Endothelial nitric oxide synthase is a key mediator of hepatocyte proliferation in response to partial hepatectomy in mice

Abstract

Endothelial nitric oxide synthase (eNOS) is a critical modulator of vascular tone and blood flow and plays major roles in liver physiology and pathophysiology. Nitric oxide is widely recognized as one of the key humoral factors important for the initiation of liver regeneration in response to partial hepatectomy. Liver regeneration in response to partial hepatectomy is dependent on the efficiency of growth factor-mediated cell cycle progression. Epidermal growth factor receptor (EGFR) is a critical mediator of multiple hepatic mitogens such as epidermal growth factor, transforming growth factor-α, amphiregulin, and heparin-binding EGF in regenerating livers. However, functional significance of eNOS expressed in hepatocytes and its potential role in EGFR-mediated hepatocyte proliferation remains unexplored. We sought to determine if eNOS is essential for hepatocyte proliferation in response to partial hepatectomy (PH). Our studies with eNOS knockout (eNOS−/−) mice suggest that eNOS activation is essential for efficient induction of early events and elicitation of a robust hepatocyte proliferative response to PH. Moreover, eNOS expression is essential for efficient early induction of matrix metalloprotease-9 (MMP-9), a known mediator of extracellular matrix remodeling and growth factor activation in regenerating livers. Our in vitro studies suggest that eNOS is a critical mediator of EGF-induced hepatocyte proliferation, potentially via its influence on induction of early growth response (Egr-1) and phosphorylation of c-Jun—known mediators of cell cycle progression. EGF-induced eNOS phosphorylation at Ser 1177 is dependent on the phosphorylation and activation of EGFR-PI3K-AKT signaling in hepatocytes. Collectively, these results highlight a hitherto unrecognized role for eNOS activation in hepatocyte proliferation with implications for targeted therapies to enhance liver regenerative response in chronic disorders.

P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy

“…Briefly, the left lateral and median lobes were individually ligated and excised, and the right lateral and caudate lobes (remnant livers) were harvested at various time points (5 min to 8 days). To assess hepatocyte proliferation in response to PH, mice subjected to hepatectomy were injected with BrdU (50 mg/kg) 2 h prior to the harvest of liver tissue and the liver sections were immunostained for BrdU incorporation (44).…”

“…Hepatocytes were isolated from 8-to 12-wk-old WT and P2Y2Ϫ/Ϫ male mice by the two-step collagenase perfusion protocol as described previously, with modifications optimized for mice (7,44,67). Hepatocyte preparations with viability over 95%, as screened by Trypan blue exclusion assay, were seeded at a low density of 200,000 cells/35 mm on Primaria tissue culture wells (Becton Dickinson Labware, Franklin Lakes, NJ) or collagen-coated glass coverslips in Williams E complete media with additives (10% fetal bovine serum, 2 mM glutamine, 400 ng/ml dexamethasone, 2.5 g/ml insulin, 4 ng/ml glucagon, 2.5 g/ml transferrin, 2.5 ng/ml sodium selenite, 10,000 U/ml penicillin, 10,000 g/ml streptomycin, 50 g/ml gentamycin) for 3 h to ensure hepatocyte adherence to plates.…”

“…Hepatocyte preparations with viability over 95%, as screened by Trypan blue exclusion assay, were seeded at a low density of 200,000 cells/35 mm on Primaria tissue culture wells (Becton Dickinson Labware, Franklin Lakes, NJ) or collagen-coated glass coverslips in Williams E complete media with additives (10% fetal bovine serum, 2 mM glutamine, 400 ng/ml dexamethasone, 2.5 g/ml insulin, 4 ng/ml glucagon, 2.5 g/ml transferrin, 2.5 ng/ml sodium selenite, 10,000 U/ml penicillin, 10,000 g/ml streptomycin, 50 g/ml gentamycin) for 3 h to ensure hepatocyte adherence to plates. Subsequently, hepatocytes were maintained in Williams E minimal media free from serum and growth factors for 24 h prior to treatment with ATP or ATP␥S (7,44,67).…”

“…Briefly, liver tissues were gently homogenized in lysis buffer (25 mM Tris buffer, pH 7.5 containing 0.5 mM EDTA, 1.5 mM MgCl 2, 420 mM NaCl, and protease inhibitors). Nuclear fractions were isolated by centrifugation at 4,000 rpm, and proteins were extracted with 25 mM Tris buffer, pH 7.5, containing 1 mM EDTA, 2 mM MgCl2, 50 mM KCl, 1 mM DTT, and protease inhibitors, by incubating at 4°C for 30 min and centrifuging at 14,000 rpm for 5 min (44). Equal amounts of total or nuclear proteins as determined by BCA protein assay (Pierce, Rockford, IL) were analyzed by Western blotting as described previously.…”

“…Equal amounts of total or nuclear proteins as determined by BCA protein assay (Pierce, Rockford, IL) were analyzed by Western blotting as described previously. Blots were probed with antibody specific for histone H3 (nuclear) or ␣-tubulin or ␤-actin (total protein) to ensure equal loading of proteins in each lane (44).…”