Korean J Physiol Pharmacol.  2019 Sep;23(5):411-417. 10.4196/kjpp.2019.23.5.411.

Humanin suppresses receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation via AMP-activated protein kinase activation

Affiliations
  • 1Department of Oral Biology, BK21 PLUS Project, Yonsei University College of Dentistry, Seoul 03722, Korea. dmshin@yuhs.ac

Abstract

Humanin (HN) is a mitochondrial peptide that exhibits cytoprotective actions against various stresses and diseases. HN has been shown to induce the phosphorylation of AMP-activated protein kinase (AMPK), which is a negative regulator of receptor activator of nuclear factor-κB ligand (RANKL). However, the role of HN in osteoclastogenesis or other skeletal disorders remains unknown. Here, we examined whether HN regulates osteoclastogenesis via AMPK activation using bone marrow-derived macrophage (BMM) cultures. Our results show that HN inhibited RANKL-induced osteoclast formation and reduced the expression of genes involved in osteoclastogenesis, including nuclear factor of activated T-cells cytoplasmic 1, osteoclast-associated receptor, cathepsin K, and tartrate-resistant acid phosphatase. Moreover, HN increased the levels of phosphorylated AMPK protein; compound C, an AMPK inhibitor, recovered HN-induced osteoclast differentiation. In addition, we found that HN significantly decreased the levels of RANKL-induced reactive oxygen species in BMMs. Therefore, these results indicate that HN plays an important role in osteoclastogenesis and may function as an inhibitor of bone disorders via AMPK activation.

Keyword

AMP-activated protein kinase; Humanin; Osteoclastogenesis; Receptor activator of nuclear factor-κB; ligand

MeSH Terms

Acid Phosphatase
AMP-Activated Protein Kinases*
Cathepsin K
Cytoplasm
Macrophages
Osteoclasts*
Phosphorylation
Reactive Oxygen Species
T-Lymphocytes
AMP-Activated Protein Kinases
Acid Phosphatase
Cathepsin K
Reactive Oxygen Species

Figure

  • Fig. 1 Effects of humanin (HN) on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation. (A) Bone marrow-derived macrophages (BMMs) were cultured in the presence of macrophage colony-stimulating factor (M-CSF) (30 ng/ml), RANKL (50 ng/ml), and the indicated concentrations of HN for 6 days. Cells were stained for tartrate-resistant acid phosphatase (TRAP). Scale bar, 500 µm. (B) Numbers of TRAP+ multinucleated cells were counted (≥ 3 nuclei). BMMs were seeded into 96-well plates and incubated with various concentrations of HN. Cell viability was measured by the MTS assay. OCs, osteoclasts.*p < 0.05 compared with control.

  • Fig. 2 Effects of humanin on the mRNA and protein levels of osteoclastogenesis-related genes. (A) The mRNA levels of receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation markers were evaluated by quantitative RT-PCR. (B) NFATc1 protein levels were examined by Western blot analyses. OSCAR, osteoclast-associated receptor; CTSK, cathepsin K; TRAP, tartrate-resistant acid phosphatase. *p < 0.05 compared with control.

  • Fig. 3 Effects of humanin (HN) on AMP-activated protein kinase (AMPK) activation during osteoclast differentiation and receptor activator of nuclear factor-κB ligand (RANKL)-induced reactive oxygen species production in bone marrow-derived macrophages (BMMs). (A) Western blot analysis of phosphor (p)-AMPK and AMPK during RANKL-induced osteoclastogenesis in untreated or HN-treated BMMs. The p-AMPK:AMPK ratio was calculated. (B) BMMs were treated with or without HN, and compound C, and stained for tartrate-resistant acid phosphatase (TRAP). Numbers of TRAP+ multinucleated cells were counted (≥ 3 nuclei). Scale bar, 500 µm. (C) BMMs were treated with RANKL (50 ng/ml) for 10 min and loaded with 10 µM dichlorofluorescein diacetate (DCF-DA). Data are expressed relative to the value for control BMMs. Scale bar, 100 µm. OCs, osteoclasts. *p < 0.05 compared with control.


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