HIF1α regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis
HIF1α regulation of Sox9 is necessary to maintain differentiation of hypoxic prechondrogenic cells during early skeletogenesis
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DOI：
10.1242/dev.008441
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发表时间：
2007-11-01
期刊：
影响因子：
4.6
通讯作者：
Zelzer, Elazar
中科院分区：
文献类型：
--
作者：
Amarilio, Roy;Viukov, Sergey V.;Zelzer, Elazar
在肢体发育的早期阶段，血管系统会发生广泛的重塑，导致软骨形成前的冷凝无血管，并且正如我们在下文中所证明的那样，缺氧。对多种细胞类型的大量研究已经报道了缺氧对细胞分化具有抑制作用。为了研究在缺氧条件下支持软骨细胞分化的机制，我们灭活了小鼠肢芽间充质中的转录因子缺氧诱导因子1 α（HIF 1 α）。对HIF 1 α缺失的肢体进行发育分析，发现在autopod中软骨和关节形成异常，这表明HIF 1 α是调节缺氧前软骨细胞分化的机制的一部分。缺氧条件下HIF 1 α耗竭的微团培养细胞中软骨形成的显著减少进一步支持了HIF 1 α在软骨形成中的调节作用。Sox 9（软骨细胞分化的关键调节因子）的表达减少，随后Sox 6、II型胶原蛋白和聚集蛋白聚糖在HIF 1 α耗尽的肢体中减少，这提出了在低氧条件下HIF 1 α调节Sox 9对于前软骨形成细胞分化为软骨细胞是必需的可能性。为了研究这种可能性，我们在微团培养物中靶向Hif 1 α表达。在低氧条件下，Sox 9表达相对于其在常氧条件下的表达增加两倍;这种增加在Hif 1 α耗尽的细胞中丢失。染色质免疫沉淀证实了HIF 1 α与Sox 9启动子的直接结合，从而支持HIF 1 α对Sox 9表达的直接调节。这项工作首次确立了HIF 1 α作为遗传程序的关键组成部分，通过调节低氧前软骨细胞中Sox 9的表达来调节软骨形成。
During early stages of limb development, the vasculature is subjected to extensive remodeling that leaves the prechondrogenic condensation avascular and, as we demonstrate hereafter, hypoxic. Numerous studies on a variety of cell types have reported that hypoxia has an inhibitory effect on cell differentiation. In order to investigate the mechanism that supports chondrocyte differentiation under hypoxic conditions, we inactivated the transcription factor hypoxia- inducible factor 1 alpha ( HIF1 alpha) in mouse limb bud mesenchyme. Developmental analysis of Hif1 alpha- depleted limbs revealed abnormal cartilage and joint formation in the autopod, suggesting that HIF1 alpha is part of a mechanism that regulates the differentiation of hypoxic prechondrogenic cells. Dramatically reduced cartilage formation in Hif1 alpha- depleted micromass culture cells under hypoxia provided further support for the regulatory role of HIF1 alpha in chondrogenesis. Reduced expression of Sox9, a key regulator of chondrocyte differentiation, followed by reduction of Sox6, collagen type II and aggrecan in Hif1 alpha- depleted limbs raised the possibility that HIF1 alpha regulation of Sox9 is necessary under hypoxic conditions for differentiation of prechondrogenic cells to chondrocytes. To study this possibility, we targeted Hif1 alpha expression in micromass cultures. Under hypoxic conditions, Sox9 expression was increased twofold relative to its expression in normoxic condition; this increment was lost in the Hif1 alpha- depleted cells. Chromatin immunoprecipitation demonstrated direct binding of HIF1 alpha to the Sox9 promoter, thus supporting direct regulation of HIF1 alpha on Sox9 expression. This work establishes for the first time HIF1 alpha as a key component in the genetic program that regulates chondrogenesis by regulating Sox9 expression in hypoxic prechondrogenic cells.