William D. Stuart

Apolipoprotein J/clusterin limits the severity of murine autoimmune myocarditis

Apolipoprotein J/clusterin (apoJ/clusterin), an intriguing protein with unknown function, is induced in myocarditis and numerous other inflammatory injuries. To test its ability to modify myosin-induced autoimmune myocarditis, we generated apoJ-deficient mice. ApoJ-deficient and wild-type mice exhibited similar initial onset of myocarditis, as evidenced by the induction of two early markers of the T cell-mediated immune response, MHC-II and TNF receptor p55. Furthermore, autoantibodies against the primary antigen cardiac myosin were induced to the same extent. Although the same proportion of challenged animals exhibited some degree of inflammatory infiltrate, inflammation was more severe in apoJ-deficient animals. Inflammatory lesions were more diffuse and extensive in apoJ-deficient mice, particularly in females. In marked contrast to wild-type animals, the development of a strong generalized secondary response against cardiac antigens in apoJ-deficient mice was predictive of severe myocarditis. Wild-type mice with a strong Ab response to secondary antigens appeared to be protected from severe inflammation. After resolution of inflammation, apoJ-deficient, but not wild-type, mice exhibited cardiac function impairment and severe myocardial scarring. These results suggest that apoJ limits progression of autoimmune myocarditis and protects the heart from postinflammatory tissue destruction.

Wnt/β-catenin signaling is required for development of the exocrine pancreas

Backgroundβ-catenin is an essential mediator of canonical Wnt signaling and a central component of the cadherin-catenin epithelial adhesion complex. Dysregulation of β-catenin expression has been described in pancreatic neoplasia. Newly published studies have suggested that β-catenin is critical for normal pancreatic development although these reports reached somewhat different conclusions. In addition, the molecular mechanisms by which loss of β-catenin affects pancreas development are not well understood. The goals of this study then were; 1] to further investigate the role of β-catenin in pancreatic development using a conditional knockout approach and 2] to identify possible mechanisms by which loss of β-catenin disrupts pancreatic development. A Pdx1-cre mouse line was used to delete a floxed β-catenin allele specifically in the developing pancreas, and embryonic pancreata were studied by immunohistochemistry and microarray analysis.ResultsPdx1-cre floxed β-catenin animals were viable but demonstrated small body size and shortened median survival. The pancreata from knockout mice were hypoplastic and histologically demonstrated a striking paucity of exocrine pancreas, acinar to duct metaplasia, but generally intact pancreatic islets containing all lineages of endocrine cells. In animals with extensive acinar hypoplasia, putative hepatocyte transdifferention was occasionally observed. Obvious and uniform pancreatic hypoplasia was observed by embryonic day E16.5. Transcriptional profiling of Pdx1-cre floxed β-catenin embryonic pancreata at E14.5, before there was a morphological phenotype, revealed significant decreases in the β-catenin target gene N-myc, and the basic HLH transcription factor PTF1, and an increase of several pancreatic zymogens compared to control animals. By E16.5, there was a dramatic loss of exocrine markers and an increase in Hoxb4, which is normally expressed anterior to the pancreas.ConclusionWe conclude that β-catenin expression is required for development of the exocrine pancreas, but is not required for development of the endocrine compartment. In contrast, β-catenin/Wnt signaling appears to be critical for proliferation of PTF1+ nascent acinar cells and may also function, in part, to maintain an undifferentiated state in exocrine/acinar cell precursors. Finally, β-catenin may be required to maintain positional identity of the pancreatic endoderm along the anterior-posterior axis. This data is consistent with the findings of frequent β-catenin mutations in carcinomas of acinar cell lineage seen in humans.

Silencing of RON Receptor Signaling Promotes Apoptosis and Gemcitabine Sensitivity in Pancreatic Cancers

The RON receptor tyrosine kinase is overexpressed in premalignant pancreatic intraepithelial neoplasia (PanIN) and in the majority of pancreatic cancers. In pancreatic cells, RON is an important K-Ras effector and RON ligand can enhance migration/invasion and apoptotic resistance. However, the pathobiological significance of RON overexpression in pancreatic cancers has yet to be fully established. In this study, we demonstrate that RON signaling mediates a unique transcriptional program that is conserved between cultured cells derived from murine PanIN or human pancreatic cancer cells grown as subcutaneous tumor xenografts. In both systems, RON signaling regulates expression of genes implicated in cancer-cell survival, including Bcl-2 and the transcription factors signal transducer and activator of transcription 3 (STAT 3) and c-Jun. shRNA-mediated silencing of RON in pancreatic cancer xenografts inhibited their growth, primarily by increasing susceptibility to apoptosis and by sensitizing them to gemcitabine treatment. Escape from RON silencing was associated with re-expression of RON and/or expression of phosphorylated forms of the related c-Met or epidermal growth factor receptors. These findings indicate that RON signaling mediates cell survival and in vivo resistance to gemcitabine in pancreatic cancer, and they reveal mechanisms through which pancreatic cancer cells may circumvent RON-directed therapies.

