POS0044 T315 SUPPRESSES OSTEOGENIC DIFFERENTIATION IN SAOS-2 CELLS BY INHIBITING PHOSPHORYLATION OF AKT
Authors: Z. Huang, X. Huang, Y. Huang, Z. Li, Q. Huang, T. Li
Journal: Annals of the Rheumatic Diseases (2021)

Background: New bone formation is common in the late stage of various inflammatory arthritis, while osteoblasts play a vital role in this process. Activation of PI3K/ Akt pathway promotes the differentiation and enhances the function of osteoblasts [1]. T315 is a novel small molecule drug, which may induce apoptosis and suppress the expression of cellular markers of chronic lymphocytic leukemia cells by disrupting PI3K/ Akt pathway [2]. However, the lack of study focuses on the influence of T31T on the other cells, except tumor cell lines. Objectives: We aimed to assess the effect of T315 on human osteoblast-like Saos-2 cells, while its potential mechanism in PI3K/ Akt pathway was evaluated as well. Methods: (1) Saos-2 was stimulated with an osteogenic reagent which contained L-ascorbic acid, β-glycerophosphoric acid, and dexamethasone. The concentration of T315 was adjusted to 0μg/ml, 1μg/ml, and 2μg/ml in the culture medium. (2) Alizarin red stain and alkaline phosphatase (ALP) stain were performed at d0, d7, d14, and d21 after being treated with T315. (3) Cellular protein was extracted at d0, d3, and d6 after being treated with T315, then ALP activity was tested based on a recommendation from the manufacturer of the kit. (4) Collagen type 1α2 Chain (Col1α2) and osteocalcin (OCN), two osteogenic markers, were measured through western blot, with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an endogenous control. (5) Phospho-phosphoinositide 3-kinase (pPI3K), phospho-protein kinase B (pAkt), and runt-related transcription factor 2 (Runx2) was tested through western blot as well. GAPDH or protein kinase (Akt) was chosen as an internal reference as appropriate. (6) Analysis of variance with the least significant difference was used to analyze the data. A P &lt;0.05 was considered statistically significant. Results: (1) The higher concentration of T315 related to the less relative mineralized area and the positive area of ALP, while longer incubation time with T315 decreased these regions as well (Figure 1A-C). (2) T315 reduced the activity of ALP accordingly (Figure 1D). (3) T315 suppressed the protein expression of Col1α2 and OCN in a dose-dependent and time-dependent manner (Figure 1E, F). (4) T315 did not alter pPI3K, but it inhibited the phosphorylation of Akt (Figure 1G, H). (5) Runx2 was reduced because of the greater dose or longer incubation time with T315 (Figure 1). Conclusion: T315 inhibits the differentiation of osteoblasts through inhibiting the phosphorylation of Akt. Surprisingly, pPI3K seldom changes in this process, so its detail mechanism should be investigated in further. References: [1]Exp Biol Med (Maywood) 2020;245(6):552-561. [2]Blood 2015;125(2):284-295. Figure 1. Effect of T315 on Saos-2 cells and PI3K/Akt pathway. (A) Alizarin red stain and ALP stain at d21. (B) Relative mineralized area in Alizarin red stain. (C) Positive area in ALP stain. (D) ALP activity. Western blot analysis and its bands at d6 for Col1α2 (E), OCN (F), pPI3K (G), pAkt (H), and Runx2 (I). Results were normalized by GAPDH or Akt. ALP: Alkaline phosphatase; Col1α2: Collagen type 1α2 Chain; GAPDH: Glyceraldehyde 3-phosphate dehydrogenase; OCN: Osteocalcin; pPI3K: Phospho-phosphoinositide 3-kinase; pAkt: Phospho-protein kinase B; Akt: Protein kinase; Runx2: Runt-related transcription factor 2. a Compared with d3 in the same concentration of T315, P &lt;0.05. b Compared with d0 in the same concentration of T315, P &lt;0.05. c Compared with 1μg/ml T315 in the same incubation time, P &lt;0.05. d Compared with 2μg/ml T315 in the same incubation time, P &lt;0.05. Disclosure of Interests: None declared.