Glycocapture-assisted global quantitative proteomics (gagQP) reveals multiorgan responses in serum toxicoproteome.

Blood is an ideal window for viewing our health and disease status. Because blood circulates throughout the entire body and carries secreted, shed, and excreted signature proteins from every organ and tissue type, it is thus possible to use the blood proteome to achieve a comprehensive assessment of multiple-organ physiology and pathology. To date, the blood proteome has been frequently examined for diseases of individual organs; studies on compound insults impacting multiple organs are, however, elusive. We believe that a characterization of peripheral blood for organ-specific proteins affords a powerful strategy to allow early detection, staging, and monitoring of diseases and their treatments at a whole-body level. In this paper we test this hypothesis by examining a mouse model of acetaminophen (APAP)-induced hepatic and extra-hepatic toxicity. We used a glycocapture-assisted global quantitative proteomics (gagQP) approach to study serum proteins and validated our results using Western blot. We discovered in mouse sera both hepatic and extra-hepatic organ-specific proteins. From our validation, it was determined that selected organ-specific proteins had changed their blood concentration during the course of toxicity development and recovery. Interestingly, the peak responding time of proteins specific to different organs varied in a time-course study. The collected molecular information shed light on a complex, dynamic, yet interweaving, multiorgan-enrolled APAP toxicity. The developed technique as well as the identified protein markers is translational to human studies. We hope our work can broaden the utility of blood proteomics in diagnosis and research of the whole-body response to pathogenic cues.

Circulating microRNAs, potential biomarkers for drug-induced liver injury.

Drug-induced liver injury is a frequent side effect of many drugs, constitutes a significant threat to patient health and has an enormous economic impact on health care expenditures. Numerous efforts have been made to identify reliable and predictive markers to detect the early signs of drug-induced injury to the liver, one of the most vulnerable organs in the body. These studies have, however, not delivered any more informative candidates than the serum aminotransferase markers that have been available for approximately 30 years. Using acetaminophen overdose-induced liver injury in the mouse as a model system, we have observed highly significant differences in the spectrum and levels of microRNAs in both liver tissues and in plasma between control and overdosed animals. Based on our survey of microRNA expression among normal tissues, some of the microRNAs, like messenger RNAs, display restricted tissue distributions. A number of elevated circulating microRNAs in plasma collected from acetaminophen-overdosed animals are highly expressed in the liver. We have demonstrated that specific microRNA species, such as mir-122 and mir-192, both are enriched in the liver tissue and exhibit dose- and exposure duration-dependent changes in the plasma that parallel serum aminotransferase levels and the histopathology of liver degeneration, but their changes can be detected significantly earlier. These findings suggest the potential of using specific circulating microRNAs as sensitive and informative biomarkers for drug-induced liver injury.

Quantitative liver-specific protein fingerprint in blood: a signature for hepatotoxicity.

We discuss here a new approach to detecting hepatotoxicity by employing concentration changes of liver-specific blood proteins during disease progression. These proteins are capable of assessing the behaviors of their cognate liver biological networks for toxicity or disease perturbations. Blood biomarkers are highly desirable diagnostics as blood is easily accessible and baths virtually all organs. Fifteen liver-specific blood proteins were identified as markers of acetaminophen (APAP)-induced hepatotoxicity using three proteomic technologies: label-free antibody microarrays, quantitative immunoblotting, and targeted iTRAQ mass spectrometry. Liver-specific blood proteins produced a toxicity signature of eleven elevated and four attenuated blood protein levels. These blood protein perturbations begin to provide a systems view of key mechanistic features of APAP-induced liver injury relating to glutathione and S-adenosyl-L-methionine (SAMe) depletion, mitochondrial dysfunction, and liver responses to the stress. Two markers, elevated membrane-bound catechol-O-methyltransferase (MB-COMT) and attenuated retinol binding protein 4 (RBP4), report hepatic injury significantly earlier than the current gold standard liver biomarker, alanine transaminase (ALT). These biomarkers were perturbed prior to onset of irreversible liver injury. Ideal markers should be applicable for both rodent model studies and human clinical trials. Five of these mouse liver-specific blood markers had human orthologs that were also found to be responsive to human hepatotoxicity. This panel of liver-specific proteins has the potential to effectively identify the early toxicity onset, the nature and extent of liver injury and report on some of the APAP-perturbed liver networks.

Hyaluronan-CD44 interaction with neural Wiskott-Aldrich syndrome protein (N-WASP) promotes actin polymerization and ErbB2 activation leading to beta-catenin nuclear translocation, transcriptional up-regulation, and cell migration in ovarian tumor cells.

