Masashi Honma

Serum metabolomics reveals γ-glutamyl dipeptides as biomarkers for discrimination among different forms of liver disease

BACKGROUND & AIMS We applied a metabolome profiling approach to serum samples obtained from patients with different liver diseases, to discover noninvasive and reliable biomarkers for rapid-screening diagnosis of liver diseases. METHODS Using capillary electrophoresis and liquid chromatography mass spectrometry, we analyzed low molecular weight metabolites in a total of 248 serum samples obtained from patients with nine types of liver disease and healthy controls. RESULTS We found that γ-glutamyl dipeptides, which were biosynthesized through a reaction with γ-glutamylcysteine synthetase, were indicative of the production of reduced glutathione, and that measurement of their levels could distinguish among different liver diseases. Multiple logistic regression models facilitated the discrimination between specific and other liver diseases and yielded high areas under receiver-operating characteristic curves. The area under the curve values in training and independent validation data were 0.952 and 0.967 in healthy controls, 0.817 and 0.849 in drug-induced liver injury, 0.754 and 0.763 in asymptomatic hepatitis B virus infection, 0.820 and 0.762 in chronic hepatitis B, 0.972 and 0.895 in hepatitis C with persistently normal alanine transaminase, 0.917 and 0.707 in chronic hepatitis C, 0.803 and 0.993 in cirrhosis type C, and 0.762 and 0.803 in hepatocellular carcinoma, respectively. Several γ-glutamyl dipeptides also manifested potential for differentiating between nonalcoholic steatohepatitis and simple steatosis. CONCLUSIONS γ-Glutamyl dipeptides are novel biomarkers for liver diseases, and varying levels of individual or groups of these peptides have the power to discriminate among different forms of hepatic disease.

RANKL subcellular trafficking and regulatory mechanisms in 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.

Function of OPG as a traffic regulator for RANKL is crucial for controlled osteoclastogenesis

The amount of the receptor activator of NF‐κB ligand (RANKL) on the osteoblastic cell surface is considered to determine the magnitude of the signal input to osteoclast precursors and the degree of osteoclastogenesis. Previously, we have shown that RANKL is localized predominantly in lysosomal organelles, but little is found on the osteoblastic cell surface, and consequently, the regulated subcellular trafficking of RANKL in osteoblastic cells is important for controlled osteoclastogenesis. Here we have examined the involvement of osteoprotegerin (OPG), which is currently recognized as a decoy receptor for RANKL, in the regulation of RANKL behavior. It was suggested that OPG already makes a complex with RANKL in the Golgi apparatus and that the complex formation is necessary for RANKL sorting to the secretory lysosomes. It was also shown that each structural domain of OPG is indispensable for exerting OPG function as a traffic regulator. In particular, the latter domains of OPG, whose physiologic functions have been unclear, were indicated to sort RANKL molecules to lysosomes from the Golgi apparatus. In addition, the overexpression of RANK‐OPG chimeric protein, which retained OPG function as a decoy receptor but lost the function as a traffic regulator, inhibited endogenous OPG function as a traffic regulator selectively in osteoblastic cells and resulted in the upregulation of osteoclastogenic ability despite the increased number of decoy receptor molecules. Conclusively, OPG function as a traffic regulator for RANKL is crucial for regulating osteoclastogenesis at least as well as that as a decoy receptor.

Regulatory Mechanisms of RANKL Presentation to Osteoclast Precursors

It is important to understand the molecular mechanisms regulating osteoclast formation, as excess activation of osteoclasts is associated with various osteopenic disorders. Receptor activator of nuclear factor kappa B (RANKL) is a central player in osteoclastogenesis. Recent findings suggest that osteocytes are the major supplier of RANKL to osteoclast precursors. It has also been suggested that osteocyte cell death upregulates the RANKL/osteoprotegerin (OPG) ratio in viable osteocytes adjacent to apoptotic osteocytes in areas of bone microdamage, thus, contributing to localized osteoclast formation. Indeed, viable osteocytes can provide RANKL through direct interactions with osteoclast precursors at osteocyte dendritic processes. In addition, OPG tightly regulates RANKL cell surface presentation in osteocytes, which contributes to the inhibition of RANKL signaling, as well as the decoy receptor function of OPG. By contrast, the physiological role of RANKL in osteoblasts is yet to be clarified, although similar mechanisms of regulation are observed in both osteocytes and osteoblasts.

