Different Roles for Colony Stimulating Factor 1 Isoforms in Anabolic Therapy for Low Bone Mass

集落刺激因子 1 同工型在低骨量合成代谢治疗中的不同作用

基本信息

批准号：
10585240
负责人：
KARL Leonard INSOGNA
金额：
$ 65.93万
依托单位：
YALE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-10 至 2028-06-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10585240
关键词：
Affinity Agonist Anabolism Animal Model Animals Attenuated B-Lymphocytes Binding Bone Resorption C57BL/6 Mouse CSF1 gene CSF1R gene Cells Coculture Techniques Colony-Stimulating Factors Data Dose Enzymes Estrogen deficiency Event Exposure to Immunophenotyping Impairment In Vitro Kinetics Macrophage Colony-Stimulating Factor Membrane Modeling Molecular Mus Osteoblasts Osteoclasts Osteogenesis Osteoporosis Paracrine Communication Patients Phospholipids Population Study Postmenopausal Osteoporosis Production Protein Isoforms Regimen Role Serum Signal Transduction T-Lymphocyte Testing Therapeutic Time Treatment Efficacy Treatment Protocols Wild Type Mouse bone bone loss bone mass density experimental study in vivo in vivo Model inhibitor insight mouse model new therapeutic target novel osteoclastogenesis paracrine pharmacologic pre-clinical receptor response skeletal skeletal disorder sphingosine 1-phosphate sphingosine kinase transcriptome transcriptome sequencing transcriptomic profiling

项目摘要

CSF1 is the principal colony stimulating activity released by osteoblasts in response to PTH treatment. Its receptor, c-fms, is more highly expressed on mature osteoclasts than any other cell in bone. We found that deleting c-fms in osteoclasts attenuates the anabolic response to PTH. This indicates that part of PTH's anabolic actions could be via a paracrine loop in which PTH stimulates expression of CSF1 in osteoblasts, which then acts on osteoclasts to induce anabolic clastokines that augment bone formation. There are two major isoforms of CSF1: soluble (sCSF1) and membrane-associated (mCSF1). We found that the anabolic response to PTH is augmented in animals only expressing the sCSF1 isoform due to a greater increase in osteoblast number in these mice compared to PTH-treated controls. In striking contrast, animals only expressing mCSF1 had no increase in bone mass in response to an anabolic PTH regimen. Importantly, sCSF1 and mCSF1 differ in the kinetics and extent to which they activate the CSF1 receptor, c-fms, suggesting that their divergent in vivo actions may be due in part, to intrinsic differences in cell-signaling. Based on these data, we hypothesize that mCSF1 inhibits PTH anabolism by opposing the actions of sCSF1 on osteoclasts. When unopposed, sCSF1 contributes to PTH anabolism by inducing production of anabolic clastokines. Specifically, single daily doses of PTH induce transient increases in sCSF1 in osteoblasts that cause bursts of anabolic clastokine production. Consistent with this, we found that sCSF1 stimulates production of the anabolic clastokine, sphingosine-1-phosphate (S-1-P) in osteoclasts. To test these hypotheses, we will, in SA1, determine if adding intermittent dosing of sCSF1 to an anabolic PTH treatment regimen augments the skeletal response to PTH in wild type animals, while adding mCSF1 to that regimen attenuates the response. We will then try to restore the anabolic response to PTH in the sCSF1-/- mice by treating with PTH plus sCSF1. These experiments will provide pharmacologic evidence that the very different response to PTH in sCSF1-/- and mCSF1-/- mice is directly due to differing actions of the two CSF1 isoforms in bone. In SA 2, we will treat wild type mice that were ovariectomized 5 months earlier, with PTH plus sCSF1 to determine if it restores bone mass to pre-OVX levels as a model of therapy for established post-menopausal osteoporosis. In SA 3, we will determine if sCSF1 and mCSF1 induce production of different clastokine profiles in mature osteoclasts. We will use osteoblast/osteoclast cocultures as a model of in vivo paracrine signaling in bone and profile the transcriptome of osteoclasts exposed to PTH-treated osteoblasts expressing only sCSF1 or only mCSF1 to identify differences in the types of clastokines produced. We will also determine differences in the transcriptomes of osteoblasts in the cocultures stimulated by these different clastokine profiles. Finally, we will examine binding kinetics of sCSF1 and mCSF1 to c-fms, and differences in downstream signaling, to gain molecular insight into the divergent in vivo and in vitro actions of these two isoforms.

CSF1是成骨细胞对PTH治疗而释放的主要菌落刺激活性。它是受体C-FMS在成熟的破骨细胞上比骨骼中的任何其他细胞更高。我们发现在破骨细胞中删除C-FMS会减弱对PTH的合成代谢反应。这表明PTH合成代谢的一部分动作可以通过旁分泌环，其中PTH刺激成骨细胞中CSF1的表达，然后作用于破骨细胞，诱导增强骨形成的合成代谢clastokines。有两个主要同工型 CSF1：可溶性（SCSF1）和膜相关（MCSF1）。我们发现合成代谢对PTH的反应由于成骨细胞的增加而仅表达SCSF1同工型的动物中增强与PTH处理的对照相比，这些小鼠的数量。在引人注目的对比中，动物只表达 MCSF1对合成代谢PTH方案的响应没有增加。重要的是，SCSF1和 MCSF1在激活CSF1受体C-FM的动力学和程度上有所不同，表明它们分歧性体内作用可能部分归因于细胞信号的固有差异。基于这些数据，我们假设MCSF1通过反对SCSF1对破骨细胞的作用来抑制PTH的变质。什么时候无反对的SCSF1通过诱导合成代谢clastokines的产生来促进PTH的变性。具体而言，每日剂量的PTH诱导成骨细胞中SCSF1的瞬态增加合成代谢clastokine生产的爆发。与此相一致，我们发现SCSF1刺激了生产破骨细胞中的合成代谢clastokine，鞘氨醇1-磷酸（S-1-P）。为了检验这些假设，我们将在 SA1，确定是否将SCSF1的间歇剂量添加到合成代谢的PTH治疗方案增加野生型动物对PTH的骨骼反应，同时将MCSF1添加到该方案中会减弱回复。然后，我们将尝试通过使用PTH来恢复SCSF1 - / - 小鼠中对PTH的合成代谢响应加上SCSF1。这些实验将提供药理证据，表明对PTH的反应非常不同 SCSF1 - / - 和MCSF1 - / - 小鼠直接是由于骨骼中两个CSF1同工型的作用不同。在SA 2中，我们将治疗5个月前卵巢切除的野生型小鼠，并使用PTH Plus SCSF1来确定是否是否它可以将骨骼质量恢复为植物前水平，作为既定的绝经后的治疗模型骨质疏松症。在SA 3中，我们将确定SCSF1和MCSF1是否诱导不同的clastokine产生成熟破骨细胞中的曲线。我们将使用成骨细胞/破骨细胞共培养作为体内旁分泌的模型骨骼和轮廓的信号传导暴露于PTH处理的成骨细胞表达的破骨细胞的转录组仅SCSF1或仅MCSF1来识别产生的clastokines类型的差异。我们还将确定这些不同的clastokine谱刺激的共培养物中成骨细胞转录组的差异。最后，我们将检查SCSF1和MCSF1与C-FMS的结合动力学，以及下游信号传导的差异，为了获得对这两种同工型的体内和体外作用的分子见解。