Investigation of the dynamics of Runx2 activation during induction of osteogenic differentiation
E. S. Gromova,
K. E. Azarkina,
D. A. Kostina,
D. A. Perepletchikova,
D. S. Smirnova,
A. B. Malashicheva https://doi.org/10.18705/2311-4495-2024-11-3-284-293
Abstract
Background. Despite the fact that Runx2 is a generally recognized marker and regulator of osteogenic differentiation, the mechanisms of its regulation, the dynamics of activation during osteogenic differentiation and the relationship with other genes and gene pathways associated with osteogenic differentiation remain unclear.
The aim is to analyze the dynamics of Runx2 activation and the effect of stable protein levels on differentiation processes.
Materials and methods. Lentiviral constructs of RUNX2 genetic isoforms were used in the work: RUNX2full (full-size gene), RUNX2delta (shortened sequence), RUNX2stop (with stop codon), a hairpin design on RUNX2 — shRUNX2 was also used to suppress its activity. The proteasome degradation inhibitor MG132 was used to maintain stable protein levels in cells. To analyze the dynamics of Runx2 activation, osteogenic differentiation was induced for different time periods. The results were analyzed by Western blotting, real-time PCR, and alizarin red staining.
Results. Stabilization of the Runx2 protein at 24 hours of induction of osteogenic differentiation contributes to its strengthening. Also, the level of Runx2 transcripts does not change, but target genes are activated.
Conclusion. During the initiation of osteogenic differentiation of gingival fibroblasts in vitro, Runx2 is regulated at both the transcriptional and post-transcriptional levels; accumulation of a small level of transcripts during induction of osteogenic differentiation and stabilization of the Runx2 protein seem to be critically important.
Supporting agencies: Research is supported by a grant from The Russian Science Foundation №23-15-00320.
About the Authors
E. S. Gromova
Institute of Cytology of the Russian Academy of Science
Russian Federation
Competing Interests:
Russian Federation
Ekaterina S. Gromova, Senior Research Laboratory Assistant at the Laboratory of Regenerative Biomedicine
Tikhoretsky Prospekt, 4, Saint Petersburg, 194064
Competing Interests:
The authors declare no conflict of interest.
K. E. Azarkina
Institute of Cytology of the Russian Academy of Science
Russian Federation
Competing Interests:
Russian Federation
Ksenia E. Azarkina, Senior Research Laboratory Assistant at the Laboratory of Regenerative Biomedicine
Saint Petersburg
Competing Interests:
The authors declare no conflict of interest.
D. A. Kostina
Institute of Cytology of the Russian Academy of Science
Russian Federation
Competing Interests:
Russian Federation
Daria A. Kostina, Researcher at the Laboratory of Regenerative Biomedicine
Saint Petersburg
Competing Interests:
The authors declare no conflict of interest.
D. A. Perepletchikova
Institute of Cytology of the Russian Academy of Science
Russian Federation
Competing Interests:
Russian Federation
Daria A. Perepletchikova, Junior Researcher at the Laboratory of Regenerative Biomedicine
Saint Petersburg
Competing Interests:
The authors declare no conflict of interest.
D. S. Smirnova
Institute of Cytology of the Russian Academy of Science
Russian Federation
Competing Interests:
Russian Federation
Darya V. Smirnova, Junior Researcher at the Laboratory of Regenerative Biomedicine
Saint Petersburg
Competing Interests:
The authors declare no conflict of interest.
A. B. Malashicheva
Institute of Cytology of the Russian Academy of Science
Russian Federation
Competing Interests:
Russian Federation
Anna B. Malashicheva, Doctor of Biological Sciences, Head of the Laboratory of Regenerative Biomedicine
Saint Petersburg
Competing Interests:
The authors declare no conflict of interest.
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Review
For citations:
Gromova E.S., Azarkina K.E., Kostina D.A., Perepletchikova D.A., Smirnova D.S., Malashicheva A.B. Investigation of the dynamics of Runx2 activation during induction of osteogenic differentiation. Translational Medicine. 2024;11(3):284-293. (In Russ.) https://doi.org/10.18705/2311-4495-2024-11-3-284-293
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