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- MATERIALS AND METHODS
Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the pre-osteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E2 (PGE2) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-β1 treatment of pre-osteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE2 increased apoptosis threefold, while treatment of pre-osteocytes with TGF-β1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival. © 2005 Wiley-Liss, Inc.
Osteoblasts (OBs) are bone-forming cells that arise from mesenchymal precursors located in bone marrow [Bodine and Komm, 2002; Lian et al., 2003]. These cells undergo a somewhat defined progression to mature-OBs that includes commitment to and proliferation of osteoprogenitors, differentiation to and maturation of pre-OBs, and finally bone matrix production. After mature-OBs have finished synthesizing the bone matrix, they have one of three fates: (i) they can become organized as quiescent lining cells that guard the bone matrix; (ii) they can differentiate into osteocytes (OCYs), which serve as mechanosensors upon entrapment within the mineralized matrix; or (iii) they can die [Bodine and Komm, 2002; Lian et al., 2003]. It has been estimated that as many as 50%–80% of cells recruited to the bone-forming surface will undergo programmed cell death (PCD) [Manolagas, 2000; Boyce et al., 2002]. Although it is thought that drugs such as estrogens and parathyroid hormone (PTH) may act in part by blocking this process [Manolagas, 2000; Boyce et al., 2002], the mechanisms by which OBs are directed towards apoptosis are not entirely clear. However, recent studies have shown the importance of canonical Wnt signaling in the control of OB and OCY PCD [Babij et al., 2003].
Some of the well-characterized molecular markers of the OB lineage are the bone-specific transcription factor Runx2, the enzyme alkaline phosphatase (ALP), and the bone matrix proteins type I collagen and osteocalcin [Bodine and Komm, 2002; Komori, 2002; Lian and Stein, 2003; Lian et al., 2003]. In addition, transcription factors like Twist [Lee et al., 1999], Msx-2 [Bidder et al., 1998], and Osterix [Nakashima et al., 2002] are also known to play important roles in regulating osteogenesis [Marie, 2001; Yang and Karsenty, 2002]. Many studies have documented the differential expression of these and other genes during OB differentiation using a variety of in vitro models [Harris and Harris, 2002; Stains and Civitelli, 2003]. Although these studies have been very informative, they do not provide insight into the extracellular signals that lead to transcription of target genes in bone. Furthermore, many studies have focused on rodent cells and less is known regarding the molecular events of human OB differentiation.
We have developed a collection of conditionally immortalized adult human OBs or HOB cell lines that contains representatives of several stages of differentiation including a pre-OB cell line, many mature-OB cell lines, and the first human OCY cell lines [Bodine and Komm, 2002]. These cells were immortalized with a temperature-sensitive simian virus (SV) 40 large T-antigen, and they exhibit a transformed phenotype at the permissive temperature (34°C) when the T-antigen mutant is active. However, in contrast to osteosarcoma cells [Stein and Lian, 1993], the HOB cell lines are faithful to the proliferation/differentiation relationship at the non-permissive temperature (≥37°C) when the T-antigen mutant is inactivated [Bodine and Komm, 2002]. Recently, we have used this collection of cell lines to characterize the osteogenic process at the molecular level with the goal of creating a more in depth understanding of how the human OB differentiates and synthesizes bone [Billiard et al., 2003]. In these studies, microarray technology was used to elucidate the differential transcription profiles of distinct stages of human OB development, and we identified 47 genes whose expression was found to change threefold or more between the pre-OB and pre-OCY stages. Although microarray utilizes small amounts of RNA, is rapid and quantitative, it is limited by RNA abundance in samples and prior gene sequence knowledge. In contrast, techniques like differential-display polymerase chain reaction (DD-PCR) analysis can identify low abundant and previously unknown mRNAs [Chang et al., 2005]. In addition, DD-PCR produces a cDNA fragment that can serve as the starting point for gene isolation.
In the current study, we have also utilized the HOB cells as models to study the molecular events associated with human OB differentiation. To identify genes associated with this process, we used known bone-forming agents and a high-throughput robotic form of DD-PCR analysis known as RADE, or rapid analysis of differential expression [Shiue, 1997]. We identified 82 differentially expressed genes, one of which was the Wnt antagonist, secreted frizzled-related protein (sFRP)-1 [Finch et al., 1997; Rattner et al., 1997] or secreted apoptosis-related protein (SARP)-2 [Melkonyan et al., 1997]. We previously reported that deletion of sFRP-1 in mice leads to increased trabecular bone formation [Bodine et al., 2004]. Here, we describe the characterization of sFRP-1 in vitro and demonstrate that it is an important regulator of human OB and OCY apoptosis.
