Growth Hormone (GH), also known as somatotrophin, is of cardinal importance in the regulation of somatic growth. Decreased GH expression results in short stature, whereas increased levels of GH lead to disorders such as gigantism or acromegaly. Mutations in the transcription factor Pit-1 have been shown to decrease GH, as well as prolactin (PRL; Parks et al., JCEM, 1999). However, understanding the regulation by selective GH transcription regulators, such as Zn16, a protein encoding 16 zinc fingers that binds to the GH promotor DNA (Wojtkiewicz et al., Endocrine, 2002), will require comparison of currently available rodent cell lines that express GH. Two current rat somatotroph cell lines are the somatotroph MtT/S and lactosomatotroph GH3 cell lines, both of which have been implemented in studies on the regulation of GH expression (Schaaf et al., Endocr Relat Cancer, 2009). MtT/S cells almost exclusively express GH, whereas GH3 cells are less differentiated and co-express PRL as well as GH. GH3 cells have been available longer, and thus are more frequently used for in vitro GH experiments, but there may be some increased utility in utilizing MtT/S cells. MtT/S cells were procured from Riken Cell Bank in Japan, and GH3 cells were acquired from the ATCC. These lines were cultured, then secretion of GH and PRL was examined after Insulin-like Growth Factor (IGF-1) treatment, which is inhibitory to GH and PRL secretion. Further, both cell lines were treated with stimulatory factors GH releasing hormone (GHRH); retinoic acid; cortisone; GH Releasing Peptide-6; and both cortisone and GHRH. GH secretion was on average higher in MtT/S cells than in GH3 cells. On the other hand, PRL secretion was extremely lower in MtT/S cells than in GH3 cells. This result confirms that the MtT/S cells are further differentiated as somatotrophs than GH3 cells. Although hormone release in response to treatment did not appear different, the overall difference in GH vs. PRL secretion may be useful for evaluating the role of Zn16 in selective control of the GH promoter. Therefore, mRNA levels of GH, PRL, Pit-1 and Zn16 in these cell lines are currently being measured using quantitative real-time PCR with ribosomal protein L19 (RPL19) expression as a standard. With the investigation of the characteristics of these differentiated pituitary cell types, we hope to advance the knowledge of GH transcription and regulation, particularly the study of Zn16 effects during pituitary development. Recently it was observed that Zn16 may have a role in a tumor suppressor gene that undergoes mutation leading to a form of colorectal cancer and childhood leukemia. This augments the utility of determining the proper cell lines to examine Zn16, and its role in tumor suppression and cell division as well as gene expression during hypophyseal development.

The ability of exogenous growth hormone (GH) to increase milk yield through insulin-like growth factor-1 (IGF-I) in dairy cows is well characterized. However, recent studies utilizing mammary epithelial cell lines indicate a direct effect of GH on mammary epithelial cells (MEC). To test if these observations are relevant in vivo and if this response differs between dairy breeds, three mammary models were utilized. Mammary explants from a lactating Jersey cow were cultured in classical lactogenic media (dexamethasone, insulin, and prolactin) with 0 or 10 ng/mL of recombinant bovine GH for 12h. Primary MEC from lactating Holstein and Jersey cows were cultured in classical lactation media with 0 or 10 ng/mL of GH for 2, 4, and 7 days. And lastly, MEC isolated from pooled Holstein or pooled Jersey milk were cultured in the same conditions as primary MEC. The response to GH was quantified by the relative abundance of mRNA for two milk protein genes (α-lactalbumin and αS1-casein), the GH receptor, IGF-I and insulin-like growth factor binding protein-3 (IGFBP-3) as determined by quantitative RT-PCR. The abundance of α-lactalbumin mRNA in explants was increased in response to GH. After 2 days, Jersey primary MEC showed an increase in GH receptor mRNA, in addition to a noteworthy trend of increasing abundance of IGFBP-3 regardless of GH treatment. After 4 days, Holstein primary cells cultured with GH had decreased IGFBP-3 mRNA. After 7 days, primary cells isolated from Holstein and Jersey mammary tissue showed a slight response to GH. Mammary cells from milk mirrored the responses to GH observed in primary cells: MEC isolated from Holsteins had decreased IGFBP-3 mRNA after 4 days of treatment with GH and MEC isolated from Jerseys showed the same trend of increasing IGFBP-3 abundance between 2 and 4 days, but with no difference between GH treatments. These results indicate that the effect of GH may differ between breeds and indicate GH has a direct effect on mammary epithelial cells, possibly including effects on the abundance of IGFBP-3 mRNA.

Acetaminophen overdose is the leading cause of acute liver failure, with one available treatment, NAC. Yet, NAC effectiveness diminishes about ten hours after APAP overdose, urging for therapeutic alternatives. Promoting intrinsic liver regeneration could address this unmet need by temporally activating key hepatocyte regenerative pathways in order to accelerate recovery from acute APAP-induced liver injury. In aim 1, we established an efficient, safe, non-integrative method to transiently express HGF and EGF in hepatocytes via nucleoside-modified mRNA-LNP delivery in mice. We confirmed specific hepatotropism of mRNA-LNP via intravenous injection of firefly luciferase encoding mRNA-LNP, with protein expression lasting about 3 days. In the liver, virtually all hepatocytes were transfected along with a subpopulation of endothelial and Kupffer cells. In homeostasis, HGF mRNA-LNP efficiently induced hepatocyte proliferation. In a chronic liver injury mouse model recapitulating nonalcoholic fatty liver disease, injections of both HGF and EGF mRNA-LNP sharply reversed steatosis and accelerated restoration of liver function. Likewise, HGF and EGF mRNA-LNP accelerated liver regeneration after APAP-induced acute liver injury with rapid return to baseline of levels of serum liver injury markers such as ALT. This study introduced mRNA-LNP as a potentially translatable safe therapeutic intervention to harness liver regeneration via controlled expression of endogenous mitogens in vivo. In aim 2, we characterized the sexually differential injury induced between males and females following APAP overdose. Consistent with the literature, our data showed higher resistance of female mice to APAP, shown by reduced liver necrosis, cell death, and detection of serum injury markers compared to males. Moreover, our single-cell RNA sequencing analyses revealed that female hepatocytes and endothelial cells express significantly higher levels of GH receptor and GH pathway activation than male cells, while males have upregulated inflammatory and cell death pathway activation. Therefore, in aim 3, we took advantage of the greater resistance of female mice and greater activation of the GH pathway to evaluate a novel treatment for APAP-induced acute liver injury utilizing growth hormone. Sex hormones and their receptors have been implicated in driving sexual dimorphism in many liver processes. Specifically, pituitary GH secretory patterns, pulsatile in males and near-continuous in females, determine the sex bias in many liver metabolic functions. In harnessing this female-specific advantage, we demonstrated that injection of recombinant human GH protein in APAP-injured mice accelerates liver recovery, promotes survival, and is superior to standard-of-care NAC. Delivery of slow-released human GH mRNA-LNP also rescues males from APAP-induced death that otherwise occurred in control mRNA-LNP-treated mice. In conclusion, this study demonstrates a sexually dimorphic liver repair advantage in females following APAP overdose, leveraged by establishing GH as an alternative treatment, delivered either as recombinant protein or mRNA-LNP, to potentially prevent liver failure and liver transplant in APAP-overdosed patients. / 2025-11-02T00:00:00Z

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