RREB1 repressed miR-143/145 modulates KRAS signaling through downregulation of multiple targets.

A lack of expression of miR-143 and miR-145 has been demonstrated to be a frequent feature of colorectal tumors. Activating KRAS mutations have been reported in 30-60% of colorectal cancers and an inverse correlation between Kras and miR-143/145 expression has been observed. Previously, we have demonstrated that oncogenic Kras leads to repression of the miR-143/145 cluster in pancreatic cancer and is dependent on the Ras responsive element (RRE) binding protein (RREB1), which negatively regulates miR-143/145 expression. In the present study, we have found that RREB1 is overexpressed in colorectal adenocarcinoma tumors and cell lines, and the expression of the miR-143/145 primary transcript is inversely related to RREB1 expression. In colorectal cancer cell lines, the miR-143/145 cluster is repressed by RREB1 downstream of constitutively active KRAS. RREB1 is activated by the MAPK pathway and negatively represses the miR-143/145 promoter through interaction with two RREs. In addition, overexpression of miR-143 or miR-145 in HCT116 cells abrogates signaling through the MAPK, PI3K and JNK pathways by downregulation of both KRAS and RREB1 in addition to downregulation of a cohort of genes in the MAPK signaling cascade. These results establish a complex network of regulation through which the miR-143/145 cluster is able to modulate KRAS signaling in colorectal cancer.

Synergistic deposition of C4d by complement-activating and non-activating antibodies in cardiac transplants.

The role of non-complement-activating alloantibodies in humoral graft rejection is unclear. We hypothesized that the non-complement-activating alloantibodies synergistically activate complement in combination with complement-activating antibodies. B10.A hearts were transplanted into immunoglobulin knock out (Ig-KO) mice reconstituted with monoclonal antibodies to MHC class I antigens. In allografts of unreconstituted Ig-KO recipients, no C4d was detected. Similarly, reconstitution with IgG1 or low dose IgG2b alloantibodies did not induce C4d deposition. However, mice administered with a low dose of IgG2b combined with IgG1 had heavy linear deposits of C4d on vascular endothelium. C4d deposits correlated with decreased graft survival. To replicate this synergy in vitro, mononuclear cells from B10.A mice were incubated with antibodies to MHC class I antigens followed by incubation in normal mouse serum. Flow cytometry revealed that both IgG2a and IgG2b synergized with IgG1 to deposit C4d. This synergy was significantly decreased in mouse serum deficient in mannose binding lectin (MBL) and in serum deficient in C1q. Reconstitution of MBL-A/C knock out (MBL-KO) serum with C1q-knock out (C1q-KO) serum reestablished the synergistic activity. This suggests a novel role for non-complement-activating alloantibodies and MBL in humoral rejection.

C4d deposition and clearance in cardiac transplants correlates with alloantibody levels and rejection in rats.

Antibody-mediated rejection of human cardiac transplants is correlated with C4d deposits and macrophage infiltrates in capillaries of endomyocardial biopsies. We produced an antibody to rat C4d to study C4d deposition and clearance in Lewis rats that were sensitized with a blood transfusion from DA rats 7, 14 or 21 days before cardiac transplantation. Cyclosporin A (CsA) immunosuppression was initiated after transplantation at a dose that inhibited graft rejection, antibody production and C4d deposition in unsensitized recipients. Blood transfusion elicited high levels of circulating IgG alloantibodies, predominantly of the complement-activating IgG2b subclass, that peaked 14 days after transplantation. At this time, macrophages accumulated in capillaries, and C4d deposits were diffuse and intense on arteries, capillaries and veins. Grafts that survived 90 days in sensitized recipients still had deposits of C4d that were associated with increased interstitial fibrosis and vasculopathy in arteries. Clearance of C4d was determined by retransplanting DA cardiac allografts from Lewis recipients back to DA recipients. C4d deposits were decreased to minimal levels within 5 days after retransplantation. Thus, C4d deposition is not limited to the capillaries, but extends throughout the arterial tree, and despite formation of a covalent bond, C4d is cleared within days.

A rat model of pregnancy-induced sensitization to transplants.