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- MATERIALS AND METHODS
Transcription profiling of numerous OB models has identified many new genes and pathways that are involved in differentiation and osteogenesis [Harris and Harris, 2002; Stains and Civitelli, 2003]. Likewise, similar studies of OBs treated with osteogenic agents like PTH [Qin et al., 2003] and BMP-2 [Locklin et al., 2001; de Jong et al., 2002; Harris and Harris, 2002; Vaes et al., 2002; Balint et al., 2003; Clancy et al., 2003] have also discovered new potential mechanisms for the bone anabolic actions of these agents. Among the important new signaling pathways that have been revealed by these studies is the Wnt pathway [de Jong et al., 2002; Doi et al., 2002; Harris and Harris, 2002; Vaes et al., 2002; Balint et al., 2003; Clancy et al., 2003; Qi et al., 2003; Roman-Roman et al., 2003; Westendorf et al., 2004]. For example, treatment of UMR-106 rat osteosarcoma cells with PTH has been reported to up-regulate sFRP-4 mRNA expression [Qin et al., 2003], and to increase Frizzled (FZD)-1 and -2 mRNA levels [Chan et al., 1992]. On the other hand, BMP-2 treatment of murine C2C12 pre-myoblastic cells has been observed to elevate Wnt inhibitory factor-1 [Vaes et al., 2002] and sFRP-2 [Vaes et al., 2002; Balint et al., 2003] messages, while treatment of mouse muscle in vivo with BMP-2 was reported to increase sFRP-1, -2, -3 and -4 as well as FZD-1 mRNAs [Clancy et al., 2003]. BMP-2 has also been observed to up-regulate Wnt-1, -3a, -5b, -7, -9b as well as FZD-1 and -9 mRNA expression in murine pluripotent mesenchymal cell lines and MC-3T3-E1 osteoblastic cells; in addition, it increases Tcf-1 and Lef-1 message levels, but suppresses low-density lipoprotein receptor-related protein (LRP)-1, LRP-5 and sFRP-2 mRNA expression in MC-3T3-E1 cells [Rawadi et al., 2003]. Finally, FZD-1 and sFRP-2 were also identified as genes whose expression changed during the differentiation of primary mouse calvarial-derived OBs cultured with ascorbic acid and β-glycerol-phosphate to promote development of the OB phenotype [Roman-Roman et al., 2003].
As noted above, Wnt antagonists like sFRPs are commonly identified in transcription profiling studies of OBs. In the current study, RADE analysis of HOB cells treated with PGE2, TGF-β1, and PTH was used to identify 82 differentially expressed genes. Of these, the Wnt antagonist sFRP-1 was the most highly regulated gene, suggesting that it plays an important role in OB differentiation and bone formation. Increased sFRP-1 expression correlated with elevated cell death, while decreased expression correlated with decreased mortality. In addition, over-expression of sFRP-1 accelerated HOB cell death and antagonized canonical Wnt signaling. Thus, regulation of Wnt activity by sFRP-1 appears to be important for the control of OB and OCY survival. This conclusion is supported by results from the sFRP-1 knockout mice, which exhibit decreased OB and OCY apoptosis when compared to wild-type controls [Bodine et al., 2004]. Moreover, the importance of the canonical Wnt pathway in the modulation of OB and OCY longevity is underscored by the suppression of bone cell apoptosis that is observed in transgenic mice that over-express the G171V gain-of-function mutation of LRP-5 [Babij et al., 2003].
In the HOB cell lines, sFRP-1 mRNA levels increased with advancing cellular differentiation and peaked in the pre-OCY stage of development. This elevated expression correlated with the highest basal cell death rate among the HOB cell lines. These data suggest that pre-OCYs are the most susceptible of the OB lineage to apoptosis, and that this process is controlled by sFRP-1. Thus, regulation of Wnt signaling by sFRP-1 may be one of the mechanisms that modulates the number of mature-OCYs that finally become entombed in mineralized bone. The observation that apoptosis increases with advancing cellular differentiation has also been observed in ROBs and implies that PCD is a fundamental component of this process [Lynch et al., 1998; Lian and Stein, 2003].
Several hormones, growth factors, and cytokines known to modulate OB differentiation and apoptosis also controlled the expression of sFRP-1 by the HOB cells. PGE2 treatment induced sFRP-1 mRNA levels in HOB cells representing pre-OBs (HOB-03-C5 cells) and mature-OBs (HOB-03-CE6 cells). This correlated with an increase in apoptosis and may relate to the ability of PGE2 to promote OB differentiation [Vrotsos et al., 2003]. Increased sFRP-1 expression could also result from a feedback mechanism to dampen the osteogenic effects of PGE2. Treatment of HOB cells representing mature-OBs with the bone-resorbing cytokine IL-1β [Mundy et al., 2003] also strongly up-regulated sFRP-1 message levels. Although we did not examine the effect of IL-1β on HOB cell viability, this too may correspond with an increase in apoptosis. Another bone-resorbing cytokine with actions similar to IL-1β, TNF-α [McCarthy et al., 2000; Mundy et al., 2003], has also been reported to increase OB PCD in vitro [Hock et al., 2001], and both IL-1β and TNF-α activate nuclear factor (NF)-κβ signaling [Baldwin, 1996]. On the other hand, treatment of pre-OBs and mature-OBs with vitamin D3 suppressed sFRP-1 message levels. Moreover, TGF-β1 treatment of HOB cells representing pre-OCYs (HOB-01-C1 cells), which have high basal levels of sFRP-1 message, down-regulated sFRP-1 gene expression and suppressed cell death. In addition, treatment of these cells with BMP-2 and IGF-1 also decreased sFRP-1 mRNA levels. This too may correspond to suppression of apoptosis, since these growth factors have been reported to decrease OB PCD in vitro [Boyce et al., 2002].
