Adipocyte-secreted factors increase osteoblast proliferation and the OPG/RANKL ratio to influence osteoclast formation

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Abstract

Several studies have reported a positive relationship of the body fat mass and bone density. However, it is not clear whether adipocyte-derived signaling molecules directly act on osteoblasts or osteoclasts. Therefore, we investigated the effect of fat cell-secreted factors on the proliferation and differentiation of preosteoblasts and the molecular mechanisms involved.

This stimulation led to an increased proliferation of MC3T3-E1 and primary preosteoblastic cells (2.8-fold and 1.5-fold, respectively; p < 0.0001), which could be reduced with inhibitors of protein tyrosine kinases, FGFR1 and PI3K. Concordantly, we found human adipocytes to secrete bFGF and bFGF to mimic the effect of adipocyte-secreted factors. The ratio of OPG/RANKL secretion in primary human preosteoblasts increased 9-fold (mRNA and protein) when stimulated with adipocyte-secreted factors. Moreover, osteoblasts which were prestimulated with adipocyte-secreted factors inhibited the formation of osteoclasts.

In conclusion, human adipocytes secrete factors that directly act on preosteoblasts and alter their crosstalk with osteoclasts. These in vitro findings reflect the higher bone mass in obese people and attribute it to effects of adipocyte-secreted factors on bone formation.

Highlights

► It is unclear whether fat mass increases bone density only by the higher weight. ► We investigate the role of adipocytes as an endocrine tissue in this context. ► Adipocyte-secreted factors include bFGF. ► These factors increase preosteoblast proliferation and OPG/RANKL ratio. ► These findings may represent an endocrine effect of adipocytes on osteoblasts.

Introduction

Osteoporosis is a generalized skeletal disorder characterized by reduced bone mass and compromised bone quality, thus, eventually leading to an increased fracture risk (NIH Consensus, 2001).

At the age of 50, the lifetime risk for an osteoporotic fracture is 39.7–53.2% for women and 13.1–22.4% for men, respectively (Johnell and Kanis, 2005). In addition, osteoporotic fractures not only cause high costs for health care systems worldwide but also lead to a significant increase in mortality (Borgstrom et al., 2006, Borgstrom et al., 2007, Burge et al., 2007, Clark et al., 2008, Haussler et al., 2007, Johnell and Kanis, 2006, Konnopka et al., 2009). The WHO anticipates a worldwide increase in osteoporotic hip fractures from 1.7 million in 1990 to 6.3. million by 2050 (WHO, Press Release 1999). Therefore, it is of great clinical and socio-economic interest to understand risk-conferring and preventing mechanisms for osteoporosis.

After a proliferation step preosteoblasts start to differentiate into mature osteoblasts (Colnot, 2005). An early differentiation marker is the alkaline phosphatase (ALP) whereas matrix mineralization with calcium deposits occurs at a later step (Hoemann et al., 2009). Mature osteoblasts also produce osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL), which have opposing effects on osteoclasts.

The association of adipose tissue mass and bone mass in humans has been investigated by several groups. The results are, however, controversial. While a few studies reported a negative association of the body mass index (BMI) with bone mineral density (BMD) (Beck et al., 2009, Travison et al., 2008, Zhao et al., 2007), the majority indicated a positive correlation between the BMI (Gnudi et al., 2009, Kinjo et al., 2007, Nguyen et al., 2005, von et al., 2007) or the body fat mass (Gnudi et al., 2007, Nguyen et al., 2005) and BMD, but the underlying mechanisms are not understood.

Adipose tissue is considered as an endocrine organ known to secrete various adipokines including leptin, adiponectin and Wnt molecules (Fischer-Posovszky et al., 2007, Rasouli and Kern, 2008, Schinner et al., 2005, Schinner et al., 2008). The effects of individual adipokines on bone are summarized elsewhere (Gomez-Ambrosi et al., 2008, Reid, 2008). In the current study we therefore investigated the direct effects of human adipocytes through secreted factors on preosteoblast proliferation as well as differentiation and OPG/RANKL expression.

Section snippets

Chemicals and proteins

Recombinant murine Sfrp1, Dkk1, RANKL and M-CSF and recombinant human bFGF were purchased from R&D Systems. I-OMe-AG 538, HNMPA-(AM)3, JAK-Inhibitor I, PD173074 and Src-Inhibitor I were purchased from Calbiochem. All other inhibitors and chemicals were from Sigma–Aldrich.

Cell culture

The murine preosteoblastic cell line MC3T3-E1 and the murine osteoclast precursor cell line RAW264.7 were purchased from ATCC, USA. All cells were cultured in α-MEM (Invitrogen) supplemented with 100 U/ml penicillin, 100 μg/ml

Adipocyte-secreted factors induce the proliferation of murine preosteoblastic cells

We assessed the effect of adipocyte-secreted factors on the proliferation of preosteoblastic cells. We treated murine preosteoblastic MC3T3-E1 cells with fat-cell conditioned-medium (FCCM) for 48 h. As shown in Fig. 1A, treatment of MC3T3-E1 cells with FCCM resulted in a highly significant 2.8-fold increase in proliferation (p < 0.0001).

The Wnt-signaling pathway is a known regulator of osteoblast proliferation (Boland et al., 2004) and we have previously identified Wnt molecules in FCCM (Schinner

Discussion

Clinical studies on the association of the BMI with BMD in humans are inconsistent. However, the majority of studies indicate a positive correlation (Gnudi et al., 2009, Kinjo et al., 2007, Nguyen et al., 2005, von et al., 2007). The correlation between BMI and BMD in the different studies might be confounded by less physical activity in very obese subjects. Another explanation is varying nutritional behavior among the participants which can also affect bone quality and strength.

The adipose

Acknowledgements

We thank Birgit Mülders-Opgenoorth for excellent technical assistance.

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