Loss of endothelial barrier integrity in mice with conditional ablation of podocalyxin (Podxl) in endothelial cells.

Podocalyxin (Podxl) has an essential role in the development and function of the kidney glomerular filtration barrier. It is also expressed by vascular endothelia but perinatal lethality of podxl(-/-) mice has precluded understanding of its function in adult vascular endothelial cells (ECs). In this work, we show that conditional knockout mice with deletion of Podxl restricted to the vascular endothelium grow normally but most die spontaneously around three months of age. Histological analysis showed a nonspecific inflammatory infiltrate within the vessel wall frequently associated with degenerative changes, and involving vessels of different caliber in one or more organs. Podxl-deficient lung EC cultures exhibit increased permeability to dextran and macrophage transmigration. After thrombin stimulation, ECs lacking Podxl showed delayed recovery of VE-cadherin cell contacts, persistence of F-actin stress fibers, and sustained phosphorylation of the ERM complex and activation of RhoA, suggesting a failure in endothelial barrier stabilization. The results suggest that Podxl has an essential role in the regulation of endothelial permeability by influencing the mechanisms involved in the restoration of endothelial barrier integrity after injury.

Human bone marrow stromal cells lose immunosuppressive and anti-inflammatory properties upon oncogenic transformation.

Because of their immunomodulatory properties, human bone marrow stromal cells (hBMSCs) represent promising stem cells for treatment of immune disorders. hBMSCs expansion precedes their clinical use, so the possibility that hBMSCs undergo spontaneous transformation upon long-term culture should be addressed. Whether hBMSCs retain immunosuppressive and anti-inflammatory properties upon oncogenic transformation remains unknown. Using sequentially mutated hBMSCs and spontaneously transformed hBMSCs, we report that, upon oncogenic transformation, hBMSCs lose immunosuppressive and anti-inflammatory properties in vitro and in vivo. Transcriptome profiling and functional assays reveal immune effectors underlying the loss of immunomodulation in transformed hBMSCs. They display a proinflammatory transcriptomic signature, with deregulation of immune and inflammatory modulators and regulators of the prostaglandin synthesis. Transformed hBMSCs lose their capacity to secrete the immunosuppressive prostacyclins prostaglandin E2 (PGE2) and PGI2 but produce proinflammatory thromboxanes. Together, the immunoregulatory profile adopted by hBMSCs largely depends on intrinsic genetic-molecular determinants triggered by genomic instability/oncogenic transformation.

Generation of mice with conditional ablation of the Cd40lg gene: new insights on the role of CD40L.

CD40 ligand (CD40L) acts as an immune modulator in activated T cells, and mutations in the extracellular domain are associated to X-linked hyper IgM syndrome. A role for platelet CD40L in mediating thrombotic and inflammatory processes in atherosclerosis has also been reported. Using the Cre/loxP recombination technology we generated four knockout lines of mice with deletion of the Cd40lg gene restricted to the hematopoietic system. Mouse lines with expression of Cre recombinase driven by the Tie2, Vav1, or CD4 promoters showed in vivo ablation of CD40L in leukocytes and platelets. In contrast, in mice with Cre expression driven by the megakaryocyte lineage-restricted Pf4 promoter, abolition of CD40L expression was observed in megakaryocytes cultured in vitro, but not in circulating platelets. Characterization of these animals revealed reduced in vivo thrombogenesis and defective activation of washed CD40L-deficient platelets, suggesting that membrane-bound CD40L is involved in the control of haemostasis acting as a platelet co-activator. In addition, we report the practically absence of CD40L in mouse and human endothelial cells, as well as the detection of an exon 3-deleted CD40L transcript in both platelets and leukocytes of mouse and human origin. Finally, compared with their corresponding littermate floxed controls, Cre+ mice carrying CD40-deficient leukocytes did not exhibit increased IgM levels, and reduction of IgA and IgG levels was statistically significant only in Tie2-Cre+ mice, suggesting that expression of CD40L in an earlier developmental step may be determinant in the regulation of the class switch recombination process.

