Hypoxia-inducible factors (HIFs) are key elements for controlling immune cell metabolism and functions. While HIFs are known to be involved in T cells and macrophages activation, their functions in B lymphocytes are poorly defined. Here, we show that hypoxia-inducible factor-1α (HIF-1α) contributes to IL-10 production by B cells. HIF-1α regulates IL-10 expression, and HIF-1α-dependent glycolysis facilitates CD1dhiCD5+ B cells expansion. Mice with B cell-specific deletion of Hif1a have reduced number of IL-10-producing B cells, which result in exacerbated collagen-induced arthritis and experimental autoimmune encephalomyelitis. Wild-type CD1dhiCD5+ B cells, but not Hif1a-deficient CD1dhiCD5+ B cells, protect recipient mice from autoimmune disease, while the protective function of Hif1a-deficient CD1dhiCD5+ B cells is restored when their defective IL-10 expression is genetically corrected. Taken together, this study demonstrates the key function of the hypoxia-associated transcription factor HIF-1α in driving IL-10 expression in CD1dhiCD5+ B cells, and in controlling their protective activity in autoimmune disease.

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Screening of the entire let-7 family of microRNAs (miRNA) by in situ hybridization identified let-7g as the only member, the diminished expression of which was significantly associated with lymph node metastasis and poor survival in breast cancer patients. Abrogation of let-7g expression in otherwise nonmetastatic mammary carcinoma cells elicited rapid metastasis from the orthotopic location, through preferential targets, Grb2-associated binding protein 2 (GAB2) and fibronectin 1 (FN1), and consequent activation of p44/42 mitogenactivated protein kinase (MAPK) and specific matrix metalloproteinases. Treatment with estrogen or epidermal growth factor specifically reduced the expression of mature let-7g through activation of p44/42 MAPK and subsequently stimulated expression of GAB2 and FN1, which, in turn, promoted tumor invasion. We thus identify let-7g as a unique member of the let-7 miRNA family that can serve as a prognostic biomarker in breast cancer and also propose a paradigm used by specific signaling molecules via let-7g to cooperatively promote breast cancer invasion and metastasis. Thus, let-7 family members neither possess equivalent clinicopathologic correlation nor function in breast cancer. Cancer Res; 71(20); 6463-74. Ó2011 AACR.

Cystic fibrosis (CF) is caused by mutations that disrupt the plasma membrane expression, stability, and function of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel. Two small molecules, the CFTR corrector lumacaftor and the potentiator ivacaftor, are now used clinically to treat CF, although some studies suggest that they have counteracting effects on CFTR stability. Here, we investigated the impact of these compounds on the instability of F508del-CFTR, the most common CF mutation. To study individual CFTR Cl− channels, we performed single-channel recording, whereas to assess entire CFTR populations, we used purified CFTR proteins and macroscopic CFTR Cl− currents. At 37 °C, low temperature-rescued F508del-CFTR more rapidly lost function in cell-free membrane patches and showed altered channel gating and current flow through open channels. Compared with purified wild-type CFTR, the full-length F508del-CFTR was about 10 °C less thermostable. Lumacaftor partially stabilized purified full-length F508del-CFTR and slightly delayed deactivation of individual F508del-CFTR Cl− channels. By contrast, ivacaftor further destabilized full-length F508del-CFTR and accelerated channel deactivation. Chronic (prolonged) co-incubation of F508del-CFTR-expressing cells with lumacaftor and ivacaftor deactivated macroscopic F508del-CFTR Cl− currents. However, at the single-channel level, chronic co-incubation greatly increased F508del-CFTR channel activity and temporal stability in most, but not all, cell-free membrane patches. We conclude that chronic lumacaftor and ivacaftor co-treatment restores stability in a small subpopulation of F508del-CFTR Cl− channels but that the majority remain destabilized. A fuller understanding of these effects and the characterization of the small F508del-CFTR subpopulation might be crucial for CF therapy development.

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Heparan sulfate (HS) is a complex linear polysaccharide that modulates a wide range of biological functions. Elucidating the structure-function relationship of HS has been challenging. Here we report the generation of a HS mutant mouse lung endothelial cell library by systematic deletion of HS genes expressing in the cell. We applied this library to answer several fundamental questions about HS biology including: 1) determining that strictly defined fine structure of HS, not its overall sulfation degree, is more important for FGF2-FGFR1 signaling; 2) defining the epitope features of commonly used anti-HS phage display antibodies; and 3) delineating the fine inter-regulation networks of HS modification and chain length by HS genes in mammalian cells and at a cell type specific level. Our mutant cell library will enable robust and systematic interrogation of the roles and related structures of HS in a cellular context.

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