Linkage analysis and haplotype mapping in interspecific mouse crosses (Mus musculus x Mus spretus) identified the gene encoding Aurora2 (Stk6 in mouse and STK15 in human) as a candidate skin tumor susceptibility gene. The Stk6 allele inherited from the susceptible M. musculus parent was overexpressed in normal cells and preferentially amplified in tumor cells from F(1) hybrid mice. We identified a common genetic variant in STK15 (resulting in the amino acid substitution F31I) that is preferentially amplified and associated with the degree of aneuploidy in human colon tumors. The Ile31 variant transforms rat1 cells more potently than the more common Phe31 variant. The E2 ubiquitin-conjugating enzyme UBE2N was a preferential binding partner of the 'weak' STK15 Phe31 variant form in yeast two-hybrid screens and in human cells. This interaction results in colocalization of UBE2N with STK15 at the centrosomes during mitosis. These results are consistent with an important role for the Ile31 variant of STK15 in human cancer susceptibility.

The Aurora-A

No abstract

Autoimmune associated congenital heart block (CHB) may result from pathogenic cross-talk between inflammatory and profibrosing pathways. Incubation of macrophages with immune complexes (IC) composed of Ro60, a target of the pathologic maternal autoantibodies necessary for CHB, hY3 ssRNA, and affinity-purified anti-Ro60 antibody induces the Toll-like receptor 7 (TLR7)-dependent generation of supernatants that provoke a fibrosing phenotype in human fetal cardiac fibroblasts. We show herein that these cells are a major source of TGF␤ and that endothelin-1 (ET-1) is one of the key components responsible for the profibrosing effects generated by stimulated macrophages. Supernatants from macrophages incubated with IC induced the fibroblast secretion of TGF␤, which was inhibited by treating the macrophages with an antagonist of TLR7. Under the same conditions, the induced fibroblast secretion of TGF␤ was decreased by inhibitors of the ET-1 receptors ETa or ETb or by an anti-ET-1 antibody but not by an isotype control. Exogenous ET-1 induced a profibrosing phenotype, whereas fibroblasts transfected with either ETa or ETb siRNA were unresponsive to the profibrosing effects of the IC-generated macrophage supernatants. Immunohistochemistry of the hearts from two fetuses dying with CHB revealed the presence of ET-1-producing mononuclear cells in the septal region in areas of calcification and fibrosis. In conclusion, these data support a novel role of ET-1 in linking TLR7 inflammatory signaling to subsequent fibrosis and provide new insight in considering therapeutics for CHB.Cardiac conduction defects detected before or at birth, in the absence of structural abnormalities, are strongly associated with maternal autoantibodies to SSA/Ro and/or SSB/La ribonucleoproteins, independent of whether the mother has systemic lupus erythematosus or Sjögren's syndrome or is asymptomatic (1, 2). The mechanism by which maternal anti-SSA/ Ro-SSB/La antibodies initiate and perpetuate inflammation with consequent scarring of the atrioventricular node (the signature lesion of CHB) 2 and endocardium (3) has not been fully defined. One proposed pathologic cascade centers apoptosis of the fetal cardiocytes as the cellular event that accounts for the surface expression of the otherwise intracellular target antigens (4 -6), facilitating subsequent opsonization by circulating maternal autoantibodies. Experimental data suggest that hY3 ssRNA associated with the SSA/Ro protein bound by affinitypurified anti-Ro60 antibody (AP60) gains access to the macrophage endosome via Fc␥R uptake with subsequent ligation of the Toll-like receptor 7 (TLR7). In clearing the opsonized apoptotic cardiomyocytes, infiltrating macrophages secrete factors that promote a scarring phenotype of the resident cardiac fibroblasts, as evidenced by their transdifferentiation to myofibroblasts and production of collagen (7,8).Immunohistochemical studies (5) of hearts from several fetuses dying with CHB demonstrate abundant TGF␤ in the septal region. TGF␤ probably contributes to s...

BACKGROUND Cardiac manifestations of neonatal lupus (cardiac NL) include congenital heart block and cardiomyopathy. Several candidate biomarkers were evaluated in cases at risk for cardiac NL on the basis of potential roles in inflammation, fibrosis, and cardiac dysfunction: C-reactive protein (CRP); NT-pro-B-type natriuretic peptide (NT-proBNP); troponin I; matrix metalloproteinase (MMP)-2; urokinase plasminogen activator (uPA); urokinase plasminogen activator receptor (uPAR); plasminogen; and vitamin D. OBJECTIVES Identification of maternal and fetal biomarkers associated with development and morbidity of cardiac NL should provide clues to pathogenesis with translational implications for management. METHODS Cord and maternal blood samples (139 each) collected during pregnancies at risk for cardiac NL were available for study. Levels of cord and maternal CRP, cord NT-proBNP, and cord troponin I were evaluated using multiplex assays. Cord and maternal vitamin D were assessed by liquid chromatography-mass spectrometry. MMP-2, uPA, uPAR, and plasminogen were evaluated using ELISA. RESULTS Cord CRP, NT-proBNP, MMP-2, uPA, uPAR, and plasminogen levels were higher in cardiac NL-affected fetuses than in unaffected cases, independent of maternal rheumatic disease, season at highest risk of cardiac NL development, and medications taken during pregnancy. These biomarkers were positively associated with a disease severity score derived from known risk factors for mortality in cardiac NL. Maternal CRP and cord troponin I levels did not differ between the groups. Cord and maternal vitamin D levels were not significantly associated with cardiac NL, but average maternal vitamin D level during pregnancy was positively associated with longer time to postnatal pacemaker placement. CONCLUSIONS These data support the association of fetal reactive inflammatory and fibrotic components with development and morbidity of cardiac NL. Following CRP and NT-proBNP levels after birth can potentially monitor severity and progression of cardiac NL. MMP-2 and the uPA/uPAR/plasminogen cascade provide therapeutic targets to decrease fibrosis. Although decreased vitamin D did not confer increased risk, given the positive influence on postnatal outcomes, maternal levels should be optimized.

