Mechanisms for GH action on epithelial cells

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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.