Breast tumor kinase/protein tyrosine kinase 6 (Brk/PTK6) activity in normal and neoplastic biliary epithelia.

BACKGROUND & AIMS: Breast tumor kinase (BRK) augments proliferation and promotes cell survival in breast cancers via interactions with SH2 and SH3 ligand-containing proteins, such as receptor tyrosine kinases (RTK; e.g. EGFR, ErbB2/neu). Since RTK contribute to cholangiocarcinoma (CC) evolution we probed BRK protein expression and function in normal and CC livers. METHODS: Immunohistochemical staining of normal livers and CC (n=93) in a tissue microarray and three CC and an immortalized human cholangiocyte cell lines (real-time PCR, Western blotting, siRNA) were used to study the functional relationships between BRK, EGFR, ErbB2, SAM68, and SPRR2a. RESULTS: BRK protein was expressed in normal human intrahepatic bile ducts; all CC cell lines and a majority of CC showed strong BRK protein expression. Multiplex immunostaining/tissue cytometry and immunoprecipitation studies showed: 1) BRK co-localized with EGFR and ErbB2/neu; 2) BRK(high)/EGFR(high)-co-expressing CC cells had significantly higher Ki67 labeling and; 3) stronger BRK protein expression was seen in perihilar and distal CC than intrahepatic CC and directly correlated with CC differentiation. In cell lines, BRK expression augmented proliferation in response to exogenous EGF, whereas BRK siRNA significantly reduced growth. The SH3 ligand-containing, SPRR2A activated pTyr342 BRK, which in turn, phosphorylated SAM68, causing nuclear localization and increased cell proliferation similar to observations in breast cancers. CONCLUSION: BRK expression in a majority of CC can interact with RTK, augmenting growth and interfering with proliferation inhibitors (SAM68). Therapeutically targeting BRK function (in addition to RTK) should be of benefit for CC treatment.

Small proline rich protein 2a in benign and malignant liver disease.

UNLABELLED: STAT3-driven expression of small proline rich protein 2a (SPRR2a), which acts as an src homology 3 (SH3) domain ligand, induces biliary epithelial cell (BEC) epithelial-mesenchymal transition (EMT), which, in turn, promotes wound healing. SPRR2a also quenches free radicals and protects against oxidative stress and DNA damage in nonneoplastic BEC. Sprr2a-induced EMT also increases local invasiveness of cholangiocarcinomas (CC), but prevents metastases. Understanding SPRR2a regulation of EMT has potential for therapeutic targeting in both benign and malignant liver disease. Molecular mechanisms responsible for SPRR2a-induced EMT were characterized, in vitro, and then evidence for utilization of these pathways was sought in human intrahepatic CC, in vivo, using multiplex labeling and software-assisted morphometric analysis. SPRR2a complexes with ZEB1 and CtBP on the microRNA (miR)-200c/141 promoter resulting in synergic suppression of miR-200c/141 transcription, which is required for maintenance of the BEC epithelial phenotype. SPRR2a induction promotes dephosphorylation and nuclear translocation of the SH3-domain containing protein GRB2 and an SH3-domain ligand in ZEB1 is required for SPRR2a-induced synergic suppression of miR-200c/141. Multiplex protein labeling of CC and morphometric analyses showed: 1) up-regulation of ZEB-1, and 2) down-regulation of CK19 in intrahepatic CC compared to nonneoplastic BEC, consistent with previous CC proteomic studies showing EMT during cholangiocarcinogenesis. CONCLUSION: SPRR2a modulates ZEB-1 signaling by way of miR-200c/141-associated EMT through SH3-domain networks and contributes to benign and malignant BEC wound-healing responses.

SPRR2A expression in cholangiocarcinoma increases local tumor invasiveness but prevents metastasis.

