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

Affiliations
  • 1Department of Food Science and Nutrition, Andong National University, 1375 Kyungdong Road, Andong, Kyungbook 36633, South Korea. iskwun@andong.ac.kr

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).


Cited by  2 articles

Cellular zinc deficiency inhibits the mineralized nodule formation and downregulates bone-specific gene expression in osteoblastic MC3T3-E1 cells
Young-Eun Cho, In-Sook Kwun
J Nutr Health. 2018;51(5):379-385.    doi: 10.4163/jnh.2018.51.5.379.

Glycyrrhiza uralensis (licorice) extracts increase cell proliferation and bone marker enzyme alkaline phosphatase activity in osteoblastic MC3T3-E1 cells
Young-Eun Cho, In-Sook Kwun
J Nutr Health. 2018;51(4):316-322.    doi: 10.4163/jnh.2018.51.4.316.


Reference

1. Coleman JE. Zinc proteins: enzymes, storage proteins, transcription factors, and replication proteins. Annu Rev Biochem. 1992; 61:897–946.
Article
2. Maret W. Zinc in cellular regulation: the nature and significance of “Zinc Signals”. Int J Mol Sci. 2017; 18(11):2285–2296.
Article
3. Tuerk MJ, Fazel N. Zinc deficiency. Curr Opin Gastroenterol. 2009; 25(2):136–143.
Article
4. Chabosseau P, Rutter GA. Zinc and diabetes. Arch Biochem Biophys. 2016; 611:79–85.
Article
5. Kim JH, Jeon J, Shin M, Won Y, Lee M, Kwak JS, Lee G, Rhee J, Ryu JH, Chun CH, Chun JS. Regulation of the catabolic cascade in osteoarthritis by the Zinc-ZIP8-MTF1 Axis. Cell. 2014; 156(4):730–743.
Article
6. da Cunha Ferreira RM, Marquiegui IM, Elizaga IV. Teratogenicity of zinc deficiency in the rat: study of the fetal skeleton. Teratology. 1989; 39(2):181–194.
Article
7. Hsieh HS, Navia JM. Zinc deficiency and bone formation in guinea pig alveolar implants. J Nutr. 1980; 110(8):1581–1588.
Article
8. Hurley LS. Teratogenic aspects of manganese, zinc, and copper nutrition. Physiol Rev. 1981; 61(2):249–295.
Article
9. Leek JC, Vogler JB, Gershwin ME, Golub MS, Hurley LS, Hendrickx AG. Studies of marginal zinc deprivation in rhesus monkeys. V. Fetal and infant skeletal effects. Am J Clin Nutr. 1984; 40(6):1203–1212.
10. Oner G, Bhaumick B, Bala RM. Effect of zinc deficiency on serum somatomedin levels and skeletal growth in young rats. Endocrinology. 1984; 114(5):1860–1863.
Article
11. Holloway WR, Collier FM, Herbst RE, Hodge JM, Nicholson GC. Osteoblast-mediated effects of zinc on isolated rat osteoclasts: inhibition of bone resorption and enhancement of osteoclast number. Bone. 1996; 19(2):137–142.
Article
12. Togari A, Arakawa S, Arai M, Matsumoto S. Alteration of in vitro bone metabolism and tooth formation by zinc. Gen Pharmacol. 1993; 24(5):1133–1140.
Article
13. Alcantara EH, Lomeda RA, Feldmann J, Nixon GF, Beattie JH, Kwun IS. Zinc deprivation inhibits extracellular matrix calcification through decreased synthesis of matrix proteins in osteoblasts. Mol Nutr Food Res. 2011; 55(10):1552–1560.
Article
14. Cho YE, Lomeda RA, Ryu SH, Lee JH, Beattie JH, Kwun IS. Cellular Zn depletion by metal ion chelators (TPEN, DTPA and chelex resin) and its application to osteoblastic MC3T3-E1 cells. Nutr Res Pract. 2007; 1(1):29–35.
Article
15. Cho YE, Lomeda RA, Ryu SH, Sohn HY, Shin HI, Beattie JH, Kwun IS. Zinc deficiency negatively affects alkaline phosphatase and the concentration of Ca, Mg and P in rats. Nutr Res Pract. 2007; 1(2):113–119.
Article
16. Kim JT, Baek SH, Lee SH, Park EK, Kim EC, Kwun IS, Shin HI. Zinc-deficient diet decreases fetal long bone growth through decreased bone matrix formation in mice. J Med Food. 2009; 12(1):118–123.
Article
17. Kwun IS, Cho YE, Lomeda RA, Shin HI, Choi JY, Kang YH, Beattie JH. Zinc deficiency suppresses matrix mineralization and retards osteogenesis transiently with catch-up possibly through Runx 2 modulation. Bone. 2010; 46(3):732–741.
Article
18. Seo HJ, Cho YE, Kim T, Shin HI, Kwun IS. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract. 2010; 4(5):356–361.
Article
19. Yamaguchi M, Goto M, Uchiyama S, Nakagawa T. Effect of zinc on gene expression in osteoblastic MC3T3-E1 cells: enhancement of Runx2, OPG, and regucalcin mRNA expressions. Mol Cell Biochem. 2008; 312(1-2):157–166.
Article
20. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G. Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell. 1997; 89(5):747–754.
Article
21. Lee MH, Kim YJ, Kim HJ, Park HD, Kang AR, Kyung HM, Sung JH, Wozney JM, Kim HJ, Ryoo HM. BMP-2-induced Runx2 expression is mediated by Dlx5, and TGF-beta 1 opposes the BMP-2-induced osteoblast differentiation by suppression of Dlx5 expression. J Biol Chem. 2003; 278(36):34387–34394.
22. Lee MH, Kwon TG, Park HS, Wozney JM, Ryoo HM. BMP-2-induced Osterix expression is mediated by Dlx5 but is independent of Runx2. Biochem Biophys Res Commun. 2003; 309(3):689–694.
Article
23. Ryoo HM, Lee MH, Kim YJ. Critical molecular switches involved in BMP-2-induced osteogenic differentiation of mesenchymal cells. Gene. 2006; 366(1):51–57.
Article
24. Suzuki T, Katsumata S, Matsuzaki H, Suzuki K. A short-term zinc-deficient diet decreases bone formation through down-regulated BMP2 in rat bone. Biosci Biotechnol Biochem. 2016; 80(7):1433–1435.
Article
25. Murshed M, Harmey D, Millan JS, McKee MD, Karsenty G. Unique coexpression in osteblasts of boadly expressed genes accounts for the apatial restrictionof ECM minralization to bone. Genes Dev. 2005; 19(9):1093–1104.
26. Katagiri T, Takahashi N. Regulatory mechanisms of osteoblast and osteoclast differentiation. Oral Dis. 2002; 8(3):147–159.
Article
27. Komori T. Regulation of osteoblast differentiation by Runx2. Adv Exp Med Biol. 2010; 658:43–49.
Article
28. Golub EE, Harrison G, Taylor AG, Camper S, Shapiro IM. The role of alkaline phosphatase in cartilage mineralization. Bone Miner. 1992; 17(2):273–278.
Article
29. Orimo H. The mechanism of mineralization and the role of alkaline phosphatase in health and disease. J Nippon Med Sch. 2010; 77(1):4–12.
Article
30. Yamaguchi M. Role of nutritional zinc in the prevention of osteoporosis. Mol Cell Biochem. 2010; 338(1-2):241–254.
Article
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