Transglutaminase differentially regulates growth signalling in rat perivenous and periportal hepatocytes

Abstract

The influence of transglutaminase 2 (TG2) activity on the proliferative effect of epidermal growth factor (EGF) and on EGF receptor affinity in periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) has been investigated using a primary culture system. PPH and PVH subpopulations have been isolated using the digitonin/collagenase perfusion technique. DNA synthesis was assessed by [3H] thymidine incorporation into hepatocytes. The assay for binding of [125I] EGF to cultured hepatocytes was analysed by Scatchard plot analysis. Pretreatment with the TG2 inhibitor monodansylcadaverine (MDC) greatly increased EGF-induced DNA synthesis in both PPH and PVH. Furthermore, [125I] EGF binding studies in PVH treated with MDC indicated that high-affinity EGF receptor expression was markedly up-regulated, whereas in PPH, there was no significant effect. Treatment with retinoic acid (RA), an inducer of TG2 expression, significantly decreased EGF-induced DNA synthesis in both PPH and PVH. Binding studies in the presence of RA revealed that the high-affinity EGF receptor was down-regulated and completely absent in both PPH and PVH. These results suggest that TG2 was involved in the differential growth capacities of PPH and PVH through down-regulation of high-affinity EGF receptors.

Figure 1

EGF‐induced DNA synthesis in primary cultured PPH and PVH. DNA synthesis induced by EGF (10⁻⁸ M) was measured by the [³H] thymidine incorporation method as described in the Materials and Methods section. [³H] thymidine was included from 0 to 24 h, 24 to 48 h, and 48 to 72 h. Each value represents the mean ± SEM of 3–4 samples. #P < 0.05, ##P < 0.01, versus respective EGF‐untreated group.

Figure 2

Effect of TG2 inhibitor (MDC) on DNA synthesis in primary cultured PPH and PVH. Hepatocytes were pretreated with 0.5 mm MDC for 30 min. DNA synthesis induced by EGF (10⁻⁸ M) was measured by the [³H] thymidine incorporation method as described in the Materials and Methods section. [³H] thymidine was included from 0 to 24 h, 24 to 48 h, and 48 to 72 h. Each value represents the mean ± SEM of 3–4 samples. #P < 0.05, ##P < 0.01, versus EGF alone group.

Figure 3

Effect of TG2 inducer (RA) on DNA synthesis in primary cultured PPH and PVH. DNA synthesis induced by EGF (10⁻⁸ M) and/or RA (5 µm) was measured by the [³H] thymidine incorporation method as described in the Materials and Methods section. [³H] thymidine was included from 0 to 24 h, 24 to 48 h, and 48 to 72 h. Each value represents the mean ± SEM of 3–4 samples. #P < 0.05, ##P < 0.01, versus EGF and RA treated group.

Figure 4

Scatchard analysis of EGF binding to primary cultured PPH and PVH. After attachment, hepatocytes were incubated for 24 h at 37 °C with serum‐free medium, and then incubated for 4 h at 4 °C with binding buffer containing various concentrations of [¹²⁵I] EGF. The inset panel shows saturation curves of [¹²⁵I] EGF‐specific binding to its receptor. (•) PPH; (○) PVH.

Figure 5

Effect of TG2 inhibitor (MDC) on Scatchard analysis of EGF binding to primary cultured PPH and PVH. After attachment, hepatocytes were incubated for 24 h at 37 °C with serum‐free medium, and were thereafter pretreated with 0.5 mm MDC for 30 min. Hepatocytes were then incubated for 4 h at 4 °C with binding buffer containing various concentrations of [¹²⁵I] EGF. The inset panel shows saturation curves of [¹²⁵I] EGF‐specific binding to its receptor. (•) PPH; (○) PVH.

Figure 6

Effect of TG2 inducer (RA) on Scatchard analysis of EGF binding to primary cultured PPH and PVH. After attachment, RA (5 µm) was added, and then hepatocytes were incubated for 24 h at 37 °C with serum‐free medium. Binding assays were then performed as described in the Materials and Methods section. The inset panel shows saturation curves of [¹²⁵I] EGF‐specific binding to its receptor. (•) PPH; (○) PVH.