Suppression of differentiation and proliferation of early chondrogenic cells by Notch
Suppression of differentiation and proliferation of early chondrogenic cells by Notch
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DOI：
10.1007/s00774-003-0428-4
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发表时间：
2003-09
影响因子：
3.3
通讯作者：
N. Watanabe;Y. Tezuka;K. Matsuno;S. Miyatani;N. Morimura;M. Yasuda;R. Fujimaki;K. Kuroda;Y. Hiraki;N. Hozumi;Ken-Ichi Tezuka
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文献类型：
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作者：
N. Watanabe;Y. Tezuka;K. Matsuno;S. Miyatani;N. Morimura;M. Yasuda;R. Fujimaki;K. Kuroda;Y. Hiraki;N. Hozumi;Ken-Ichi Tezuka
Notch是一种参与细胞命运决定的跨膜蛋白。在本研究中，我们观察到Notch1在发育中的软骨中在时间和空间上受限的表达。 Notch1从胚胎小鼠前肢的间充质凝结阶段开始定位。有趣的是，虽然在增殖的软骨细胞中无法检测到定位，但明显的免疫反应性表明其表达保留在软骨周区域。接下来，我们研究了软骨形成细胞系 ATDC5 细胞中 Notch1 和相关分子的表达。 Notch1、Delta-like (Dll)1、Deltex2 和 Deltex3 在 6 天胰岛素治疗后共表达。随后表达Hairy 和分裂同源物(HES)-1 的增强子。这些结果表明Notch可能在软骨形成的早期阶段发挥作用。为了评估Notch激活的效果，我们将ATDC5细胞与组成型表达Dll1（Notch的配体）的骨髓瘤克隆一起培养。我们还使用腺病毒载体来表达组成型活性 Notch1 胞内结构域 (NIC)。通过细胞的阿尔新蓝染色和软骨细胞分化标记物评估，激活内源性或外源性Notch受体显着抑制ATDC5细胞的软骨形成细胞分化。最后，我们研究了NIC对ATDC5细胞增殖的影响。腺病毒表达的 NIC 强烈抑制胸苷掺入。这些结果表明Notch在软骨形成细胞分化的初始阶段表达，并且当被激活时对细胞的分化和增殖具有强烈的抑制作用。随着软骨分化的进行，Notch 的表达减少；然而，Notch1 持续表达的细胞群会变成软骨膜细胞。考虑到软骨膜作为成骨细胞和软骨细胞的干细胞来源，Notch1可能通过抑制分化和增殖在这些细胞的形成中发挥作用。
Notch is a transmembrane protein involved in cell fate determination. In the present study, we observed temporally and spatially restricted expression of Notch1 in developing cartilage. Notch1 was localized starting from the mesenchymal condensation stage of embryonic mouse forelimbs. Interestingly, although localization could not be detected in the proliferating chondrocytes, obvious immunoreactivity indicating its expression was retained in the perichondrial region. Next, we investigated the expression of Notch1 and related molecules in a chondrogenic cell line, ATDC5 cells. Notch1, Delta-like (Dll)1, Deltex2, and Deltex3 were coexpressed after 6-day insulin treatment. Expression of Hairy and Enhancer of split homologue (HES)-1 followed thereafter. These results suggest that Notch may have a role in the early stage of chondrogenesis. To assess the effect of Notch activation, we cultured ATDC5 cells with a myeloma clone constitutively expressing Dll1, a ligand of Notch. We also used an adenovirus vector to express the constitutively active Notch1 intracellular domain (NIC). Activating either the endogenous or exogenous Notch receptor dramatically inhibited chondrogenic cell differentiation of ATDC5 cells, as assessed by Alcian blue staining of the cells and chondrocyte differentiation markers. Last, we investigated the effect of NIC on the proliferation of the ATDC5 cells. Expression of NIC by the adenovirus strongly suppressed thymidine incorporation. These results indicate that Notch is expressed in the initial stage of chondrogenic cell differentiation and has a strong inhibitory effect on both differentiation and proliferation of the cells when activated. The expression of Notch decreases as chondrogenic differentiation proceeds; however, a population of the cells with sustained expression of Notch1 become perichondrial cells. Considering that the perichondrium acts as a stem cell source of osteoblasts and chondrocytes, Notch1 may have a role in the formation of these cells by suppressing both differentiation and proliferation.