Control of cell adhesion and migration by podocalyxin. Implication of Rac1 and Cdc42.

Podocalyxin (PODXL) is a type I membrane sialomucin, originally described in the epithelial cells (podocytes) of kidney glomeruli. PODXL is also found in extra-renal tissues and in certain aggressive tumors, but its precise pathophysiological role is unknown. Expression of PODXL in CHO cells enhances their adhesive, migratory and cell-cell interactive properties in a selectin and integrin-dependent manner. We aimed at defining the PODXL domains responsible for those cell responses. For this purpose we have analyzed the cell adhesion/migration responses to deletion mutants of human PODXL, and the correlation with the activities of Rac1 and Cdc42 GTPases. The results obtained indicate that integrity of the PODXL ectodomain is essential for enhancing cell adhesion but not migration, while the integrity of the cytoplasmic domain is required for both adhesion and migration. Deletion of the carboxy-terminal DTHL domain (PODXL-ΔDTHL) limited only cell adhesion. The activities of Rac1 and Cdc42 GTPases parallel the PODXL-induced variations in cell adhesion and migration. Moreover, silencing the rac1 gene virtually abolished the effect of PODXL in enhancing cell adhesion.

Histone H1 poly[ADP]-ribosylation regulates the chromatin alterations required for learning consolidation.

Memory formation requires changes in gene expression, which are regulated by the activation of transcription factors and by changes in epigenetic factors. Poly[ADP]-ribosylation of nuclear proteins has been postulated as a chromatin modification involved in memory consolidation, although the mechanisms involved are not well characterized. Here we demonstrate that poly[ADP]-ribose polymerase 1 (PARP-1) activity and the poly[ADP]-ribosylation of proteins over a specific time course is required for the changes in synaptic plasticity related to memory stabilization in mice. At the molecular level, histone H1 poly[ADP]-ribosylation was evident in the hippocampus after the acquisition period, and it was selectively released in a PARP-1-dependent manner at the promoters of cAMP response element-binding protein and nuclear factor-κB dependent genes associated with learning and memory. These findings suggest that histone H1 poly[ADP]-ribosylation, and its loss at specific loci, is an epigenetic mechanism involved in the reprogramming of neuronal gene expression required for memory consolidation.

Sirtuin 1 regulation of developmental genes during differentiation of stem cells.

The longevity-promoting NAD+-dependent class III histone deacetylase Sirtuin 1 (SIRT1) is involved in stem cell function by controlling cell fate decision and/or by regulating the p53-dependent expression of NANOG. We show that SIRT1 is down-regulated precisely during human embryonic stem cell differentiation at both mRNA and protein levels and that the decrease in Sirt1 mRNA is mediated by a molecular pathway that involves the RNA-binding protein HuR and the arginine methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). SIRT1 down-regulation leads to reactivation of key developmental genes such as the neuroretinal morphogenesis effectors DLL4, TBX3, and PAX6, which are epigenetically repressed by this histone deacetylase in pluripotent human embryonic stem cells. Our results indicate that SIRT1 is regulated during stem cell differentiation in the context of a yet-unknown epigenetic pathway that controls specific developmental genes in embryonic stem cells.

Cancer genes hypermethylated in human embryonic stem cells.

Developmental genes are silenced in embryonic stem cells by a bivalent histone-based chromatin mark. It has been proposed that this mark also confers a predisposition to aberrant DNA promoter hypermethylation of tumor suppressor genes (TSGs) in cancer. We report here that silencing of a significant proportion of these TSGs in human embryonic and adult stem cells is associated with promoter DNA hypermethylation. Our results indicate a role for DNA methylation in the control of gene expression in human stem cells and suggest that, for genes repressed by promoter hypermethylation in stem cells in vivo, the aberrant process in cancer could be understood as a defect in establishing an unmethylated promoter during differentiation, rather than as an anomalous process of de novo hypermethylation.