Cholangiocarcinoma morbidity and mortality is attributable to local invasiveness and regional lymph node and distant organ metastasis. Cholangiocarcinoma progression follows a series of sequential events that resemble wound healing reactions: local invasion resembles the epithelial migration phase involving epithelial-mesenchymal transition (EMT); colonization at distant sites resembles epithelial restitution seen during the reverse process, mesenchymal-epithelial transition (MET). In this study we compare the in vivo local and metastatic growth potential of cholangiocarcinoma cell lines with respect to expression of a novel pSTAT3-dependent, biliary epithelial cell wound healing protein, small proline-rich protein 2A (SPRR2A). SPRR2A has been associated with local aggressiveness, but decreased metastatic capabilities in other cancers. Stable SPRR2A transfection into two cholangiocarcinoma cell lines (SG231 and HuCCT-1), previously shown by us to induce permanent EMT, resulted in local aggressiveness but an inability to form metastases. In contrast, SPRR2A-negative epithelial control cells showed relatively poor local aggressiveness, but readily formed metastatic tumors. Post-intrasplenic injection cell tracking showed that: (a) mesenchymal (SPRR2A+) cells were not trapped in the liver, but were rapidly cleared through mesenteric lymph nodes and did not form metastases; whereas (b) epithelial (SPRR2A-) controls were primarily entrapped within MUC-1-associated liver "micro-infarcts" that later evolved into metastatic colonies. SPRR2A-associated tumor behavior was mimicked by MUC1 shRNA, which induced EMT and, like SPRR2A+ cells, showed reduced metastatic capabilities. Cholangiocarcinoma local invasion involves EMT processes, whereas MET and MUC1 expression promote metastasis. A better understanding of disease progression should help target treatment for this deadly neoplasm.

Gut bacteria drive Kupffer cell expansion via MAMP-mediated ICAM-1 induction on sinusoidal endothelium and influence preservation-reperfusion injury after orthotopic liver transplantation.

Bacteria in the gut microbiome shed microbial-associated molecule patterns (MAMPs) into the portal venous circulation, where they augment various aspects of systemic immunity via low-level stimulation. Because the liver is immediately downstream of the intestines, we proposed that gut-derived MAMPs shape liver immunity and affect Kupffer cell (KC) phenotype. Germ-free (GF), antibiotic-treated (AVMN), and conventional (CL) mice were used to study KC development, function, and response to the significant stress of cold storage, reperfusion, and orthotopic transplantation. We found that a cocktail of physiologically active MAMPs translocate into the portal circulation, with flagellin (Toll-like receptor 5 ligand) being the most plentiful and capable of promoting hepatic monocyte influx in GF mice. In MAMP-deficient GF or AVMN livers, KCs are lower in numbers, have higher phagocytic activity, and have lower major histocompatibility complex II expression. MAMP-containing CL livers harbor significantly increased KC numbers via induction of intercellular adhesion molecule 1 on liver sinusoidal endothelium. These CL KCs have a primed yet expected phenotype, with increased major histocompatibility complex class II and lower phagocytic activity that increases susceptibility to liver preservation/reperfusion injury after orthotopic transplantation. The KC number, functional activity, and maturational status are directly related to the concentration of gut-derived MAMPs and can be significantly reduced by broad-spectrum antibiotics, thereby affecting susceptibility to injury.

Preexisting epithelial diversity in normal human livers: a tissue-tethered cytometric analysis in portal/periportal epithelial cells.

UNLABELLED: Routine light microscopy identifies two distinct epithelial cell populations in normal human livers: hepatocytes and biliary epithelial cells (BECs). Considerable epithelial diversity, however, arises during disease states when a variety of hepatocyte-BEC hybrid cells appear. This has been attributed to activation and differentiation of putative hepatic progenitor cells (HPC) residing in the canals of Hering and/or metaplasia of preexisting mature epithelial cells. A novel analytic approach consisting of multiplex labeling, high-resolution whole-slide imaging (WSI), and automated image analysis was used to determine if more complex epithelial cell phenotypes preexist in normal adult human livers, which might provide an alternative explanation for disease-induced epithelial diversity. "Virtually digested" WSI enabled quantitative cytometric analyses of individual cells displayed in a variety of formats (e.g., scatterplots) while still tethered to the WSI and tissue structure. We employed biomarkers specifically associated with mature epithelial forms (HNF4α for hepatocytes, CK19 and HNF1ß for BEC) and explored for the presence of cells with hybrid biomarker phenotypes. The results showed abundant hybrid cells in portal bile duct BEC, canals of Hering, and immediate periportal hepatocytes. These bipotential cells likely serve as a reservoir for the epithelial diversity of ductular reactions, appearance of hepatocytes in bile ducts, and the rapid and fluid transition of BEC to hepatocytes, and vice versa. CONCLUSION: Novel imaging and computational tools enable increased information extraction from tissue samples and quantify the considerable preexistent hybrid epithelial diversity in normal human liver. This computationally enabled tissue analysis approach offers much broader potential beyond the results presented here.

