Oophorectomy-induced bone loss is attenuated in MAGP1-deficient mice

Abstract

Microfibril-associated glycoprotein-1 (MAGP1), together with the fibrillins, are constitutive components of vertebrate microfibrils. Mice deficient in MAGP1 (murine MAGP1 knockout animals (Mfap2(-/-)); MAGP1Δ) is appropriate develop progressive osteopenia and reduced whole bone strength, and have elevated numbers of osteoclasts lining the bone surface. Our previous studies suggested that the increased osteoclast population was associated with elevated levels of receptor activator of NF-κB ligand (RANKL), a positive regulator of osteoclast differentiation. To explore the relationship between RANKL expression and osteoclast differentiation in MAGP1 deficiency, oophorectomy (OVX) was used to stimulate RANKL expression in both WT and MAGP1Δ animals. Bone loss following OVX was monitored using whole body DEXA and in vivo µCT. While WT mice exhibited significant bone loss following OVX, percent bone loss was reduced in MAGP1Δ mice. Further, serum RANKL levels rose significantly in OVX WT mice, whereas, there was only a modest increase in RANKL following OVX in the mutant mice due to already high baseline levels. Elevated RANKL expression was normalized when cultured MAGP1Δ osteoblasts were treated with a neutralizing antibody targeting free TGFβ. These studies provide support for increased RANKL expression associated with MAGP1 deficiency and provide a link to altered TGF-β signaling as a possible causative signaling pathway regulating RANKL expression in MAGP1Δ osteoblasts.

Figure 1

MAGP1Δ mice show a blunted response to OVX-induced bone loss. Trabecular BMD (A) and BV/TV (B) pre- and post-surgery were determined by vivaCT. N=4 (WT-sham), 6 (WT-OVX), 4 (MAGP1Δ-sham), 3 (MAGP1Δ-OVX). Data are presented as AVG±SD, *=p<0.05

Figure 2

OVX-induced bone loss stabilizes in MAGP1Δ mice by 2 weeks post-OVX. Trabecular BMD (A) and BV/TV (B) pre- and post-surgery were determined by vivaCT. N=7 (WT-OVX) and 6 (MAGP1Δ-OVX). Data are presented as AVG±SD, *=p<0.05, NS=not significantly different

Figure 3

Serum RANKL is elevated in MAGP1Δ mice. Serum was collected 6 weeks post-surgery. N= 6 (WT-sham), 7 (WT-OVX), 5 (MAGP1Δ-sham), 8 (MAGP1Δ-OVX). Data are presented as AVG±SE, *=p<0.05

Figure 4

MAGP1 binds TGFβ1 and regulates its bioavailability. 4a) Surface plasmon resonance (BIAcore) was utilized to determine MAGP1’s affinity for pro-osteoclastogenic factors TGFβ1, RANKL, TNFα. 4b-c) RFL-6 cells were stably transfected with full-length MAGP1 or control vector (VC). Cells were cultured for 7 days to allow development of ECM devoid (VC) or enriched with MAGP1. 4c) Basal (unstimulated) or exogenous TGFβ1-induced SMAD-2 activation was determined by SDS-PAGE and immunoblotting with antibodies specific to phosphorylated SMAD-2 (p-smad2) or total smad-2 (tot-smad2).

Figure 5

Elevated RANKL expression is linked to increased free TGFβ. RANKL transcript was determined by RT-qPCR. Calvaria osteoblasts were cultured for 4 days in the presence of 300ng/mL neutralizing antibodies targeting TGFβ(-1,-2,-3) or TNFα. N=3 per group. Data are presented as RU (RANKL normalized to cyclophilin) AVG±SD, *=p<0.05