Regulation of Expression of the Complement Factor H Gene in a Murine Liver Cell Line by Interferon‐γ

Abstract

Factor H is a regulatory protein of the alternative pathway of complement activation that is synthesized mainly in the liver. The authors used the +/+ Li murine liver cell line as a model for examining its regulation. When +/+ Li cells were incubated with IFN-gamma, the levels of factor H mRNA increased in a dose-dependent manner, achieving a maximal response at a concentration of 50-100 units/ml. The increase in factor H mRNA levels was paralleled by an increase in factor H secretion. The kinetics of induction of factor H mRNA were slow, with the response reaching near maximal levels at 24 h. The increase in factor H mRNA by IFN-gamma was dependent on protein synthesis, as cycloheximide abolished the response. The presence of IFN-gamma was required for the entire incubation period in order to produce a maximal response. The luciferase system was used in an attempt to identify an interferon-responsive element. Luciferase constructs containing from 807 to 236 bp of upstream sequence responded to IFN-gamma with a twofold induction of luciferase activity, whereas a construct containing 83 bp of 5' sequence did not. Thus, IFN-gamma stimulates factor H mRNA transcription through a protein intermediary that interacts with the promoter between positions -83 and -236.

Constitutive Expression and Regulation of Rat Complement Factor H in Primary Cultures of Hepatocytes, Kupffer Cells, and Two Hepatoma Cell Lines

“…Although it had not been shown before in these cell types, the IFN-␥-mediated up-regulation that was observed here for HC and KC was not unexpected because this phenomenon had formerly been observed in HUVEC (Brooimans et al, 1989;Dauchel at al, 1990;Lappin et al, 1992;Ripoche et al, 1988b;Schlaf et al, 2001), in primary myoblasts and in two rhabdomyosarcoma cell lines (CRL 1558 and HTB 153) (Legoedec et al, 1995), in primary fibroblasts (Friese et al, 1999;Katz and Strunk, 1988;Lappin et al, 1992;Schwaeble et al, 1991), in fibroblast-like L cells (Munoz-Canoves et al, 1989), in monocytes (Lappin et al, 1992), in the murine liver cell line ϩ/ϩLi (Vik, 1996), and in the human Hep3b hepatoma cell line (Luo and Vik, 1999) and may therefore be a common feature. In the present study, the proinflammatory cytokines TNF-␣, IL-6, and IL-1␤ did not upregulate FH.…”

“…It has been claimed that complement FH also is mainly produced by this organ, although experimental evidence using primary cells of the liver has not yet been provided. Many hepatoma cells of human or murine origin such as Hep3b (Luo and Vik, 1999;Schwaeble et al, 1991), HepG2 (Lappin at al, 1992;Schwaeble et al, 1991), HepG4 (Schwaeble et al, 1991), H4 (Schwaeble et al, 1991), HUH7 (Friese et al, 1999), or ϩ/ϩ Li (Vik, 1996) have been investigated with respect to the expression of FH. Most of the analyzed hepatoma cell lines (HepG2, HepG3, H4) have been found not to express the FH protein constitutively, whereas the human line HUH7 (Friese et al, 1999) and the murine line ϩ/ϩ Li (Vik, 1996) have been found to produce FH in the absence of stimulation.…”

“…In contrast to these findings, Luo and Vik (1999) reported that Hep3b cells that did not express FH constitutively could be stimulated to express FH protein upon treatment with IFN-␥. The only human hepatoma cell line that constitutively expressed FH (HUH 7) exhibited an IFN-␥-dependent up-regulation of this molecule (Friese et al, 1999) similar to the murine ϩ/ϩ Li cell line (Vik, 1996). This inconsistent profile of the investigated cell lines prompted us to investigate the constitutive expression and regulation of FH in cultures of primary rat HC and KC.…”

“…Because IFN-␥ had been found to be an effective up-regulator of FH protein secretion in several primary and malignantly transformed cell lines (Brooimans et al, 1989;Luo and Vik, 1999;Ripoche et al, 1988b;Schwaeble et al, 1991;Vik, 1996), its potential effects were investigated in primary rat HC and the rat hepatoma cell lines H4IIE and FAO. Using conventional RT-PCR, it was demonstrated that only primary HC up-regulated FH-specific mRNA after exposure to IFN-␥ (Fig.…”

“…These cell types may function as local sources of FH and thereby reduce tissue damage caused by local complement activation. As a model for the investigation of the hepatic synthesis and regulation of FH, various hepatoma cell lines of mouse (ϩ/ϩ Li) (Vik, 1996) and man [Hep3b (Luo and Vik, 1999;Schwaeble et al, 1991), HepG2 (Lappin et al, 1992;Schwaeble et al, 1991), and HepG3, HepG4, H4 (Schwaeble et al, 1991)] have been used in the past. In the present study, we show the first approach to investigate the constitutive expression of FH in primary, cultured liver cells, ie, in hepatocytes (HC) and in Kupffer cells (KC) of rat as well as in two rat hepatomaderived cell lines (FAO and H4IIE).…”