Fig 2: Regulation of SOST by histone deacetylase 4/5 (HDAC4/5) activation. Integrin-disintegration, due to fluid flow shear stress (FFSS) in the extracellular matrix (ECM), activates focal adhesion kinase (FAK), which subsequently phosphorylates HDAC4/5. Phosphorylated HDAC4/5 binds to protein 14-3-3 leading to nuclear translocation where, by binding to myocyte-specific enhancer factor 2C (MEF2C) and histone deacetylation at the evolutionarily conserved region 5 (ECR5) site, prevents SOST expression. Lack of FFSS precludes HDAC4/5 phosphorylation and thus nuclear translocation, which promotes MEF2C binding to ECR5 and SOST upregulation. SOST downregulation = black arrow down; SOST upregulation = black arrow up. Created with BioRender.com, accessed on 15 February 2022.

Fig 3: Runx2 expression in MDA-MB-231 cells is required for inhibition of osteoblast differentiation. (A) Western blot showing stable MDA-MB-231 cells knocked down for Runx2 or CBFß. The left panel shows total cell lysates derived from stables MDA-MB-231 cells, transfected with a shRNA non-specific (shNS) or shRNA for Runx2 (shRunx2) or CBFß (shCBFß). The lower panel is a Tubulin loading control. Graphs show that expression SOST is significantly reduced in the cell lines. Data are presented as mean ± standard deviation (SD) (n = 3). Statistical evaluation of significant differences was performed using the Student's t-test. Asterisk (*) indicates P < 0.05 when compared to control (shNS). (B) Knock-down of Runx2 in MDA-MB-231 cells permits the differentiation of osteoblasts. BMSCs were differentiated with normal differentiation media (Diff), conditioned media from stable shNS cells (MDAshNS) or shRunx2 cells (MDAshRunx2). Cells grown in non-differentiation media (Non-diff) were used as negative control of differentiation. (C) Alizarin red was quantified following elution and total RNAs from cells treated as in (B) were used to analyse expression of the osteoblast differentiation marker, osteocalcin. Data are presented as mean ± standard deviation (SD) (n = 3). One-way analysis of variance was used and multiple comparisons between individual groups were assessed by the method of Tukey. * indicates P < 0.05 compared to Non-Diff by analysis of variance; # indicates P < 0.05 compared to Diff+MDA shNS by analysis of variance. (D) Diagram depicting the proposed role of Runx2 in the modulation of bone cell functions by regulating secreted factors. Other genes involved in invasion are omitted for clarity.

Fig 4: Runx2 is essential for the expression of secreted sclerostin and GM-CSF in MDA-MB-231 cells. (A and B) Runx2 regulates the activity of the SOST and CSF-2 promoter. MDA-MB-231 cells transfected with siRunx2 or a non-specific siRNA (siNS) were transfected with a plasmid containing either the SOST (A) or CSF-2 (B) promoter. Luciferase activities are presented as mean values ± SD All values are relative to the activity of the Renilla luciferase reporter. Insets show Western blots of Runx2 expression when transfected with siRNAs as indicated. (C and D) MDA-MB-231 cells transfected with the luciferase reporter constructs containing, either the SOST (C) or CSF-2 (D) wild-type promoter (WT), or the Runx site mutant (Mut). Data are presented as mean ± standard deviation (SD) (n = 3). All values are relative to the activity of the Renilla luciferase reporter. (E) ELISA showing sclerostin and GM-CSF in the media of MDA-MB-231 cells. Data are presented as mean ± standard deviation (SD) (n = 3). Statistical evaluation of significant differences was performed on all graphical data using the Student's t-test. Asterisk (*) indicates P < 0.05 when compared to controls.

Fig 5: Interaction between sclerostin and the low-density lipoprotein receptor-related protein 6 (LRP6). Loop 2 of sclerostin binds to the E1 ectodomain of LRP6, while a part of the C-terminal arm interacts with the E2 ectodomain. Image created in the Research Collaboratory for Structural Bioinformatics—Protein Data Bank with the Mol* Viewer [64], based on the crystal structure LRP6 E1-E2-SOST complex, PDB-ID: 6L6R [63].