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RANKL is biosynthesized as a single-pass transmembrane protein, and soluble molecular species are produced by enzymatic cleavage at the cell surface. Recent studies have revealed that the transmembrane form of RANKL is a major contributor to the induction of mature osteoclasts under physiological conditions in vivo. In osteoblasts and osteocytes, most newly synthesized RANKL forms a protein complex with OPG and is selectively sorted to lysosomes. Only the small proportion of newly synthesized RANKL that does not form a complex with OPG is transported to the cell surface. Then, the transmembrane RANKL is delivered to the surface of osteoclast precursors to stimulate RANK, and induces the activation of a downstream signaling pathway. The ability of osteocytes to support the formation of mature osteoclasts appears to depend upon the amount of RANKL molecules present on their cell surfaces. However, the way in which osteocytes, which are embedded in the bone matrix, deliver transmembrane RANKL to the cell surfaces of osteoclast precursors, which are localized in the bone marrow cavity, remains to be elucidated. Further studies are needed to clarify the mechanisms underlying this process.

Receptor activator of nuclear factor-kappa B (RANK) ligand (RANKL) binds RANK on the surface of osteoclast precursors to trigger osteoclastogenesis. Recent studies have indicated that osteocytic RANKL has an important role in osteoclastogenesis during bone remodelling; however, the role of osteoblastic RANKL remains unclear. Here we show that vesicular RANK, which is secreted from the maturing osteoclasts, binds osteoblastic RANKL and promotes bone formation by triggering RANKL reverse signalling, which activates Runt-related transcription factor 2 (Runx2). The proline-rich motif in the RANKL cytoplasmic tail is required for reverse signalling, and a RANKL(Pro29Ala) point mutation reduces activation of the reverse signalling pathway. The coupling of bone resorption and formation is disrupted in RANKL(Pro29Ala) mutant mice, indicating that osteoblastic RANKL functions as a coupling signal acceptor that recognizes vesicular RANK. RANKL reverse signalling is therefore a potential pharmacological target for avoiding the reduced bone formation associated with inhibition of osteoclastogenesis. Osteoclasts secrete small extracellular vesicles that stimulate osteoblasts, promoting bone formation via receptor activator of nuclear factor-kappa B ligand (RANKL), thereby linking bone formation and resorption.

RANKL is a key molecule that bridges the bone and immune systems. RANKL stimulation activates a signaling pathway downstream of RANK, thereby determining the extent of bone resorption by inducing osteoclast maturation. The signaling pathway also regulates the development of different lymphoid organs, including the thymus, lymph nodes, and Peyer's patches, and plays an essential role in the establishment of immune tolerance. Such characteristics have continued to attract the attention of many researchers, even though it is now more than 20 years since RANKL was identified as a novel member of the TNF superfamily. Recently, we found that RANKL can function not only as a signal input molecule but also as a signal receptor to activate the RANKL reverse signaling pathway, which mediates the coupling between bone resorption and formation. This new finding may provide an important basis for elucidating the complex physiological roles played by RANKL.

We recently found that the membrane-bound receptor activator of NF-κB ligand (RANKL) on osteoblasts works as a receptor to stimulate osteoblast differentiation, however, the reason why the RANKL-binding molecules stimulate osteoblast differentiation has not been well clarified. Since the induction of cell-surface receptor clustering is known to lead to cell activation, we hypothesized that the induction of membrane-RANKL clustering on osteoblasts might stimulate osteoblast differentiation. Immunoblotting showed that the amount of RANKL on the membrane was increased by the RANKL-binding peptide OP3-4, but not by osteoprotegerin (OPG), the other RANKL-binding molecule, in Gfp-Rankl-transfected ST2 cells. Observation under a high-speed atomic force microscope (HS-AFM) revealed that RANKL molecules have the ability to form clusters. The induction of membrane-RANKL-OPG-Fc complex clustering by the addition of IgM in Gfp-Rankl-transfected ST2 cells could enhance the expression of early markers of osteoblast differentiation to the same extent as OP3-4, while OPG-Fc alone could not. These results suggest that the clustering-formation of membrane-RANKL on osteoblasts could stimulate early osteoblast differentiation.

