Mechanisms for GH action on epithelial cells

项目摘要 累积的受损DNA，如果不修复，可能会破坏组织的稳态，使环境有利于 组织变性、癌症和代谢功能障碍。DNA损伤反应和修复途径是 随着衰老而减弱，也可能导致细胞衰老，具有衰老相关的分泌表型 （SASP）促进邻近细胞中的DNA损伤和细胞增殖，或促进细胞凋亡。非垂体生长 在外周组织中局部合成的一种激素（npGH）被广泛表达的GH受体识别 (GHR)，并通过自分泌/旁分泌机制发挥作用。我们的初步结果表明，npGH是诱导 通过老年人结肠组织中的DNA损伤，在诱导多能干细胞（iPSC）衍生的人结肠3D中， 在缺乏内分泌GH的垂体切除大鼠结肠和衰老结肠细胞中的类器官。反过来，npGH 抑制DNA修复，进一步加强DNA损伤并导致染色体不稳定。我们提出 支持局部上皮生长激素作为年龄相关DNA的不利决定因素的新假说 损伤累积和作为上皮老化的标志。 我们将评估老年结肠类器官中的npGH诱导和DNA损伤， 微流体装置我们将研究npGH表达的人结肠组织标本来自年龄- 确定的队列，评估与表达npGH的细胞结合的空间基因组谱，并阐明 npGH是染色体不稳定的原因。我们将探讨npGH调节DNA的信号机制 损伤反应活性、衰老途径和DNA损伤修复。体外上皮GHR信号传导将 用GHR拮抗剂pegvisomant、JAK 2抑制剂和二甲双胍阻断。我们将评估NPGH的行动 在来自结肠特异性和通用GHR敲除小鼠的衰老组织中， 转基因小鼠，并评估npGH旁分泌对结肠上皮细胞体细胞拷贝数改变的影响 （sCNA）和受sCNA影响的相应基因。我们将研究npGH是否在衰老细胞中表达， 是否影响邻近细胞DNA损伤和衰老。我们将测试旁分泌npGH对衰老的影响 在与人结肠成纤维细胞npGH转染子共培养的结肠肠芯片装置中，并将证实 在体外导致过表达GH的转基因模型或GHR敲除小鼠。 我们主要关注于经验证的人类结肠上皮模型，但本提案中的实验设计 阐明局部npGH增强上皮年龄相关DNA损伤的普遍机制 导致组织变性和/或瘤形成。

PROJECT SUMMARY Accumulated damaged DNA, if left unrepaired, may disrupt tissue homeostasis, enabling a milieu favoring tissue degeneration, cancer, and metabolic dysfunction. DNA damage response and repair pathways are attenuated with aging and may also result in cell senescence, with senescence-associated secretory phenotype (SASP) promoting DNA damage and cell proliferation in neighboring cells, or in apoptosis. Non-pituitary growth hormone (npGH), synthesized locally in peripheral tissues, is recognized by the widely expressed GH receptor (GHR), and acts through autocrine/paracrine mechanisms. Our preliminary results show that npGH is induced by DNA damage in aged human colon tissues, in induced pluripotent stem cell (iPSC)-derived human colon 3D organoids, in hypophysectomized rat colon lacking endocrine GH, and in senescent colon cells. In turn, npGH suppresses DNA repair, further reinforcing DNA damage and resulting in chromosomal instability. We propose a novel hypothesis supporting the role of local epithelial GH as an adverse determinant of age-associated DNA damage accumulation and as a marker of epithelial aging. We will assess npGH induction and DNA damage in aged colon organoids, and Colon Intestine-Chip microfluidic devices. We will examine npGH expression in human colon tissue specimens derived from age- determined cohorts, assess spatial genomic profiles in conjunction with cells expressing npGH, and elucidate npGH as a cause of chromosomal instability. We will explore signaling mechanisms for npGH regulation of DNA damage response activity, senescence pathways, and DNA damage repair. Epithelial GHR signaling in vitro will be blocked with the GHR antagonist pegvisomant, a JAK2 inhibitor, and metformin. We will assess npGH actions in aged tissues derived from colon-specific and universal GHR knockout mice, and in GH overexpressing transgenic mice, and also assess npGH paracrine effects on colon epithelial somatic copy number alteration (sCNA) and respective genes affected by sCNA. We will study whether npGH is expressed in senescent cells, and whether it affects adjacent cell DNA damage and senescence. We will test paracrine npGH effects on senescence in Colon Intestine-Chip devices co-cultured with human colon fibroblast npGH transfectants, and will confirm in vitro results in transgenic models overexpressing GH or in GHR knockout mice. We largely focus on validated human colon epithelial models, yet experiments in this proposal are designed to elucidate a universal mechanism whereby local npGH reinforces epithelial age-associated DNA damage leading to tissue degeneration and or neoplasia.