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

• 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
• 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

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是成骨细胞在甲状旁腺素治疗后释放的主要集落刺激活性。它的 C-FMS受体在成熟的破骨细胞上的表达比骨骼中的任何其他细胞都高。我们发现 在破骨细胞中删除c-fms可减弱对甲状旁腺素的合成代谢反应。这表明甲状旁腺素的部分合成代谢 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，诱导促进骨形成的同化破骨因子。有两种主要的亚型 CSF1：可溶性(SCSF1)和膜结合(MCSF1)。我们发现对甲状旁腺素的合成代谢反应 在仅表达sCSF1亚型的动物中由于成骨细胞的较大增加而增强 在这些小鼠中与PTH处理的对照组进行比较。与之形成鲜明对比的是，动物只表达 MCSF1对合成代谢甲状旁腺素方案没有增加骨量。重要的是，sCSF1和 MCSF1在激活CSF1受体c-fms的动力学和程度上有所不同，这表明他们的 体内作用的不同可能部分是由于细胞信号的内在差异。根据这些数据，我们 假设mCSF1通过对抗sCSF1对破骨细胞的作用而抑制PTH合成代谢。什么时候 非相反，sCSF1通过诱导合成的碎裂因子的产生而促进PTH的合成代谢。 特别是，每天单次剂量的甲状旁腺素诱导成骨细胞中sCSF1的一过性增加，从而导致 一连串合成代谢的碎裂因子产生。与此一致，我们发现sCSF1刺激了 破骨细胞中合成的破骨细胞因子-1-磷酸鞘氨醇(S-1-P)。为了检验这些假设，我们将在 SA1，确定在合成代谢甲状旁腺激素治疗方案中加入间歇剂量的sCSF1是否会增加 野生型动物对甲状旁腺素的骨骼反应，同时在该方案中加入mCSF1可减弱 回应。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，证明对甲状旁腺素的不同反应 SCSF1-/-和mCSF1-/-小鼠是由于两种CSF1亚型在骨骼中的不同作用直接造成的。在SA 2中，我们 将用PTH加sCSF1治疗5个月前切除卵巢的野生型小鼠，以确定是否 它将骨量恢复到去卵巢前的水平，作为绝经后的治疗模式 骨质疏松。在SA 3中，我们将确定sCSF1和mCSF1是否诱导产生不同的clastokine 成熟破骨细胞的特征。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。 骨信号转导与甲状旁腺素作用下破骨细胞转录组的表达 仅使用sCSF1或仅使用mCSF1来识别产生的碎裂因子类型的差异。我们还将确定 在这些不同的碎裂因子谱刺激的共培养中，成骨细胞转录本的差异。 最后，我们将研究sCSF1和mCSF1与c-fms的结合动力学，以及下游信号转导的差异， 从分子水平深入了解这两种异构体在体内和体外的不同作用。