J Bone Metab.  2016 Feb;23(1):8-15. 10.11005/jbm.2016.23.1.8.

Deficiency of Lipocalin-2 Promotes Proliferation and Differentiation of Osteoclast Precursors via Regulation of c-Fms Expression and Nuclear Factor-kappa B Activation

• Kim HJ
• Ohk B
• Kang WY
• Seong SJ
• Suk K
• Lim MS
• Kim SY
• Yoon YR

• KMID: 2156281
• DOI: http://doi.org/10.11005/jbm.2016.23.1.8

Abstract

BACKGROUND
Lipocalin-2 (LCN2), a small glycoprotein, has a pivotal role in diverse biological processes such as cellular proliferation and differentiation. We previously reported that LCN2 is implicated in osteoclast formation induced by receptor activator of nuclear factor-kappa B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF). In the present study, we used a knockout mouse model to further investigate the role of LCN2 in osteoclast development.
METHODS
Osteoclastogenesis was assessed using primary bone marrow-derived macrophages. RANKL and M-CSF signaling was determined by immunoblotting, cell proliferation by bromodeoxyuridine (BrdU) enzyme-linked immunosorbent assay (ELISA), and apoptosis by cell death detection ELISA. Bone morphometric parameters were determined using a micro-computed tomography system.
RESULTS
Our results showed that LCN2 deficiency increases tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclast formation in vitro, a finding that reflects enhanced proliferation and differentiation of osteoclast lineage cells. LCN2 deficiency promotes M-CSF-induced proliferation of bone marrow macrophages (BMMs), osteoclast precursors, without altering their survival. The accelerated proliferation of LCN2-deficient precursors is associated with enhanced expression and activation of the M-CSF receptor, c-Fms. Furthermore, LCN2 deficiency stimulates the induction of c-Fos and nuclear factor of activated T cells c1 (NFATc1), key transcription factors for osteoclastogenesis, and promotes RANKL-induced inhibitor of kappa B (IkappaBalpha) phosphorylation. Interestingly, LCN2 deficiency does not affect basal osteoclast formation in vivo, suggesting that LCN2 might play a role in the enhanced osteoclast development that occurs under some pathological conditions.
CONCLUSIONS
Our study establishes LCN2 as a negative modulator of osteoclast formation, results that are in accordance with our previous findings.

Keyword

c-Fms; LCN2; NF-kappaB; Osteoclast

MeSH Terms

Acid Phosphatase
Animals
Apoptosis
Biological Processes
Bone Marrow
Bromodeoxyuridine
Cell Death
Cell Proliferation
Enzyme-Linked Immunosorbent Assay
Glycoproteins
Immunoblotting
Macrophage Colony-Stimulating Factor
Macrophages
Mice
Mice, Knockout
NF-kappa B
Osteoclasts*
Phosphorylation
RANK Ligand
T-Lymphocytes
Transcription Factors
Acid Phosphatase
Bromodeoxyuridine
Glycoproteins
Macrophage Colony-Stimulating Factor
NF-kappa B
RANK Ligand
Transcription Factors

Figure

• 

  Fig. 1 Lipocalin-2 (LCN2) deficiency increases osteoclast formation. Wild-type (WT) and LCN2-/- bone marrow macrophages were cultured with macrophage colony-stimulating factor (10 ng/mL) and the indicated concentrations of receptor activator of nuclear factor-kappa B ligand. (A) After 4 days, cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP). (B) Statistical analysis of WT and LCN2-/- TRAP-positive multinucleated cells/well at day 4. Data are expressed as the mean standard deviation. *P<0.05. WT, wild-type; LCN2, lipocalin-2; RANKL, receptor activator of nuclear factor-kappa B ligand; TRAP, tartrate-resistant acid phosphatase; MNC, more than three nuclie.

• 

  Fig. 2 Lipocalin-2 (LCN2) deficiency promotes precursor proliferation by enhancing expression and activation of c-Fms. (A) Wild-type (WT) and LCN2-/- bone marrow macrophages (BMMs) were cultured with macrophage colony-stimulating factor (M-CSF) (30 ng/mL). Apoptosis was determined as a function of DNA fragmentation. (B) WT and LCN2-/- BMMs were cultured with indicated concentrations of M-CSF. After 3 days, bromodeoxyuridine corporation was measured. Data are expressed as the mean standard deviation. *P<0.05, **P<0.001. (C) Serum-starved WT and LCN2-/- BMMs were stimulated with M-CSF (50 ng/mL) for the indicated time. Phosphorylated and total c-Fms were detected by immunoblotting; beta-actin served as a loading control. Phosphorylation of extracellular signal-regulated kinase (ERK) and Akt was determined by immunoblotting; total ERK and Akt levels served as loading controls. BrdU, bromodeoxyuridine; WT, wild-type; LCN2, lipocalin-2; M-CSF, macrophage colony-stimulating factor; ERK, extracellular signal-regulated kinase.

• 

  Fig. 3 Lipocalin-2 (LCN2) deficiency enhances receptor activator of nuclear factor-kappa B ligand (RANKL)-induced c-Fos expression and inhibitor of kappa B alpha phosphorylation. (A and B) wild-type (WT) and LCN2-/- bone marrow macrophages (BMMs) were cultured in osteoclastogenic media for the indicated days, and the expression of the indicated genes was analyzed by real-time polymerase chain reaction (PCR) (A) or immunoblotting (B). Data are expressed as the mean standard deviation. *P<0.05, ***P<0.001. (C) The expression of LCN2 receptors was determined by teal-time-PCR. (D) WT and LCN2-/- BMMs were treated with RANKL (50 ng/mL) for the indicated time. Immunoblots of total protein extracts were performed with the indicated antibodies; beta-actin and total p38 levels served as loading controls. WT, wild-type; LCN2, lipocalin-2; NFAT, nuclear factor-activated T cells; TRAP, tartrate-resistant acid phosphatase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; RANKL, receptor activator of nuclear factor-kappa B ligand; IκBα, inhibitor of kappa B alpha.

• 

  Fig. 4 Bone phenotype of lipocalin-2 (LCN2)-/- mice. (A) Micro-computed tomography (µCT) images of distal femurs of wild-type (WT) and LCN2-/- mice. (B) µCT analysis of trabecular bone and the serum collagen type 1 fragment levels of WT and LCN2-/- mice. (C) Histological analysis of the proximal tibiae of WT and LCN2-/- mice; hematoxylin and eosin staining (top) and tartrate-resistant acid phosphatase staining (bottom). WT, wild-type; LCN2, lipocalin-2; TRAP, tartrate-resistant acid phosphatase; H&E, hematoxylin and eosin; BV, bone volume; TV, tissue volume; Tb.N, trabecular number; Tb.Sp, trabecular space; CTX-1, C-terminal telopeptides of type I collagen.

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