Ron receptor regulates Kupffer cell‐dependent cytokine production and hepatocyte survival following endotoxin exposure in mice

Previous studies demonstrated that targeted deletion of the Ron receptor tyrosine kinase (TK) domain in mice leads to marked hepatocyte protection in a well‐characterized model of lipopolysaccharide (LPS)‐induced acute liver failure in D‐galactosamine (GalN)‐sensitized mice. Hepatocyte protection in TK−/− mice was observed despite paradoxically elevated serum levels of tumor necrosis factor alpha (TNF‐α). To understand the role of Ron in the liver, purified populations of Kupffer cells and hepatocytes from wildtype (TK+/+) and TK−/− mice were studied. Utilizing quantitative reverse‐transcription polymerase chain reaction (RT‐PCR), we demonstrated that Ron is expressed in these cell types. Moreover, we also recapitulated the protected hepatocyte phenotype and exaggerated cytokine production observed in the TK−/− mice in vivo through the use of purified cultured cells ex vivo. We show that isolated TK−/− Kupffer cells produce increased levels of TNF‐α and select cytokines compared to TK+/+ cells following LPS stimulation. We also show that conditioned media from LPS‐treated TK−/− Kupffer cells was more toxic to hepatocytes than control media, suggesting the exaggerated levels of cytokines produced from the TK−/− Kupffer cells are detrimental to wildtype hepatocytes. In addition, we observed that TK−/− hepatocytes were more resistant to cell death compared to TK+/+ hepatocytes, suggesting that Ron functions in both the epithelial and inflammatory cell compartments to regulate acute liver injury. These findings were confirmed in vivo in mice with hepatocyte and macrophage cell‐type‐specific conditional Ron deletions. Mice with Ron loss selectively in hepatocytes exhibited less liver damage and increased survival compared to mice with Ron loss in macrophages. Conclusion: We dissected cell‐type‐specific roles for Ron such that this receptor modulates cytokine production from Kupffer cells and inhibits hepatocyte survival in response to injury. (HEPATOLOGY 2011;)

RON RECEPTOR TYROSINE KINASE NEGATIVELY REGULATES TNFα PRODUCTION IN ALVEOLAR MACROPHAGES BY INHIBITING NF-κB ACTIVITY AND ADAM17 PRODUCTION

The Ron receptor tyrosine kinase (TK) plays a regulatory role in the inflammatory response to acute lung injury induced by intranasal administration of bacterial LPS. Previously, we have shown that mice with a targeted deletion of the TK signaling domain of the Ron receptor exhibited more severe lung injury in response to intranasal LPS administration as evidenced by an increased leakage of albumin in the lungs and a greater thickening of the alveolar septa compared with wild-type mice. In addition, lung injury in the Ron TK-deficient (TK−/−) mice was associated with increased activation of the transcription factor, nuclear factor-&kgr;B (NF-&kgr;B), and significantly increased intrapulmonary expression of TNF&agr;. TNF&agr;, a multifunctional proinflammatory cytokine, is a central mediator in several disease states, including rheumatoid arthritis and sepsis. On the basis of the observation that TNF&agr; production is increased in the Ron TK−/− mice and that macrophages are a major source of this cytokine, we hypothesized that the alterations observed in the Ron TK−/− mice may be due, in part, to Ron signaling, specifically in alveolar macrophages. To test this hypothesis, we used the wild-type and Ron TK−/− primary alveolar macrophages and the murine alveolar macrophage cell line, MH-S, to examine the effects of Ron activation on LPS-induced TNF&agr; production and NF-&kgr;B activity. Here, we reported that Ron is expressed on alveolar macrophages and MH-S cells. Activation of Ron by its ligand, hepatocyte growth factor-like protein, decreases TNF&agr; production in alveolar macrophages after LPS challenge. Decreased TNF&agr; is associated with hepatocyte growth factor-like protein-induced decreases in NF-&kgr;B activation and increases in the NF-&kgr;B inhibitory protein, I&kgr;B. We also provided the first evidence for Ron as a negative regulator of Adam17, the metalloprotease involved in TNF&agr; processing. These results indicate that Ron plays a critical role in regulation of alveolar macrophage signaling and validates this receptor as a target in TNF&agr;-mediated pulmonary pathologies.ABBREVIATIONS-HGFL-hepatocyte growth factor-like protein; TK-tyrosine kinase; NF-&kgr;B-nuclear factor &kgr;B; Adam17-a disintegrin and metalloprotease

Local synthesis of apolipoprotein J in the eye

Apolipoprotein J (apoJ), a secretory glycoprotein known to transport lipids and to regulate terminal complement function, is present in the human eye in both aqueous and vitreous, as well as in the retina. Ocular apoJ is the product of local synthesis, rather than plasma contamination, as demonstrated by its distinct structural properties and the presence of abundant apoJ mRNA in retina and retina pigment epithelium. ApoJ mRNA is also present in mouse eye, with a developmentally regulated pattern of expression. In fetal mouse, apoJ mRNA is present in retina, lens and cornea. In contrast, adult eye apoJ mRNA is present in retina and ciliary body. We propose that apoJ is important in tissue remodeling and in stabilizing hydrophobic molecules which are required for vision and/or which would otherwise be deleterious and membrane-active.

