Proliferation Of Cultured Hepatocytes Research Articles

Evidence That Cyclin D1 Mediates Both Growth and Proliferation Downstream of TOR in Hepatocytes

Signaling through the target of rapamycin is required for increased protein synthesis, cell growth, and proliferation in response to growth factors. However, the downstream mediators of these responses, and the elements linking growth and proliferation, have not been fully elucidated. Rapamycin inhibits hepatocyte proliferation in culture and liver regeneration in vivo. In cultured rat hepatocytes, rapamycin prevented the up-regulation of cyclin D1 as well as proteins acting downstream in the cell cycle. Transfection with cyclin D1 or E2F2, but not cyclin E or activated Akt, overcame the rapamycin-mediated cell cycle arrest. Rapamycin also inhibited the induction of global protein synthesis after growth factor stimulation, and cyclin D1 overcame this inhibition. Rapamycin inhibited hepatocyte proliferation and cyclin D1 expression in the mouse liver after 70% partial hepatectomy. In rapamycin-treated mice, transfection with cyclin D1 induced hepatocyte proliferation, increased hepatocyte cell size, and promoted growth of the liver. These results suggest that cyclin D1 is a key mediator of increased protein synthesis, cell growth, and proliferation downstream of target of rapamycin in mitogen-stimulated hepatocytes.

Rat stellate cells regulate proliferation of rat hepatocytes in culture

This study was aimed to reveal the stellate cell derived factors that regulate hepatocyte proliferation in culture. Rat hepatocytes and stellate cells were cultured in serum free Williams-E medium. We used hepatocyte monoculture and two different co-cultures of hepatocytes and stellate cells ; 1) co-culture on the same surface (Co-mix.) and 2) co-culture without contact between hepatocytes and stellate cells using a culture insert (Co-sep.) . Changes in the number and the DNA synthesis of hepatocytes was estimated. Although the number of hepatocyte decreased to 76% of the original at 48 h after starting mono-culture, it was kept at 106% in Co-mix and increased to 135% in Co-sep. The hepatocyte DNA synthesis was enhanced by carbenoxolone, gap junction blocker, in Co-mixo and reduced by NK 1 in each coculture. PD 153035, an EGF receptor specific inhibitor, had no effect. HeparitinaseI (20 mU/ml) and sodium chrolate (25 mM) reduced the hepatocyte DNA synthesis in Co-sepo to 71%. Activation of MAPK was induced in hepatocytes stimulated by conditioned mediums. These results indicated that hepatocyte proliferation was stimulated in the presence of stellate cells through HGF, extracellular heparan sulfate, and heparan sulfate proteoglycan, and might be negatively regulated by gap junction dependent mechanism.

Differential regulation of hepatocyte DNA synthesis by cAMP in vitro in vivo.

Adenosine 3',5'-cyclic monophosphate (cAMP) prevents epidermal growth factor (EGF)-induced DNA synthesis in many types of cultured cells, including hepatocytes, but its effects on cellular proliferation in vivo are unknown. This study compares the effects of supplemental cAMP on hepatocyte proliferation induced in vivo by 70% partial hepatectomy (PH) and in vitro by EGF and determines the effects of cAMP on AP-1, a family of growth-regulatory transcription factors, and the kinase cascades that normally activate AP-1. Although injection of dibutyryladenosine 3',5'-cyclic monophosphate (30 mg/kgip) at the time of PH increased liver cAMP concentrations at least 100-fold for several hours, it did not inhibit hepatic incorporation of [3H]thymidine or proliferating cell nuclear antigen expression 24 h after PH. cAMP treatment led to a complete inhibition of extracellular signal-related kinase (ERK) activity and transiently reduced NH2-terminal Jun nuclear kinase (JNK) activity after PH but did not decrease the expression of c-jun mRNA or protein. Consistent with the known cAMP stimulation of jun-B in cultured cells, cAMP treatment increased jun-B mRNA, protein, and DNA binding activity post-PH. Surprisingly, cAMP treatment enhanced Raf kinase activity after PH in rats. In primary hepatocyte cultures, supplemental cAMP inhibited JNK and ERK activity, total AP-1 and c-Jun transcriptional activities, and DNA synthesis. Thus elevated cAMP inhibited ERK and JNK activity in culture and in vivo and inhibited hepatocyte proliferation in culture but not in vivo. This suggests that in vivo mechanisms compensate for cAMP inhibition of certain growth-related signaling cascades and emphasizes potential risks of extrapolating from simple cell culture systems to explain physiology in intact animals.

Effect of iron and hydroxypyridin-4-ones on rat hepatocyte proliferation in culture

Effect of iron and hydroxypyridin-4-ones on rat hepatocyte proliferation in culture

Fetal Growth Factors as Determinants of Intrauterine Hepatic Growth

To understand mechanisms at the cellular level that may lead to the selective organomegaly seen in fetuses of diabetic mothers, we examined the role of insulin and autocrine-paracrine growth factors in the regulation of hepatic growth in the fetal rat. Analyses of fetal liver from the last one-third of gestation demonstrated the presence of specific mRNAs for the transforming growth factors (TGFs) TGF-alpha and TGF-beta. TGF-alpha, a homologue of epidermal growth factor (EGF), acts through EGF receptors. Levels of mRNA for TGF-alpha increased dramatically postnatally, whereas EGF receptor number increased just before term. In contrast, levels of mRNA for TGF-beta, an inhibitor of epithelial cell growth, were greater in fetal liver than in adult liver, as was TGF-beta-receptor binding. Other analyses demonstrated increases in tyrosine kinase activities of the insulin receptor, EGF receptor, and insulinlike growth factor I receptor as term approached. Proliferation of fetal rat hepatocytes in primary culture did not require mitogens or serum, consistent with production and activity of autocrine-paracrine growth factors. TGF-beta was a potent inhibitor of fetal hepatocyte proliferation in culture, whereas insulin potentiated fetal hepatocyte growth above "mitogen-independent" levels. The regulatory mechanisms controlling fetal hepatic growth involve a complex interaction between stimulatory and inhibitory factors. Growth factor expression, receptor expression, receptor tyrosine kinase activity, and postreceptor signal transmission represent potential loci for insulin action that might be involved in the pathogenesis of fetal macrosomia seen in diabetic pregnancies.

L-Proline is an essential amino acid for hepatocyte growth in culture

For improvement of the culture conditions of adult rat hepatocytes in primary culture in collagen coated dishes, effects of various commercial culture media on the induction of replicative DNA synthesis of the cells stimulated by insulin plus epidermal growth factor were studied. Proline-deficient media, such as Leibovitz's L-15, Eagle's minimal essential medium and Dulbecco's modified minimal essential medium, did not induce DNA synthesis in hepatocytes, whereas proline-rich media, such as Williams medium E, McCoy's 5A and Ham's F-12, induced markedly hepatocyte proliferation. Moreover, when the proline-deficient media were supplemented with L-proline, the cells synthesized DNA in response to the two hormones. Cis-4-hydroxyl-L-proline strongly inhibited the induction of DNA synthesis, without affecting protein synthesis of the cells or showing any cytotoxicity. This inhibition was recovered completely by adding excess proline to the medium. Addition of other amino acids not present in the medium did not promote DNA synthesis. These findings indicate that L-proline is essential for induction of hepatocyte proliferation in culture, through its affect on synthesis of intracellular collagen.