EDR-1 PHOSPORYLATION & ESTROGEN REGULATED M-CSF

增强破骨细胞（OC）的形成是雌激素（E2）缺乏导致骨质流失的关键机制。E2缺乏通过改变成熟基质细胞（SC）的表型特征刺激破骨细胞（OC）的形成。因此，来自卵巢切除（ovx）小鼠的成熟SC产生巨噬细胞集落刺激因子（M-CSF）水平增加，巨噬细胞集落刺激因子是OC前体增殖和分化所必需的细胞因子。来自ovx小鼠的SC产生高水平的M-CSF，因为转录启动子Sp-1与M-CSF启动子中Sp位点的结合增加。我们最近发现转录因子Egr-1抑制Sp-1诱导的M-CSF基因表达，而自身不与DNA结合。我们还发现Egr-1直接与Sp-1相互作用，形成一种新的Egr 1/Sp- 1复合物。Ovx增加ckii依赖的Egr-1磷酸化，导致Egr-1与Sp-1的关联降低，并增加了能够反激活M-CSF基因的未结合Sp-1的可用性。因此，E2缺乏通过增加Egr-1磷酸化，降低Egr-1和Sp-1之间的关联。这些事件导致可与M-CSF启动子结合和反激活的“自由”Sp-1水平增加。这些发现是假设Egr-1（以及调节其磷酸化的因子）在E2阻断OC形成和防止骨质流失的机制中发挥关键作用的基础。为了验证这一假设，我们提出：1)利用Egr-1敲除小鼠，确定Egr-1对E2抑制M-CSF SC生成和M-CSF诱导的破骨细胞生成、骨吸收和骨丢失的贡献。2)利用酵母2杂交系统绘制对Egr-1/Sp-1复合物形成至关重要的Egr-1和Sp-1区域，通过pull - down-Western分析和转染Egr-1缺陷小鼠SC中的Egr-1突变体。3)。确定CKII在ovx和E2充满小鼠SC中磷酸化Egr-1的位点，并探讨Egr-1磷酸化在降低Egr-1与Sp-1结合中的作用。这将通过使用质谱法来绘制HPLC纯化的Egr-1蛋白水解片段，这些片段来自ovx和E2充满小鼠的SC磷酸化。pull - down-Western分析和转染Egr-1缺陷小鼠SC中的Egr-1突变体将用于确定每个Egr-1磷酸化残基在调节Egr-1与Sp-1关联中的功能作用。

Enhanced osteoclast (OC) formation is a key mechanisms by which estrogen (E2) deficiency induces bone loss. E2 deficiency stimulates osteoclast (OC) formation by altering the phenotypic characteristics of mature stromal cells (SC). As a result, mature SC from ovariectomized (ovx) mice produce increased levels of macrophage colony-stimulating factor (M-CSF), a cytokine essential for the proliferation and differentiation of OC precursors. SC from ovx mice produce high M-CSF levels because of increased binding of the transcriptional initiator Sp-1 to a Sp site in the M-CSF promoter. We have recently discovered that the transcription factor Egr-1 inhibits Sp-1 induced M-CSF gene expression without itself binding to DNA. We have also found that Egr-1 interacts directly with Sp-1 forming a novel Egr 1/Sp- 1 complex. Ovx increases CKII-dependent Egr-1 phosphorylation, leading to decreased association of Egr-1 with Sp-1, and increased availability of unbound Sp-1, capable of transactivating the M-CSF gene. Thus, E2 deficiency, by increasing Egr-1 phosphorylation, decreases association between Egr-1 and Sp-1. These events result in increased levels of "free" Sp-1 available for binding to, and transactivation of, the M-CSF promoter. These findings are the basis for the hypothesis that Egr-1 (and factors which regulate its phosphorylation) play a critical role in the mechanism by which E2 blocks OC formation and prevents bone loss. In order to test this hypothesis, we propose 1) To determine the contribution of Egr-1 to the inhibitory effects of E2 on SC production of M-CSF and M-CSF induced osteoclastogenesis, bone resorption and bone loss in vivo using Egr-1 knock out mice. 2) To map the regions of Egr-1 and Sp-1 critical for the formation of Egr-1/Sp-1 complex using a yeast 2 hybrid system, a pull down-Western analysis and transfections of Egr-1 mutants in SC from Egr-1 deficient mice. 3). To determine the sites of Egr-1 phosphorylated by CKII in SC from ovx and E2 replete mice, and to investigate the role of Egr- 1 phosphorylation in decreasing the binding of Egr-1 to Sp-1. This will be accomplished by using mass spectrometry to map HPLC purified proteolytic fragments of Egr-1 phosphorylated by SC originating from ovx and E2 replete mice. A pull down-Western analysis and transfections of Egr-1 mutants in SC harvested from Egr-1 deficient mice will be used to determine the functional role of each Egr-1 phosphorylated residue in regulating the association of Egr-1 with Sp-1.