SPRR2A enhances p53 deacetylation through HDAC1 and down regulates p21 promoter activity.

BACKGROUND: Small proline rich protein (SPRR) 2A is one of 14 SPRR genes that encodes for a skin cross-linking protein, which confers structural integrity to the cornified keratinocyte cell envelope. New evidence, however, shows that SPRR2A is also a critical stress and wound repair modulator: it enables a variety of barrier epithelia to transiently acquire mesenchymal characteristics (EMT) and simultaneously quench reactive oxygen species during wound repair responses. p53 is also widely recognized as the node in cellular stress responses that inhibits EMT and triggers cell-cycle arrest, apoptosis, and cellular senescence. Since some p53-directed processes would seem to impede wound repair of barrier epithelia, we hypothesized that SPRR2A up regulation might counteract these effects and enable/promote wound repair under stressful environmental conditions. RESULTS: Using a well characterized cholangiocarcinoma cell line we show that levels of SPRR2A expression, similar to that seen during stressful biliary wound repair responses, disrupts acetylation and subsequent p53 transcriptional activity. p53 deacetylation is accomplished via two distinct, but possibly related, mechanisms: 1) a reduction of p300 acetylation, thereby interfering with p300-p53 binding and subsequent p300 acetylation of K382 in p53; and 2) an increase in histone deacetylase 1 (HDAC1) mRNA and protein expression. The p300 CH3 domain is essential for both the autoacetylation of p300 and transference of the acetyl group to p53 and HDAC1 is a component of several non-p300 complexes that enhance p53 deacetylation, ubiquitination, and proteosomal degradation. HDAC1 can also bind the p300-CH3 domain, regulating p300 acetylation and interfering with p300 mediated p53 acetylation. The importance of this pathway is illustrated by showing complete restoration of p53 acetylation and partial restoration of p300 acetylation by treating SPRR2A expressing cells with HDAC1 siRNA. CONCLUSION: Up-regulation of SPRR2A, similar to that seen during barrier epithelia wound repair responses reduces p53 acetylation by interfering with p300-p53 interactions and by increasing HDAC1 expression. SPRR2A, therefore, functions as a suppressor of p53-dependent transcriptional activity, which otherwise might impede cellular processes needed for epithelial wound repair responses such as EMT.

Cooperation of p300 and PCAF in the control of microRNA 200c/141 transcription and epithelial characteristics.

Epithelial to mesenchymal transition (EMT) not only occurs during embryonic development and in response to injury, but is an important element in cancer progression. EMT and its reverse process, mesenchymal to epithelial transition (MET) is controlled by a network of transcriptional regulators and can be influenced by posttranscriptional and posttranslational modifications. EMT/MET involves many effectors that can activate and repress these transitions, often yielding a spectrum of cell phenotypes. Recent studies have shown that the miR-200 family and the transcriptional suppressor ZEB1 are important contributors to EMT. Our previous data showed that forced expression of SPRR2a was a powerful inducer of EMT and supports the findings by others that SPRR gene members are highly upregulated during epithelial remodeling in a variety of organs. Here, using SPRR2a cells, we characterize the role of acetyltransferases on the microRNA-200c/141 promoter and their effect on the epithelial/mesenchymal status of the cells. We show that the deacetylase inhibitor TSA as well as P300 and PCAF can cause a shift towards epithelial characteristics in HUCCT-1-SPRR2a cells. We demonstrate that both P300 and PCAF act as cofactors for ZEB1, forming a P300/PCAF/ZEB1 complex on the miR200c/141 promoter. This binding results in lysine acetylation of ZEB1 and a release of ZEB1 suppression on miR-200c/141 transcription. Furthermore, disruption of P300 and PCAF interactions dramatically down regulates miR-200c/141 promoter activity, indicating a PCAF/P300 cooperative function in regulating the transcriptional suppressor/activator role of ZEB1. These data demonstrate a novel mechanism of miRNA regulation in mediating cell phenotype.

Adding value to liver (and allograft) biopsy evaluation using a combination of multiplex quantum dot immunostaining, high-resolution whole-slide digital imaging, and automated image analysis.