In this study we have investigated the interaction of hyaluronan (HA) and CD44 with the neuronal Wiskott-Aldrich syndrome protein (N-WASP) in regulating actin polymerization and ErbB2/beta-catenin signaling in human ovarian tumor cells (SK-OV-3.ipl cells). Biochemical and immunological analyses indicate that N-WASP is expressed in SK-OV-3.ipl cells and that the binding of HA stimulates N-WASP association with CD44 and Arp2/Arp3 leading to filamentous actin formation and ovarian tumor cell migration. In addition, HA binding promotes CD44-N-WASP association with ErbB2 and activates ErbB2 kinase activity that in turn increases phosphorylation of the cytoskeletal protein, beta-catenin. Subsequently, phosphorylated beta-catenin is transported into the nucleus leading to beta-catenin-mediated TCF/LEF-transcriptional co-activation. Because HA-induced beta-catenin phosphorylation, nuclear translocation, and TCF/LEF transcriptional activation is effectively blocked by the ErbB2 inhibitor, AG825, we conclude that HA/CD44-N-WASP-associated ErbB2 activation is required for beta-catenin-mediated signaling events. Transfection of SK-OV-3.ipl cells with N-WASP-VCA (verpolin homology, cofilin homology, and acidic domain) fragment cDNA not only blocks HA/CD44-induced N-WASP-Arp2/3 complex formation but also inhibits actin polymerization/F-actin assembly and tumor cell migration. Overexpression of the N-WASP-VCA domain also significantly reduces HA-induced ErbB2 recruitment to CD44, diminishes beta-catenin phosphorylation/nuclear translocation, and abrogates TCF/LEF-specific transcriptional co-activation by beta-catenin. Taken together, our findings strongly suggest that N-WASP plays a pivotal role in regulating HA-mediated CD44-ErbB2 interaction, beta-catenin signaling, and actin cytoskeleton functions that are required for tumor-specific behaviors and ovarian cancer progression.

Hyaluronan-CD44 interaction stimulates Rac1 signaling and PKN gamma kinase activation leading to cytoskeleton function and cell migration in astrocytes.

Both hyaluronan [HA, the major glycosaminoglycans in the extracellular matrix (ECM)] and CD44 (a primary HA receptor) are associated with astrocyte activation and tissue repair following central nervous system (CNS) injury. In this study we investigated the question of whether HA-CD44 interaction influences astrocyte signaling and migration. Our data indicated that HA binding to the cultured astrocytes stimulated Rac1 signaling and cytoskeleton-mediated migration. To determine the cellular and molecular basis of these events, we focused on PKN gamma, a Rac1-activated serine/threonine kinase in astrocytes. We determined that HA binding to astrocytes stimulated Rac1-dependent PKN gamma kinase activity which, in turn, up-regulated the phosphorylation of the cytoskeletal protein, cortactin, and attenuated the ability of cortactin to cross-link F-actin. Further analyses indicated that the N-terminal antiparallel coiled-coil (ACC) domains of PKN gamma interacted with Rac1, and transfection of astrocytes with PKN gamma-ACCcDNA inhibited PKN gamma activity. Over-expression of the PKN gamma-ACC domain also functions as a dominant-negative mutant to block HA/CD44-mediated PKN gamma activation of cortactin and astrocyte migration. Taken together, these findings strongly suggest that hyaluronan/CD44 interaction with Rac1-PKN gamma plays a pivotal role in cytoskeleton activation and astrocyte migration. These newly discovered HA/CD44-induced astrocyte function may provide important insight into novel therapeutic treatments for tissue repair following CNS injury.

Hyaluronan-CD44 interaction with leukemia-associated RhoGEF and epidermal growth factor receptor promotes Rho/Ras co-activation, phospholipase C epsilon-Ca2+ signaling, and cytoskeleton modification in head and neck squamous cell carcinoma cells.

In this study we have examined the interaction of CD44 (a major hyaluronan (HA) receptor) with a RhoA-specific guanine nucleotide exchange factor (leukemia-associated RhoGEF (LARG)) in human head and neck squamous carcinoma cells (HNSCC-HSC-3 cell line). Immunoprecipitation and immunoblot analyses indicate that CD44 and the LARG protein are expressed in HSC-3 cells and that these two proteins are physically associated as a complex. HA-CD44 binding induces LARG-specific RhoA signaling and phospholipase C epsilon (PLC epsilon) activity. In particular, the activation of RhoA-PLC epsilon by HA stimulates inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, and the up-regulation of Ca2+/calmodulin-dependent kinase II (CaMKII), leading to phosphorylation of the cytoskeletal protein, filamin. The phosphorylation of filamin reduces its interaction with filamentous actin, promoting tumor cell migration. The CD44-LARG complex also interacts with the EGF receptor (EGFR). Most importantly, the binding of HA to the CD44-LARG-EGFR complex activates the EGFR receptor kinase, which in turn promotes Ras-mediated stimulation of a downstream kinase cascade including the Raf-1 and ERK pathways leading to HNSCC cell growth. Using a recombinant fragment of LARG (the LARG-PDZ domain) and a binding assay, we have determined that the LARG-PDZ domain serves as a direct linker between CD44 and EGFR. Transfection of the HSC-3 cells with LARG-PDZcDNA significantly reduces LARG association with CD44 and EGFR. Overexpression of the LARG-PDZ domain also functions as a dominant-negative mutant (similar to the PLC/Ca2+-calmodulin-dependent kinase II (CaMKII) and EGFR/MAPK inhibitor effects) to block HA/CD44-mediated signaling events (e.g. EGFR kinase activation, Ras/RhoA co-activation, Raf-ERK signaling, PLC epsilon-mediated inositol 1,4,5-triphosphate production, intracellular Ca2+ mobilization, CaMKII activity, filamin phosphorylation, and filamin-actin binding) and to abrogate tumor cell growth/migration. Taken together, our findings suggest that CD44 interaction with LARG and EGFR plays a pivotal role in Rho/Ras co-activation, PLC epsilon-Ca2+ signaling, and Raf/ERK up-regulation required for CaMKII-mediated cytoskeleton function and in head and neck squamous cell carcinoma progression.