Reg3β promotes chondrocyte proliferation and ECM metabolism during acetabular roof remodeling in a rat model of DDH‑induced residual dysplasia

Abstract

Residual acetabular dysplasia (RAD) is a common complication after the successful management of developmental dysplasia of the hip (DDH). RAD remodeling is important for predicting the outcome of the affected hip, and optimal treatment can be chosen accordingly. Regenerating islet‑derived protein 3‑β (Reg3β), a multifaceted cytokine, is a prognostic marker for inflammation and cardiac disease. Nevertheless, the roles of Reg3β in RAD remain unclear. Consequently, the aim of the present study was to assess the role of Reg3β in RAD and explore its related functions in chondrocytes in vitro. First, remodeling of the affected hip after fixation removal was observed in a neonatal rat DDH model, which simulated the process of RAD. Reg3β expression in RAD was upregulated at weeks 1, 2 and 4, as determined by western blot analysis. The serum concentration of Reg3β was greater than that of normal rats at 2 weeks and returned to normal levels at 4 weeks. Subsequently, it was found that Reg3β promoted cell proliferation and extracellular matrix (ECM) metabolism via the Jak2/Stat3/Socs3 signaling pathway through gene knockdown and addition of recombinant Reg3β protein. These findings suggest that Reg3β is a novel potent prognostic biomarker for the remodeling of RAD via regulation of chondrocyte proliferation and metabolism of the ECM.

Keywords: anti‑apoptosis; chondrocytes; extracellular matrix metabolism; proliferation; regenerating islet‑derived protein 3β; residual dysplasia of the hip.

Figure 1.

Morphology of a hip with residual dysplasia and the expression of Reg3β. (A) Success of the DDH model after 2 days of fixation (scale bar, 1 mm) and percentage of failed reduction of the affected hip after removal of fixation (n=20 in each group). (B) Dislocated hip was reduced at 2 weeks with F3D and F5D fixation. By contrast, the affected hip was unreducible along with the thickness of the acetabular roof after F7D, which is consistent with the morphology of RAD (scale bar, 1 cm). In addition, Reg3β in the acetabular roof was expressed at a higher level in the 7-day fixation group than in the 3-day and 5-day fixation groups and was expressed at lower levels in the 3-day and 5-day fixation groups than in the control group (n=3 in each group). (C) Reg3β expression was greater in the 7-day fixation group than in the normal group at weeks 1, 2 and 4, and Reg3β expression was transiently upregulated in the serum at week 2 but returned to the normal level at week 4. Student's t-tests were used to compare the two groups between each time point. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Reg3β, regenerating islet-derived protein 3-β; DDH, developmental dysplasia of the hip; F3D, 3-day fixation; F5D, 5-day fixation; F7D, 7-day fixation; RAD, residual acetabular dysplasia; CON, control.

Figure 2.

Western blot analysis of Reg3β expression, proliferation and apoptosis after Reg3β knockdown. (A) Clear cell death was observed with the S1 sequence, which exhibited the most effective knockdown compared with the other two sequences. One-way analysis of variance was used to compare these groups followed by Dunnett's post hoc test. Scale bar, 100 µm. (B) CCK-8 assay revealed that the proliferation of chondrocytes was inhibited after 72 h. (C) A colony formation assay with Alcian blue staining revealed that few cells remained after 1 week of S1 infection. (D) Cleaved caspase-3 and caspase-3, along with Bax, were increased after Reg3β knockdown at 72 h, whereas the level of Bcl-2 was significantly decreased, and the level of caspase-9 remained essentially unchanged. Student's t-tests were conducted to assess the statistical difference between the two groups in Fig. 2B-D. **P<0.01 and ***P<0.001. Reg3β, regenerating islet-derived protein 3-β; CCK-8, Cell Counting Kit-8; NC, negative control; OD, optical density.

Figure 3.

Anabolic and catabolic changes after Reg3β knockdown. Aggrecan, Mmp13 and Adamts-5 were downregulated, whereas Col2a1 exhibited no obvious change. Student's t-tests were performed to evaluate the statistical difference between the two groups. **P<0.01. Reg3β, regenerating islet-derived protein 3-β; NC, negative control; ns, not significant; Mmp13, matrix metallopeptidase 13; Col2a1, collagen type II alpha 1 chain; Adamts-5, ADAM metallopeptidase with thrombospondin type 1 motif 5.

Figure 4.

Jak2/Stat3/Socs3 signaling pathway is inhibited after Reg3β knockdown. Jak2/Stat3 and the corresponding phosphorylated proteins, as well as Extl3 and Socs3, were downregulated. Student's t-tests were performed to evaluate the statistical difference between the two groups. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Reg3β, regenerating islet-derived protein 3-β; Extl3, exostosin like glycosyltransferase 3; Socs3, suppressor of cytokine signaling 3; NC, negative control.

Figure 5.

Recombinant Reg3β protein promotes the activity of chondrocytes and the metabolism of the extracellular matrix. (A) The optimum concentration of recombinant Reg3β protein for subsequent experiments was determined. (B) Changes in Reg3β expression over time after the addition of recombinant Reg3β protein. (C) Aggrecan and corresponding degradative enzymes, such as Adamts-5 and Mmp13, were upregulated significantly after stimulation with the recombinant Reg3β protein, whereas Col2a1 expression did not change following transient downregulation at 24 h. (D) Stat3 and Socs3 were activated after Reg3β protein stimulation. ANOVA with Dunnett's post hoc test were performed. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. Reg3β, regenerating islet-derived protein 3-β; CCK-8, Cell Counting Kit-8; OD, optical density; CON, control; rReg3β, recombinant Reg3β; Mmp13, matrix metallopeptidase 13; Col2a1, collagen type II alpha 1 chain; Adamts-5, ADAM metallopeptidase with thrombospondin type 1 motif 5; Socs3, suppressor of cytokine signaling 3.