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首页> 外文期刊>Journal of Cell Science >Inhibition of chondrocyte differentiation in vitro by constitutive and inducible overexpression of the c-fos proto-oncogene
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Inhibition of chondrocyte differentiation in vitro by constitutive and inducible overexpression of the c-fos proto-oncogene

机译：c-fos原癌基因的组成型和诱导型过表达抑制软骨细胞体外分化

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We have investigated the role of c-Fos in chondrocyte differentiation in vitro using both constitutive and inducible overexpression approaches in ATDC5 chondrogenic cells, which undergo a well-defined sequence of differentiation from chondroprogenitors to fully differentiated hypertrophic chondrocytes. Initially, we constitutively overexpressed exogenous c-fos in ATDC5 cells. Several stable clones expressing high levels of exogenous c-fos were isolated and those also expressing the cartilage marker type II collagen showed a marked decrease in cartilage nodule formation. To investigate further whether c-Fos directly regulates cartilage differentiation independently of potential clonal variation, we generated additional clones in which exogenous c-fos expression was tightly controlled by a tetracycline-regulatable promoter. Two clones, DT7.1 and DT12.4 were capable of nodule formation in the absence of c-fos, However, upon induction of exogenous c-fos, differentiation was markedly reduced in DT7.1 cells and was virtually abolished in clone DT12.4. Pulse experiments indicated that induction of c-fos only at early stages of proliferation/differentiation inhibited nodule formation, and limiting dilution studies suggested that overexpression of c-fos decreased the frequency of chondroprogenitor cells within the clonal population. Interestingly, rates of proliferation and apoptosis were unaffected by c-fos overexpression under standard conditions, suggesting that these processes do not contribute to the observed inhibition of differentiation. Finally, gene expression analyses demonstrated that the expression of the cartilage markers type II collagen and PTH/PTHrP receptor were downregulated in the presence of exogenous c-Fos and correlated well with the differentiation status. Moreover, induction of c-fos resulted in the concomitant increase in the expression of fra-1 and c-jun, further highlighting the importance of AP-1 transcription factors in chondrocyte differentiation. These data demonstrate: that c-fos overexpression directly inhibits chondrocyte differentiation in vitro, and therefore these cell lines provide very useful tools for identifying novel c-Fos-responsive genes that regulate the differentiation and activity of chondrocytes. [References: 82]
机译：我们已经研究了AT-DC5软骨细胞中本构和诱导型过表达方法在体外软骨细胞分化中c-Fos的作用，这些细胞经历了从软骨生成细胞到完全分化的肥大性软骨细胞的明确分化序列。最初，我们在ATDC5细胞中组成型过表达外源性c-fos。分离了几个表达高水平外源性c-fos的稳定克隆，也表达了II型胶原标记物的那些克隆显示出软骨结节形成的明显减少。为了进一步研究c-Fos是否独立于潜在的克隆变异而直接调节软骨分化，我们产生了另外的克隆，其中外源c-fos的表达受四环素可调节的启动子严格控制。在不存在c-fos的情况下，两个克隆DT7.1和DT12.4能够形成结节。但是，在诱导外源性c-fos时，DT7.1细胞中的分化明显减少，并且在克隆DT12中实际上被废除了。 4。脉冲实验表明，仅在增殖/分化的早期才诱导c-fos抑制结节的形成，而有限稀释研究表明c-fos的过表达降低了克隆种群中软骨生成细胞的频率。有趣的是，在标准条件下c-fos过表达不会影响增殖和凋亡的速率，这表明这些过程对观察到的分化抑制没有贡献。最后，基因表达分析表明，在存在外源性c-Fos的情况下，II型胶原胶原标记和PTH / PTHrP受体的表达下调，并且与分化状态密切相关。此外，c-fos的诱导导致fra-1和c-jun表达的同时增加，进一步突显了AP-1转录因子在软骨细胞分化中的重要性。这些数据证明：c-fos的过度表达直接在体外抑制软骨细胞的分化，因此，这些细胞系为鉴定调节软骨细胞分化和活性的新型c-Fos反应基因提供了非常有用的工具。 [参考：82]

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