In contrast to published reports indicating that pharmacological concentrations of glucocorticoids increase OB and OCY apoptosis [Manolagas, 2000; Hock et al., 2001; Boyce et al., 2002], treatment of HOB cells representing pre-OBs and mature-OBs with Dex down-regulated sFRP-1 mRNA levels and inhibited PCD. Other studies have also reported that Dex treatment suppresses OB apoptosis in vitro [Zalavras et al., 2003]. A potential explanation for these discrepancies is that glucocorticoids promote OB apoptosis in vitro when cell culture conditions do not fully support differentiation (e.g., sub-confluent cultures), while they suppress in vitro PCD when OB differentiation is enhanced (e.g., confluent cultures) [Zalavras et al., 2003]. In the case of the HOB cell lines, in vitro differentiation of these cells is promoted when the cultures are incubated at non-permissive temperatures, and the temperature-sensitive T-antigen is inactivated [Bodine and Komm, 2002].
Finally, although estrogens and PTH have been reported to decrease OB and OCY apoptosis [Manolagas, 2000; Hock et al., 2001; Boyce et al., 2002], treatment of the HOB cells with these hormones did not affect sFRP-1 gene expression or cell viability.
sFRP-1 gene expression is also up-regulated by PGE2 and IL-11 treatment of murine stromal cells and OBs; however, incubation of these cells with Dex, vitamin D3, PTH, and IL-1, -4, -10, and -18 had no effect on mRNA levels of the Wnt antagonist [Hausler et al., 2004]. Thus, mechanisms controlling sFRP-1 expression appear to vary among species.
Wang et al.  recently reported that pharmacological concentrations of Dex increased sFRP-1 expression in primary rat MSCs in vitro and in rat OBs and chondrocytes in vivo. In this study, increased sFRP-1 expression correlated with decreased cytosolic β-catenin levels, reduced osteogenesis and increased apoptosis. Moreover, the authors reported that in vivo administration of recombinant human sFRP-1 to rats decreased proximal femur bone mineral density and trabecular bone volume, which is consistent with an inhibitory effect of the protein on bone formation [Bodine et al., 2004].
In addition to suppressing PCD, deletion of sFRP-1 also increases OB proliferation, differentiation, and function [Bodine et al., 2004]. Although loss of sFRP-1 did not appear to affect bone resorption in vivo, it did result in increased osteoclastogenesis in vitro [Bodine et al., 2004]. This may be caused by increased osteoblastogenesis [Manolagas, 2000], which also occurs in the sFRP-1 knockout mice [Bodine et al., 2004]. However, another potential mechanism is the ability of sFRP-1 to bind and antagonize receptor activator of NF-κβ ligand [Chuman et al., 2004; Hausler et al., 2004], which is an important stimulator of osteoclastogenesis [Manolagas, 2000].
Other sFRPs have also been observed to affect OB physiology, mineral metabolism, and skeletal apoptosis. Using MC-3T3-E1 mouse OBs, Chung et al.  recently reported that treatment of the cells with sFRP-3 decreased proliferation and increased differentiation. The authors proposed that these effects occur via regulation of β-catenin-independent pathways. Berndt et al.  have observed that treatment of opossum renal epithelial cells in vitro with sFRP-4 inhibited sodium-dependent phosphate transport, while treatment of rats and mice in vivo with sFRP-4 elevated renal fractional phosphate excretion and suppressed renal phosphate re-absorption. These data indicate that sFRP-4 has phosphatonin-like properties, promoting phosphaturia and hypophosphatemia, and may therefore control bone mineralization. Finally, James et al.  have reported that sFRP-4 is highly expressed in chondrocytes from patients with osteoarthritis, while its expression is negligible in cells from normal cartilage. Moreover, the authors observed a correlation between sFRP-4 expression and chondrocyte apopotosis.
In summary, DD-PCR analysis of HOB cell lines in three stages of differentiation treated with three osteogenic agents has identified sFRP-1 as a highly differentially expressed gene. Expression of sFRP-1 is associated with OB differentiation and PCD, and it is regulated by hormones, growth factor and cytokines that are known to modulate these processes. These data lend support to other studies indicating the importance of sFRP-1 in controlling Wnt regulation of apoptosis and bone metabolism.