A stimulus needed for the study of membrane traffic in hepatocytes†

A major function carried out by the liver is maintenance of the blood chemistry by the continuous secretion of many different serum proteins into the blood sinusoid, transport of solutes and xenobiotics from blood to bile, and filtration of the blood through active internalization of many different receptor-ligand complexes as well as unwanted viral and bacterial pathogens. These primary hepatic secretory and endocytic functions depend on an elaborate cytoskeletal-based vesicle trafficking system that can be modified to meet the specific tasks of moving cargo outward versus inward. Despite the seminal importance of these cell biological processes to the central function of the hepatocyte and general liver physiology, their molecular mechanisms remain poorly defined and remarkably understudied by the hepatology community. Here we provide a short overview of a few important cell biological processes that are performed by hepatocytes and require our attention to better understand liver physiology and disease. The important cell biological processes described above require a highly polarized structural scaffold upon which cell signaling, membrane traffic, protein secretion, and bile transport are organized. Although hepatocyte polarity is well described morphologically, we know very little about the molecules required for polarity formation or their temporal expression because there has been no ideal system for study. A working model for the development of epithelial cell polarity postulates that extracellular signals generated from cell-cell and cell-substrate interactions initiate the multiple cellular processes that must occur to form a polarized epithelium.1 These processes include (1) an organized and stabilized cytoskeleton, (2) sorted apical and basolateral components, (3) an arrangement of intracellular organelles, and (4) the formation of adherens junctions and tight junctions to act as a diffusion barrier. Most epithelial cells exhibit a simple polarity, consisting of single apical and basal surfaces that are opposite to each other and lateral surfaces that participate in cell-cell associations to form a sheet or tube structure. In contrast, hepatocytes are polygonal and multipolar, with at least two basal surfaces facing the circulation and a branched network of grooves that forms between adjacent cells and constitutes the apical, or bile canalicular, surface. Recent studies have increasingly pointed to the adherens and tight junctions as important macromolecular complexes whose formation helps to establish the apical and basolateral poles and which serve as landmarks for vesicle targeting in epithelial cells.2 Given the tremendous flux of solutes and macromolecules into and out of hepatocyte, how is the polarized organization of the cell maintained? We know that protein cargo destined for secretion, localization to a specific plasma membrane (PM) domain, or endocytosis contains structural signals (e.g., short sequences, lipid anchors, and carbohydrate modifications) recognized by cellular machinery that mediates the formation of vesicles. These vesicles are then targeted to their appropriate destination. Morphological, biochemical, and genetic approaches have successfully identified many molecules involved in vesicle targeting, a complex series of molecular events that minimally include docking and fusion. These basic mechanisms are conserved throughout the biosynthetic and endocytic pathways and in organisms ranging from yeast to humans. The players so far identified fall into two broad categories: some are used repeatedly throughout the pathways, and others belong to discrete protein families in which one or a few members act at one or a few transport sites. Members of three protein families are central players in vesicle targeting and fusion and include (1) the soluble NSF attachment protein receptors (SNAREs), which in general are a group of cytoplasmically oriented integral membrane proteins that are present on vesicles (vesicle SNAREs) or target membranes (target SNAREs); (2) the Sec1/Munc18 proteins; and (3) low-molecular-weight guanosine triphosphate–binding proteins (the rabs). Enormous progress over the past decade has increased our understanding of how these complex systems of many different proteins interact synergistically to establish and maintain a unique and complex polarized organization that supports liver function. In comparison with the impressive recent advances in our understanding of sorting signals, recognition machinery, and pathways in simple epithelial cells, our knowledge of PM protein trafficking in hepatocytes remains rudimentary (reviewed by Braiterman and Hubbard3). We pose two important questions that need to be addressed. First, are the apical sorting signals in each multi–transmembrane domain (multi-TMD) protein unique? The formation of bile is a central function of hepatocytes and a process requiring many multi-TMD apical PM proteins.4 Additional export processes are carried