Alternative Splicing of CEACAM1 by Hypoxia-Inducible Factor-1α Enhances Tolerance to Hepatic Ischemia in Mice and Humans

Although alternative splicing (AS) drives transcriptional responses and cellular adaptation to environmental stresses, its contributions in organ transplantation have not been appreciated. We have shown that carcinoembryonic antigen-related cell adhesion molecule (Ceacam1; CD66a), a transmembrane biliary glycoprotein expressed in epithelial, endothelial, and immune cells, determines donor liver transplant quality. Here, we studied how AS of Ceacam1 affects ischemia-reperfusion injury (IRI) in mouse and human livers. We found that the short cytoplasmic isoform Ceacam1-S increased during early acute and late resolution phases of warm IRI injury in mice. Transfection of Ceacam1-deficient mouse hepatocytes with adenoviral Ceacam1-S mitigated hypoxia-induced loss of cellular adhesion by repressing the Ask1/p-p38 cell death pathway. Nucleic acid-blocking morpholinos, designed to selectively induce Ceacam1-S, protected hepatocyte cultures against temperature-induced stress in vitro. Luciferase and chromatin immunoprecipitation assays identified direct binding of hypoxia-inducible factor-1α (Hif-1α) to the mouse polypyrimidine tract binding protein 1 (Ptbp1) promoter region. Dimethyloxalylglycine protected mouse livers from warm IR stress and hepatocellular damage by inhibiting prolyl hydroxylase domain-containing protein 1 and promoting AS of Ceacam1-S. Last, analysis of 46 human donor liver grafts revealed that CEACAM1-S positively correlated with pretransplant HIF1A expression. This also correlated with better transplant outcomes, including reduced TIMP1, total bilirubin, proinflammatory MCP1, CXCL10 cytokines, immune activation markers IL17A, and incidence of delayed complications from biliary anastomosis. This translational study identified mouse Hif-1α-controlled AS of Ceacam1, through transcriptional regulation of Ptbp1 promoter region, as a functional underpinning of hepatoprotection against IR stress and tissue damage in liver transplantation

Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing

<p>Abstract</p> <p>Background</p> <p>Despite tremendous progress in understanding the mechanisms of constitutive and alternative splicing, an important and widespread step along the gene expression pathway, our ability to deliberately regulate gene expression at this step remains rudimentary. The present study was performed to investigate whether a theophylline-dependent "splice switch" that sequesters the branchpoint sequence (BPS) within RNA-theophylline complex can regulate alternative splicing.</p> <p>Results</p> <p>We constructed a series of pre-mRNAs in which the BPS was inserted within theophylline aptamer. We show that theophylline-induced sequestering of BPS inhibits pre-mRNA splicing both in vitro and in vivo in a dose-dependent manner. Several lines of evidence suggest that theophylline-dependent inhibition of splicing is highly specific, and thermodynamic stability of RNA-theophylline complex as well as the location of BPS within this complex affects the efficiency of splicing inhibition. Finally, we have constructed an alternative splicing model pre-mRNA substrate in which theophylline caused exon skipping both in vitro and in vivo, suggesting that a small molecule-RNA interaction can modulate alternative splicing.</p> <p>Conclusion</p> <p>These findings provide the ability to control splicing pattern at will and should have important implications for basic, biotechnological, and biomedical research.</p

Heme Oxygenase-1 in liver transplant ischemia-reperfusion injury: From bench-to-bedside.

Hepatic ischemia-reperfusion injury (IRI), a major risk factor for early allograft dysfunction (EAD) and acute or chronic graft rejection, contributes to donor organ shortage for life-saving orthotopic liver transplantation (OLT). The graft injury caused by local ischemia (warm and/or cold) leads to parenchymal cell death and release of danger-associated molecular patterns (DAMPs), followed by reperfusion-triggered production of reactive oxygen species (ROS), activation of inflammatory cells, hepatocellular damage and ultimate organ failure. Heme oxygenase 1 (HO-1), a heat shock protein-32 induced under IR-stress, is an essential component of the cytoprotective mechanism in stressed livers. HO-1 regulates anti-inflammatory responses and may be crucial in the pathogenesis of chronic diseases, such as arteriosclerosis, hypertension, diabetes and steatosis. An emerging area of study is macrophage-derived HO-1 and its pivotal intrahepatic homeostatic function played in IRI-OLT. Indeed, ectopic hepatic HO-1 overexpression activates intracellular SIRT1/autophagy axis to serve as a key cellular self-defense mechanism in both mouse and human OLT recipients. Recent translational studies in rodents and human liver transplant patients provide novel insights into HO-1 mediated cytoprotection against sterile hepatic inflammation. In this review, we summarize the current bench-to-bedside knowledge on HO-1 molecular signaling and discuss their future therapeutic potential to mitigate IRI in OLT

Microbiota in organ transplantation: An immunological and therapeutic conundrum?

