Pieles Oliver, Reichert Torsten E, Morsczeck Christian
Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.
Department of Oral and Maxillofacial Surgery, University Hospital Regensburg, Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany.
Arch Oral Biol. 2022 Jun;138:105409. doi: 10.1016/j.archoralbio.2022.105409. Epub 2022 Mar 16.
The aim of this study was to investigate the mechanisms of how protein kinase A (PKA) is activated during bone morphogenetic protein 2 (BMP2)-induced osteogenic differentiation in dental follicle stem cells.
Human dental follicle stem cells were cultured and treated with a BMP2-containing osteogenic differentiation medium or differentiation medium without BMP2. Specific siRNAs and substances/proteins were used to modulate pathways. PKA activity and activity of alkaline phosphatase were determined. Expression of targets was measured by Western Blots and reverse transcription-quantitative polymerase chain reaction, while protein interactions were investigated by immunoprecipitation. Immunofluorescence staining was used for subcellular target localization.
PKA activity is stimulated after osteogenic induction by BMP2. Differentiation medium without BMP2 strongly induces BMP2 gene expression, which correlates with downstream target expression. Elevation of cAMP levels does not affect alkaline phosphatase activity and PKA does not directly interact with Smad 4. However, PKA activation requires expression of parathyroid hormone-related protein (PTHrP), which is stimulated after BMP2-induced differentiation. Furthermore, neither supplementation with PTHrP nor with the receptor antagonist parathyroid hormone (7-34) affects PKA activity. Thus, endogenous PTHrP expression is required for PKA activation and immunofluorescence staining shows that PTHrP is mainly located in the nucleus of dental follicle stem cells. Beyond, knockdown of PKA stimulates the BMP2 signaling pathway and down-stream expression of PTHrP.
BMP2-induced osteogenic differentiation activates PKA in dental follicle stem cells via endogenous expression of PTHrP. Additionally, PKA inhibits BMP2 signaling and expression of PTHrP in a negative feedback loop.
本研究旨在探讨在骨形态发生蛋白2（BMP2）诱导牙囊干细胞成骨分化过程中蛋白激酶A（PKA）被激活的机制。
培养人牙囊干细胞，并用含BMP2的成骨分化培养基或不含BMP2的分化培养基进行处理。使用特异性小干扰RNA（siRNAs）和物质/蛋白质来调节信号通路。测定PKA活性和碱性磷酸酶活性。通过蛋白质免疫印迹法和逆转录-定量聚合酶链反应测量靶标的表达，同时通过免疫沉淀研究蛋白质相互作用。采用免疫荧光染色进行亚细胞靶标定位。
BMP2诱导成骨后PKA活性受到刺激。不含BMP2的分化培养基强烈诱导BMP2基因表达，这与下游靶标表达相关。环磷酸腺苷（cAMP）水平升高不影响碱性磷酸酶活性，且PKA不直接与Smad 4相互作用。然而，PKA激活需要甲状旁腺激素相关蛋白（PTHrP）的表达，BMP2诱导分化后PTHrP表达受到刺激。此外，补充PTHrP或其受体拮抗剂甲状旁腺激素（7 - 34）均不影响PKA活性。因此，内源性PTHrP表达是PKA激活所必需的，免疫荧光染色显示PTHrP主要位于牙囊干细胞的细胞核中。此外，敲低PKA可刺激BMP2信号通路及PTHrP的下游表达。
BMP2诱导的成骨分化通过内源性PTHrP表达激活牙囊干细胞中的PKA。此外，PKA在负反馈环路中抑制BMP2信号传导及PTHrP的表达。
