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The isoflavone genistein enhances osteoblastogenesis: signaling pathways involved

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Abstract

Phytoestrogens have been proposed as a natural therapy for prevention of bone loss. In this work, we studied the mechanism of action of genistein on osteoblast differentiation. Primary cell cultures of calvarial osteoblasts isolated from female Wistar rats were in vitro exposed to genistein. Osteoblast differentiation markers were measured. Genistein stimulated osteoblast migration (71–257% above control). An earlier upregulation of estrogen receptor alpha gene expression and an enhancement of mRNA levels of the Runt-related transcription factor 2 were detected after 3 days of culture. The isoflavone significantly increased osteocalcin expression, extracellular collagen deposition, and alkaline phosphatase activity. The mechanism displayed by genistein involved estrogen receptor and nitric oxide pathway participation, since cell preincubation with the estrogen receptor antagonist ICI 182780, or the nitric oxide synthase inhibitor L-NAME, suppressed the phytoestrogen action. Evidence of MAPK and PI3K transduction systems participation on the stimulatory action of genistein on extracellular collagen deposition and alkaline phosphatase activity was also obtained. Genistein favored monocyte adhesion to osteoblasts (77% above control) in an ER; NOS; and MAPK kinase–dependent and PI3K-dependent manner. Co-cultured osteoblast-monocyte long term exposed (21 days) to genistein exhibited a high number of multinucleated and tartrate-resistant acid phosphatase–positive cells added to osteoblasts, suggesting that the phytoestrogen promotes osteoclast differentiation. In conclusion, genistein promoted osteoblastogenesis through the participation of ER and NOS pathways, and the contribution of ERK or PI3K signal transduction pathways, and also stimulates osteoclast differentiation from its mononuclear progenitor.

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Acknowledgments

We thank the researchers Dr. Adrián Campelo for his counsel in osteocalcin expression evaluation and Dr. Pablo De Genaro for technical assistance for flow cytometry assays.

Funding

This work was supported by Universidad Nacional del Sur (UNS), Bahía Blanca, Argentina (grant number 24/B247); and Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, Argentina, grant number PIP D-4061).

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Authors and Affiliations

  1. Instituto de Ciencias Biológicas y Biomédicas del Sur (INBIOSUR), Universidad Nacional del Sur (UNS), Consejo de Investigaciones Científicas y Técnicas (CONICET), Departamento de Biología, Bioquímica y Farmacia, San Juan 670, B8000ICN, Bahía Blanca, Argentina

    Sabrina B. Cepeda, Marisa J. Sandoval, María Carla Crescitelli, María Belén Rauschemberger & Virginia L. Massheimer

Authors
  1. Sabrina B. Cepeda
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  2. Marisa J. Sandoval
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  3. María Carla Crescitelli
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  4. María Belén Rauschemberger
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  5. Virginia L. Massheimer
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Contributions

S.B.C and M.J.S: conceptualization; methodology; investigation; writing original draft. M.C.C. and M.B.R: methodology; resources. V.L.M: writing-review and editing; supervision; project administration; funding acquisition.

Corresponding author

Correspondence to Virginia L. Massheimer.

Ethics declarations

All the procedures involving animals were carried out in accordance with the guidelines published in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Animal Care Use Committee approved the protocols employed in this study (Protocol number 028/2017).

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The authors declare that they have no conflict of interest.

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UNS and CONICET had no role in the design, analysis, or writing of this article.

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Keypoints

The study provides cellular and molecular evidence of the osteoblastogenesis action of genistein.

The isoflavone enhances osteoblast differentiation marker expression.

Nitric oxide pathway plays a key role on bone phytoestrogen action.

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Cepeda, S.B., Sandoval, M.J., Crescitelli, M.C. et al. The isoflavone genistein enhances osteoblastogenesis: signaling pathways involved. J Physiol Biochem 76, 99–110 (2020). https://doi.org/10.1007/s13105-019-00722-3

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  • DOI: https://doi.org/10.1007/s13105-019-00722-3

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