Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP /1/A

NON-GENOMIC STEROID RECEPTOR SIGNALING PATHWAYS Tímea Berki and Ferenc Boldizsár Signal transduction Manifestation of Novel Social Challenges of the European Union in the Teaching Material of Medical Biotechnology Master’s Programmes at the University of Pécs and at the University of Debrecen Identification number: TÁMOP /1/A

TÁMOP /1/A Glucocorticoid action The name glucocorticoid (glucose + cortex + steroid) derives from their role in the regulation of the metabolism of glucose, their synthesis in the adrenal cortex, and their steroidal structure. The glucocorticoid receptor is present in all vertebrate cell type DexamethasoneCortisol

TÁMOP /1/A Basics Some steroid effects can be detected within minutes eg. ion-currents change, membrane changes, phosphorylation changes Treatment of acute conditions: asthma, allergies, shock – high dose steroids exert rapid effects Apoptosis-inducing capacity

TÁMOP /1/A GR signaling pathways RE Plasma membrane Nucleus Other cytoplasmic signaling proteins Hormone mReceptor Mitochondrion Gene expression ? cReceptor Cytoplasm HSP90

TÁMOP /1/A GR signaling pathways Genomic (“classical”) Direct membrane effect Membrane GR Interaction with cytoplasmic signaling proteins Mitochondrial translocation

TÁMOP /1/A Genomic steroid actions GRE Milliseconds (?)Seconds-minutes (?)Hours-days Multiple co- regulators TFs Nucleus Dimerization Binding Molecular assembly ? ? Levels of regulation CBG binding in blood MDR in the membrane Metabolism and nuclear receptor fate Transcription GRE RE RNAs Proteins TFs G-ptotein coupled receptor Ion channelIonotropic receptor Ion pump Nucleus MR/GRSteroidG-proteinNeurotransmitter

TÁMOP /1/A Slow Medium slow Rapid Transmembrane currents Phosphorylation events Calcium levels Transmembrane currents Phosphorylation events Calcium levels Plasma membrane Cytoplasm Glucocorticoid mGR cGR Specific cGR dependent effects Specific mGR dependent effects Nonspecific GC effects GRE TF nGREpGRE Transrepression Transactivation Genomic GC effects Nongenomic GC effects Genomic and non-genomic GC effects

TÁMOP /1/A Evidences Binding of corticosteroids to the glucocorticoid receptor (GR) stimulates PIP3K and protein kinase AKT, leading to eNOS activation and vasorelaxation Membrane associated GR has been shown to mediate lymphocytolysis In addition, some glucocorticoids have been shown to rapidly inhibit the release of the inflammatory prostaglandin PGE2 A multi-protein complex composed of the unliganded glucocorticoid receptor, Hsp90, and the tyrosine kinases LCK and FYN is recruited to the antigen activated T cell receptor (TCR). This GR complex is necessary for TCR signalling. On binding of glucocorticoids to GR, this multi-protein complex dissociates blocking TCR signalling

TÁMOP /1/A Direct membrane effects Human RBC High-dose steroid treatment influenced the membrane lipid mobility in mammary cancer cell line Increased membrane lipid mobility in LPS treated B lymphocytes Inhibited membrane transport of Na + and Ca 2+, and increased the H + uptake into the mitochondria In canine kidney epithel cell system there is a direct effect of DX on tight junction formation 20 min cortisol treatment caused changes in the excitability of principal basolateral amygdala neurons GCs, especially at high doses, could change plasma membrane physico-chemical properties due to their lipid soluble nature.

TÁMOP /1/A Membrane GR Rodent and human lymphoid cell lines Amphibian brain Correlation between the mGR expression and the cell cycle-dependent GC-induced apoptosis sensitivity of a human leukaemia cell line Presence of the mGR correlates with GC- resistance of a cell type (Sionov) Human blood monocytes and B cells mGR+ monocyte frequency increased in rheumatoid arthritis, SLE and ankylosing spondylitis patients What pathways are activated by the mGR???

TÁMOP /1/A Evidences for mitochondrial GR Upon ligand binding the glucocorticoid receptor can directly translocate to the mitochondria in both lymphoid and non- lymphoid cells where it can initiate the apoptotic cascade Ligand-induced mitochondrial GR translocation showed a close correlation with the GC-induced apoptosis sensitivity of several cell types In CD4 + CD8 + (DP) thymocytes the GR translocates to the mitochondria rather than to the nucleus upon short-term in vitro GC treatment correlating with the high GC- induced apoptosis sensitivity of this cell type

TÁMOP /1/A Mitochondrial GR actions In the mitochondria, the GR might act through diverse mechanisms: Act as mitochondrial transcription factor Interaction with other mitochondrial transcription factors Interaction with pro- and anti-apoptotic proteins (eg. Bcl-2 family proteins) Decreasing the mitochondrial membrane potential

TÁMOP /1/A Ligand induced mitochondrial GR translocation in DP thymocytesDIC CD4CD8 GR CMX - Ros GR+CMX-Ros DIC CD4CD8 GR GR+CMX-Ros KontrollDX Number of mitochondria-GR colocalised pixels * Ctrl DX

TÁMOP /1/A Mitochondrial GR translocation Mitochondrion cReceptor HSP90 Membrane potential ↓ DNA Bcl-2 family Otherproteins APOPTOSIS Transcriptionfactors

TÁMOP /1/A Summary of genomic and non- genomic glucocorticoid effects RE Plasma membrane Nucleus Other cytoplasmic signaling proteins Hormone mReceptor Mitochondrion Gene expression ? cReceptor Cytoplasm HSP90