HnRNPL inhibits the osteogenic differentiation of PDLCs stimulated by SrCl2 through repressing Setd2

Abstract

Osteoporosis has been shown to intensify bone loss caused by periodontitis and both share common risk factors. One strategy utilized to manage the disease has been via the release of Sr ions by Strontium Ranelate having a direct effect on preventing osteoclast activation and promoting osteoblast differentiation. Previously we have developed and characterized porous Sr-mesoporous bioactive glass (Sr-MBG) scaffolds and demonstrated their ability to promote periodontal regeneration when compared to MBG alone. Our group further discovered a splicing factor, heterogeneous nuclear ribonucleoprotein L (hnRNPL), was drastically down-regulated in periodontal ligament stem cells (PDLCs) stimulated by Sr through the activation of AKT pathway. Furthermore, hnRNPL restrained the osteogenic differentiation of PDLCs through down-regulating H3K36me3-specific methyltransferase Setd2. The goal of the present study was to investigate the mechanism of periodontal regeneration stimulated by Sr It was first found that the epigenetic mechanism of splicing factor hnRNPL participated in the osteogenesis processing of PDLCs stimulated by SrCl₂ . Meanwhile, the different role of hnRNPL and SET domain containing 2 (Setd2) may provide some implication of the treatment of periodontitis patients simultaneously suffering from osteoporosis.

Keywords: RNA splicing; bioengineering; hnRNPL; osteoporosis; periodontal ligament stem cells.

Figure 1

Regenerative potential and expression of hnRNPL, Setd2 and H3K36me3 in the healing of bone defects filled with MBG and Sr‐MBG. (A, B) Masson staining; (C‐J) immunohistochemistry staining with Runx2‐antibody (C, D), hnRNPL‐antibody (E, F), Setd2‐antibody (G, H) and H3K36me3‐antibody (I, J) in tissues from control and Sr‐MBG groups. scale bar = 20 μm; (K‐O) Quantitative analysis of new bone formation (K) and immuno‐histochemical staining of Runx2(L), hnRNPL (M), Setd2 (N) and H3K36me3 (O) positive cells between groups. *P ＜ 0.05; **P ＜ 0.01; ***P ＜ 0.001

Figure 2

Role of various concentrations of SrCl₂ (0, 0.01, 0.1, 1, 3 mmol/L) on PDLCs osteogenic differentiation. (A) ALP staining of PDLCs cultured in osteoblast differentiation media with or without SrCl₂ at 7, 14 and 21 d. (B) Alizarin red staining of PDLCs at 21 d. (C, D) Quantification of ALP staining at 7 d (C) and alizarin red staining (D) of PDLCs stimulated by SrCl₂ in different concentrations. (E)Relative expression of osteogenic differentiation markers of ALP, OCN and BSP of PDLCs stimulated by SrCl₂ in different concentrations. (F) Cell proliferation of PDLCs assessed by CCK8 assay. *P ＜ 0.5; **P ＜ 0.01; ***P ＜ 0.001

Figure 3

AKT pathway was activated by SrCl₂ stimulation in PDLCs. (A‐C) PDLCs were first starved for 24 h, then stimulated by 1 mmol/L SrCl₂ at different time periods ranging from 0 to 240 min. Then the total protein (A), cytoplasm protein (B) and nuclear protein (C) were extracted and immunoblotted using antibodies using AKT, hnRNPL and Setd2 antibody separately. (D) PDLCs were pretreated by MK2206 for 12 h to inhibit AKT signaling pathway, followed by stimulation with 1 mmol/L SrCl₂. Total protein was collected and detected by immunoblot analysis of hnRNPL and Setd2. (E) PDLCs pretreated with MK2206 for 12 h were then induced towards osteogenic differentiation and alizarin red staining was performed on day 21. (F) Quantification of alizarin red staining. *P ＜ 0.5; **P ＜ 0.01; ***P ＜ 0.001

Figure 4

HnRNPL has negative effect on PDLCs osteogenic differentiation. (A) PDLCs were transfected by lentivirus to silence hnRNPL and real‐time PCR and western blot were utilized to detect the knock‐down proficiency of hnRNPL. (B‐D) Expression level of osteogenic genes, Runx2 (B), ALP (C), OCN (D) of PDLCs after hnRNPL knockdown and induction towards osteogenic differentiation with or without SrCl₂ stimulation. Alizarin red staining (E) and quantification of mineralization (F), ALP staining (G) and quantification of ALP activity (H) of PDLCs transfected by lentivirus with scrambled and hnRNPL‐shRNA vectors after osteogenic induction with or without SrCl₂. (I) Expression level of Setd2 of PDLCs after hnRNPL knockdown and induction towards osteogenic differentiation with or without SrCl₂ stimulation. *P ＜ 0.5; **P ＜ 0.01; ***P ＜ 0.001

Figure 5

Setd2 promotes PDLCs osteogenic differentiation stimulated by Sr. PDLCs were transfected by lentivirus to knockdown or over‐express Setd2, with satisfactory expressions confirmed by real‐time PCR (A) and western blot (B) analysis. Alizarin red staining (C) and quantification of mineralization (D) of PDLCs transfected by lentivirus with scrambled, Setd2‐shRNA and Setd2‐overexpressing vectors following osteogenic differentiation with or without SrCl₂. ALP staining (E) and quantification of ALP activity (F) of PDLCs transfected by lentivirus with scrambled, Setd2‐shRNA and Setd2‐overexpressing vectors following osteogenic differentiation with or without SrCl₂. (G) Expression level of osteogenic genes, Runx2, ALP, OCN of PDLCs after Setd2 knockdown or over‐expression and induction towards osteogenic differentiation with or without SrCl₂ stimulation. *P ＜ 0.5; **P ＜ 0.01; ***P ＜ 0.001

Figure 6

Schematic diagram of the mechanism involved in Sr promoted‐PDLCs osteogenic differentiation