The receptor activator of the NF-κB ligand (RANKL) is the central player in the regulation of osteoclastogenesis, and the quantity of RANKL presented to osteoclast precursors is an important factor determining the magnitude of osteoclast formation. Because osteoblastic cells are thought to be a major source of RANKL, the regulatory mechanisms of RANKL subcellular trafficking have been studied in osteoblastic cells. However, recent reports showed that osteocytes are a major source of RANKL presentation to osteoclast precursors, prompting a need to reinvestigate RANKL subcellular trafficking in osteocytes. Investigation of molecular mechanisms in detail needs well-designed in vitro experimental systems. Thus, we developed a novel co-culture system of osteoclast precursors and osteocytes embedded in collagen gel. Experiments using this model revealed that osteocytic RANKL is provided as a membrane-bound form to osteoclast precursors through osteocyte dendritic processes and that the contribution of soluble RANKL to the osteoclastogenesis supported by osteocytes is minor. Moreover, the regulation of RANKL subcellular trafficking, such as OPG-mediated transport of newly synthesized RANKL molecules to lysosomal storage compartments, and the release of RANKL to the cell surface upon stimulation with RANK are confirmed to be functional in osteocytes. These results provide a novel understanding of the regulation of osteoclastogenesis.

The ATP-binding cassette, subfamily G, member 2 (ABCG2/BCRP) gene encodes a well-known transporter, which exports various substrates including nucleotide analogs such as 3'-azido-3'-deoxythymidine (AZT). ABCG2 is also located in a gout-susceptibility locus (MIM 138900) on chromosome 4q, and has recently been identified by genome-wide association studies to relate to serum uric acid (SUA) and gout. Becuase urate is structurally similar to nucleotide analogs, we hypothesized that ABCG2 might be a urate exporter. To demonstrate our hypothesis, transport assays were performed with membrane vesicles prepared from ABCG2-overexpressing cells. Transport of estrone-3-sulfate (ES), a typical substrate of ABCG2, is inhibited by urate as well as AZT and ES. ATP-dependent transport of urate was then detected in ABCG2-expressing vesicles but not in control vesicles. Kinetic analysis revealed that ABCG2 is a high-capacity urate transporter that maintained its function even under high-urate concentration. The calculated parameters of ABCG2-mediated transport of urate were a Km of 8.24 ± 1.44 mM and a Vmax of 6.96 ± 0.89 nmol/min per mg of protein. Moreover, the quantitative trait locus (QTL) analysis performed in 739 Japanese individuals revealed that a dysfunctional variant of ABCG2 increased SUA as the number of minor alleles of the variant increased (p = 6.60 × 10(-5)). Because ABCG2 is expressed on the apical membrane in several tissues, including kidney, intestine, and liver, these findings indicate that ABCG2, a high-capacity urate exporter, has a physiological role of urate homeostasis in the human body through both renal and extrarenal urate excretion.

Aim: Multidrug resistance protein 3 (MDR3/ABCB4), located on the bile canalicular membrane of hepatocytes, is responsible for the translocation of phosphatidylcholine across the plasma membrane, and its hereditary defect causes liver disorders, such as progressive familial intrahepatic cholestasis type 3. We aimed to identify the proteins responsible for the surface expression of human ABCB4. Methods: We performed yeast two-hybrid screening with the cytoplasmic linker region of ABCB4 against a human liver cDNA library. This screening allowed us to identify the receptor for activated C-kinase 1 (RACK1) as a novel binding partner of ABCB4. The association of RACK1 with the linker region of ABCB4 was further confirmed by GST-pulldown assay, although we could not find out the interaction of full length of ABCB4 and RACK1 in co-immunoprecipitation assay in HeLa cells. Results: Down-regulation of endogenous RACK1 expression by siRNA in HeLa cells resulted in the localization of ABCB4 in the cytosolic compartment as well as reduced protein expression of ABCB4, although mRNA expression and the protein stability of ABCB4 were not affected by the suppression of endogenous RACK1. Similar alterations in cellular localization of ABCB4 were also found by suppressing endogenous RACK1 expression in HepG2 cells. Consequently, ABCB4-mediated phosphatidylcholine translocation activity was significantly reduced when endogenous RACK1 expression was suppressed in HeLa cells. In contrast, the membrane surface localization and the protein expression of ABCB1 were not affected by the suppression of endogenous RACK1 expression. Conclusion: These results suggest that RACK1 may have a functional significance as a regulatory cofactor of ABCB4 and is indispensable for the plasma membrane localization and translocation function of ABCB4.

