HBV, a causative agent for acute/chronic hepatitis and HCC, is transmitted through contact with infected blood or other body fluids and triggers immune-mediated liver diseases of varying severity and duration. HBV has a circular, partly double-stranded, enveloped DNA genome that selectively enters hepatocytes where it delivers its genome, thereby initiating a multifaceted process of viral replication (Iannacone and Guidotti 2022). The HBV envelope contains three forms of the HBV surface protein (HBsAg), namely the large (L), middle (M), and small (S) proteins. Importantly, the preS1 domain of the large envelope protein has been identified as an essential structure for HBV attachment and entry (Sureau et al., 1993). However, the functional receptor mediating HBV entry into hepatocytes remained unknown for more than 20 years since the discovery of the virus, until NTCP was identified as being critical for preS1 binding and HBV infection (Yan et al., 2012).

NTCP is located on the basolateral membrane domain (blood-side) of hepatocytes and is broadly expressed in humans, rats, and monkeys, among other species (Qiu et al., 2013). NTCP is expressed exclusively in the liver. Human NTCP (hNTCP, encoded by the SLC10A1 gene) is a protein of 349 amino acids with an apparent mass of 56 kDa (Dawson et al., 2009; Watashi and Wakita 2015). Although the crystal structure of hNTCP has not been solved, a series of modeling, mutagenesis, and biochemical analyses suggest that this protein has a putative nine transmembrane domains with a topology predicted to consist of an extracellular N-terminus and an intracellular C-terminus (Hu et al., 2011; Fukano et al., 2019; Appelman et al., 2021) (Figure 1). Which region(s) of NTCP is essential for viral attachment and the subsequent triggering of internalization remains incompletely understood. Current evidence suggests that hNTCP residues 157 to 165 (KGIVISLVL) are important for preS1 binding and, thus, HBV infection (Yan et al., 2012; Ni et al., 2014). By analyzing the susceptibility of NTCP variants to HBV attachment, Müller et al. (2018) showed that a single amino acid at position 158 of NTCP is critical for HBV binding and subsequent infection. Similarly, Junko and others demonstrated that the sequence of amino acid 158 was a determining factor for the attachment of the HBV envelope protein to the host cell (Takeuchi et al., 2019). In addition, it was found that the molecular determinants for the transporter function of NTCP overlapped with those for its ability to support HBV entry (Yan et al., 2014). Mutations in the amino acids of NTCP that are critical for bile salt binding (N262A, Q293A/L294A) abrogated both the binding of NTCP to the preS1 peptide and infection by HBV. The S267F variant of NTCP could neither bind to the preS1 region nor support HBV infection in cell culture, thereby supporting the role of NTCP as the cellular receptor for HBV infection in humans (Peng et al., 2015; Lee et al., 2017; An et al., 2018). A recent study demonstrated that hepatocytes expressing the NTCP-S267F variant, introduced into the SLC10A site via CRISPR editing, are resistant to HBV infection (Uchida et al., 2021). However, patients homozygous for S267F can still be infected by HBV, suggesting the existence of alternative receptors that allow viral entry in the absence of functional NTCP (Hu et al., 2016).

HBV primarily infects humans, chimpanzees, and several other great apes, but not monkeys or lower primates. Viral infections in non-susceptible species are known to be mainly restricted at the entry level as viral replication can be achieved in cells from the relevant species when the cells are transfected with the viral genome (Yan et al., 2013). Lempp and others reported that hepatocytes from cynomolgus macaques, rhesus macaques, and pigs expressing hNTCP become fully susceptible to HBV with efficiencies comparable to that for human hepatocytes. However, when expressed in rodent or canine hepatocytes, hNTCP can support HBV attachment but not the establishment of HBV infection (Lempp et al., 2017). Moreover, the substitution of residues 85–87 of murine NTCP (mNTCP) with those of human NTCP is sufficient to facilitate HBV entry into the cell but not to support infection (Yan et al., 2013). These observations imply that other host factors besides NTCP are required for HBV internalization in hepatocytes of these animals and that the differences between species, such as NTCP structure and (co)receptors, affect HBV susceptibility and infection efficiency.

