Growth Hormone Signaling Pathway Leading to the Induction of DNA Synthesis and Proliferation in Primary Cultured Hepatocytes of Adult Rats

Abstract

Background: We investigated the signal transduction pathway associated with growth hormone (GH)-stimulated DNA synthesis and proliferation in primary cultured hepatocytes. Methods: Adult rat hepatocytes were isolated from normal livers by two-step in situ collagenase perfusion to facilitate disaggregation of the adult rat liver. Then hepatocytes were cultured in serum-free Williams’ medium E supplemented with GH (1-100 ng/ml) in the presence or absence of test reagents. GH-induced hepatocyte DNA synthesis and proliferation were determined, and the phosphorylation activities of Janus kinase (JAK) 2 (JAK2) (p125 kDa), p95-kDa RTK, and ERK1/2 were measured by western blotting. Results: Hepatocytes grown in serum-free defined medium proliferated within 5 h of culture in the presence of GH (100 ng/ml) in a concentration- and time-dependent manner (EC50 75 ng/ml). These proliferative effects of GH were almost completely blocked by an anti-GH receptor monoclonal antibody (85 ng/ml) and an anti-insulin-like growth factor (IGF)-I receptor monoclonal antibody. In addition, the proliferative effects of GH were significantly blocked by a JAK2 inhibitor (TG101209, 10−6 M), as well as specific signal-transducing inhibitors of phospholipase C (PLC; U-73122, 10−6 M), RTK (AG538, 10−6 M), phosphoinositide 3-kinase (PI3K; LY294002, 10−6 M), mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK; PD98059, 10−6 M), and mammalian target of rapamycin (mTOR; rapamycin, 10 ng/ml). GH significantly induced the phosphorylations of JAK2 (p125 kDa), p95-kDa IGF-I receptor tyrosine kinase (RTK), and ERK2 in this order according to western blotting analysis. Conclusions: The proliferative action of GH is mediated by two main signaling pathways. One includes activation of the GH receptor/JAK2/PLC/Ca2+ pathway, and the other involves activation of the p95-kDa IGF-I RTK/PI3K/ERK2/mTOR pathway in primary cultures of adult rat hepatocytes.

Overactivation of hepatic mechanistic target of rapamycin kinase complex 1 (mTORC1) is associated with low transcriptional activity of transcription factor EB and lysosomal dysfunction in dairy cows with clinical ketosis

“…Previous studies and our results show that dairy cows with ketosis have higher blood concentrations of TNF-α, IL-18, and GH (Song et al, 2016;El-Deeb and El-Bahr, 2017;Du et al, 2018), which could activate mTORC1 in a different way (Zhang et al, 2008;El-Darawish et al, 2018;Kimura et al, 2021). In addition, activity of AMP-activated protein kinase, one of the upstream targets of and a negative regulator of mTORC1, was decreased in calf hepatocytes treated with BHB and in mouse hepatocytes treated with FFA (Deng et al, 2016;Xu et al, 2021a).…”

Role of Hepatocyte Growth Regulators in Liver Regeneration

“…GH promotes the autocrine secretion of IGF-I from the hepatocytes via the GH receptor/JAK2/PLC/Ca 2+ pathway. We then found that the secreted IGF-I directly promotes DNA synthesis and hepatocyte proliferation (Figure 5) [15,16,23].…”

“…We also found that there are many factors that promote hepatocyte proliferation, but there are few factors that can strongly inhibit the effects of these hepatocyte growthpromoting factors (Table 1). Recently, we were also able to confirm the hepatocyte proliferation-promoting effects of 5-HT and growth hormone (GH) in culture [14][15][16][17][18]. Therefore, we investigated the associated intracellular signaling pathways and found that these regulatory factors are indirect mitogens that act via the secretion of autocrine factors (transforming growth factor-α (TGF-α), insulin-like growth factor-I (IGF-I), etc.).…”

Differential Inhibite Effect of Xanthohumol on HepG2 Cells and Primary Hepatocytes

“…Rats were anesthetized by intraperitoneal injection of 45 mg/kg sodium pentobarbital. Primary hepatocytes were isolated and enriched by a two-step in situ Seglen procedure 10 , 11 with minor modifications as previously described. 12 Fresh hepatocytes (5.0 × 10 4 cells/cm 2 ) were placed onto 12.5 cm 2 cell culture flasks coated with collagen type II and incubated in Williams’ medium E containing 10% fetal bovine serum.…”