The ATP-binding cassette transporter family C 2 (Abcc2) is a member of efflux transporters involved in the biliary excretion of organic anions from hepatocytes. Posttranslational regulation of Abcc2 has been implicated, although the molecular mechanism is not fully understood. In the present study, we performed yeast two-hybrid screening to identify novel protein(s) that particularly interacts with the linker region of Abcc2 located between the NH2-terminal nucleotide binding domain and the last membrane-spanning domain. The screening resulted in the identification of a series of small ubiquitin-like modifier (SUMO)-related enzymes and their substrates. In yeast experiments, all of these interactions were abolished by substituting the putative SUMO consensus site in the linker region (IKKE) in Abcc2 to IRKE. In vitro SUMOylation experiments confirmed that the Abcc2 linker was a substrate of Ubc9-mediated SUMOylation. It was also found that the IKKE sequence is the target of SUMOylation, since a mutant with IKKE is substituted by IRKE was not SUMOylated. Furthermore, we demonstrated for the first time that Abcc2, endogenously expressed in rat hepatoma-derived McARH7777 cells, is SUMOylated. Suppression of endogenous Ubc9 by small interfering RNA resulted in a selective 30% reduction in Abcc2 protein expression in the postnuclear supernatant, whereas subcellular localization of Abcc2 confirmed by semiquantitative immunofluorescence analysis was minimally affected. This is the first demonstration showing the regulation of ABC transporter expression by SUMOylation.

Objective Previous studies have suggested an association between gout susceptibility and common dysfunctional variants in ATP-binding cassette transporter subfamily G member 2/breast cancer resistance protein (ABCG2/BCRP), including rs72552713 (Q126X) and rs2231142 (Q141K). However, the association of rare ABCG2 variants with gout is unknown. Therefore, we investigated the effects of rare ABCG2 variants on gout susceptibility in this study. Methods We sequenced the exons of ABCG2 in 480 patients with gout and 480 healthy controls (Japanese males). We also performed functional analyses of non-synonymous variants of ABCG2 and analysed the correlation between urate transport function and scores from the protein prediction algorithms (Sorting Intolerant from Tolerant (SIFT) and Polymorphism Phenotyping v2 (PolyPhen-2)). Stratified association analyses and multivariate logistic regression analysis were performed to evaluate the effects of rare and common ABCG2 variants on gout susceptibility. Results We identified 3 common and 19 rare non-synonymous variants of ABCG2. SIFT scores were significantly correlated with the urate transport function, although some ABCG2 variants showed inconsistent scores. When the effects of common variants were removed by stratified association analysis, the rare variants of ABCG2 were associated with a significantly increased risk of gout (OR=3.2, p=6.4×10−3). Multivariate logistic regression analysis revealed that the size effect of these rare ABCG2 variants (OR=2.7, p=3.0×10−3) was similar to that of the common variants, Q126X (OR=3.4, p=3.2×10−6) and Q141K (OR=2.3, p=2.7×10−16). Conclusions This study revealed that multiple common and rare variants of ABCG2 are independently associated with gout. These results could support both the ‘Common Disease, Common Variant’ and ‘Common Disease, Multiple Rare Variant’ hypotheses for the association between ABCG2 and gout susceptibility.

