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 是成骨细胞响应 PTH 治疗而释放的主要集落刺激活性。它是受体 c-fms 在成熟破骨细胞上的表达水平高于骨中任何其他细胞。我们发现删除破骨细胞中的 c-fms 会减弱对 PTH 的合成代谢反应。这表明 PTH 的合成代谢部分作用可能是通过旁分泌环，其中 PTH 刺激成骨细胞中 CSF1 的表达，然后作用于破骨细胞，诱导合成代谢破骨因子，促进骨形成。有两种主要的异构体 CSF1：可溶性（sCSF1）和膜相关性（mCSF1）。我们发现对 PTH 的合成代谢反应在仅表达 sCSF1 同种型的动物中，由于成骨细胞的增加更多与 PTH 处理的对照组相比，这些小鼠的数量。与此形成鲜明对比的是，动物只表达 mCSF1 骨量没有因合成代谢 PTH 方案而增加。重要的是，sCSF1 和 mCSF1 在激活 CSF1 受体 c-fms 的动力学和程度方面有所不同，这表明它们不同的体内作用可能部分归因于细胞信号传导的内在差异。根据这些数据，我们假设 mCSF1 通过对抗 sCSF1 对破骨细胞的作用来抑制 PTH 合成代谢。什么时候毫无疑问，sCSF1 通过诱导合成代谢因子的产生来促进 PTH 合成代谢。具体来说，每日单剂量的 PTH 会诱导成骨细胞中 sCSF1 的短暂增加，从而导致合成代谢因子的激增。与此一致的是，我们发现 sCSF1 刺激产生破骨细胞中的合成代谢因子、1-磷酸鞘氨醇 (S-1-P)。为了检验这些假设，我们将在 SA1，确定在合成代谢 PTH 治疗方案中添加间歇性给药 sCSF1 是否会增强野生型动物的骨骼对 PTH 的反应，而在该方案中添加 mCSF1 会减弱回复。然后，我们将尝试通过 PTH 治疗来恢复 sCSF1-/- 小鼠对 PTH 的合成代谢反应加上sCSF1。这些实验将提供药理学证据，证明不同个体对 PTH 的反应截然不同。 sCSF1-/- 和 mCSF1-/- 小鼠的出现直接归因于两种 CSF1 亚型在骨中的不同作用。在 SA 2 中，我们将使用 PTH 加 sCSF1 治疗 5 个月前切除卵巢的野生型小鼠，以确定是否它将骨量恢复到 OVX 之前的水平，作为已确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 的产生成熟破骨细胞的概况。我们将使用成骨细胞/破骨细胞共培养物作为体内旁分泌的模型骨中的信号传导并分析暴露于 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 clastokine 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，获得对这两种异构体不同的体内和体外作用的分子洞察。