The Developmentally Regulated Osteoblast Phosphodiesterase GDE3 Is Glycerophosphoinositol-specific and Modulates Cell Growth
The Developmentally Regulated Osteoblast Phosphodiesterase GDE3 Is Glycerophosphoinositol-specific and Modulates Cell Growth
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DOI：
10.1074/jbc.m109.035444
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发表时间：
2009-09-11
影响因子：
4.8
通讯作者：
Mariggio, Stefania
中科院分区：
文献类型：
--
作者：
Corda, Daniela;Kudo, Takahiro;Mariggio, Stefania
参与甘油磷酸二酯水解的甘油磷酸二酯磷酸二酯酶家族已经在细菌中表征，并且最近在哺乳动物中鉴定。在这里，我们已经确定了七种哺乳动物酶之一GDE 3的活性和功能。GDE 3在成骨细胞分化过程中上调，并可影响细胞形态。我们表明，GDE 3是一种甘油磷酸肌醇（GroPIns）磷酸二酯酶，水解GroPIns，产生肌醇1-磷酸和甘油，从而表明这种酶在GroPIns代谢中的特定作用。底物特异性分析显示，野生型GDE 3选择性水解GroPIn超过甘油磷酸胆碱、甘油磷酸乙醇胺和甘油磷酸丝氨酸。GDE 3催化结构域中的单点突变（GDE 3R 231 A）导致GroPIns酶促水解的损失，鉴定出对GDE 3活性至关重要的精氨酸残基。在HEK 293 T细胞中异源GDE 3表达后，在细胞外培养基中检测到磷酸二酯酶活性，对细胞内GroPIns库没有影响。与GDE 3活性所需的钙的毫摩尔浓度一起，这预测了具有细胞外催化结构域的酶拓扑结构。有趣的是，在来自鼠成骨细胞系的稳定克隆中检测到GDE 3细胞外活性，进一步证实了GDE 3在更生理背景下的活性。最后，野生型GDE 3在成骨细胞中的过表达促进肌动蛋白应力纤维的分解，生长速率降低，碱性磷酸酶活性和钙含量增加，表明GDE 3在诱导分化中的作用。因此，我们已经将GDE 3底物GroPIns鉴定为成骨细胞增殖的候选介质，这与先前在上皮细胞中观察到的GroPIns活性一致。
The glycerophosphodiester phosphodiesterase enzyme family involved in the hydrolysis of glycerophosphodiesters has been characterized in bacteria and recently identified in mammals. Here, we have characterized the activity and function of GDE3, one of the seven mammalian enzymes. GDE3 is up-regulated during osteoblast differentiation and can affect cell morphology. We show that GDE3 is a glycerophosphoinositol (GroPIns) phosphodiesterase that hydrolyzes GroPIns, producing inositol 1-phosphate and glycerol, and thus suggesting specific roles for this enzyme in GroPIns metabolism. Substrate specificity analyses show that wild-type GDE3 selectively hydrolyzes GroPIns over glycerophosphocholine, glycerophosphoethanolamine, and glycerophosphoserine. A single point mutation in the catalytic domain of GDE3 (GDE3R231A) leads to loss of GroPIns enzymatic hydrolysis, identifying an arginine residue crucial for GDE3 activity. After heterologous GDE3 expression in HEK293T cells, phosphodiesterase activity is detected in the extracellular medium, with no effect on the intracellular GroPIns pool. Together with the millimolar concentrations of calcium required for GDE3 activity, this predicts an enzyme topology with an extracellular catalytic domain. Interestingly, GDE3 ectocellular activity is detected in a stable clone from a murine osteoblast cell line, further confirming the activity of GDE3 in a more physiological context. Finally, overexpression of wild-type GDE3 in osteoblasts promotes disassembly of actin stress fibers, decrease in growth rate, and increase in alkaline phosphatase activity and calcium content, indicating a role for GDE3 in induction of differentiation. Thus, we have identified the GDE3 substrate GroPIns as a candidate mediator for osteoblast proliferation, in line with the GroPIns activity observed previously in epithelial cells.