Drug transfer into milk is of concern due to the unnecessary exposure of infants to drugs. Proposed prediction methods for such transfer assume only passive drug diffusion across the mammary epithelium. This study reorganized data from the literature to assess the contribution of carrier-mediated transport to drug transfer into milk, and to improve the predictability thereof. Milk-to-plasma drug concentration ratios (M/Ps) in humans were exhaustively collected from the literature and converted into observed unbound concentration ratios (M/Punbound,obs). The ratios were also predicted based on passive diffusion across the mammary epithelium (M/Punbound,pred). An in vitro transport assay was performed for selected drugs in breast cancer resistance protein (BCRP)-expressing cell monolayers. M/Punbound,obs and M/Punbound,pred values were compared for 166 drugs. M/Punbound,obs values were 1.5 times or more higher than M/Punbound,pred values for as many as 13 out of 16 known BCRP substrates, reconfirming BCRP as the predominant transporter contributing to secretory transfer of drugs into milk. Predictability of M/P values for selected BCRP substrates and non-substrates was improved by considering in vitro-evaluated BCRP-mediated transport relative to passive diffusion alone. The current analysis improved the predictability of drug transfer into milk, particularly for BCRP substrates, based on an exhaustive data overhaul followed by focused in vitro transport experimentation.

Drug transfer into milk is a general concern during lactation. So far, breast cancer resistance protein (Bcrp) is the only transporter known to be involved in this process, whereas participation of other transporters remains unclear. We investigated the importance of organic cation transporter (Oct) in drug transfer into milk in mice. The mammary glands of lactating versus nonlactating FVB strain mice revealed elevated mRNA levels of Oct1 and Bcrp, whereas Oct2 and Oct3 mRNA levels were decreased. Specific uptake of cimetidine, acyclovir, metformin, and terbutaline was observed in human embryonic kidney 293 cells transfected with murine Oct1 or Oct2. The milk-to-plasma concentration ratio (M/P) values of cimetidine and acyclovir were significantly decreased in Bcrp knockout and Oct1/2 double-knockout (DKO) mice compared with control FVB mice, whereas the M/P values of terbutaline and metformin were significantly decreased in Oct1/2 DKO mice alone. These are the first to suggest that Oct1 might be involved in secretory transfer of substrate drugs into milk.

Recent studies have revealed that osteocytes play multiple important physiological roles. To analyze osteocyte functions in detail, an in vitro experimental system for primary osteocytes would be useful. Unfortunately, osteocytes tend to dedifferentiate and acquire osteoblast-like features even when the cells are cultured in three-dimensional (3D) collagen gel. Therefore, it is desirable to establish osteocyte culture conditions that prevent dedifferentiation over longer periods. In this study, we obtained systematic information about the influence of culture conditions on osteocyte differentiation states. Fetal bovine serum (FBS) concentrations from 0.1 to 0.5 % in 3D culture matrix did not significantly influence the expression of osteocyte markers. On the other hand, addition of Matrigel to the culture matrix significantly enhanced the expression of Rankl and late osteocyte markers such as Sost and Fgf23. Matrigel addition also inhibited upregulation of Opg and early osteocyte markers such as Dmp1 and Gp38. These effects on osteocyte properties were maximal at a Matrigel culture matrix content of 50 %. Matrigel addition to the matrix also increased dendritic process extension by osteocytes. In addition, Matrigel addition significantly stimulated tartrate-resistant acid phosphatase activity in co-culture with bone marrow macrophages. Among the conditions tested, 50 % Matrigel and 0.2 % FBS in type I collagen matrix were optimal for culture of primary osteocytes.

The receptor activator of the NF-κB ligand (RANKL) is the central player in the regulation of osteoclastogenesis, and the quantity of RANKL presented to osteoclast precursors is an important factor determining the magnitude of osteoclast formation. Because osteoblastic cells are thought to be a major source of RANKL, the regulatory mechanisms of RANKL subcellular trafficking have been studied in osteoblastic cells. However, recent reports showed that osteocytes are a major source of RANKL presentation to osteoclast precursors, prompting a need to reinvestigate RANKL subcellular trafficking in osteocytes. Investigation of molecular mechanisms in detail needs well-designed in vitro experimental systems. Thus, we developed a novel co-culture system of osteoclast precursors and osteocytes embedded in collagen gel. Experiments using this model revealed that osteocytic RANKL is provided as a membrane-bound form to osteoclast precursors through osteocyte dendritic processes and that the contribution of soluble RANKL to the osteoclastogenesis supported by osteocytes is minor. Moreover, the regulation of RANKL subcellular trafficking, such as OPG-mediated transport of newly synthesized RANKL molecules to lysosomal storage compartments, and the release of RANKL to the cell surface upon stimulation with RANK are confirmed to be functional in osteocytes. These results provide a novel understanding of the regulation of osteoclastogenesis.