Off-Target Serine/Threonine Kinase 10 Inhibition by Erlotinib Enhances Lymphocytic Activity Leading to Severe Skin Disorders

Skin disorders are among the most common adverse events related to treatment with epidermal growth factor receptor (EGFR) kinase inhibitors, and of these, erlotinib is known to cause more frequent and severe skin disease than other agents in this class. Although previous reports have shown that cutaneous manifestations are triggered by the inhibition of multiple EGFR-related homeostatic functions of the skin, this mechanism alone cannot explain the differences in frequency and severity of skin disorders caused by different kinase inhibitors. In this study, we focused on the relationship between the off-target kinase inhibition and aggravation of skin disorders. Based on calculations using reported Kd values and plasma drug concentrations, serine/threonine kinase 10 (STK10) and Ste20-like kinase (SLK) were selected as candidates preferentially inhibited by erlotinib over gefitinib. In vitro experiments confirmed that STK10 and SLK kinase activity are inhibited by erlotinib at clinical concentrations, whereas only STK10 is slightly inhibited by gefitinib. It was also shown that erlotinib up-regulated lymphocytic responses such as interleukin (IL)-2 secretion and cell migration at clinical concentrations, whereas gefitinib did not affect lymphocyte activity. Moreover, small interfering RNA experiments revealed that STK10 plays a major role in up-regulation of the lymphocytic responses induced by erlotinib treatment. Finally, the role of erlotinib-induced lymphocyte activation was assessed in vivo using irritant hypersensitivity models. The results indicated that erlotinib aggravates cutaneous inflammatory reactions through the activation of lymphocytic responses such as IL-2 secretion and cell migration. These results demonstrated that off-target inhibition of STK10 by erlotinib enhances lymphocytic responses, which lead to the aggravation of skin inflammation.

Quantitative Prediction of in Vivo Profiles of CYP3A4 Induction in Humans from in Vitro Results with a Reporter Gene Assay

Although primary human hepatocytes are commonly used for induction studies, the evaluation method is associated with several problems. More recently, a reporter gene assay has been suggested to be an alternative, although the contribution of only transfected nuclear receptors can be evaluated. The aim of the present study was to establish a method by which the extent of in vivo CYP3A4 induction in humans can be quantitatively predicted based on in vitro results with a reporter gene assay. From previous reports, we calculated in vivo induction ratios (Rin vivo) caused by prototypical inducers based on the alterations in the hepatic intrinsic clearance of probe drugs. Next, we derived equations by which these Rin vivo values can be predicted from the results of a reporter gene assay. To use the data obtained from a reporter gene assay, rifampicin was used as a reference drug. The correction coefficient (CC), which is used to quantitatively correlate the activity of inducers between in vitro and in vivo situations, was calculated by comparing the predicted data with the observed Rin vivo values for rifampicin. With the calculated CC value, good correlations were found between the predicted and observed Rin vivo values for other inducers such as phenobarbital, phenytoin, and omeprazole. Taken together, with the equations derived in the present study, we have been able to predict the extent of in vivo induction of human CYP3A4 by inducers in a time-dependent and quantitative manner from in vitro data.

Rab27a and Rab27b are involved in stimulation-dependent RANKL release from secretory lysosomes in osteoblastic cells.