HBV infection into host hepatocytes involves multiple steps. In the initiation of infection, HBV reversibly attaches to heparan sulfate proteoglycans on the cell surface via a highly conformational determinant region 1) of the HBsAg glycoprotein (Sureau and Salisse 2013). HBV subsequently interacts with its specific receptor with high affinity, thereby triggering viral internalization. After endocytosis-mediated internalization, the virus fuses with the cellular membrane compartment. However, the precise mechanisms are not yet fully understood (Watashi et al., 2014a).

Data collected to date clearly indicate that NTCP can serve as an HBV receptor, especially in vitro (Li and Tong 2015). NTCP mediates the specific binding of HBV to the host cell surface with high affinity by interacting with the preS1 region of the large surface protein (LHB) of HBV. NTCP confers HBV susceptibility to human hepatic cell lines such as HepG2, Huh7, or undifferentiated HepaRG cells, which are originally non-susceptible to infection (Iwamoto et al., 2014; Ni et al., 2014). This easily manipulated model of cell infection has been used to decipher the early steps of HBV entry. However, the expression of NTCP alone is not sufficient for efficient HBV internalization into hepatocytes, and additional host factors are likely to be required for susceptibility to HBV infection, potentially through the formation of a complex and a multistep entry process. For example, host epidermal growth factor receptor (EGFR) was reported to interact with NTCP and mediate HBV internalization (Iwamoto et al., 2019), a finding that potentially accounts for the low rate of infection in the HepG2 cell line, in which EGFR expression is undetectable (Zhao et al., 2013). Kinesin family member 4 (KIF4) regulates the levels of surface NTCP via the anterograde transport of NTCP to the cell surface, potentially also mediating HBV entry (Gad et al., 2021).

Most studies on NTCP transcriptional regulation are based on rodent models and engineered HepG2-NTCP or other cells because NTCP shows minimal endogenous expression in hepatocellular cell models and is rapidly lost from primary human hepatocytes following their isolation (Xia et al., 2017). Key transcriptional regulators of NTCP identified to date include farnesoid X receptor (FXR)/small heterodimer partner (SHP), the homeodomain protein hepatocyte nuclear factor 1 alpha (HNF-1α), HNF-4α, retinoid X receptor-alpha (RXRα), HNF-3β, and signal transducer and activator of transcription 5 (STAT5) (Ganguly et al., 1997; Lu et al., 2019). Other factors, such as proinflammatory cytokines, hormones, and several chemical compounds, are also suggested to participate in the transcriptional regulation of NTCP (Figure 2).

NTCP mRNA regulation is mainly linked to bile acid concentration, which serves as an adaptive response to block excessive bile acid accumulation in hepatocytes under pathophysiological conditions (Xia et al., 2017). NTCP is downregulated during cholestasis. Bile acid-induced, FXR-mediated induction of the nuclear repressor SHP has been proposed to be a key mechanism underlying the reduction in NTCP expression through interfering with the RXR-retinoic acid receptor (RAR) heterodimer, for which there is a binding site within the NTCP promoter (Anwer 2004). FXR is a bile acid-activated nuclear receptor (BAR) and is mainly expressed in the liver and intestine. In the liver, NTCP was reported to be strongly repressed in wild-type (WT) mice fed a colic acid-containing diet. This repression was largely retained in SHP-null mice, in agreement with the proposed role of SHP in the negative regulation of NTCP expression. This phenomenon also indicated that SHP-independent mechanisms may be involved in mediating NTCP repression in cholestasis (Wang et al., 2003). In the distal ileum, FXR activation also instigates the production of fibroblast growth factor 19 (FGF19; FGF15 in mice), which then reaches hepatocytes via the portal circulation and activates the fibroblast growth factor receptor 4 (FGFR4)/β-Klotho complex, thereby triggering intracellular signaling pathways in the liver to maintain the bile acid balance. The administration of recombinant hFGF19 downregulates NTCP mRNA by ∼50% in WT mice, indicating that FXR further modulates NTCP mRNA expression via FGF15/19 signaling (Inagaki et al., 2005; Slijepcevic et al., 2017). In addition, the glucocorticoid receptor (GR), a ligand-activated transcription factor, can directly bind and activate the NTCP promoter. Human NTCP expression is upregulated by the GR ligand dexamethasone in Huh-7 cells and augmented by peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), while GR-mediated activation is inhibited by SHP downstream of FXR (Eloranta et al., 2006). Furthermore, it was reported that FXR expression is regulated by hepatic sirtuin 1 (SIRT1), a NAD-dependent deacetylase that has been found to couple the deacetylation of many transcription factors and cofactors, such as NF-κB, PGC-1α and FXR, to NAD⁺ hydrolysis (Yu et al., 2016).