ABCG2, also known as BCRP, is a high-capacity urate exporter, the dysfunction of which raises gout/hyperuricemia risk. Generally, hyperuricemia has been classified into urate 'overproduction type' and/or 'underexcretion type' based solely on renal urate excretion, without considering an extra-renal pathway. Here we show that decreased extra-renal urate excretion caused by ABCG2 dysfunction is a common mechanism of hyperuricemia. Clinical parameters, including urinary urate excretion, are examined in 644 male outpatients with hyperuricemia. Paradoxically, ABCG2 export dysfunction significantly increases urinary urate excretion and risk ratio of urate overproduction. Abcg2-knockout mice show increased serum uric acid levels and renal urate excretion, and decreased intestinal urate excretion. Together with high ABCG2 expression in extra-renal tissues, our data suggest that the 'overproduction type' in the current concept of hyperuricemia be renamed 'renal overload type', which consists of two subtypes—'extra-renal urate underexcretion' and genuine 'urate overproduction'—providing a new concept valuable for the treatment of hyperuricemia and gout., Hyperuricemia, or gout, is thought to arise either from urate overproduction or from decreased renal excretion of urate. Ichida et al. show that the extra-renal excretion of urate also has a role in the pathogenesis of hyperuricemia, and propose a new classification for patients with this disease.

In a previous report, we identified the receptor for activated C-kinase 1 (RACK1) as a positive regulator of the cellular localization and expression of ATP-binding cassette B4, a phosphatidylcholine translocator expressed on the bile canalicular membrane. In the present study, we focused on the role of RACK1 on ATP-binding cassette G2 (ABCG2), which is responsible for the cellular extrusion of compounds including antitumor drugs. Protein expression of ABCG2 was up-regulated by RACK1 overexpression, although mRNA expression of ABCG2 was not dependent on RACK1. The effect of RACK1 on the expression of ABCG2 on the cell surface was confirmed by the uptake of [(3)H]estrone sulfate, an ABCG2 substrate, into isolated membrane vesicles. The expression of RACK1 affected cellular resistance to mitoxantrone, an anticancer drug excreted by ABCG2, and this effect of RACK1 was abolished in the presence of fumitremorgin C, a selective ABCG2 inhibitor. These results suggest that RACK1 has functional significance as a regulatory cofactor of ABCG2 and is indispensable for the cell surface expression and excretion function of ABCG2. The precise mechanism for RACK1-dependent expression of ABCG2 remains to be clarified, because the results of N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132) and chloroquine treatment and those of metabolic labeling experiments did not give us clear evidence whether the reduction of ABCG2 expression in RACK1-knocked down cells may be caused by the suppression of ABCG2 protein synthesis or by acceleration of its degradation.

Gout based on hyperuricemia is a common disease with a genetic predisposition, which causes acute arthritis. The ABCG2/BCRP gene, located in a gout-susceptibility locus on chromosome 4q, has been identified by recent genome-wide association studies of serum uric acid concentrations and gout. Urate transport assays demonstrated that ABCG2 is a high-capacity urate secretion transporter. Sequencing of the ABCG2 gene in 90 hyperuricemia patients revealed several nonfunctional ABCG2 mutations, including Q126X. Quantitative trait locus analysis of 739 individuals showed that a common dysfunctional variant of ABCG2, Q141K, increases serum uric acid. Q126X is assigned to the different disease haplotype from Q141K and increases gout risk, conferring an odds ratio of 5.97. Furthermore, 10% of gout patients (16 out of 159 cases) had genotype combinations resulting in more than 75% reduction of ABCG2 function (odds ratio, 25.8). Our findings indicate that nonfunctional variants of ABCG2 essentially block gut and renal urate excretion and cause gout.