ENDOTHELIN-1 IN NORMAL AND PATHOLOGICAL BONE REMODELING

描述(申请人提供)：内皮素-1(ET-1)可引起成骨细胞骨转移，通过激活成骨细胞上的内皮素A受体(ETAR)刺激失调的新骨形成。ET-1在正常骨发育、生长和改建中的作用尚不清楚。我们的初步数据表明：1)ET-I是体内成骨细胞活性和新骨形成的有力刺激因子；2)肿瘤产生的ET-1通过ETAR刺激成骨细胞的转移；3)ETAR拮抗剂对骨量具有性别类固醇依赖的效应：最显著的是，ETAR阻断剂减少了性腺下部的骨量，但增加了完整雌性小鼠的骨量。4)ET-1增加成骨细胞骨活性因子IL-6和Cyr61的表达，降低Dkk1的表达，增加转录因子C/EBPDelta和TSC-22的表达。ET-1不改变EGFR活化和血管内皮细胞生长因子。 我们将检验以下假设： 1)ET-1在正常骨重建中起重要作用。 2)ET-1通过改变旁分泌调节因子1L-6、Dkk1和潜在的Cyr61的分泌而介导对成骨细胞的作用。 3)ET-1增加与Runx2相互作用的成骨细胞转录因子C/EBP\Delta，并受TGFβ调节。 4)Etar阻断可导致成骨细胞成骨减少，破骨细胞活性增强，加速破骨细胞性激素缺乏所致的破骨细胞性骨吸收和继发性骨丢失。 5)雌激素抑制ET-1的合成。在雌激素缺乏状态下，升高的ET-1维持骨量 提出了三个具体目标： 目的1：确定ET-1和ETAR在正常骨发育、生长和改建中的作用。用Etar缺失的成骨细胞测定小鼠的骨表型 1B.用ET-L高表达的成骨细胞测定小鼠的骨表型 目的2：确定ET-1和ETAR在性激素缺乏状态骨重建中的作用 2a.性激素与ET-1在成骨细胞新骨形成中的相互作用 2B。测定联合抑制吸收和ETAR信号对骨量的影响 2C。用Etar缺失或ET-1++g成骨细胞测定性腺功能低下小鼠的骨表型 目的3：确定ET-1对骨作用的分子机制 3A。确定ET-1是否通过分泌IL-6、Dkk1或Cyr61介导其对成骨细胞的影响 3B。测定ET-1对成骨细胞转录因子C/EBPDelta和TSC-22的影响 3C。测定成骨细胞中ET-1激活的主要信号通路 3D。评估性类固醇与骨细胞内ET-1的相互作用 ET-1和ETAR在正常骨稳态中的生理作用有待研究。Etar阻滞剂正在对许多疾病状态进行临床试验，但对骨骼的后果尚不清楚。接受慢性Etar阻断治疗的癌症患者，除了性激素缺乏所致的骨质流失外，还可能遭受更多的骨质流失。我们将确定这种作用的机制，并确定这种影响是否可以通过双膦酸类药物治疗而减弱。了解ET-1在成骨细胞中对Etar的激活可能为治疗低骨量提供新的合成代谢疗法。建议的实验将使用体内和体外方法来测试成骨细胞ETAR的ET-1激活在骨生长和重建中的作用。

DESCRIPTION (provided by applicant): Endothelin-1 (ET-1) can cause osteoblastic bone metastases, stimulating dysregulated new bone formation by activating the endothelin A receptor (ETAR) on the osteoblast. The role of ET-1 in normal bone development, growth and remodeling is unclear. Our preliminary data indicate that: 1) ET-I is a potent stimulator of osteoblast activity and new bone formation in vivo; 2) Tumor-produced ET-1 stimulates osteoblastic metastases via ETAR; 3) ETAR blockade has sex steroid-dependent effects on bone mass: most strikingly, ETAR blockade reduces bone mass in hypogonadal but increases bone mass in intact female mice. 4) ET-1 increases expression of bone active factors IL-6 and Cyr61 and decreases that of Dkkl in osteoblasts, while increasing expression of the transcription factors C/EBPdelta and TSC-22. ET-1 did not alter EGFR activation or VEGF. The following hypotheses will be tested: 1) ET-1 plays an important role in normal bone remodeling. 2) ET-1 mediates effects on osteoblasts by changing the secretion of paracrine regulators 1L-6, Dkk1 and potentially Cyr61. 3) ET-1 increases the osteoblast transcription factor C/EBP\delta that interacts with Runx2, and TGFbeta-regulated TSC-22. 4) ETAR blockade results in reduced osteoblastic bone formation and increased osteoclast activity and may accelerates the osteoclastic bone resorption and subsequent bone loss associated with sex steroid deficiency. 5) Estrogen suppresses ET-1 synthesis. In estrogen-deficient states, increased ET-1 maintains bone mass Three Specific Aims are proposed: Aim 1: Determine the role of ET-1 and ETAR in normal bone development, growth, and remodeling 1A. Determine bone phenotype of mice with ETAR-null osteoblasts 1B. Determine bone phenotype of mice with ET-l- overexpressing osteoblasts Aim 2: Determine the role of ET-1 and ETAR in bone remodeling in sex-steroid deficient states 2A. Determine interactions between sex steroids and ET-1 in osteoblastic new bone formation 2B. Determine effects of combined inhibition of resorption and ETAR signaling on bone mass 2C. Determine bone phenotype of hypogonadal mice with ETAR-null or ET-1 ++g osteoblasts Aim 3: Determine the molecular mechanisms of ET-1 effects on bone 3A. Determine if ET-1 mediates its effects on osteoblasts via secreted IL-6, Dkk1, or Cyr61 3B. Determine effects of ET-1 on osteoblast transcription factors C/EBPdelta and TSC-22 3C. Determine major signaling pathways activated by ET-1 in osteoblasts 3D. Assess the interactions of sex steroids with ET-1 in bone cells The physiological role of ET-1 and ETAR in normal bone homeostasis needs investigation. 1, ETAR blockade is in clinical trials for many disease states, but the consequences for bone are unknown. Cancer patients treated with chronic ETAR blockade may suffer increased bone loss, in addition to that caused by sex-steroid deficiency. We will define the mechanisms and determine if this effect can be attenuated by bisphosphonate treatment. 2, an understanding of ET-1 activation of ETAR in the osteoblast may lead to new anabolic therapies for low bone mass. The experiments proposed will use in vivo and in vitro approaches to test the role of ET-1 activation of osteoblast ETAR in bone growth and remodeling.