Phorbol ester modulates interleukin 6- and interleukin 1-regulated expression of acute phase plasma proteins in hepatoma cells

Abstract

Interleukin 6 (IL 6) and interleukin 1 (IL-1) regulate the expression of acute phase plasma proteins in rat and human hepatoma cells. Phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), partially mimics the stimulatory effect of IL-6 but reduces that effect of IL-1. TPA and IL-6 act synergistically. These regulatory properties of TPA are also manifested in HepG2 cells transiently transfected with an indicator gene construct carrying the IL-1/IL-6 regulatory enhancer element of the rat alpha 1-acid glycoprotein gene. IL-6 and IL-1 act independently of TPA-inducible kinase C, and of changes in intracellular Ca2+ concentrations. However, prolonged pretreatment of HepG2 cells with TPA results in a drastically reduced cytokine response that is proportional to the loss of cell surface binding activity for the cytokine. These data suggest that hormones activating protein kinase C probably play a contributing role in stimulating the expression of acute phase plasma protein genes but they may be crucial in controlling the responsiveness of liver cells to inflammatory cytokines during subsequent stages of the hepatic acute phase reaction.

Fig. 1. Effect of TPA and IL-6 on H-35

H-35 cells were treated for 8 h with serum-free MEM containing 1 μM dexamethasone alone or with 0.1 μM TPA and/or 250 units/ml IL-6. Aliquots of cellular RNA (15 μg) were analyzed by Northern blot hybridization for the levels of α-fibrinogen (α-FB), γ-fibrinogen (γ-FB), thiostatin (TST), and AGP. The autoradiograms were exposed for 24 h. The ethidium bromide (EtBr)-stained RNA pattern of the α-fibrinogen analysis is pictured to demonstrate equal loading of RNA.

Fig. 2. Effect of TPA and IL-6 on HepG2 cells

HepG2 cells were treated for 8 h with MEM containing 0.1 μM dexamethasone alone or with 0.15 μM TPA and/or 100 units/ml IL-6. Cellular RNA (15 μg) were analyzed by Northern blot hybridization for haptoglobin (HP), AGP, and α₁-antichymotrypsin (ACH) mRNA. Autoradiogram of the mature RNA bands are shown after 3-day (HP, AGP) and 1-day (ACH) exposures.

Fig. 3. Regulated expression of a rat AGP-CAT gene construct in HepG2 cells

HepG2 cells in 6-well cluster plates were transfected with plasmid DNA mixture (18 μg of pAGP(3xDRE)-140-CAT and 1 μg of pIE-MUP/ml). After a recovery period of 24 h and culture in serum-free MEM for 16 h, the cells were treated for 8 h with MEM containing the indicated factors (0.15 μM TPA, 100 units/ml IL-6, 250 units/ml IL-1α; 0.1 μM dexamethasone (Dex)). The thin layer chromatography pattern of the chloramphenicol acetyltransferase (CAT) activity in 10 μl of cell extract after 20 h autoradiography is reproduced. The rocket immunoelectrophoresis of the MUP secreted by the same cells during the final 24 h period is shown at the top. The specific chloramphenicol acetyltransferase activity was determined by using 0.3–30 μl of the cell extract and the values were related to the amount of MUP produced.

Fig. 4. Gel electrophoretic pattern and activity of ¹²⁵I-IL-6

In A, an aliquot of IL-6 labeled with the ¹²⁵I-Bolton Hunger reagent (65,000 cpm) was separated together with molecular weight standard (Std) on an 11% sodium dodecyl sulfate polyacrylamide gel. The fluorogram shown was exposed for 3 h. (The ¹²⁵I-IL-6 protein band at M_r 24,000 yielded 59,800 cpm.) In B, medium (MEM, 1% fetal calf serum, 1 μM dexamethasone) containing 10, 1, or 0.1 ng/ml of unlabeled IL-6 or ¹²⁵I-IL-6 (lanes 1, 2, and 3, respectively), was tested on HepG2 cells. The stimulated production of haptoglobin (HP) and fibrinogen (FB) was measured by rocket Immunoelectrophoresis (20). Control represents HepG2 cells treated with medium containing the buffer used for chromatography of ¹²⁵I-IL-6 (50 mM sodium phosphate, pH 7.5, 0.25% gelatin; Ref. 41). The amount of buffer added was the same as used in the experimental assays.

Fig. 5. Binding of IL-1 and IL-6 to HepG2 cells

Confluent monolayers of HepG2 cells were pretreated for 24 h with serum-free MEM alone (○), or with MEM containing either 0.1 μM dexamethasone (△), 0.15 μM TPA (●), or dexamethasone and TPA (▲). The cells were washed once with binding buffer and then incubated for 4 h at 4 °C with increasing concentrations of ¹²⁵I IL-1 or ¹²⁵I-IL-6. The binding of label to the 4 cm² (IL-1) or 10 cm² (IL-6) monolayer cultures was expressed according to Scatchard (55). The mean values of duplicate cultures are shown.