The Ron Receptor Tyrosine Kinase Negatively Regulates Mammary Gland Branching Morphogenesis

The Ron receptor tyrosine kinase is expressed in normal breast tissue and is overexpressed in approximately 50% of human breast cancers. Despite the recent studies on Ron in breast cancer, nothing is known about the importance of this protein during breast development. To investigate the functional significance of Ron in the normal mammary gland, we compared mammary gland development in wild-type mice to mice containing a targeted ablation of the tyrosine kinase (TK) signaling domain of Ron (TK-/-). Mammary glands from RonTK-/- mice exhibited accelerated pubertal development including significantly increased ductal extension and branching morphogenesis. While circulating levels of estrogen, progesterone, and overall rates of epithelial cell turnover were unchanged, significant increases in phosphorylated MAPK, which predominantly localized to the epithelium, were associated with increased branching morphogenesis. Additionally, purified RonTK-/- epithelial cells cultured ex vivo exhibited enhanced branching morphogenesis, which was reduced upon MAPK inhibition. Microarray analysis of pubertal RonTK-/- glands revealed 393 genes temporally impacted by Ron expression with significant changes observed in signaling networks regulating development, morphogenesis, differentiation, cell motility, and adhesion. In total, these studies represent the first evidence of a role for the Ron receptor tyrosine kinase as a critical negative regulator of mammary development.

Ron receptor signaling is protective against DSS-induced colitis in mice.

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders of the intestine that result in painful and debilitating complications. Currently no cure exists for IBD, and treatments are primarily aimed at reducing inflammation to alleviate symptoms. Genome-wide linkage studies have identified the Ron receptor tyrosine kinase (TK) and its ligand, hepatocyte growth factor-like protein (HGFL), as genes highly associated with IBD. However, only scant information exists on the role of Ron or HGFL in IBD. Based on the linkage of Ron to IBD, we directly examined the biological role of Ron in colitis. Wild-type mice and mice lacking the TK signaling domain of Ron (TK-/- mice) were utilized in a well-characterized model of chronic colitis induced by cyclic exposure to dextran sulfate sodium. In this model, TK-/- mice were more susceptible to injury as judged by increased mortality compared with control mice and developed more severe colitis. Loss of Ron led to significantly reduced body weights and more aggressive clinical and histopathologies. Ron loss also resulted in a dramatic reduction in colonic epithelial cell proliferation and increased proinflammatory cytokine production, which was associated with alterations in important signaling pathways known to regulate IBD. Examination of human gene expression data further supports the contention that loss of Ron signaling is associated with IBD. In total, our studies point to important functional roles for Ron in IBD by regulating healing of the colonic epithelium and by controlling cytokine secretion.

Ron receptor overexpression in the murine prostate induces prostate intraepithelial neoplasia.

Previous studies have shown that the Ron receptor is overexpressed in prostate cancer and Ron expression increases with disease severity in humans and the mouse TRAMP model. Here, the causal role of Ron overexpression in the murine prostate was examined in the development and progression of prostate cancer. Transgenic mouse strains were generated which selectively overexpressed Ron in the prostate epithelium and prostate histopathology was evaluated and compared to wild type controls. Ron overexpression led to the development of prostate intraepithelial neoplasia (mPIN) with local invasion and was associated with increases in prostate cell proliferation and decreases in cell death.

Quantitation of plasma apolipoprotein J.

Publisher Summary Apolipoprotein J (apoJ),* a fascinating secretory protein present in most, if not all, physiological fluids, including plasma, semen, cerebrospinal fluid, aqueous humor, breast milk, synovial fluid, and urine has been linked with chronic degenerative diseases in humans. Notable among these are atherosclerosis, glomerulonephritis and other renal pathologies, Alzheimers disease and other neurological pathologies, and immune complex-related skin disorders. Although it has not been unequivocally established that apoJ is an apolipoprotein, the available evidence favors this classification. On the basis of gene structure and the sequences of potential amphipathic helices, apoJ is clearly not a member of the apolipoprotein gene family, including the apoAs, apoCs, and apoE. However, where evaluated, for example, in plasma and cerebrospinal fluid (B. Murphy, unpublished results, 1994), apoJ is associated with lipid. Moreover, the protein is secreted by cultured hepatocytes as a lipoprotein, in fact considerably enriched in triglyceride compared to the plasma form. The most compelling evidence is the finding by Argraves and co-workers TM that apoJ is recognized as a ligand by gp330, a member of the LDL receptor gene family. Internalization and degradation of apoJ by cultured F9 cells 18 is mediated by gp330.