Nutr Res Pract.  2017 Jun;11(3):190-197. 10.4162/nrp.2017.11.3.190.

Melanin extract from Gallus gallus domesticus promotes proliferation and differentiation of osteoblastic MG-63 cells via bone morphogenetic protein-2 signaling

• Yoo HS
• Chung KH
• Lee KJ
• Kim DH
• An JH

• KMID: 2407766
• DOI: http://doi.org/10.4162/nrp.2017.11.3.190

Abstract

BACKGROUND/OBJECTIVES
Gallus gallus domesticus (GD) is a natural mutant breed of chicken in Korea with an atypical characterization of melanin in its tissue. This study investigated the effects of melanin extracts of GD on osteoblast differentiation and inhibition of osteoclast formation.
MATERIALS/METHODS
The effects of the melanin extract of GD on human osteoblast MG-63 cell differentiation were examined by evaluating cell viability, osteoblast differentiation, and expression of osteoblast-specific transcription factors such as bone morphogenetic protein 2 (BMP-2), small mothers against decapentaplegic homologs 5 (SMAD5), runt-related transcription factor 2 (RUNX2), osteocalcin and type 1 collagen (COL-1) by reverse transcription-polymerase chain reaction and western blotting analysis. We investigated the inhibitory effect of melanin on the osteoclasts formation through tartrate-resistant acid phosphatase (TRAP) activity and TRAP stains in Raw 264.7 cell.
RESULTS
The melanin extract of GD was not cytotoxic to MG-63 cells at concentrations of 50-250 µg/mL. Alkaline phosphatase (ALP) activity and bone mineralization of melanin extract-treated cells increased in a dose-dependent manner from 50 to 250 µg/mL and were 149% and 129% at 250 µg/mL concentration, respectively (P < 0.05). The levels of BMP-2, osteocalcin, and COL-1 gene expression were significantly upregulated by 1.72-, 4.44-, and 2.12-fold in melanin-treated cells than in the control cells (P < 0.05). The levels of RUNX2 and SMAD5 proteins were higher in melanin-treated cells than in control vehicle-treated cells. The melanin extract attenuated the formation of receptor activator of nuclear factor kappa-B ligand-induced TRAP-positive multinucleated RAW 264.7 cells by 22%, and was 77% cytotoxic to RAW 264.7 macrophages at a concentration of 500 µg/mL.
CONCLUSIONS
This study provides evidence that the melanin extract promoted osteoblast differentiation by activating BMP/SMADs/RUNX2 signaling and regulating transcription of osteogenic genes such as ALP, type I collagen, and osteocalcin. These results suggest that the effective osteoblastic differentiation induced by melanin extract from GD makes it potentially useful in maintaining bone health.

Keyword

Melanin; alkaline phosphatase; osteoblast; cell differentiation

MeSH Terms

Acid Phosphatase
Alkaline Phosphatase
Blotting, Western
Bone Morphogenetic Protein 2
Calcification, Physiologic
Cell Differentiation
Cell Survival
Chickens*
Collagen Type I
Coloring Agents
Gene Expression
Humans
Korea
Macrophages
Melanins*
Osteoblasts*
Osteocalcin
Osteoclasts
RAW 264.7 Cells
Smad Proteins
Smad5 Protein
Transcription Factors
Acid Phosphatase
Alkaline Phosphatase
Bone Morphogenetic Protein 2
Collagen Type I
Coloring Agents
Melanins
Osteocalcin
Smad Proteins
Smad5 Protein
Transcription Factors

Figure

• 

  Fig. 1 Characterization of melanin extracted from Gallus gallus domesticus (GD) skin. (A) Electron ionization mass spectrometry (EI-MS) spectra of melanin. (B) Attenuated total reflection (ATR)-Fourier transform infrared (FTIR) spectra of melanin. (C) Carbon-13 nuclear magnetic resonance (13C-NMR) of melanin.

• 

  Fig. 2 Effect of melanin extracted from Gallus gallus domesticus (GD) on viability and alkaline phosphatase (ALP) activity of MG-63 osteoblast cells during differentiation. Cells were treated with GD melanin at 0, 50, 150, and 250 µg/mL for 24 h, and 96 h for ALP activity. (A) Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B) ALP activity, as described in the materials and methods section. Values are mean ± SD (n = 3). Values with different letters were significantly different using Duncan's multiple range test (P < 0.05).

• 

  Fig. 3 Mineralization of MG-63 cells treated with melanin extracted from Gallus gallus domesticus (GD) determined using alizarin red S staining after 4 days of differentiation. (A) Mineralized matrix was stained with alizarin red S. Melanin (250 µg/mL) significantly increased intracellular calcium. (B) Quantitation of mineralization using alizarin red S staining, as described in the materials and methods section. Values are mean ± SD (n = 3). Values with different letters were significantly different using Duncan's multiple range test (P < 0.05).

• 

  Fig. 4 Effect of melanin extracted from Gallus gallus domesticus (GD) on viability and tartrate-resistant acid phosphatase (TRAP) activity of RAW 264.7 cells during their differentiation. Cells were treated with GD melanin at 0, 50, 150, 250 and 500 µg/mL for 2 days, and 4 days for TRAP activity. (A) Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (B) TRAP activity, as described in materials and methods section. (C) TRAP staining using TRAP stain kit. Each value is mean ± SD (n = 3). Values with different letters were significantly different using Duncan's multiple range test (P < 0.05).

• 

  Fig. 5 Gene expression of bone morphogenetic protein-2 (BMP-2), osteocalcin and type 1 collagen (COL-1) using reverse transcription-polymerase chain reaction (RT-PCR). Cells were differentiated for 4 days in differentiation medium in the presence or absence of melanin (250 µg/mL). (A) The mRNA expression of osteoblast differentiation factors such as BMP-2, COL-1, and osteocalcin. (B) Relative expression was quantified using image J program and calculated according to the reference bands of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Values are mean ± SD (n = 3). Values with different letters were significantly different using the Duncan's multiple range test (P < 0.05).

• 

  Fig. 6 Effects of melanin extracted from Gallus gallus domesticus (GD) on bone morphogenetic protein-2 (BMP) signal pathway in MG-63 cells. Cells were differentiated for 4 days in differentiation medium in the presence or absence of melanin (250 µg/mL). (A) Protein expression of BMP signaling pathway-related markers such as BMP-2, runt-related transcription factor 2 (RUNX2), and small mothers against decapentaplegic homologs 5 (SMAD5) was detected by Western blot. (B) Relative expression was quantified by image J and calculated according to the reference bands of β-actin. Each value is expressed as mean ± SD (n = 3). Values with different letters were found to be significantly different using the Duncan's multiple range test (P < 0.05).

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