Various technologies including nucleic acid, protein, and metabolic array analyses of blood, liver tissue, and bile are emerging as powerful tools in the study of hepatic pathophysiology. The entire lexicon of liver disease, however, has been written using classical hematoxylin-eosin staining and light microscopic examination. The authors' goal is to develop new tools to enhance histopathologic examination of liver tissue that would enrich the information gained from liver biopsy analysis, enable quantitative analysis, and bridge the gap between various "-omics" tools and interpretation of routine liver biopsy results. This article describes the progress achieved during the past 2 years in developing multiplex quantum dot (nanoparticle) staining and combining it with high-resolution whole-slide imaging using a slide scanner equipped with filters to capture 9 distinct fluorescent signals for multiple antigens. The authors first focused on precise characterization of leukocyte subsets, but soon realized that the data generated were beyond the practical limits that could be properly evaluated, analyzed, and interpreted visually by a pathologist. Therefore, the authors collaborated with the open source FARSIGHT image analysis project (http://www.farsight-toolkit.org). FARSIGHT's goal is to develop and disseminate the next-generation toolkit of automated image analysis methods to enable quantification of molecular biomarkers on a cell-by-cell basis from multiparameter images. The resulting data can be used for histocytometric studies of the complex and dynamic tissue microenvironments that are of biomedical interest. The authors envisage that these tools will eventually be incorporated into the routine practice of surgical pathology and precipitate a revolution in the specialty.

Estrogen stimulates female biliary epithelial cell interleukin-6 expression in mice and humans.

UNLABELLED: Females are more susceptible than males to several biliary tract diseases. Interleukin-6 (IL-6) is critical to triggering autoimmune reactions and contributes substantially to biliary epithelial cell (BEC) barrier function and wound repair, and estrogen differentially regulates IL-6 expression in various cell types. We hypothesized that estrogen might stimulate BEC IL-6 production. Exposure to physiologic levels of estradiol, in vitro, increased female mouse BEC (mBEC) IL-6 messenger RNA (mRNA) and protein expression, but either inhibited or had no effect on male mBECs. Female mBECs expressed higher concentrations of estrogen receptor-alpha (ERalpha) mRNA and protein and were also more dependent on estradiol for survival, in vitro. In vivo, elevated estrogen during estrous cycling in mice, and estrogen treatment of mice harboring an ERalpha(+) human cholangiocarcinoma resulted in increased BEC IL-6 mRNA and tumor viability, respectively. Both responses could be blocked by an ERalpha antagonist. Human cholangiocarcinoma cell lines differentially expressing ERalpha were treated with specific ERalpha and ERbeta agonists/antagonists to further test the relationship between estrogen stimulation, ERalpha expression, and IL-6 production. Results show that ERalpha, and not the underlying BEC sex, was responsible for estrogen-induced IL-6 production. Estrogen-induced proliferation of ERalpha-expressing cholangiocarcinoma was blocked by anti-IL-6 antibodies, indicating that at least some of the estrogen-trophic effects are mediated via IL-6. Finally, an association between ERalpha, IL-6, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) signaling was shown in female-predominant polycystic livers using immunohistochemical analyses, including multiplex quantum dot labeling. CONCLUSION: Estrogens stimulate IL-6 production in non-neoplastic female BECs and in neoplastic BECs expressing ERalpha. An association between these signaling pathways was demonstrated for female-predominant polycystic livers and might also influence autoimmune hepatitis, primary biliary cirrhosis, and cholangiocarcinogenesis.

Small proline-rich proteins (SPRR) function as SH3 domain ligands, increase resistance to injury and are associated with epithelial-mesenchymal transition (EMT) in cholangiocytes.

BACKGROUND/AIMS: Deficient biliary epithelial cell (BEC) expression of small proline-rich protein (SPRR) 2A in IL-6(-/-) mice is associated with defective biliary barrier function after bile duct ligation. And numerous gene array expression studies show SPRR2A to commonly be among the most highly up-regulated genes in many non-squamous, stressed and remodeling barrier epithelia. Since the function of SPRR in these circumstances is unknown, we tested the exploratory hypothesis that BEC SPRR2A expression contributes to BEC barrier function and wound repair. METHODS: The effect of SPRR2A expression was studied in primary mouse BEC cultures; in a BEC cell line after forced overexpression of SPRR2A; and in human livers removed at the time of liver transplantation. RESULTS: Forced SPRR2A overexpression showed that it functions as a SH3 domain ligand that increases resistance to oxidative injury and promotes wound restitution by enhancing migration and acquisition of mesenchymal characteristics. Low confluency non-neoplastic mouse BEC cultures show a phenotype similar to the stable transfectants, as did spindle-shaped BEC participating in atypical ductular reactions in primary biliary cirrhosis. CONCLUSIONS: These observations suggest that SPRR2A-related BEC barrier modifications represent a novel, but widely utilized and evolutionarily conserved, response to stress that is worthy of further study.

