Dental Research Laboratory, Faculty of Dental Medicine, Institute of Dental Sciences, The Hebrew University of Jerusalem, Hadassah, Jerusalem, Israel.
Rambam Health Care Campus, 8 Ha'Aliyah Street, 35254, Haifa, Israel.
J Mol Neurosci. 2019 May;68(1):135-143. doi: 10.1007/s12031-019-01292-1. Epub 2019 Mar 21.
Nerve growth factor (NGF) promotes pleiotropic gene transcription-dependent biological effects, in neuronal and non-neuronal cells, including survival, proliferation, differentiation, neuroprotection, pain, and angiogenesis. It is hypothesized that during odontogenesis, NGF may be implicated in morphogenetic and mineralization events by affecting proliferation and/or differentiation of dental cells. Tuftelin belongs to the enamel associated teeth proteins and is thought to play a role in enamel mineralization. We previously reported that tuftelin transcript and protein, which are ubiquitously expressed in various tissues of embryos, adults, and tumors, were significantly upregulated during NGF-induced PC12 differentiation. To further confirm the involvement of tuftelin in the differentiation process, we established a tuftelin-knockdown neuronal PC12 cell model, using a non-cytotoxic siRNA directed towards sequences at the 3' UTR of the tuftelin gene. Using real-time PCR, we quantified tuftelin mRNA expression and found that tuftelin siRNA, but not scrambled siRNA or transfection reagents, efficiently depleted about 60% of NGF-induced tuftelin mRNA transcripts. The effect of tuftelin siRNA was quantified up to 6 days of NGF-induced differentiation. Using immunofluorescence and western blot analyses, we also found a direct correlation between reduction of 60-80% in tuftelin protein expression and inhibition of about 50-70% in NGF-induced differentiation of the cells, as was detected after 3-6 days of treatment. These results demonstrate an important role for tuftelin in NGF-induced differentiation of PC12 cells. Tuftelin could be a useful target for drug development in disease where neurotrophin therapy is required.
神经生长因子（NGF）促进多效基因转录依赖性的生物学效应，在神经元和非神经元细胞中，包括存活、增殖、分化、神经保护、疼痛和血管生成。据推测，在牙发生过程中，NGF 可能通过影响牙细胞的增殖和/或分化而参与形态发生和矿化事件。Tuftelin 属于釉质相关牙齿蛋白，被认为在釉质矿化中发挥作用。我们之前报道过，tuftelin 转录本和蛋白在胚胎、成体和肿瘤的各种组织中广泛表达，在 NGF 诱导的 PC12 分化中显著上调。为了进一步证实 tuftelin 在分化过程中的参与，我们建立了一种 tuftelin 敲低的神经元 PC12 细胞模型，使用针对 tuftelin 基因 3'UTR 的非细胞毒性 siRNA。通过实时 PCR，我们量化了 tuftelin mRNA 的表达，发现 tuftelin siRNA，但不是 scramble siRNA 或转染试剂，有效地耗尽了约 60%的 NGF 诱导的 tuftelin mRNA 转录本。tuftelin siRNA 的效果在 NGF 诱导的分化的 6 天内被量化。通过免疫荧光和 western blot 分析，我们还发现，tuftelin 蛋白表达减少 60-80%与 NGF 诱导的细胞分化抑制之间存在直接相关性，在处理 3-6 天后检测到约 50-70%的抑制。这些结果表明 tuftelin 在 NGF 诱导的 PC12 细胞分化中起着重要作用。tuftelin 可能成为神经生长因子治疗所需的神经保护治疗药物开发的有用靶点。