out at the canalicular PM by other multi-TMD proteins (e.g., the Cu-ATPase ATP7B and drug transporters). The apical transporters studied to date appear to have unique and complex sorting signals, the recognition systems are only beginning to be identified, and the targeting mechanisms are still largely unknown. More study is needed, particularly of patient missense mutations whose protein phenotypes (in hepatic cells) may give important clues about these processes (eg, see Braiterman et al.5). Second, are several apical PM protein recognition systems missing from hepatocytes? The clear difference in the biosynthetic pathways taken by all tested glycosylphosphatidylinositol-anchored and single-TMD apical PM proteins in hepatic cells (transcytotic) versus those in simple epithelial cells (direct) suggests that the former cells lack certain key recognition mechanisms operating in the latter.3 The relevant proteins need to be studied. EGFR, epidermal growth factor receptor; ESCRT, endosomal sorting complex required for transport; HBV, hepatitis B virus; HCV, hepatitis C virus; LD, lipid droplet; PM, plasma membrane; SNARE, soluble NSF attachment protein receptor; TfR, transferrin receptor; TMD, transmembrane domain. A major function of the hepatocyte is the transport of various solutes such as bile acids, drugs, and other xenobiotics from the circulation into the cell, where biotransformation may occur prior to secretion across the bile canaliculus into bile. Transport proteins that mediate these processes have been identified. For the most part, these proteins have multiple TMDs and polarized localization to either the basolateral (sinusoidal) or apical (bile canalicular) domains of the PM. These transporters have also been found to be associated with intracellular vesicles, representing a pool of proteins that can be recruited to the PM to augment transport activity when needed. Alterations in the recruitment to and retrieval from the cell surface represent a potentially important and novel means to regulate transporter function. That is, increased internalization of the transporter will result in a net reduction in its PM localization and consequently reduced ability to transport substrate. For example, if all transporter resides in intracellular vesicles, transport function across the PM will cease. Such alterations have been seen in the subcellular distribution of the bile canalicular transporter for bile acids (bile salt export pump) and may be a cause of cholestasis. Similarly, regulation of the trafficking of basolateral PM transporters can influence the ability of the hepatocyte to take up compounds from the circulation and could be a determinant in the first-pass clearance of drugs, resulting in unexpectedly altered concentrations of drugs in the peripheral circulation. Recent studies have shown that transporter-containing intracellular vesicles can move along microtubules, powered by specific members of the molecular motor families, dynein and kinesin.6, 7 Although exact mechanisms still need to be discovered, the direction of movement can be regulated by the activity of specific vesicle-associated kinases and rabs. Interestingly, the proteins and motors associated with these vesicles appear to differ, depending on the transporter. Future discovery of the proteins and mechanisms involved in these transporter trafficking regulatory pathways may provide new insights into key points that are perturbed in disease processes and may provide new areas for therapeutic intervention. Iron homeostasis is vital, and the liver plays a central regulatory role in part by dual receptor cascades [transferrin receptor 1(TfR1) and TfR2] that act to both internalize iron-coupled transferrin and stimulate peptide signals to iron transporters in other cells and tissues. TfR2 is expressed almost exclusively in the liver, whereas TfR1, the classic receptor generally called TfR and expressed widely, is present in modest amounts in the liver.8 Importantly, these related receptors appear to be regulated differently, stimulate distinct downstream effectors, and are internalized by distinct mechanisms. TfR1 is internalized by hepatocytes via a clathrin-dependent mechanism, whereas TFR2 localizes to lipid rafts and interacts with caveolin-1.9 Furthermore, TfR1 is recycled back to the surface, but TfR2 is delivered to multivesicular bodies and subsequently trafficked to the lysosome for degradation.10 Additional studies are needed to elucidate the molecular machinery that regulates the different endocytic pathways used by the two TfR types. For example, it is important to define how the related receptors are sequestered into distinct endocytic containers and whether TfR2, similar to epidermal growth factor receptor (EGFR), is ubiquitinated; TfR2 would therefore be targeted for entry into the degradative pathway. Ligand-induced internalization and degradation of receptor tyrosine kinases such as EGFR and hepatocyte growth factor receptor are very important for maintenance versus attenuation of growth signaling