HNF-1α and HNF-3β are also essential regulators of bile acid metabolism. HNF-1α can bind and transactivate the rat Ntcp promoter, but not that of humans or mice, whereas HNF-3β mediates the transcriptional repression of the NTCP/Ntcp promoter in all three species via directly binding to its response elements (Jung et al., 2004; Zhao et al., 2021). HNF-1α^−/− livers exhibit decreased NTCP expression in the basolateral membrane, leading to impaired portal bile acid uptake and elevated plasma bile acid concentrations (Yu et al., 2016). HNF-1α also regulates the expression of the nuclear receptor FXR, a key regulator of NTCP expression (Shih et al., 2001). As mentioned above, SIRT1 modulates FXR transcriptional regulation through HNF-1α, and SIRT1 deficiency in the liver was found to decrease the binding of HNF-1α to the FXR promoter and thus reduce its expression, resulting in impaired bile acid metabolism (Purushotham et al., 2012). HNF-4α directly binds to the mouse Ntcp promoter through functional HNF-4α response elements while PGC-1α potentiates HNF-4α-induced NTCP transactivation (Geier et al., 2008). Importantly, as HNF-1α was shown to control HNF-4α transcription, repressed NTCP expression in HNF-1α deficiency may potentially reflect an HNF-4α-mediated mechanism (Odom et al., 2004).

STAT5 was reported to directly bind to interferon-gamma (IFN-γ)-activated sequence-like elements (GLEs) in the Ntcp promoter as well as mediate the upregulation of Ntcp expression by prolactin in the rat (Ganguly et al., 1997). Notably, however, Ntcp expression was found to be reduced in pregnant rats in late gestation despite the increase in the level of STAT5. This suggests that a STAT5-independent pathway that is activated during pregnancy exerts a greater effect than STAT5 on Ntcp expression (Zhao et al., 2021). Bu and others investigated the impact of STAT5 signaling on the expression of the mouse and human Ntcp/NTCP gene using berberine, an inhibitor of STAT signaling. Through assessing the binding of phospho-STAT5 protein to STAT5 response elements in the NTCP promoter, the authors determined that STAT5 regulates the expression of both mouse and human Ntcp/NTCP (Bu et al., 2017), as previously also reported for the rat.

Cholestasis can trigger the release of cytokines such as IL-6, IL-1β, and TNF-α, which, in turn, repress NTCP transcription. IL-6 is an important cytokine with roles in inflammation and liver regeneration, and NTCP expression is known to be strongly regulated by IL-6. A decrease of up to 98% in NTCP mRNA steady-state levels was detected in primary human hepatocyte (PHH) and HepaRG cells after IL-6 treatment (Bouezzedine et al., 2015). The effect of IL-6 on NTCP has also been investigated in mice through the injection of turpentine, which induces an acute phase response with IL-6 production. Turpentine treatment resulted in the downregulation of NTCP mRNA expression in WT mice, but not in IL-6 KO animals (Siewert et al., 2004). In addition, Yan et al. (2019) found that there was a sharp rise in IL-6 mRNA levels in mice injected with LPS, an effect that was accompanied by a reduction in NTCP transcript levels. However, NTCP mRNA levels showed a partial recovery when serum IL-6 was neutralized with IL-6 antibodies, indicating that IL-6 can downregulate the expression of NTCP in inflammation at the transcriptional level. Mechanistic studies indicated that the downregulation of NTCP could be mediated by the activation of c-Jun N-terminal kinase (JNK), which suppresses the expression and binding activity of multiple nuclear factors such as HNF-1α and HNF-4α (Geier et al., 2007). Similarly, a marked downregulation of NTCP mRNA expression was detected in HepaRG cells in response to 24 h of exposure to IL-1β at the concentration of 1 ng/ml (Le Vee et al., 2008). Moreover, this effect was shown to be mediated via the suppression of the RAR/RXR complex in a JNK pathway-dependent manner, resulting in the downregulation of NTCP promoter activity and transport activity (Li et al., 2002). The effects of IL-1β and TNFα have been investigated in rats, and the inactivation of either cytokine was found to prevent the downregulation of NTCP mRNA expression. In contrast to the predominant role of IL-1β in a complex signaling network involved in NTCP regulation in cholestatic liver injury, TNF-α represents the master cytokine responsible for the HNF1-dependent reduction of NTCP levels in CCl4-induced toxic liver injury (Geier et al., 2003). Another cytokine within the IL-6 family, oncostatin M (OSM), also dose-dependently induced the downregulation of NTCP expression, an effect that was highly correlated with that of IL-6 (Le Vee et al., 2011).