The gastrointestinal (GI) tract microbiota is an environmental factor that regulates host immunity in allo-transplantation (allo-Tx). It is required for the development of resistance against pathogens and the stabilization of mucosa-associated lymphoid tissue. The gut-microbiota axis may also precipitate allograft rejection by producing metabolites that activate host cell-mediated and humoral immunity. Here, we discuss new insights into microbial immunomodulation, highlighting ongoing attempts to affect commensal colonization in an attempt to ameliorate allograft rejection cascade. Recent progress on the use of antibiotics to modulate GI microbiota diversity and innate-adaptive immune interface are discussed. Our focus on the microbiota's influence of endoplasmic reticulum (ER) stress and autophagy signaling through hepatic EP4/CHOP/LC3B platforms reveals a novel molecular pathway and potential biomarkers determining the progression of allo-Tx damage. Understanding and harnessing the potential of microbiome/bacteriophage therapies may offer safe and effective means for personalized treatment to reduce risks of infections and immunosuppression in allo-Tx

Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing-6

Ning mutations within core TBA). Cells were treated with buffer (lanes 4 and 6) or with 1 mM theophylline (lanes 5 and 7). The product of RT-PCR assays of total RNA isolated from each well was analyzed on a 2.5% agarose gel to estimate the effect of theophylline on the efficiency of exon 2 alternative splicing. The PCR amplified bands corresponding to exon 2 included or excluded mRNA is indicated. (B) The internal exon 2 exclusion: inclusion ratio for ABT4M or ABT4Mmu in the absence (open box) or presence of theophylline (shaded box) is shown. The data represent mean ± SEM. * < 0.04 versus mutant is significant.<p><b>Copyright information:</b></p><p>Taken from "Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing"</p><p>http://www.biomedcentral.com/1471-2199/9/23</p><p>BMC Molecular Biology 2008;9():23-23.</p><p>Published online 12 Feb 2008</p><p>PMCID:PMC2275289.</p><p></p

Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing-3

He encircled adenosine residue represents branch nucleotide. The distorted hairpin loop structure is the simplified secondary structure of theophylline aptamer. The horizontal lines in aptamer lower stem represent hydrogen bonds between complementary bases. (B) The splicing of AdBPT15AG pre-mRNA with lower aptamer stem of 4 bp (shown in Fig. 1A) was compared with substrates in which the stem was either decreased to a single bp (AdBPT15AG-1S) or increased to eight bp (AdBPT15AG-8S). P-labeled pre-mRNAs were subjected to in vitro splicing for 2 h in the absence (lanes 1, 3 and 5) or presence of theophylline (lanes 2, 4 and 6) as described in Fig. 1. The extracted RNAs were fractionated on a 13% denaturing polyacrylamide gel. The % splicing calculated as in Fig. 1 and values are expressed as mean ± SEM.<p><b>Copyright information:</b></p><p>Taken from "Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing"</p><p>http://www.biomedcentral.com/1471-2199/9/23</p><p>BMC Molecular Biology 2008;9():23-23.</p><p>Published online 12 Feb 2008</p><p>PMCID:PMC2275289.</p><p></p

Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing-4

In the lower aptamer stem and the length of stem is 8 bp. (B) P-labeled AdBPT15AG-LS pre-mRNA was incubated in HeLa nuclear extract for 2 h in the absence (lanes 1) or presence of theophylline (lanes 2) as described in Fig. 1. The extracted RNA was fractionated on a 13% polyacrylamide denaturing gel. Percent splicing calculated as in Fig. 1 and values are expressed as mean ± SEM.<p><b>Copyright information:</b></p><p>Taken from "Ligand-induced sequestering of branchpoint sequence allows conditional control of splicing"</p><p>http://www.biomedcentral.com/1471-2199/9/23</p><p>BMC Molecular Biology 2008;9():23-23.</p><p>Published online 12 Feb 2008</p><p>PMCID:PMC2275289.</p><p></p