclear what functional TNFs, what TNF receptors, and what TNF-antagonists are expressed by human osteoblasts. The exception is that TNF regulation of osteoclast formation by osteoblasts is well studied. Osteoprotegerin (OPG) is a soluble or “decoy” TNF receptor produced by osteoblasts that is of key importance in osteoclast differentiation [1], [2], [3], [4]. OPG binds the TNF-family protein, RANKL, which activates the receptor activator of nuclear factor-κB, RANK, and mediates osteoclast differentiation [5]. RANKL is produced by osteoblasts, mesenchymal cells, and T-lymphocytes [5], [6], [7], and RANKL knockout mice have osteopetrosis [8].

On the other hand, the role of TNFs and TNF-receptors in osteoblast survival and differentiation is not clear. Bone loss in arthritis is related to expression of TNFα, leading to the hypothesis that TNF controls osteoblast survival. But many studies conclude that osteoblasts are resistant to TNF-mediated apoptosis. Polymorphisms in TNFα are linked to the RA-HLA-susceptibility locus [9] and a TNF-family receptor locus [10]; additionally, and TNFα-blocking produces sustained improvement in rheumatoid arthritis [11]. However, because TNFα is also implicated in osteoclast differentiation [12], it is not clear that TNFα effects on bone loss are osteoblast related. Rodent osteoblasts do undergo apoptosis in response to TNFα and serum starvation [13], [14], but human osteoblasts are resistant to TNF-mediated apoptosis [15], although reports vary [16]. Further, TNF-ligands other than TNFα and RANKL occur in osteoblasts, notably the TNF-related apoptosis inducing ligand, TRAIL, although TRAIL did not cause osteoblast apoptosis [15]. Because osteoblast apoptosis is an important cause of focal bone loss [17], the regulation of apoptosis by TNF-family members in osteoblasts is an important unresolved point.

There is also uncertainty regarding the mechanisms that protect bone in arthritis. Soluble “decoy” receptors may be important. Several decoy receptors in addition to OPG exist, which may regulate TNF-family proteins in bone, but their occurrence is not well studied. OPG, in addition, binds at least one TNF besides RANKL, TRAIL [18]. OPG deficiency is important in the progress of TNFα-mediated arthritis [19], while OPG reduces osteoclastic activity in animal models of arthritis [20], [21]. Anti-TNFα therapy normalizes RANKL and serum OPG [22], which may reflect changes in osteoclast formation. However, TNFα upregulates OPG in human mesenchymal stem cells [23], and continuing joint damage occurs in rheumatoid arthritis despite elevated OPG [24].

These results suggest that TNF-family proteins and receptors play complex roles in bone, but because data on occurrence and function of TNFs, TNF receptors, and decoy receptors are not consistent it is difficult to develop plausible mechanisms beyond a few well-established paradigms such as RANKL supporting osteoclast differentiation. We undertook to analyze occurrence and function of TNF-related proteins in human osteoblasts and osteosarcoma cells without a priori assumptions and with more comprehensive analytical tools than have been used previously. We began with unamplified gene-screening assays, and pursued functional studies of TNF effects on human osteoblasts in tissue culture when indicated by expression data. Because osteoblasts have several phases of growth and function, including fibroblast-like proliferation, epithelium-like bone-lining cells, and nodular matrix synthesis, we studied effects of TNFs on osteoblasts grown in conditions favoring different cell behaviors. By this approach, we hoped to clarify why some studies have reported TNF effects and others have not. The results indicate that nontransformed and transformed human osteoblasts contain multiple TNF-receptors and produce several nonsignaling TNF-receptors. Synthesis of TNF-family ligands in most cases is low, although the cells are capable of producing several TNFs including RANKL, TNFα, and TRAIL.

Section snippets

Cell cultures

The human osteosarcoma MG63 was from the American Type Culture Collection, Baltimore, MD, grown in RPMI 1640 or MEM, supplemented with 10% fetal calf serum unless indicated. MG63 cells were used at passages 83–86. As with many cell lines, stability can be a problem, so expression screening of MG63 mRNAs (described below) was done on three separate clonal isolates of these cells. Human mesenchymal stem cells were from Cambrex/Bio-Whittaker, East Rutherford, NJ, and were differentiated to

Expression of TNF receptors, decoy receptors, and ligands

RNA based screening was performed to survey the TNF receptors in three independent clonal isolates of MG63 cells and in nontransformed osteoblasts made from mesenchymal stem cells. These results (Table 1) showed remarkable consistency of expression of several important TNF receptors, decoy receptors, and TNFs between the four individual gene screen assays, although expression of important TNFs known to be produced by osteoblasts was in some cases unclear (such as for RANKL). Osteoblast

Discussion

The discovery that TNF-family cytokines are critically important in normal bone turnover, RANKL in particular [4], [5], [6], [8], and in the development of rheumatoid arthritis, TNFα in particular [11], [12], made occurrence and function of TNF-family proteins and receptors in bone an important issue. The situation is complicated by the degeneracy of TNF interactions. For example, the 55 kD TNF-R1 binds two minimally homologous substrates, TNFα and lymphotoxinα [29], and the decoy receptor OPG

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    Grant support: Supported in part by the U.S. National Institutes of Health, AG12951 and AR47700, and by the Department of Veterans Affairs.

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