The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease.

RNA functions through the dynamic formation of complexes with RNA-binding proteins (RBPs) in all clades of life. We determined the RBP repertoire of beating cardiomyocytic HL-1 cells by jointly employing two in vivo proteomic methods, mRNA interactome capture and RBDmap. Together, these yielded 1,148 RBPs, 391 of which are shared with all other available mammalian RBP repertoires, while 393 are thus far unique to cardiomyocytes. RBDmap further identified 568 regions of RNA contact within 368 RBPs. The cardiomyocyte mRNA interactome composition reflects their unique biology. Proteins with roles in cardiovascular physiology or disease, mitochondrial function, and intermediary metabolism are all highly represented. Notably, we identified 73 metabolic enzymes as RBPs. RNA-enzyme contacts frequently involve Rossmann fold domains with examples in evidence of both, mutual exclusivity of, or compatibility between RNA binding and enzymatic function. Our findings raise the prospect of previously hidden RNA-mediated regulatory interactions among cardiomyocyte gene expression, physiology, and metabolism.

Mice deficient in heparanase exhibit impaired dendritic cell migration and reduced airway inflammation.

Heparanase is a ß-d-endoglucuronidase that cleaves heparan sulphate, a key component of the ECM and basement membrane. The remodelling of the ECM by heparanase has been proposed to regulate both normal physiological and pathological processes, including wound healing, inflammation, tumour angiogenesis and cell migration. Heparanase is also known to exhibit non-enzymatic functions by regulating cell adhesion, cell signalling and differentiation. In this study, constitutive heparanase-deficient (Hpse(-/-) ) mice were generated on a C57BL/6 background using the Cre/loxP recombination system, with a complete lack of heparanase mRNA, protein and activity. Although heparanase has been implicated in embryogenesis and development, Hpse(-/-) mice are anatomically normal and fertile. Interestingly, consistent with the suggested function of heparanase in cell migration, the trafficking of dendritic cells from the skin to the draining lymph nodes was markedly reduced in Hpse(-/-) mice. Furthermore, the ability of Hpse(-/-) mice to generate an allergic inflammatory response in the airways, a process that requires dendritic cell migration, was also impaired. These findings establish an important role for heparanase in immunity and identify the enzyme as a potential target for regulation of an immune response.

The NF-κB transcription factor c-Rel is required for Th17 effector cell development in experimental autoimmune encephalomyelitis.

Experimental autoimmune encephalomyelitis (EAE) is a T cell-mediated autoimmune disease involving effector Th subsets such as Th1 and Th17. In this study, we demonstrate that mice lacking the NF-κB transcription factor family member c-Rel (rel(-/-)), which are known to be resistant to EAE, show impaired Th17 development. Mixed bone marrow chimeras and EAE adoptive transfer experiments show that the deficiency of effector Th17 cells in rel(-/-) mice is T cell intrinsic. Consistent with this finding, c-Rel was activated in response to TCR signaling in the early stages of Th17 development and controlled the expression of Rorc, which encodes the Th17 transcription factor retinoic acid-related orphan receptor γt. CD28, but not IL-2, repression of Th17 development was dependent on c-Rel, implicating a dual role for c-Rel in modulating Th17 development. Adoptive transfer experiments also suggested that c-Rel control of regulatory T cell differentiation and homeostasis influences EAE development and severity by influencing the balance between Th17 and regulatory T cells. Collectively, our findings indicate that in addition to promoting Th1 differentiation, c-Rel regulates the development and severity of EAE via multiple mechanisms that impact on the generation of Th17 cells.

Regulation of mouse Heparanase gene expression in T lymphocytes and tumor cells.

Heparanase (HPSE) is an endoglycosidase that cleaves heparan sulfate (HS) and plays an important role in tumor metastasis, angiogenesis and inflammation. The regulation of HPSE expression and function is tightly controlled and the increasing use of the mouse as an animal model to define the role of HPSE in many physiological and pathological settings, makes understanding the regulatory mechanisms of HPSE in this species of fundamental importance. However, the expression distribution of the mouse Hpse gene and the mechanisms that regulate its transcription are poorly defined. In this study, the mouse Hpse gene was determined to encode for two mRNA transcripts of 1.9 and 3.2 kb in length with identical open reading frames that showed similar tissue expression distribution to the human HPSE. The mouse Hpse promoter was cloned and a 478-bp minimal promoter was identified that contained regulatory elements responsible for both basal promoter activity in mouse tumor cells as well as inducible activity in T cells. Mutagenesis and transactivation studies identified a functional site in the minimal promoter region for the transcription factor Early growth response gene 1 (Egr1). Interestingly, Egr1 acted differentially in mouse tumor cells, functioning in an activating or repressive manner in breast carcinoma or melanoma cells, respectively. Furthermore, the proximal region of the promoter, identified as important in the regulation of Hpse transcription, was shown to become accessible in T cells upon cell activation. Significantly, the maximal accessibility of the promoter occurred at 16 h post-stimulation, which correlated with the induction kinetics of Hpse mRNA expression. In summary, this study demonstrates that mouse Hpse is expressed and regulated in a similar manner to human HPSE and also provides some novel insights into mechanisms of Hpse gene regulation that are likely to be relevant to control of the human gene.