J Nutr Health.  2018 Feb;51(1):23-30. 10.4163/jnh.2018.51.1.23.

Zinc upregulates bone-specific transcription factor Runx2 expression via BMP-2 signaling and Smad-1 phosphorylation in osteoblasts

• Cho YE
• Kwun IS

• KMID: 2410322
• DOI: http://doi.org/10.4163/jnh.2018.51.1.23

Abstract

PURPOSE
Runx2 (runt-related transcription factor 2), a bone-specific transcription factor, is a key regulator of osteoblast differentiation and its expression is induced by the activation of BMP-2 signaling. This study examined whether zinc modulates BMP-2 signaling and therefore stimulates Runx2 and osteoblast differentiation gene expression.
METHODS
Two osteoblastic MC3T3-E1 cell lines (subclones 4 as a high osteoblast differentiation and subclone 24 as a low osteoblastic differentiation) were cultured in an osteogenic medium (OSM) as the normal control, Zn− (1 µM Zn) or Zn+ (15 µM Zn) for 24 h. The genes and proteins for BMP-2 signaling (BMP-2, Smad-1/p-Smad-1), transcription factors (Runx2, osterix), and osteoblast differentiation marker proteins were assessed.
RESULTS
In both cell lines, BMP-2 mRAN and protein expression and extracellular BMP-2 secretion all decreased in Zn−. The expression of Smad-1 (downstream regulator of BMP-2 signaling) and p-Smad-1 (phosphorylated Smad-1) also downregulated in Zn−. Furthermore, the expression of the bone-specific transcription factors, Runx2 and osterix, decreased in Zn−, which might be due to the decreased BMP-2 expression and Smad-1 activation (p-Smad-1) by Zn−, because Runx2 and osterix both are downstream in BMP-2 signaling. Bone marker gene expression, such as alkaline phosphatase (ALP), collagen type I (COLI), osteocalcin, and osteopontin were also downregulated in Zn−.
CONCLUSION
The results suggest that a zinc deficiency in osteoblasts suppresses the BMP-2 signaling pathway via the suppression of Smad-1 activation, and this suppressed BMP-2 signaling can cause poor osteoblast differentiation.

Keyword

Zinc; MC3T3-E1 cells; BMP-2; Smad-1/pSmad-1; Runx2; osterix

MeSH Terms

Alkaline Phosphatase
Cell Line
Collagen Type I
Gene Expression
Osteoblasts*
Osteocalcin
Osteopontin
Phosphorylation*
Transcription Factors*
Zinc*
Alkaline Phosphatase
Collagen Type I
Osteocalcin
Osteopontin
Transcription Factors
Zinc

Figure

• 

  Fig. 1 Cell viability, ALP activity and collagen synthesis by Zn in MC3T3-E1 cells. (A) Both MC3T3-E1 cell lines (SC 24, low osteoblasts differentiation and SC 4, high osteoblast differentiation) were cultured with Zn− (1 µM) or Zn+ (15 µM) along with normal osteogenic differentiation media (OSM) as normal control for 24 hours. Cell viability was measured by MTT assay. (B, C) The enzyme activity of ALP within cells (unit of mg PNP/mg protein/min) and outside cells in media (unit of mg PNP/mL/min) were measured. Different superscripts mean significantly different among zinc treatment groups at p < 0.05 by Tukey, ANOVA. mean ± SEM (n = 4).

• 

  Fig. 2 BMP-2 expression and Smad 1 activation by Zn in MC3T3-E1 cells. (A-C) The cellular secretion (A) and the expression (B & C) of BMP-2 by Zn were measured. (D) Smad1 and p-Smad1 protein expression by zinc. Cells were cultured for 24 hours with the designated Zn level and mRNA and protein expression measured by real time PCR and immunoblotting, respectively. Data reported as concentration values or relative quantification as 1 at OSM control (mRNA and protein). Different superscripts mean significantly different at p < 0.05 by Tukey, ANOVA. mean ± SEM (n = 4).

• 

  Fig. 3 mRNA and protein expression of Runx2 and Osterix by Zn. (A) Nuclear Runx2 and osterix mRNA levels were measured by quantitative real-time PCR analysis in 24 hours under zinc-treated culture. (B) Immunoblot analysis of Runx2 and osterix. The values are presented as 1 at OSM. Different superscripts mean significantly different among Zn treatments at p < 0.05 by Tukey, ANOVA. mean ± SEM (n = 4).

• 

  Fig. 4 Bone marker and osteoblast differentiation gene and protein expression in MC3T3-E1 cells by Zn. (A) mRNA transcription level for bone markers (ALP, COL I) and osteoblast differentiation genes and proteins (osteoclacin, osteopontin) were measured using quantitative real time PCR (qRT-PCR) after culturing cells with zinc treatment for 24 hours. (B) ALP and COL-1 protein expression was detected using Western blots. Values are presented as fold changes or relative quantification of target mRNA and protein expression as 1 at OSM. Different superscripts mean significantly different among Zn treatments at p < 0.05 by Tukey, ANOVA. mean ± SEM (n = 4).

• 

  Fig. 5 A suggested scheme for zinc modulation via BMP-2 signaling in osteoblasts. OC (osteocalcin), OPN (osteopontin). Solid line (confirmed in this study), dot line (to be expected).

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