Thymosin β4 regulates the expression of bone sialoprotein (BSP) and dentin sialophosphoprotein (DSPP) in odontoblast

Abstract

Thymosin β4(Tβ4)는 포유류 세포에서 상처치유, 혈관생성, 세포 증식, 이동, 분화에 관여한다. Tβ4가 과발현된 형질전환 생쥐는 법랑질형성부전을 보였고, Tβ4 knockdown은 치배의 성장을 억제시켰다. 또한 Tβ4는 구강상피세포 증식에 관여했고, 상아질모세포 분화와 광화에 관련된 단백질들의 발현과 분비를 조절했다. 선행된 연구결과들로 보아 Tβ4는 상아질모세포에서 상아기질 형성에 관련된 인자들의 발현을 조절할 것으로 생각되지만, 이들 인자들의 발현과 연관된 Tβ4의 신호전달체계에 관한 연구는 미흡한 실정이다. 따라서 본 연구에서는 상아질모세포주인 MDPC-23 세포에서 상아기질형성 조절인자 중 bone sialoprotein(BSP)과 dentin sialoprotein(DSP)의 발현과 관련된 Tβ4의 신호전달경로를 규명하고자 하였다. Membrane hybridization 실험 결과, 합성된 antisense Tβ4 probe가 sense Tβ4 probe에 특이적으로 결합했고, antisense Tβ4 probe와 상아질모세포 내 Tβ4 mRNA 사이에 hybridization을 확인했다. In situ hybridization 방법을 통해 발생 중인 구치 조직의 상아질모세포에서 Tβ4 mRNA는 후기 종시기인 postnatal day 5(PN5)에 급격하게 증가했다. Tβ4가 처리된 MDPC-23 세포(MDPC-23/Tβ4)에서 BSP 발현은 대조군에 비해 증가했지만, DSP 발현은 감소했다. MDPC-23/Tβ4에 대한 면역형광 결과에서도 세포질 내 BSP 발현은 증가했지만, DSP는 감소했다. MDPC-23/Tβ4에서 ERK1/2 인산화, 세포질과 핵 내 Runx2 그리고 BSP 발현은 대조군에 비해 증가됐으나 DSP 발현은 감소됐다. MEK inhibitor인 PD98059가 처리된 MDPC-23 세포(MDPC-23/PD98059)에서 ERK1/2 인산화, 세포질과 핵 내 Runx2 그리고 BSP 발현은 MDPC-23/Tβ4에 비해 감소했지만, DSP의 감소된 발현 경향은 유지됐다. MDPC-23/Tβ4에서 β-catenin 인산화는 대조군에 비해 증가했고, 세포질 내 β-catenin은 감소했지만, β-catenin 핵 이동이 일어났으며 BSP 발현은 대조군에 비해 증가됐으나, DSP 발현은 감소했다. 또한 MDPC-23/PD98059에서 β-catenin 인산화는 MDPC-23/Tβ4에 비해 감소했지만, 세포질 내 β-catenin 및 핵 이동은 증가했다. MDPC-23/Tβ4에서 인산화된 Smad3는 핵으로 이동했으며, β-catenin 인산화와 세포질 내 β-catenin, Runx2 그리고 BSP 발현 경향은 ERK1/2 경로와 유사했다. Smad3의 inhibitor인 SIS3가 처리된 MDPC-23 세포(MDPC-23/SIS3)에서 Smad3 인산화는 MDPC-23/Tβ4에 비해 감소했고 β-catenin 인산화, β-catenin, Runx2와 BSP 그리고, DSP 단백질 발현 경향은 MDPC-23/PD98059와 유사했다. MDPC-23/Tβ4 에서 BSP promoter activity를 측정한 결과 음성대조군(pGL3-Luc)에 비해 크게 증가했지만, PD98059와 SIS3를 처리하면 Tβ4 처리군에 비해 감소했다. 또한 SIS3를 처리한 군의 promoter activity는 PD98059를 처리한 군보다 더 감소했다. 결론적으로, Tβ4는 상아질모세포에서 BSP 발현 증가와 DSP 발현 감소를 통해 상아질형성을 조절하고, Tβ4에 의한 BSP 발현 증가는 ERK1/2/Runx2, ERK1/2/β-catenin, Smad3, Smad3/Runx2 또는 Smad3/β-catenin 경로를 통해 일어나지만, DSP 발현 감소는 ERK1/2 또는 Smad3가 아닌 다른 신호전달 기작이 있을 것으로 생각된다.|Thymosin β4 (Tβ4) is involved in wound healing, angiogenesis, proliferation, migration and differentiation in mammalians. Tβ4-overexpressed transgenic mice present enamel hyperplasia and the knockdown of Tβ4 inhibits the growth of tooth germs. In addition, Tβ4 is involved in the proliferation of oral epithelial cells, and regulates the expression and secretion of the proteins related to differentiation and mineralization. Based on the previous results, Tβ4 regulates the expression of the factors related with dentinogenesis, but the signal transduction pathway of Tβ4 is insufficient in relation to the expression of these factors. Therefore, this study investigates the signal transduction pathway of Tβ4 associated with the expression of bone sialoprotein (BSP) and dentin sialophosphoprotein (DSPP), among the regulating factors to dentinogenesis, in odontoblastic cell lines, MDPC-23 cells. As a result of membrane hybridization, a synthetic antisense Tβ4 probe bound to specifically with a sense Tβ4 probe and identified the hybridization between antisense Tβ4 probe and the intracellular Tβ4 mRNA in odontoblasts. In in situ hybridization, the Tβ4 mRNA was increased rapidly in the odontoblasts of molar tissue at postnatal day 5 (PN5) of the advanced bell stage. In Tβ4-treated MDPC-23 cells (MDPC-23/Tβ4), the expression of BSP was increased, but DSP expression was decreased compared to the control. In addition, in immunofluorescence, the expression of cytoplasmic BSP was increased in MDPC-23/Tβ4, but DSP expression was decreased. The phosphorylation of ERK1/2, cytoplasmic or nuclear Runx2 and BSP expression were increased MDPC-23/Tβ4 compared to that of the control, but DSP expression was decreased. In PD98059-treated MDPC-23 cells (MDPC-23/PD98059), the phosphorylation of ERK1/2, cytoplasmic or nuclear Runx2, and BSP expression were decreased, but the reduced expression pattern of DSP was similar to that of MDPC-23/Tβ4. In MDPC-23/Tβ4, the phosphorylation of β-catenin was increased, and the nuclear translocation of β-catenin was induced with the reduction of cytoplasmic β-catenin compared to that of control, and the expression of BSP was increased, but DSP expression decreased. In addition, in MDPC-23/PD98059, the phosphorylation of β-catenin was decreased and cytoplasmic β-catenin was increased with the induction of β-catenin nuclear translocation. In MDPC-23/Tβ4, phosphorylated Smad3 translocated into the nucleus and the pattern of the phosphorylation of β-catenin, cytoplasmic β-catenin, Runx2, and BSP expression were similar to that of ERK1/2 pathway. In SIS3-treated MDPC-23 cells (MDPC-23/SIS3), the phosphorylation of Smad3 was decreased compared to that of MDPC-23/Tβ4, and the pattern of phosphorylation of β-catenin, β-catenin, Runx2, BSP, and DSP expression were similar to MDPC-23/PD98059. In MDPC-23/Tβ4, the BSP promoter activity was increased significantly compared to the negative control (pGL3-Luc), but it was decreased in pGL3-BSP/PD98059 and pGL3-BSP/SIS3 compared to that of pGL3-BSP-Luc. In addition, the promoter activity of pGL3-BSP-Luc/SIS3 was more decreased than pGL3-BSP-Luc/PD98059. In conclusion, the increase of BSP expression was induced through ERK1/2/Runx2, ERK1/2/β-catenin, Smad3, Smad3/Runx2, or Smad3/β-catenin signaling pathways, but decreased DSP expression was not affected by MEK and pSmad3 inhibitors, indicating that DSP expression by Tβ4 is not related with ERK or Smad3 signaling pathways in MDPC-23 cells. This result suggests that Tβ4 may regulate dentin matrix formation with the increase of BSP through ERK1/2 or Smad3 signaling pathways in odontoblasts.