Gut-derived commensal bacterial products inhibit liver dendritic cell maturation by stimulating hepatic interleukin-6/signal transducer and activator of transcription 3 activity.

UNLABELLED: Intraorgan dendritic cells (DCs) monitor the environment and help translate triggers of innate immunity into adaptive immune responses. Liver-based DCs are continually exposed, via gut-derived portal venous blood, to potential antigens and bacterial products that can trigger innate immunity. However, somehow the liver avoids a state of perpetual inflammation and protects central immune organs from overstimulation. In this study, we tested the hypothesis that hepatic interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) activity increases the activation/maturation threshold of hepatic DCs toward innate immune signals. The results show that the liver nuclear STAT3 activity is significantly higher than that of other organs and is IL-6-dependent. Hepatic DCs in normal IL-6 wild-type (IL-6(+/+)) mice are phenotypically and functionally less mature than DCs from IL-6-deficient (IL-6(-/-)) or STAT3-inhibited IL-6(+/+) mice, as determined by surface marker expression, proinflammatory cytokine secretion, and allogeneic T-cell stimulation. IL-6(+/+) liver DCs produce IL-6 in response to exposure to lipopolysaccharide (LPS) and cytidine phosphate guanosine oligonucleotides (CpG) but are resistant to maturation compared with IL-6(-/-) liver DCs. Conversely, exogenous IL-6 inhibits LPS-induced IL-6(-/-) liver DC maturation. IL-6/STAT3 signaling influences the liver DC expression of toll-like receptor 9 and IL-1 receptor associated kinase-M. The depletion of gut commensal bacteria in IL-6(+/+) mice with oral antibiotics decreased portal blood endotoxin levels, lowered the expression of IL-6 and phospho-STAT3, and significantly increased liver DC maturation. CONCLUSION: Gut-derived bacterial products, by stimulating hepatic IL-6/STAT3 signaling, inhibit hepatic DC activation/maturation and thereby elevate the threshold needed for translating triggers of innate immunity into adaptive immune responses. Manipulating gut bacteria may therefore be an effective strategy for altering intrahepatic immune responses.

Multiplicity of expression of FXYD proteins in mammalian cells: dynamic exchange of phospholemman and gamma-subunit in response to stress.

Functional properties of Na-K-ATPase can be modified by association with FXYD proteins, expressed in a tissue-specific manner. Here we show that expression of FXYDs in cell lines does not necessarily parallel the expression pattern of FXYDs in the tissue(s) from which the cells originate. While being expressed only in lacis cells in the juxtaglomerular apparatus and in blood vessels in kidney, FXYD1 was abundant in renal cell lines of proximal tubule origin (NRK-52E, LLC-PK1, and OK cells). Authenticity of FXYD1 as a part of Na-K-ATPase in NRK-52E cells was demonstrated by co-purification, co-immunoprecipitation, and co-localization. Induction of FXYD2 by hypertonicity (500 mosmol/kgH(2)O with NaCl for 48 h or adaptation to 700 mosmol/kgH(2)O) correlated with downregulation of FXYD1 at mRNA and protein levels. The response to hypertonicity was influenced by serum factors and entailed, first, dephosphorylation of FXYD1 at Ser(68) (1-5 h) and, second, induction of FXYD2a and a decrease in FXYD1 with longer exposure. FXYD1 was completely replaced with FXYD2a in cells adapted to 700 mosmol/kgH(2)O and showed a significantly decreased sodium affinity. Thus dephosphorylation of FXYD1 followed by exchange of regulatory subunits is utilized to make a smooth transition of properties of Na-K-ATPase. We also observed expression of mRNA for multiple FXYDs in various cell lines. The expression was dynamic and responsive to physiological stimuli. Moreover, we demonstrated expression of FXYD5 protein in HEK-293 and HeLa cells. The data imply that FXYDs are obligatory rather than auxiliary components of Na-K-ATPase, and their interchangeability underlies responses of Na-K-ATPase to cellular stress.

