Structure and function of a dual antagonist of the human growth hormone and prolactin receptors with site-specific PEG conjugates.

Human growth hormone (hGH) is a pituitary-derived endocrine protein that regulates several critical postnatal physiologic processes including growth, organ development, and metabolism. Following adulthood, GH is also a regulator of multiple pathologies like fibrosis, cancer, and diabetes. Therefore, there is a significant pharmaceutical interest in developing antagonists of hGH action. Currently, there is a single FDA-approved antagonist of the hGH receptor (hGHR) prescribed for treating patients with acromegaly and discovered in our laboratory almost 3 decades ago. Here, we present the first data on the structure and function of a new set of protein antagonists with the full range of hGH actions-dual antagonists of hGH binding to the GHR as well as that of hGH binding to the prolactin receptor. We describe the site-specific PEG conjugation, purification, and subsequent characterization using MALDI-TOF, size-exclusion chromatography, thermostability, and biochemical activity in terms of ELISA-based binding affinities with GHR and prolactin receptor. Moreover, these novel hGHR antagonists display distinct antagonism of GH-induced GHR intracellular signaling in vitro and marked reduction in hepatic insulin-like growth factor 1 output in vivo. Lastly, we observed potent anticancer biological efficacies of these novel hGHR antagonists against human cancer cell lines. In conclusion, we propose that these new GHR antagonists have potential for development towards multiple clinical applications related to GH-associated pathologies.

Increased Fibrosis in White Adipose Tissue of Male and Female bGH Transgenic Mice Appears Independent of TGF-ß Action.

Fibrosis is a pathological state caused by excess deposition of extracellular matrix proteins in a tissue. Male bovine growth hormone (bGH) transgenic mice experience metabolic dysfunction with a marked decrease in lifespan and with increased fibrosis in several tissues including white adipose tissue (WAT), which is more pronounced in the subcutaneous (Sc) depot. The current study expanded on these initial findings to evaluate WAT fibrosis in female bGH mice and the role of transforming growth factor (TGF)-ß in the development of WAT fibrosis. Our findings established that female bGH mice, like males, experience a depot-dependent increase in WAT fibrosis, and bGH mice of both sexes have elevated circulating levels of several markers of collagen turnover. Using various methods, TGF-ß signaling was found unchanged or decreased-as opposed to an expected increase-despite the marked fibrosis in WAT of bGH mice. However, acute GH treatments in vivo, in vitro, or ex vivo did elicit a modest increase in TGF-ß signaling in some experimental systems. Finally, single nucleus RNA sequencing confirmed no perturbation in TGF-ß or its receptor gene expression in any WAT cell subpopulations of Sc bGH WAT; however, a striking increase in B lymphocyte infiltration in bGH WAT was observed. Overall, these data suggest that bGH WAT fibrosis is independent of the action of TGF-ß and reveals an intriguing shift in immune cells in bGH WAT that should be further explored considering the increasing importance of B cell-mediated WAT fibrosis and pathology.