Background and previous results
The MR is a ligand-dependent transcription factor that can mediate pathophysiological processes, including inflammation and fibrosis in the cardiovascular system. An important aspect of the deleterious action of MR is the requirement of additional, poorly understood factors for “pathological” MR activation. Several studies indicate that a para-inflammatory micro-milieu, created by a shift in redox state and/or altered N- or O-centred oxidant formation (RNS, ROS), represents such a trigger. During ageing, vascular cells are increasingly exposed to this para-inflammatory micro-milieu and the resulting increase in MR signalling may amplify age-associated inflammation. Our previous work focused on molecular mechanisms underlying pathophysiological MR actions in the cardiovascular system. We investigated novel MR-protein interactions, MR-specific binding sites, enzymatic MR modifications as well as the influence of RNS on MR signalling and function. We found that ONOO– acts as a ligand-independent MR-activating factor at the CDEF domain, while NO attenuates MR signalling.
This project aims to investigate ageing-relevant alterations of MR signalling and function related to non-enzymatic modifications (Cys-nitrosylation, Tyr-nitration, oxidation) with special emphasis on radicals/radical-generating factors.
Identification of non-enzymatic MR modifications during ageing: Non-enzymatic modifications of MR will be identified by peptide arrays and by mass spectrometry in vascular cells of old and young mice and will be compared to those of the glucocorticoid receptor. Modifications will be validated by co-immunoprecipitation (Co-IP)/Western Blot and site-directed mutagenesis experiments. Signalling of modified MR: Signalling of
modified MR will be investigated by looking at ligand binding (surface plasmon resonance), nuclear translocation (time-lapse fluorescence microscopy), dimerisation (Co-IP, FRET, pull-down assays), DNA binding (TF-ELISA, EMSA, surface plasmon resonance) and MR degradation (pulse-chase experiments) in vitro (recombinant MR, cell extracts/lysates) and in vascular cells (using ONOO– and NO donors and mutated MR). Deletion constructs of different MR domains are available for additional analysis.
Consequences of modifications for MR function: Cellular function of modified MR will be assessed by GRE reporter gene assays, gene expression analysis with droplet digital PCR and monitoring of non-genomic MR signalling (MAPKinases, ratiometric Ca2+- imaging). Pathological relevance of modified MR will be investigated in vascular cells (smooth muscle and endothelial cells) by looking at cell migration, hypertrophy, proliferation, differentiation (EMT/MET) and cell viability (apoptosis/necrosis). Furthermore, markers of inflammation, ROS- and NO-generating enzymes, as well as cell adhesion and extracellular matrix proteins will be assessed and angiogenesis assays will be performed. Collaborations are planned with respect to characterisation of vascular cells with SP8 (experimental models, AGEs), SP9 (cellular stress resistance) and SP13 (endothelial cell function).