Wound healing in the biliary tree of liver allografts.

An increasing need for liver transplantation requires evaluation and triage of organs harvested from "extended criteria" donors. Although there is currently no widely accepted definition, most would agree that "extended criteria" includes organs donated by individuals that are old (>65 years), obese, infected with HBV or HCV, non-heart beating (NHBD), or had an unstable blood pressure before harvesting or the organ experienced a long cold ischemic time. These organs carry a statistical risk of dysfunction early after transplantation, but in the majority of recipients, hepatic parenchymal function recovers. Later, however, a small but significant percentage of extended criteria donors develop biliary strictures within several months after transplantation. The strictures occur primarily because of preservation injury that leads to "ischemic cholangitis" or deep wounding of the bile duct wall. Subsequent partial wound healing and wound contraction, but failed restitution of the biliary epithelial cell (BEC) lining, result in biliary tract strictures that cause progressive biliary fibrosis, increased morbidity, and decreased organ half-life. Better understanding of the pathophysiologic mechanisms that lead to biliary strictures in extended criteria donors provides an ideal proving ground for regenerative medicine; it also can provide insights into other diseases, such as extrahepatic biliary atresia and primary sclerosing cholangitis, that likely share certain pathogenic mechanisms. Possible points of therapeutic intervention include limiting cold and warm ischemic times, donor and/or donor organ treatment, ex vivo, to minimize the ischemic/preservation injury, maximize blood flow after transplantation, promote BEC wound healing, and limit myofibroblasts activation and proliferation in the bile duct wall. The pathobiology of biliary wound healing and therapeutic potential of interleukin-6 (IL-6) are highlighted.

Biliary wound healing, ductular reactions, and IL-6/gp130 signaling in the development of liver disease.

Basic and translational wound healing research in the biliary tree lag significantly behind similar studies on the skin and gastrointestinal tract. This is at least partly attributable to lack of easy access to the biliary tract for study. But clinical relevance, more interest in biliary epithelial cell (BEC) pathophysiology, and widespread availability of BEC cultures are factors reversing this trend. In the extra-hepatic biliary tree, ineffectual wound healing, scarring and stricture development are pressing issues. In the smallest intra-hepatic bile ducts either impaired BEC proliferation or an exuberant response can contribute to liver disease. Chronic inflammation and persistent wound healing reactions in large and small bile ducts often lead to liver cancer. General concepts of wound healing as they apply to the biliary tract, importance of cellular processes dependent on IL-6/gp130/STAT3 signaling pathways, unanswered questions, and future directions are discussed.

Modulation of Na, K-ATPase activity by prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin.

Adenylyl cyclase is activated by prostaglandin E and inhibited by mu-opioids. Since cAMP-related events influence the activity of the Na Pump and its biochemical correlate Na,K-ATPase in many systems, we tested the hypothesis that prostaglandin E1 and [D-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO), a mu-opioid agonist, have opposing actions on Na,K-ATPase activity. Studies were conducted with alamethicin-permeabilized SH-SY5Y human neuroblastoma cells. Prostaglandin E1 (1 microM) transiently inhibited Na,K-ATPase activity for 10-15 min. A direct activator of protein kinase A, 8-Br-cAMP (150 and 500 microM), also inhibited, but more rapidly and for a shorter duration. Both DAMGO (1 microM) and Rp-adenosine 3',5'-cyclic monophosphorothioate (500 microM), a protein kinase A-inhibitor, reversed the inhibitory effect of prostaglandin E1. DAMGO alone (1 microM) stimulated Na,K-ATPase activity up to nearly three-fold control activity. The stimulatory action of DAMGO was blocked by cyclosporine A (2 microM), an inhibitor of calcineurin, and was dependent on Ca2+ entry through nifedipine-sensitive Ca2+ channels. In the presence of 1 mM EGTA, DAMGO inhibited Na,K-ATPase activity. DAMGO-induced inhibition was blocked by the inositol 1,4,5-trisphosphate receptor antagonist xestospongin C (1 microM). Na,K-ATPase is poised to modulate neuronal excitability through its roles in maintaining the membrane potential and transmembrane ion gradients. The differential effects of prostaglandin E1 and opioids on Na,K-ATPase activity may be related to their actions in hyperalgesia.

Buccal mucosal cell immunohistochemistry: a simple method of determining the ABH phenotype of baboons, monkeys, and pigs.