The quantity of the receptor activator of NF‐κB ligand (RANKL) expressed at the cell surface of osteoblastic cells is an important factor regulating osteoclast activation. Previously, RANKL was found to be localized to secretory lysosomes in osteoblastic cells and to translocate to the cell surface in response to stimulation with RANK‐Fc‐conjugated beads. However, the in vivo significance of stimulation‐dependent RANKL release has not been elucidated. In this study we show that small GTPases Rab27a and Rab27b are involved in the stimulation‐dependent RANKL release pathway in osteoblastic cells. Suppression of either Rab27a or Rab27b resulted in a marked reduction in RANKL release after stimulation. Slp4‐a, Slp5, and Munc13‐4 acted as effector molecules that coordinated Rab27a/b activity in this pathway. Suppression of Rab27a/b or these effector molecules did not inhibit accumulation of RANKL in lysosomal vesicles around the stimulated sites but did inhibit the fusion of these vesicles to the plasma membrane. In osteoblastic cells, suppression of the effector molecules resulted in reduced osteoclastogenic ability. Furthermore, Jinx mice, which lack a functional Munc13‐4 gene, exhibited a phenotype characterized by increased bone volume near the tibial metaphysis caused by low bone resorptive activity. In conclusion, stimulation‐dependent RANKL release is mediated by Rab27a/b and their effector molecules, and this mechanism may be important for osteoclast activation in vivo.

Vps33a Mediates RANKL Storage in Secretory Lysosomes in Osteoblastic Cells

Previous studies have indicated that the amount of RANKL expressed on the cell surface of osteoblasts or bone marrow stromal cells (BMSCs) is considered an important factor determining the extent of osteoclast activation. However, subcellular trafficking of RANKL and its regulatory mechanisms in osteoblastic cells is still unclear. In this study, we showed that RANKL is predominantly localized in lysosomal organelles, but little is found on the cell surface of osteoblastic cells. We also showed that RANKL is relocated to the plasma membrane in response to stimulation with RANK‐Fc–coated beads, indicating that the lysosomal organelles where RANKL is localized function as secretory lysosomes. In addition, using a protein pull‐down method, we identified vacuolar protein sorting (Vps)33a as interacting with the cytoplasmic tail of RANKL. Furthermore, knockdown of Vps33a expression reduced the lysosomal storage of RANKL and caused the accumulation of newly synthesized RANKL in the Golgi apparatus, indicating that Vps33a is involved in transporting RANKL from the Golgi apparatus to secretory lysosomes. We also showed that suppression of Vps33a affects the cell surface expression level of RANKL and disrupts the regulated behavior of RANKL. These results suggest that RANKL storage in secretory lysosomes is important to control osteoclast activation and to maintain bone homeostasis.

Organic Cation Transporter/Solute Carrier Family 22a is Involved in Drug Transfer into Milk in Mice

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.

Metabolic profiling to identify potential serum biomarkers for gastric ulceration induced by nonsteroid anti-inflammatory drugs.

Nonsteroid anti-inflammatory drugs (NSAIDs) are among the most frequently prescribed drugs currently available. The most frequently reported serious side effects associated with NSAIDs are gastric mucosal ulceration and gastric hemorrhage. Presently, these side effects are only detectable by endoscopy, however, and no biomarkers have yet been identified. The ability to identify serum biomarkers would likely improve the safety of NSAID use. In this study we performed capillary electrophoresis-mass spectrometry (CE-MS)-based metabolomic profiling in stomach extract and serum from rats administered NSAIDs. Results showed drug-induced decreases in levels of citrate, cis-aconitate, succinate, 3-hydroxy butanoic acid, o-acetyl carnitine, proline, and hydroxyproline. We consider that these changes are due to NSAID-induced depression of mitochondrial function and activation of collagenase by lesions in the stomach. In addition, four of these changes in metabolite levels in the stomach were significantly correlated with changes in the serum. While further study is needed to clarify the mechanism of change in the level of these biomarkers, limitation of indications, and extrapolation to humans, these new serum biomarker candidates of gastric injury may be useful in the monitoring of NSAID-induced tissue damage.