cascades. A disruption of this degradative pathway can lead to amplified signaling cascades and tumor growth. It is now known that the attachment of a ubiquitin moiety to proteins plays a major role in lysosomal targeting and degradation and is required for the passage of certain cargo molecules into and out of vesicles of the endocytic pathway as well as signaling processes.11-16 Ubiquitin-mediated down-regulation of receptor tyrosine kinases has been observed for a number of different receptors, with the best studied example being EGFR. Importantly, this ubiquitin-dependent sorting to the lysosome is mediated in part by a series of multiprotein complexes termed endosomal sorting complexes required for sorting (ESCRTs). Interestingly, this complex appears to be used by multiple viruses, including the human immunodeficiency virus, to exit the cell. As the specifics of this ESCRT pathway have been largely defined in yeast, more work is needed in epithelial cells such as the hepatocyte to better understand receptor signaling pathways as they pertain to liver growth, regeneration, and neoplasia. It is well known that the liver plays an important role in the internalization of lipid-rich particles from the blood. How this endocytic internalization of lipids supports the formation of numerous large lipid droplets (LDs) that form during liver steatosis is remarkably undefined, just as the subsequent trafficking steps that lead to LD formation are unclear. Nascent LDs are believed to bud from the endoplasmic reticulum, in which they incorporate newly synthesized neutral fatty acids and increase in size, but the exact mechanisms of this growth are poorly understood. Current models for this process include mutual fusion, fusion and fission cycles in contact with the endoplasmic reticulum, and direct synthesis of lipid esters at LDs. As LDs have been shown to be associated with specific vesicle trafficking proteins such as caveolin-1 and rab proteins, it has been predicted but not demonstrated that they traffic through an endosomal pathway. However, it is not understood how alterations in trafficking may affect LD formation, accumulation, and composition. Thus, it will be important to determine how these proteins are recruited to the LD surface and how the hepatocyte distinguishes between the levels of neutral lipids entering the LD/storage versus the secretory pathway. Remarkably, mechanistic details that provide insights into how LDs form, mature, and exactly contribute to lipid homeostasis in the cell are mostly missing. Human pathogens such as hepatitis B virus (HBV) and hepatitis C virus (HCV) have evolved mechanisms to subvert parts of the cell's normal membrane trafficking machinery to accomplish their own internalization, propagation, and exocytic release. For example, HBV is hypothesized to enter the hepatocyte via clathrin-coated pits after binding to a receptor on the sinusoidal membrane, possibly the TfR, immunoglobulin A, interleukin-6, and/or asialoglycoprotein receptor, although this remains undefined. How the virus is transported through the endocytic pathway and released for replication without entering the lysosome is unclear. Furthermore, how the host secretory pathway is used for the propagation, packaging, and release of the nascent virus is also largely unknown. Interestingly, the ESCRT complex described previously and used at the late endosome to traffic ubiquitinylated receptor cargo has been implicated in the final stages of HBV assembly and maturation. As cellular models to study HCV infection are even more difficult to come by, less is known about the entry, traffic, and exit mechanisms of this virus. Understanding basic mechanisms by which the hepatocyte functions is essential for elucidation of the complex perturbations that result in disease. This discussion has focused on mechanisms of protein and vesicle trafficking through the hepatocyte. These trafficking and transport mechanisms are required for health but are also essential for diseases caused by viruses, toxins, and some drugs. It was not too long ago that hepatocytes were said to be devoid of microtubules. We now know that they are abundant and provide railroad tracks for trafficking events. Although much has been learned in recent years, much remains to be discovered. It is likely that the elucidation of mechanisms that control the activity of the important cellular processes that have been discussed in this brief review may provide new approaches and insights for the care and treatment of patients with disorders of hepatobiliary function. It is our view that additional attention to the cell biology of the liver should be a priority for this journal, the American Association for the Study of Liver Diseases, and the National Institutes of Health by a variety of approaches. Some might include increased support of basic cell biological research as well as single-topic conferences and scholarly reviews and an increased presence at the annual meeting of the American Association for the Study of Liver Diseases.