Atomic Force Microscopy-Based Nanoscopy of Chondrogenically Differentiating Human Adipose-Derived Stem Cells: Nanostructure and Integrin ß1 Expression.

Integrin ß1 is known to be involved in differentiation, migration, proliferation, wound repair, tissue development, and organogenesis. In order to analyze the binding probability between integrin ß1 ligand and cluster of differentiation 29 (CD29) receptors, atomic force microscopy (AFM) was used to detect native integrin ß1-coupled receptors on the surface of human adipose-derived stem cells (hADSc). The binding probability of integrin ß1 ligand-receptor interaction was probed by integrin ß1-functionalized tips on hADSc during early chondrogenic differentiation at the two-dimensional cell culture level. Cell morphology and ultrastructure of hADSc were measured by AFM, which demonstrated that long spindled cells became polygonal cells with decreased length/width ratios and increased roughness during chondrogenic induction. The binding of integrin ß1 ligand and CD29 receptors was detected by ß1-functionalized tips for living hADSc. A total of 1200 curves were recorded at 0, 6, and 12 days of chondrogenic induction. Average rupture forces were, respectively, 61.8 ± 22.2 pN, 60 ± 20.2 pN, and 67.2 ± 22.0 pN. Rupture events were 19.58 ± 1.74%, 28.03 ± 2.05%, and 33.4 ± 1.89%, respectively, which demonstrated that binding probability was increased between integrin ß1 ligand and receptors on the surface of hADSc during chondrogenic induction. Integrin ß1 and the ß-catenin/SOX signaling pathway were correlated during chondrogenic differentiation. The results of this investigation imply that AFM offers kinetic and visual insight into the changes in integrin ß1 ligand-CD29 receptor binding on hADSc during chondrogenesis. Changes in cellular morphology, membrane ultrastructure, and the probability of ligand-transmembrane receptor binding were demonstrated to be useful markers for evaluation of the chondrogenic differentiation process.

Transcriptional regulation of Runx2 by HSP90 controls osteosarcoma apoptosis via the AKT/GSK-3ß/ß-catenin signaling.

Osteosarcoma (OS) is the most malignant primary bone tumor in children and adolescents with limited treatment options and poor prognosis. Recently, aberrant expression of Runx2 has been found in OS, thereby contributing to the development, and progression of OS. However, the upstream signaling molecules that regulate its expression in OS remain largely unknown. In the present study, we first confirmed that the inhibition of HSP90 with 17-AAG caused significant apoptosis of OS cells via a caspase-3-dependent mechanism, and that inhibition or knockdown of HSP90 by 17-AAG or siRNAs significantly suppressed mRNA and protein expression of Runx2. Furthermore, we provided evidence that Runx2 was transcriptionally regulated by HSP90 when using MG132 and CHX chase assay. We also demonstrated that ß-catenin was overexpressed in OS tissue, and that knockdown of ß-catenin induced pronounced apoptosis of OS cells in the presence or absence of 17-AAG. Interestingly, this phenomenon was accompanied with a significant reduction of Runx2 and Cyclin D1 expression, indicating an essential role of Runx2/Cyclin D1 in 17-AAG-induced cells apoptosis. Moreover, we demonstrated that the apoptosis of OS cells induced by 17-AAG did require the involvement of the AKT/GSK-3ß/ß-catenin signaling pathway by using pharmacological inhibitor GSK-3ß (LiCl) or siGSK-3ß. Our findings reveal a novel mechanism that Runx2 is transcriptionally regulated by HSP90 via the AKT/GSK-3ß/ß-catenin signaling pathway, and by which leads to apoptosis of OS cells.