Hepatocyte growth factor increases uptake of estradiol 17β-d-glucuronide and Oatp1 protein level in rat hepatocytes

Abstract

Hepatocyte growth factor (HGF) ameliorates liver injuries in hepatectomized cholestatic rats. On the other hand, the protein level of organic anion-transporting polypeptide (Oatp1), which is responsible for the uptake of bile salts into hepatocytes, decreases in cholestatic humans and rats. However, the relationship between the ameliorative effects of HGF and the decrease in Oatp1 levels in cholestasis remains to be understood. Therefore, in order to investigate this relationship, we evaluated the effects of HGF on the function and protein level of Oatp1. HGF treatment significantly increased the uptake of radiolabeled estradiol 17β-d-glucuronide ([³H]E₂17βG), a predominant Oatp1 substrate, in primary cultured rat hepatocytes. Additionally, there was an increase in the Oatp1 protein levels. The increased [³H]E₂17βG uptake was significantly inhibited by simultaneous incubation with the HGF receptor antibody and treatment with non-radiolabeled E₂17βG. However, inhibition by taurocholic acid, a Na⁺-taurocholate co-transporting polypeptide (Ntcp) substrate, was weaker than that caused by non-radiolabeled E₂17βG. Further, the increase was not altered by replacing Na⁺ in the medium with Li⁺. In the inhibition study, the increased [³H]E₂17βG uptake was inhibited by Oatp1 substrates, including bromosulfophthalein, ochratoxin A, and ouabain, but not by digoxin, which is an Oatp2-specific substrate. Furthermore, HGF did not alter the Oatp1 mRNA expression. In contrast, HGF treatment suppressed the ubiquitination of Oatp1 protein. In conclusion, this is the first report suggesting that HGF regulates Oatp1 protein level and that the ameliorative effects of HGF in cholestasis was induced, at least in part, by correcting the down-regulation of the Oatp1 protein level.

Introduction

Hepatocyte growth factor (HGF) was originally isolated from rat platelets (Nakamura et al., 1987) and plasma from patients with fulminant liver failure (Gohda et al., 1988). HGF promotes hepatocyte proliferation and liver regeneration. Consistent with this is the transient increase in the plasma levels of HGF in patients undergoing partial hepatectomy (Nishizaki et al., 1995, Selden et al., 1986). However, in contrast, it has been reported that HGF down-regulates the expression of cytochrome P450 (CYP) isozymes, which directly affect the pharmacokinetic, in primary cultures of human hepatocytes (Donato et al., 1998). The hepatocyte is the major cell involved in the metabolism. Hence, the HGF-induced down-regulation of CYP expression causes a decrease in the detoxification capacity; this is an impediment because of the potential therapeutic use of HGF in certain situations involving liver regeneration or repair. Therefore, it is important to consider the influence of HGF on other factors that affect the metabolic process, such as transport protein, involved in the uptake of endogenous and exogenous compounds into hepatocytes in order to investigate the physiological and pathophysiological roles and the therapeutic effects of HGF.

Several transport proteins in hepatocytes are involved in the generation of bile flow. Defects in these transporters can cause cholestasis (Geier et al., 2007). Bile acid transporters are typically grouped into two major categories—the canalicular transport system and the basolateral transport system. The canalicular transport system plays the role of an efflux pump that excretes bile salts into the bile duct. On the other hand, the basolateral transport system is responsible for the uptake of bile salts into hepatocytes; it can be further sub-divided into two groups—the Na⁺-dependent taurocholate co-transporting polypeptide (rodent, Ntcp; human, NTCP) (Meier and Stieger, 2002) and the Na⁺-independent organic anion-transporting polypeptide (rodent, Oatps; human OATPs) families of transporters. Ntcp is responsible for the uptake of the most parts of taurocholic acid (Hata et al., 2003, Kouzuki et al., 1998). Oatps/OATPs are multispecific transporters that mediate hepatic uptake of numerous organic anions in addition to that of taurocholic acid. In the rat liver, Oatp1 (Oatp1a1, Slco1a1), Oatp2 (Oatp1a4, Slco1a4), and Oatp4 (Oatp1b2, Slco1b2) are expressed at the basolateral membrane of hepatocytes (Cattori et al., 2001). Fickert et al. reported the down-regulation of Oatp1 and Ntcp after cholate feeding in mice (Fickert et al., 2001). Rost et al. reported decreased Oatp1 levels in cholic acid (CA)-fed rats; further, the decrease of Oatp1 was greater than that of Oatp4. In contrast, Oatp2 increased in the CA-fed rats (Rost et al., 2003). Additionally, Geier et al. reported that Oatp1 decreased in cholestasis model rats, however, Oatp4 decreased slightly (Geier et al., 2003). Furthermore, OATPs and NTCP have been reported to decrease in humans with cholestasis (Oswald et al., 2001, Zollner et al., 2001). However, in contrast, the up-regulation of the expression of the transporters involved in the excretion of bile salts at the canalicular plasma membrane has been reported in an endotoxemic rat liver (Vos et al., 1998). The up-regulation may be a protective pathophysiological phenomenon occurring in cholestasis. However, under pathophysiological conditions involving decreased expression of the influx pumps including Oatp1, the protective phenomenon does not adequately ameliorate bile flow dysfunction. Hence, of all the modulations involving Oatps expression, decrease in the levels of expression of Oatp1 is considered to be an important pathophysiological factor in the development of cholestasis.

Recently, HGF has been reported to increase in cholestatic mice (Wang et al., 2005). Accordingly, because HGF is an essential survival factor (Huh et al., 2004, Ueki et al., 1999) and mitogen for the hepatocytes (Fausto et al., 1995, Stolz et al., 1999), there is attenuation of the liver injury associated with cholestatic liver diseases. Furthermore, Yoshikawa et al. reported that pre-operative HGF supply not only promoted liver regeneration but also ameliorated hyperbilirubinemia in hepatectomized cholestatic rats (Yoshikawa et al., 1998). However, the mechanisms by which HGF ameliorates cholestasis remain to be understood. Therefore, considering the abovementioned reports regarding HGF, transporters, and cholestasis, we hypothesized that the beneficial effects of HGF in cholestasis may be induced via an improvement in transporter dysfunction. To validate this hypothesis, in the present study, we evaluated the HGF effects on the level of Oatp1 protein and the kinetic properties of estradiol-17β-d-glucuronide (E₂17βG), a predominant substrate of Oatp1, uptake into primary cultured rat hepatocytes. Further, we investigated the transcriptional and post-transcriptional pathways of Oatp1 protein after HGF treatment to identify the mechanisms underlying the effects of HGF on the Oatp1 expression.