Previous pregnancy is a known risk factor for alloantibody production and graft rejection in clinical transplantation. However, in previous rat models, immune responses to RT1.A antigens induced by allogeneic pregnancy resulted in prolonged survival of subsequent allografts. This study was designed to investigate the effects of a previous pregnancy on alloantibody response, complement activation, and allograft survival in a highly immunogenic rat strain combination. C6-sufficient and -deficient female PVG.1U (RT1.A(u)B(u)) rats were mated with allogeneic PVG.R8 (RT1.A(a)B(u)) males or control isogeneic PVG.1U (RT1.A(u)B(u)) males. Three weeks after parturition, experimental and control females received cardiac allografts from female PVG.R8 donors. A low dose of cyclosporine (CsA, 5 mg/kg on alternate days) was used for immunosuppression after transplantation. Allogeneic, but not control isogeneic, pregnancy elicited a weak, transient IgG alloantibody response that declined before transplantation. Experimental female recipients produced a rapid, vigorous IgM and IgG alloantibody response to the transplant despite CsA treatment. C6-sufficient recipients rejected their transplants at an accelerated rate (5 days, n = 6) compared with control animals (7 days, n = 5). In contrast, allografts to C6-deficient recipients functioned until sacrifice at 90 days in both the experimental group (n = 7) and control group (n = 4). Most experimental C6-deficient recipients continued to produce strong IgG alloantibodies for 90 days. Complement activation resulting from the alloantibody response was evidenced by the diffuse deposition of C3d on the vascular endothelium of the grafts. In summary, previous pregnancy leads to memory alloantibody responses that accelerate allograft rejection even with immunosuppression. Membrane attack complex is required for accelerated rejection induced by previous pregnancy.

Iron deficiency enhances cholesterol gallstone formation.

BACKGROUND: Cholesterol gallstones occur most commonly in multiparous women, but the causes for this phenomenon remain unclear. This same patient population is prone to chronic iron deficiency anemia. In addition, iron is known to play an important role in hepatic enzyme metabolism. Therefore, we tested the hypotheses that iron deficiency would alter hepatic cholesterol metabolism and enhance gallstone formation. METHODS: Forty adult prairie dogs were fed either a control iron-supplemented (200 ppm), an iron-deficient (8 ppm), a 0.4% cholesterol iron-supplemented (200 ppm), or a 0.4% cholesterol iron-deficient (8 ppm) diet. After 8 weeks gallbladder bile, serum, and liver were harvested. Gallbladder bile was examined for cholesterol crystals and gallstones. Bile lipids and hepatic enzymes were measured, and a cholesterol saturation index (CSI) was calculated. RESULTS: Animals receiving the iron-deficient diet were more likely to have cholesterol crystals in their bile than were animals on the control diet (80% vs. 20%; p < 0.05). Animals on the 0.4% cholesterol iron-deficient diet had more cholesterol crystals per high-powered field (79 +/- 10 vs. 49 +/- 9; p = 0.07), a higher molar % cholesterol (6.0 +/- 0.3 vs 4.4 +/- 0.5; p < 0.05), and a higher CSI (1.27 +/- 0.10 vs. 0.91 +/- 0.07; p < 0.05) compared to animals receiving the 0.4% cholesterol iron supplemented diet. The 7 alpha-hydroxylase levels were lower in the animals on the iron-deficient diet compared to those receiving the control diet (0.42 +/- 0.08 vs 1.17 +/- 0.40 pmol/mg per minute; p = 0.07). CONCLUSIONS: These data suggest that an iron-deficient diet (1) alters hepatic enzyme metabolism, which, in turn, (2) increases gallbladder bile cholesterol and promotes cholesterol crystal formation. We conclude that iron deficiency plays a previously unrecognized role in the pathogenesis of cholesterol gallstone formation in women.

Cholesterol nucleates rapidly from mixed micelles in the prairie dog.

Current theory suggests that the nucleation of cholesterol in human bile requires the aggregation and fusion of cholesterol-enriched phospholipid vesicles. This theory is based on observations which do not exclude the precipitation of cholesterol from mixed micelles. The present study examines the role of mixed micelles and vesicles in the formation of cholesterol monohydrate crystals in the prairie dog. The intermicellar bile salt concentration of prairie dog gallbladder bile was determined using equilibrium dialysis. Model bile equivalent to gallbladder bile from cholesterol-fed prairie dogs was used as dialysant yielding the intermicellar (dialysate) concentration of 9 mM. Cholesterol carriers in gallbladder bile from 11 cholesterol-fed animals were then separated by Sephacryl S200 gel filtration chromatography using eluant buffer containing the intermicellar bile salt concentration. Gel filtration chromatography of fresh bile demonstrated that 100% of cholesterol was carried in the mixed micellar fraction with no vesicles observed in any of the 11 animals. The gallbladder bile nucleation time was 2.0 +/- 0.3 days for the cholesterol-fed animals. Gel filtration chromatography immediately after nucleation again revealed a single mixed micellar peak. These data indicate that cholesterol is carried exclusively in and nucleates rapidly from mixed micelles in the cholesterol-fed prairie dog and that cholesterol-phospholipid vesicles are not required in this process.