Effect of Activins AB and B on DNA Synthesis Stimulated by Epidermal Growth Factor in Primary Cultured Rat Hepatocytes.

Abstract

Activins are members of the transforming growth factor-b superfamily and are homodimeric proteins composed of two inhibin/activin b-subunits linked by a disulfide bridge. Activin was originally identified as a stimulator of FSH (follicle-stimulating hormone) secretion. [6][7][8] Later, activins were shown to be expressed in various organs during development and in acquired conditions, and to have a wide variety of biological functions, including regulation of cell proliferation and differentiation, and induction of mesodermal tissues during amphibian development. 12,13) Recent studies have shown that activin A plays several roles in rat liver in vivo and in vitro. [13][14][15][16][17] It inhibits mitogeninduced DNA synthesis in vitro, 14) induces hepatocellular apoptosis in vivo and vitro, [15][16][17] and stimulates glycogenolysis in vitro.18) Activin C has been reported to have no effect on DNA synthesis by liver tumor cell line HepG2.11) However, there have been no reports on the actions of activin AB and B in rat liver either in vivo or in vitro. It, therefore, seemed meaningful to examine the effect of activins AB and B on the liver functions that have been shown to be regulated by activin A. In this regard, we were especially interested in mitogen-stimulated DNA synthesis as a model system for its biological significance, because expression of b A and b B mRNA in rat liver has been found to change markedly in rat models of liver regeneration. 14,19) In this paper, we examined the effect of activins AB and B on DNA synthesis stimulated by epidermal growth factor (EGF) in primary cultured rat hepatocytes and compared their effect with that of activin A. MATERIALS AND METHODS MaterialsThe materials used for cell isolation and cell culture were the same as reported by Tanaka et al. 20) Recombinant human (rh) activin A was kindly provided by Dr. Y. Eto (Ajinomoto Co., Inc., Japan). Porcine activin A, activin AB, and activin B were purchased from Wako (Osaka, Japan). EGF was obtained from Toyobo (Osaka). [3 H]thymidine (89.9 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA, U.S.A.).Cell Isolation and Monolayer Culture Parenchymal hepatocytes were isolated from adult male Wistar strain rats weighing 180-200 g by in situ perfusion of the liver with collagenase, 20) and they were suspended, 2ϫ10 5 cells/ml, in Williams' E medium containing 5% fetal bovine serum, 10 nM insulin, and 1 nM dexamethasone, and cultured at 0.2ϫ10 5 cells/cm 2 in a 48-well and 6-well microplate (IWAKI) precoated with collagen type-1 AC in a humidified chamber at 37°C under 5% CO 2 and 30% O 2 in air. Cells plated in the 48-well and 6-well plate were used to measure DNA synthesis and [ 125 I]rh activin A binding, respectively. After 2.5 h, the medium was replaced with serum-and hormone-free medium containing aprotinin (1 mg/ml). Measurement of [ 3 H]thymidine IncorporationAfter 15 h, the medium was replaced with hormone-free medium containing aprotinin (1 mg/ml) and 0.1% bovine serum albumin (BSA), and various hormones we...

Activins and follistatins: Emerging roles in liver physiology and cancer

“…Activin beta B mRNA was induced in stellate cells of CCl4 treated rat livers [47] and exposure to the peroxisome proliferator di-n-butyl phthalate led to a transient surge of beta B mRNA expression 6 h after treatment [69] . With respect to biological activities, recombinant activin B, in contrast to activins A and AB, did not inhibit EGF induced DNA synthesis in primary rat hepatocytes [70] . In contrast to the rat, beta A and beta B transcripts are expressed to similar levels in human liver (RodgarkiaDara, unpublished observation).…”

Activins and activin antagonists in hepatocellular carcinoma

“…Exposure to the peroxisome proliferator di-n-butyl phthalate led to a transient surge of βB mRNA expression 6 h after treatment in rat livers [67] . With respect to biological activities, recombinant activins A and AB but not activin B inhibited EGF induced DNA synthesis in primary rat hepatocytes [68] . In normal human liver the βB transcript is readily detectable by RT-PCR (M.G.…”

Expression of Annexin A3 in Primary Cultured Parenchymal Rat Hepatocytes and Inhibition of DNA Synthesis by Suppression of Annexin A3 Expression Using RNA Interference

“…The cells were then cultured for another 24 h. [ 3 H]thymidine incorporation was measured as described previously. 25) The difference between the radioactivity in the hot-trichloroacetic acid soluble fraction with and without aphidicolin was calculated as dpm/mg protein.…”