Decreased MORF leads to prolonged endoplasmic reticulum stress in periodontitis-associated chronic inflammation

Abstract

The association between inflammation and endoplasmic reticulum (ER) stress has been described in many diseases. However, if and how chronic inflammation governs the unfolded protein response (UPR) and promotes ER homeostasis of chronic inflammatory disease remains elusive. In this study, chronic inflammation resulted in ER stress in mesenchymal stem cells in the setting of periodontitis. Long-term proinflammatory cytokines induced prolonged ER stress and decreased the osteogenic differentiation of periodontal ligament stem cells (PDLSCs). Interestingly, we showed that chronic inflammation decreases the expression of lysine acetyltransferase 6B (KAT6B, also called MORF), a histone acetyltransferase, and causes the upregulation of a key UPR sensor, PERK, which lead to the persistent activation of the UPR in PDLSCs. Furthermore, we found that the activation of UPR mediated by MORF in chronic inflammation contributes to the PERK-related deterioration of the osteogenic differentiation of PDLSCs both in vivo and in vitro. Taken together, our results suggest that chronic inflammation compromises UPR function through MORF-mediated-PERK transcription, which is a previously unrecognized mechanism that contributes to impaired ER function, prolonged ER stress and defective osteogenic differentiation of PDLSCs in periodontitis.

Figure 1

Activation of the UPR target genes in MSCs in periodontitis-associated chronic inflammation. (a and b) Transmission electron microscopy (TEM) images of the endoplasmic reticulum (ER) in H-PDLSCs and P-PDLSCs. The overall view of cells with a low magnification (left column, scale bar, 5 μm) and a high magnification (right column, scale bar, 1 μm). The morphology of ERs is shown in the red rectangle. The results showed more dilated and abundant ERs in P-PDLSCs. (c) The expressions of UPR target genes (PERK, GRP78, GRP94, CHOP) in consecutive passages of H-PDLSCs and P-PDLSCs (Passage 2–4) were determined by real-time RT-PCR. The results were normalized to GAPDH mRNA expression. The representative results were from three independent experiments. The error bars represent the S.D. from the mean values.*P<0.05, **P<0.01, ***P<0.001

Figure 2

Chronic inflammation triggers the UPR and impairs the osteogenic differentiation of H-PDLSCs. (a and b) The expressions of UPR target genes (PERK, GRP78, GRP94, CHOP) were measured in H-PDLSCs cultured with IL-1β and TNF-α, respectively, for 7 days (Passage 2), and in consecutive passages of H-PDLSCs (Passage 3 and 4) with the removal of the stimulus. The results were normalized to GAPDH mRNA expression. (c and d) Western blot analysis showed the protein levels of Runx2 and OCN in H-PDLSCs treated with IL-1β (5 ng/ml) and TNF-α (10 ng/ml) after osteogenic induction for 14 days. β-Actin was used as an internal control. (e and f) Alizarin red staining of H-PDLSCs incubated with IL-1β and TNF-α was performed and quantified after 28 days of osteogenic induction. Scale bar, 50 μm, n=3. The representative results were from three independent experiments. The error bars represent the S.D. from the mean values. *P<0.05, **P<0.01, ***P<0.001

Figure 3

Periodontitis-associated chronic inflammation triggers UPR activation with MORF mediation. (a and b) The expressions of UPR target genes (PERK, GRP78, GRP94, CHOP) were measured in H-PDLSCs treated with IL-1β and TNF-α for 0, 3, 6, 9, 12 and 24 h. (c) Western blot showed that MORF expression was decreased after transfection with MORF siRNA. (d) The expressions of UPR genes in H-PDLSCs and H-PDLSCs transfected with MORF siRNA were measured by real-time RT-PCR. The expressions of PERK, ATF4 and CHOP were elevated after the knockdown of MORF in H-PDLSCs. The results were normalized to GAPDH mRNA expression. (e) Transmission electron microscopy (TEM) images of the endoplasmic reticulum (ER) in H-PDLSCs and H-PDLSCs transfected with MORF siRNA. The overall view of cells with a low magnification (left column, scale bar, 5 μm) and a high magnification (right column, scale bar, 1 μm). The morphology of the ERs is shown in the red rectangle. The results showed more dilated and abundant ERs in H-PDLSCs transfected with MORF siRNA. (f) Western blot showed that MORF expression was increased after transfection with MORF overexpression vector. β-Actin was used as an internal control. (g) The expressions of UPR genes in H-PDLSCs and H-PDLSCs transfected with the MORF overexpression vector were measured by real-time RT-PCR. The expressions of PERK, ATF4 and CHOP were downregulated after the overexpression of MORF in H-PDLSCs. The results were normalized to GAPDH mRNA expression. The representative results were from three independent experiments. The error bars represent the S.D. from the mean values.*P<0.05, **P<0.01, ***P<0.001