BACKGROUND: Baboons and monkeys fail to express ABH antigens on red blood cells (RBCs), and the A or H antigens are expressed only weakly on the surface of pig RBCs. Baboons and monkeys have been previously blood typed by detection of ABH antigens in the saliva after administration of pilocarpine. A reliable method to ABH type pigs is by immunohistochemical staining of renal distal tubules in kidney biopsies. We describe a simple and efficient method to blood type baboons, monkeys, and pigs. METHODS: Baboons (n = 14) and cynomolgus monkeys (n = 8) were blood typed by staining of buccal mucosal smears and by determining the presence of serum anti-A or B antibodies following human type O adsorption. Pigs (n = 11) were tested for ABH type by immunohistochemistry for the presence of A, B, and H antigens using monoclonal antibodies on (i) renal biopsies, (ii) RBCs, and (iii) buccal mucosal smears, without pilocarpine administration, in addition, (iv) after adsorption on human type O RBCs to remove anti-human antibodies, the pig sera were typed by hemagglutination assay for the presence of anti-A or B antibodies using human A and B RBCs. RESULTS AND CONCLUSIONS: There was complete consistency among the results obtained using all of the above methods, except that no determination could be made from staining of RBCs in one pig. Staining of buccal mucosal cells proved to be the preferred method in all three species because: (i) expression of A or H antigen is weak on pig RBCs, making an accurate blood type determination difficult, and A, B, and H expression is non-existent on baboon and monkey RBCs, (ii) neither venepuncture nor organ biopsy is necessary, (iii) time-consuming adsorption of anti-human antibodies from the sera of the test animal is not required, and (iv) it proved a quick method of evaluation.

Small proline-rich proteins 2 are noncoordinately upregulated by IL-6/STAT3 signaling after bile duct ligation.

Small proline-rich proteins 2 (SPRR2) are coordinately expressed with other epidermal differential complex (EDC) genes in the skin. They function as crosslinking proteins that form bridges between other proteins that comprise the cornified cell envelope, which is the major barrier against the environment. IL-6 is invariably produced at sites of biliary tract injury and IL-6-deficient (IL-6(-/-)) mice show impaired barrier function after bile duct ligation (BDL). Screening microarray analysis identified noncoordinate expression of SPRR2 as a candidate gene that is: (a) expressed in biliary epithelial cells (BEC); (b) IL-6 responsive; and (c) potentially related to biliary barrier function. Therefore, we studied in detail the regulation of BEC SPRR2A expression, in vitro; and tested the hypothesis that if BEC SPRR2 expression contributes to biliary barrier function, it should be increased after BDL in IL-6-wild type (IL-6(+/+)) mice and not in IL-6(-/-) mice. In vitro studies confirmed that IL-6/gp130-signaling, mediated primarily by signal transducer and activator of transcription 3 (STAT3), stimulated noncoordinate BEC SPRR2 expression. In vivo, noncoordinate upregulation of BEC SPRR2 expression after BDL was seen in the IL-6(+/+) mice and was unrelated to squamous metaplasia. IL-6(-/-) mice showed deficient BEC SPRR2 expression after BDL associated with impaired barrier function, as evidenced by smaller diameters of obstructed ducts, decreased bile volume, and an inability to form 'white bile' compared to IL-6(+/+) mice at 12 weeks after BDL. IL-6 replacement therapy reversed the barrier defect in IL-6(-/-) mice after BDL, coincident with recovery of SPRR2A expression. SPRR2 in diseased mouse and human liver localized subjacent to the apical plasma membrane of BEC lining bile ducts, but was more diffusely expressed throughout the cytoplasm of cholangioles. In conclusion, BEC IL-6/gp130/STAT3 signaling noncoordinately upregulates BEC SPRR2 that appears to contribute to modification of the biliary barrier under conditions of stress.

Regulation and function of trefoil factor family 3 expression in the biliary tree.

