Ott Claus-Eric, Bauer Sebastian, Manke Thomas, Ahrens Susan, Rödelsperger Christian, Grünhagen Johannes, Kornak Uwe, Duda Georg, Mundlos Stefan, Robinson Peter N
Institute for Medical Genetics, Charité Universitätsmedizin Berlin, Berlin, Germany.
J Bone Miner Res. 2009 Jul;24(7):1247-62. doi: 10.1359/jbmr.090206.
Whereas mechanical stimulation is essential for bone homeostasis, straining of larger magnitude promotes bone regeneration by directing cell differentiation and proliferation and influencing the gene expression patterns of osteoblasts, which play a vital role in fracture healing by producing and mineralizing osteoid matrix. To elucidate the molecular mechanisms underlying the response of osteoblasts to mechanical strains comparable to those occurring during bone regeneration, MC3T3 S4 (MC4) osteoblast-like cells were stretched in vitro. Analysis based on microarray expression profiling during the first 8 h after straining showed 674 differentially expressed genes. The response to mechanical strain can be divided in an immediate-early response (IER) and later responses. Examination of the approximately 40 genes differentially expressed within the first 60 min, including 11 involved in regulating gene transcription, showed both promiscuous IER genes such as Fos that are upregulated by multiple extracellular stimuli, as well as a number of genes previously shown in neurons to be induced preferentially by depolarization (IPD-IER). Selected differentially expressed genes were validated after mechanical straining and KCl-induced depolarization. The effects of inhibitors for protein kinase A, mitogen-activated protein kinase, and calcineurin pathways were assessed in separate experiments by quantitative RT-PCR and shown to have differential effects on the response of MC4 cells and primary calvaria osteoblasts to both mechanical straining and KCl-induced depolarization. Therefore, our results showed the existence of two distinct pathways that mediate the IER of osteoblasts to large-magnitude mechanical straining and suggest that the IER to depolarizing stimuli is conserved in cell types as different as osteoblasts and neurons.
虽然机械刺激对骨稳态至关重要，但更大强度的拉伸通过引导细胞分化和增殖以及影响成骨细胞的基因表达模式来促进骨再生，而成骨细胞通过产生类骨质基质并使其矿化在骨折愈合中发挥着至关重要的作用。为了阐明成骨细胞对与骨再生过程中出现的机械应变相当的应变作出反应的分子机制，在体外对MC3T3 S4（MC4）成骨样细胞进行拉伸。基于拉伸后最初8小时内的微阵列表达谱分析显示有674个差异表达基因。对机械应变的反应可分为早期即时反应（IER）和后期反应。对在最初60分钟内差异表达的约40个基因进行检测，其中包括11个参与调节基因转录的基因，结果显示既有受多种细胞外刺激上调的混杂IER基因，如Fos，也有一些先前在神经元中显示优先由去极化诱导的基因（IPD-IER）。在机械拉伸和氯化钾诱导的去极化后，对选定的差异表达基因进行了验证。通过定量逆转录-聚合酶链反应在单独的实验中评估了蛋白激酶A、丝裂原活化蛋白激酶和钙调神经磷酸酶途径抑制剂的作用，结果显示它们对MC4细胞和原代颅骨成骨细胞对机械拉伸和氯化钾诱导的去极化的反应有不同影响。因此，我们的结果表明存在两条不同的途径介导成骨细胞对大强度机械拉伸的IER，并表明对去极化刺激的IER在成骨细胞和神经元等不同细胞类型中是保守的。
