Falconi Dominic, Aubin Jane E
Department of Medical Biophysics, University of Toronto. Toronto, Ontario, Canada.
J Bone Miner Res. 2007 Aug;22(8):1289-300. doi: 10.1359/jbmr.070417.
LIF arrests osteogenesis in fetal rat calvaria cells in a differentiation stage-specific manner. Differential display identified HAS2 as a LIF-induced gene and its product, HA, modulated osteoblast differentiation similarly to LIF. Our data suggest that LIF arrests osteoblast differentiation by altering HA content of the extracellular matrix.
Leukemia inhibitory factor (LIF) elicits both anabolic and catabolic effects on bone. We previously showed in the fetal rat calvaria (RC) cell system that LIF inhibits osteoblast differentiation at the late osteoprogenitor/early osteoblast stage.
To uncover potential molecular mediators of this inhibitory activity, we used a positive-negative genome-wide differential display screen to identify LIF-induced changes in the developing osteoblast transcriptome.
Although LIF signaling is active throughout the RC cell proliferation-differentiation sequence, only a relatively small number of genes, in several different functional clusters, are modulated by LIF specifically during the LIF-sensitive inhibitory time window. Based on their known and predicted functions, most of the LIF-regulated genes identified are plausible candidates to be involved in the LIF-induced arrest of osteoprogenitor differentiation. To test this hypothesis, we further analyzed the function of one of the genes identified, hyaluronan synthase 2 (HAS2), in the LIF-induced inhibition. Synthesis of hyaluronan (HA), the product of HAS enzymatic activity, was stimulated by LIF and mimicked the HAS2 expression profile, with highest expression in early/proliferative and late/maturing cultures and lowest levels in intermediate/late osteoprogenitor-early osteoblast cultures. Exogenously added high molecular weight HA, the product of HAS2, dose-dependently inhibited osteoblast differentiation, with pulse-treatment effective in the same differentiation stage-specific inhibitory window as seen with LIF. In addition, however, pulse treatment with HA in early cultures slightly increased bone nodule formation. Treatment with hyaluronidase, on the other hand, stimulated bone nodule formation in early cultures but caused a small dose-dependent inhibition of osteoblast differentiation in the LIF- and HA-sensitive late time window.
Together the data suggest that osteoblast differentiation is acutely sensitive to HA levels and that LIF inhibits osteoblast development at least in part by stimulating high molecular weight HA synthesis through HAS2.
白血病抑制因子（LIF）以分化阶段特异性方式抑制胎鼠颅骨细胞的成骨作用。差异显示法鉴定出透明质酸合酶2（HAS2）为LIF诱导基因，其产物透明质酸（HA）对成骨细胞分化的调节作用与LIF相似。我们的数据表明，LIF通过改变细胞外基质的HA含量来抑制成骨细胞分化。
白血病抑制因子（LIF）对骨骼具有合成代谢和分解代谢作用。我们之前在胎鼠颅骨（RC）细胞系统中表明，LIF在晚期骨祖细胞/早期成骨细胞阶段抑制成骨细胞分化。
为了揭示这种抑制活性的潜在分子介质，我们使用全基因组正负差异显示筛选法来鉴定LIF诱导的成骨细胞转录组变化。
尽管LIF信号在整个RC细胞增殖-分化序列中都有活性，但在LIF敏感的抑制时间窗口内，只有少数几个不同功能簇中的基因受到LIF的特异性调节。根据它们已知和预测的功能，大多数鉴定出的LIF调节基因可能参与了LIF诱导的骨祖细胞分化停滞。为了验证这一假设，我们进一步分析了其中一个鉴定出的基因——透明质酸合酶2（HAS2）在LIF诱导抑制中的功能。透明质酸（HA）是HAS酶活性的产物，其合成受到LIF的刺激，并模仿了HAS2的表达谱，在早期/增殖期和晚期/成熟期培养物中表达最高，在中期/晚期骨祖细胞-早期成骨细胞培养物中水平最低。外源性添加的高分子量HA（HAS2的产物）剂量依赖性地抑制成骨细胞分化，脉冲处理在与LIF相同的分化阶段特异性抑制窗口有效。然而，此外，在早期培养物中用HA进行脉冲处理会轻微增加骨结节形成。另一方面，用透明质酸酶处理在早期培养物中刺激骨结节形成，但在LIF和HA敏感的晚期时间窗口中导致成骨细胞分化出现小剂量依赖性抑制。
这些数据共同表明，成骨细胞分化对HA水平高度敏感，并且LIF至少部分通过HAS2刺激高分子量HA合成来抑制成骨细胞发育。
