Melanin extract fromGallus gallus domesticuspromotes proliferation and differentiation of osteoblastic MG-63 cells via bone morphogenetic protein-2 signaling

Abstract

BACKGROUND/OBJECTIVESGallus 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/METHODSThe 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.RESULTSThe 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.CONCLUSIONSThis 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.

Icariin attenuates titanium particle‐induced inhibition of osteogenic differentiation and matrix mineralization via miR‐21‐5p

“…ALP, which is expressed by osteoblasts, is a crucial indicator of bone mineralization (Kim et al, 2016;Yoo et al, 2017). The differentiated osteoblasts exhibit enhanced ALP activity and bone mineralization (Yoo et al, 2017). Alizarin Red staining is commonly used to evaluate mineralization (Choi et al, 2015;Yusa et al, 2016).…”

Protective Effects of Fermented Oyster Extract against RANKL-Induced Osteoclastogenesis through Scavenging ROS Generation in RAW 264.7 Cells

“…In order to measure TRAP activity, the culture medium was collected, and the activity of TRAP was measured using a TRAP assay kit (Sigma-Aldrich Chemical Co.) at 450 nm with an ELISA microplate reader. TRAP activity was calculated as a percentage of the control, in accordance with the method from a previous study [52].…”

Distinct anti-proliferative effects of herbal melanin on human acute monocytic leukemia THP-1 cells and embryonic kidney HEK293 cells

“…The anti-proliferative effects of high concentrations (500-1000 μg/mL) of HM were also reported in human carcinoma cells [30]. Previously, studies on the effect of melanin on cell growth were performed using melanin extracts from sources other than Nigella sativa L. Contradicting results were reported, demonstrating both positive and negative effects of melanin on osteoblastic MG-63 cells, a skin cancer cell line and normal human dermal cell viability, respectively [39][40][41] [42]. Down-regulation of these cell cycle-related proteins was observed after epidermal cell treatment with 50-70 and 100 μg/mL of melanin [42].…”