Figure 4

MORF represses PERK and impairs MSC osteogenic differentiation in periodontitis-associated chronic inflammation. (a) A decreased expression of MORF in consecutive passages of P-PDLSCs was shown by real-time RT-PCR compared with H-PDLSCs (Passage 2–4). (b and c) Decreased expressions of MORF in H-PDLSCs treated with IL-1β and TNF-α for 7 days and in consecutive passages with the removal of the stimulus were shown compared with H-PDLSCs (Passage 2–4). The results were normalized to GAPDH mRNA expression. (d) Decreased expressions of osteogenic protein Runx2 and OCN were confirmed by western blot analysis after knockdown of MORF in H-PDLSCs. β-Actin was used as an internal control. (e) Alizarin red staining showed decreased mineralized nodules after knockdown of MORF in H-PDLSCs after 28 days of osteogenic induction. Scale bar, 50 μm, n=3. (f) H-PDLSCs, P-PDLSCs, H-PDLSCs transfected with MORF siRNA and H-PDLSCs transfected with MORF overexpression vector were subjected to chromatin immunoprecipitation (ChIP) assays with the binding of MORF and rabbit IgG to PERK promoters. The PERK primers for the locus indicated were CHIP-ed with anti-MORF or IgG control. (g) The increased expression of PERK in H-PDLSCs transfected with MORF siRNA and the decreased expression of PERK in H-PDLSCs with MORF overexpression vector were measured by western blot analysis. β-Actin was used as an internal control. The representative results were from three independent experiments. The error bars represent the S.D. from the mean values. *P<0.05, ***P<0.001

Figure 5

UPR activation leads to the decreased osteogenic differentiation of PDLSCs. (a and b) Increased expressions of UPR genes were found in H-PDLSCs treated with 1 μg/ml Tm and 1 μmol/l Tg for 6 h. The results were normalized to GAPDH mRNA expression. (c) Increased expressions of UPR genes were found in P-PDLSCs treated with 5 mmol/l 4-PBA for 6 h. (d and e) Decreased expressions of osteogenic protein Runx2 and OCN were shown by western blot analysis in H-PDLSCs treated with Tm and Tg after osteogenic induction. β-Actin was used as an internal control. (f and g) Alizarin red staining showed decreased numbers of mineralized nodules in H-PDLSCs treated with Tm and Tg after 28 days of osteogenic induction. Scale bar, 50 μm, n=3. (h) Increased expressions of the osteogenic proteins Runx2 and OCN were shown by western blot analysis in P-PDLSCs treated with 4-PBA after osteogenic induction. β-Actin was used as an internal control. (i) Alizarin red staining showed increased numbers of mineralized nodules in P-PDLSCs treated with 4-PBA after 28 days of osteogenic induction. Scale bar, 50 μm, n=3. The representative results were from three independent experiments. The error bars represent the S.D.from the mean values. *P<0.05, **P<0.01, ***P<0.001

Figure 6

UPR activation regulates the osteogenic differentiation of PDLSCs through the PERK pathway. (a and b) The expression of PERK was downregulated in P-PDLSCs after being transfected with PREK siRNA as assayed by real-time RT-PCR and western blot. (c) The osteogenic proteins Runx2 and OCN were decreased in P-PDLSCs after knockdown of PERK as assessed by western blot. β-Actin was used as an internal control. (d) Alizarin red staining showed the increased formation of mineralized nodules in P-PDLSCs transfected with PERK siRNA after 28 days of osteogenic induction. Scale bar, 50 μm, n=3. (e) The alveolar bone loss of SD rats (n=3) was determined by micro-CT and H&E staining. Four sites for two molars (one site for each root of one tooth) were analyzed morphometrically. The results of micro-CT and HE staining showed more alveolar bone loss in both the Tm (0.1 mg/ml) and Tg (0.1 mmol/l) groups compared with the saline (10 μl) group. Injection of GSK2606414 (10 μmol/l, PERK inhibitor) rescued the bone loss caused by Tm (0.1 mg/ml). Scale bar, 200 μm, n=3. The representative results were from three independent experiments. The error bars represent the S.D. from the mean values. *P<0.05, **P<0.01, ***P<0.001

Figure 7

Schematic diagram shows that the mechanisms of decreased MORF lead to prolonged ER stress in periodontitis-associated chronic inflammation. Inflammatory cytokines compromise UPR function through MORF-mediated-PERK transcription, which contributes to impaired ER function, prolonged ER stress and defective osteogenic differentiation of PDLSCs in periodontitis-associated chronic inflammation