Quantitative aspects of the effects of insulin, epidermal growth factor and dexamethasone on DNA synthesis in cultured adult rat hepatocytes

Abstract

Abstract. Epidermal growth factor (EGF) and insulin in combination have previously been shown to initiate S-phase in primary cultures of adult rat hepatocytes. We here describe the detailed time course and dosedependency of the effects of EGF and insulin on DNA synthesis in cultured hepatocytes. The DNA synthesis was assessed either biochemically or autoradiographically with a fairly good correlation between the two methods. DNA synthesis started 24–30 h after plating of the cells and peaked at approximately 70 h. Up to 70% of the cells entered DNA synthesis during this period. EGF and insulin acted synergistically on the DNA synthesis. Dexamethasone raised the DNA synthesis slightly, maximal effect occurred at concentrations above 2.5 nm and this agent was routinely used in the experiments with EGF and insulin. In the presence of 0.4 μm insulin from the time of plating, EGF dose-dependently increased the DNA synthesis with maximal effect at 5–15 nm. When added in combination with 1.7 nm EGF, insulin enhanced the DNA synthesis over the concentration range from 0.1 to 3 nm. These studies show that primary cultures of hepatocytes are useful in assessing the quantitative aspects of the interactions between the growth stimulating effects of hormones.

Inhibitory Effects of Dexamethasone on Hepatocyte Growth Factor–Induced DNA Synthesis and Proliferation in Primary Cultures of Adult Rat Hepatocytes

“…This demonstrates that high-dose dexamethasone (10 −8 M) inhibits the HGF-induced hepatocyte DNA synthesis and proliferation downstream of ERK2. The present results support the notion that dexamethasone (10 −8 M) suppresses HGF-induced DNA synthesis and proliferation (i.e., cell cycle progression) at the nuclear level (27). We also found that dexamethasone dose-dependently inhibited EGF-and PDGF-induced hepatocyte DNA synthesis and proliferation (accepted for publication).…”

“…Glucocorticoids such as dexamethasone and hydrocortisone have been reported to affect cell attachment and viability, but not proliferation (5,15). However, the concentrations (about 10 −9 -10 −5 M) and timing of glucocorticoid addition to cultures has varied among investigators (5,6,27,28). Especially, these doses are higher compared with our experimental condition.…”

Dexamethasone modulates ErbB tyrosine kinase expression and signaling through multiple and redundant mechanisms in cultured rat hepatocytes

“…To determine whether dexamethasone altered the ErbB expression profile, we cultured cells in the presence of 10 Ϫ8 or 10 Ϫ6 M dexamethasone for 72 h. We cultured the cells in both insulin (100 nM) and a submitogenic dose of TGF-␣ (0.1 nM) to enhance cell viability. Insulin was also added because it is required to preserve maximal TGF-␣ responsiveness of hepatocytes after 24 h of culture (30). Lysates were immunoblotted for the four ErbB proteins at 24, 48, and 72 h. Confirming our previous experience, no ErbB4 was detected.…”

“…Given the temporal changes of ErbB protein expression in vitro, we further speculated that the overall influence of dexamethasone on the hepatocellular response to EGF-type ligands depends on the time of growth factor addition. Our analysis of prior work suggested that the stimulatory effects of dexamethasone occurred when EGF and dexamethasone were added simultaneously at the beginning of the culture period (30). In contrast, the inhibitory action occurred when isolated hepatocytes were plated in a high concentration of dexamethasone for at least 24 h before EGF exposure (35).…”

“…Likewise, dexamethasone has been reported to inhibit the proliferation of rat hepatocytes (34,35) and liver-derived cell lines (24). In primary hepatocyte culture, however, other investigators have reported that dexamethasone enhances epidermal growth factor (EGF)-stimulated DNA synthesis (26,30). We hypothesized that these divergent observations result from differential effects of dexamethasone on one or more components of EGF receptor signaling.…”