Hormones such as estrogen, adrenal glucocorticoid, prolactin, growth hormone, and thyroid hormone are also suggested to participate in the regulation of NTCP expression (Simon et al., 2004; Cheng et al., 2007; Rose et al., 2011). For example, Rose et al. (2011) demonstrated that ex vivo glucocorticoid treatment increases NTCP expression in a GR-dependent manner in both the human and mouse liver. Meanwhile, chemical compounds such as dioxin, rifampicin, cholestyramine, phenobarbital, and oltipraz have also been shown to participate in the regulation of NTCP expression (Cheng et al., 2007; Zhao et al., 2021). However, the mechanisms underlying the effects of these hormones and compounds on NTCP expression need further investigation.

NTCP, an integral membrane glycoprotein, is localized to the plasma membrane and endocytic vesicles. The localization and membrane expression of NTCP is controlled by post-translational mechanisms involving phosphorylation or dephosphorylation, translocation to and retrieval from the plasma membrane, and degradation by the ubiquitin-proteasome system (UPS) (Anwer 2014). These post-translational regulatory mechanisms are mediated via signaling pathways involving cyclic adenosine monophosphate (cAMP), calcium, phosphoinositide-3-kinase (PI3K), protein kinase C (PKC), nitric oxide, and protein phosphatases (Anwer and Stieger 2014). For example, cAMP stimulates the dephosphorylation and consequent translocation of NTCP to the plasma membrane by dephosphorylating the Ser-226 site in the third cytoplasmic loop of NTCP (Anwer et al., 2005). Moreover, cAMP can enhance taurocholate uptake by promoting the insertion of NTCP into the plasma membrane via increases in intracellular Ca²⁺ concentrations and the subsequent activation of protein phosphatase 2B (PP2B) via Ca²⁺-calmodulin kinase (Mukhopadhayay et al., 1997; Anwer and Stieger 2014).

The PI3K-signaling pathway, which comprises three downstream effectors (protein kinase B [PKB/AKT]), P70 S6 kinase [p70^S6K], and PKC) plays a role in the cAMP-mediated translocation of NTCP. The effect of cAMP is not mediated via p70^S6K because treatment with rapamycin, an inhibitor of p70^S6K, does not inhibit cAMP-mediated stimulation of Na⁺-taurocholate uptake (Webster and Anwer 1999; Webster et al., 2000). Transfection with constitutively active PKB was shown to increase PKB activity, taurocholate uptake, and NTCP translocation, whereas inhibiting PKB blocked cell swelling- and cAMP-induced increases in taurocholate uptake and NTCP translocation (Webster et al., 2002). PKC is expressed as multiple isoforms, among which PKCδ and PKCζ have been suggested to play a role in the cAMP-induced translocation of NTCP to the cell surface. The PKCδ isoform mediates cAMP-induced translocation of NTCP via the activation of Rab4, which is associated with early endosomes and is involved in vesicular trafficking. These effects of PKCδ are mediated by its plasma membrane localization rather than its kinase activity (Park et al., 2012). Moreover, PKCζ activation enhances the cAMP-promoted motility of NTCP-containing vesicles, while specific inhibition of PKCζ exerts the opposite effect, indicating that PKCζ is specifically required for the cAMP-induced intracellular movement of vesicles that contain the NTCP transporter (Sarkar et al., 2006).

In addition, maturing NTCP is degraded by the ubiquitin-proteasome system at the level of ER-associated degradation (ERAD), and an imbalance in NTCP synthesis and degradation can result in intracellular NTCP deposits and subsequently lead to cholestasis (Kühlkamp et al., 2005). In summary, PP2B and PI3K/PKB/PKC axis facilitate the intracellular movement of NTCP toward the plasma membrane following cAMP activation, while the ubiquitin-proteasome system controls NTCP protein abundance (Figure 2).