Microarray analysis identified trefoil factor family 3 (TFF3) as a gene expressed in biliary epithelial cells (BECs), regulated by interleukin (IL)-6, and potentially involved in biliary pathophysiology. We therefore studied the regulation and function of BEC TFF3, in vitro and in vivo in IL-6(+/+) and IL-6(-/-) mice subjected to chronic bile duct ligation for 12 weeks. In vitro studies showed that IL-6 wild-type (IL-6(+/+)) BECs expressed higher TFF3 mRNA and protein levels than IL-6-deficient (IL-6(-/-)) BECs. BEC TFF3 expression is dependent primarily on signal transducer and activator of transcription (STAT3) signaling, but the reciprocal negative regulation known to exist between the intracellular IL-6/gp130 signaling pathways, STAT3 and mitogen-activated protein kinase (MAPK), importantly contributes to BEC TFF3 expression. Specifically blocking STAT3 activity with a dominant-negative molecule or treatment with a growth factor such as hepatocyte growth factor, which increases MAPK signaling, decreases BEC TFF3 expression. In contrast, specifically blocking MAPK activity with PD98059 significantly increased BEC TFF3 expression. Higher BEC TFF3 levels in IL-6(+/+) BECs were associated with significantly better migration than IL-6(-/-) BECs in a wound-healing assay and defective IL-6(-/-) BEC migration was reversed with exogenous TFF3. In vivo, hepatic TFF3 mRNA and protein expression was limited to BECs and dependent significantly on STAT3 signaling, but was influenced by other factors present after bile duct ligation. Comparable results were obtained in normal and diseased human tissue samples. In conclusion the regulation and function of BEC TFF3 expression is similar to the colon. BEC TFF3 expression depends primarily on gp130/STAT3 and contributes to BEC migration and wound healing. Therefore, use of recombinant IL-6 or TFF3 peptides should exert a therapeutic role in preventing biliary strictures in liver allografts.

Production of alpha 1,3-galactosyltransferase-deficient pigs.

The enzyme alpha1,3-galactosyltransferase (alpha1,3GT or GGTA1) synthesizes alpha1,3-galactose (alpha1,3Gal) epitopes (Galalpha1,3Galbeta1,4GlcNAc-R), which are the major xenoantigens causing hyperacute rejection in pig-to-human xenotransplantation. Complete removal of alpha1,3Gal from pig organs is the critical step toward the success of xenotransplantation. We reported earlier the targeted disruption of one allele of the alpha1,3GT gene in cloned pigs. A selection procedure based on a bacterial toxin was used to select for cells in which the second allele of the gene was knocked out. Sequencing analysis demonstrated that knockout of the second allele of the alpha1,3GT gene was caused by a T-to-G single point mutation at the second base of exon 9, which resulted in inactivation of the alpha1,3GT protein. Four healthy alpha1,3GT double-knockout female piglets were produced by three consecutive rounds of cloning. The piglets carrying a point mutation in the alpha1,3GT gene hold significant value, as they would allow production of alpha1,3Gal-deficient pigs free of antibiotic-resistance genes and thus have the potential to make a safer product for human use.

Immature and mature CD8alpha+ dendritic cells prolong the survival of vascularized heart allografts.

CD8alpha+ and CD8alpha- dendritic cells (DCs) arise from committed bone marrow progenitors and can induce or regulate immune reactivity. Previously, the maturational status of CD8alpha-(myeloid) DCs has been shown to influence allogeneic T cell responses and allograft survival. Although CD8alpha+ DCs have been implicated in central tolerance and found to modulate peripheral T cell function, their influence on the outcome of organ transplantation has not been examined. Consistent with their equivalent high surface expression of MHC and costimulatory molecules, sorted mature C57BL/10J (B10; H2(b)) DCs of either subset primed naive, allogeneic C3H/HeJ (C3H; H2(k)) recipients for Th1 responses. Paradoxically and in contrast to their CD8alpha- counterparts, mature CD8alpha+ B10 DCs given systemically 7 days before transplant markedly prolonged B10 heart graft survival in C3H recipients. This effect was associated with specific impairment of ex vivo antidonor T cell proliferative responses, which was not reversed by exogenous IL-2. Further analyses of possible underlying mechanisms indicated that neither immune deviation nor induction of regulatory cells was a significant contributory factor. In contrast to the differential capacity of the mature DC subsets to affect graft outcome, immature CD8alpha+ and CD8alpha- DCs administered under the same experimental conditions significantly prolonged transplant survival. These observations demonstrate for the first time the innate capacity of CD8alpha+ DCs to regulate alloimmune reactivity and transplant survival, independent of their maturation status. Mobilization of such a donor DC subset with capacity to modulate antidonor immunity may have significant implications for the therapy of allograft rejection.