Rationale Binding of maternal anti-Ro/La antibodies to cognate antigen expressed on apoptotic cardiocytes decreases clearance by healthy cardiocytes, which may contribute to the development of autoimmune associated congenital heart block and fatal cardiomyopathy. Objective Given recent evidence implicating the urokinase plasminogen activator receptor (uPAR) as a “don’t eat me” signal during efferocytosis, experiments addressed whether surface bound anti-Ro antibodies inhibit apoptotic cell removal via an effect on the expression/function of the urokinase-type plasminogen activator protease (uPA)/uPAR system. Methods and Results As assessed by flow cytometry and confocal microscopy, uPAR colocalizes and interacts with Ro60 on the surface of apoptotic human fetal cardiocytes. Blocking of uPAR enhances phagocytosis of apoptotic cardiocytes by healthy cardiocytes and reverses the anti-Ro60 dependent impaired clearance of apoptotic cardiocytes. Binding of anti-Ro60 antibodies to apoptotic cardiocytes results in increased uPAR expression as well as enhanced uPA activity. The binding of anti-Ro60 did not alter other surface molecules involved in cell recognition (calreticulin, CD31 or CD47). Conclusions These data suggest that increased uPAR expression and uPA activity induced by anti-Ro60 binding to the apoptotic fetal cardiocyte provide a molecular basis by which these antibodies inhibit efferocytosis and ultimately lead to scar of the fetal conduction system and working myocardium.

In congenital heart block (CHB), binding of maternal anti-SSA/Ro antibodies to fetal apoptotic cardiocytes impairs their removal by healthy cardiocytes and increases uPA/uPAR-dependent plasmin activation. Since the uPA/uPAR system plays a role in TGF beta activation, we evaluated whether anti-Ro binding to apoptotic cardiocytes enhances plasmin-mediated activation of TGF beta thereby promoting a profibrosing phenotype. Supernatants from co-cultures of healthy cardiocytes and apoptotic cardiocytes bound by IgG from a mother whose child had CHB (apo-CHB-IgG) exhibited significantly increased levels of active TGF beta compared to supernatants from co-cultures of healthy cardiocytes and apoptotic cardiocytes preincubated with IgG (apo-nl-IgG) from a healthy donor. Treatment of the culture medium with anti-TGF beta antibody or TGF beta inhibitor (SB431542) abrogated the luciferase response thereby confirming TGF beta dependency. Increased uPA levels and activity were present in supernatants generated from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes compared to healthy cardiocytes and and apo nl-IgG cardiocytes, respectively. Treatment of apo-CHB-IgG cardiocytes with anti-uPAR or anti-uPA antibodies or plasmin inhibitor aprotinin prior to coculturing with healthy cardiocytes attenuated TGF beta activation. Supernatants derived from cocultures of healthy cardiocytes and apo-CHB-IgG cardiocytes promoted Smad2 phosphorylation and fibroblast transdifferentiation as evidenced by increased SMAc and collagen expression, which decreased when fibroblasts were treated with supernatants from cocultures pretreated with uPAR antibodies. These data suggest that binding of anti-Ro antibodies to apoptotic cardiocytes triggers TGF beta activation, by virtue of increasing uPAR-dependent uPA activity, thus initiating and amplifying a cascade of events that promote myofibroblast transdifferentiation and scar.

Aurora-A is an important regulator of mitosis and is frequently amplified in human cancer. Ectopic expression of Aurora-A in mammalian cells induces centrosome amplification, genomic instability and transformation. A common genetic variant in Aurora-A (F31I) is preferentially amplified and is associated with the occurrence and the status of colon, oesophageal and breast cancers. Here we demonstrate that the N-terminal domain of Aurora-A Phe-31 variant exhibits an intrinsic ubiquitin ligase activity. Mutation of cysteines 8, 33 and 49 of Aurora-A abolishes the ubiquitin ligase activity of the protein. Aurora-A in a complex with UBE2N/MMS2 catalyses polyubiquitination of IkappaBalpha in vitro and in vivo.