Non-genomic steroid receptor signaling pathways

1. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identificationnumber: TÁMOP-4.1.2-08/1/A-2009-0011

2. Manifestation of Novel Social Challenges of the European Unionin the Teaching Material ofMedical Biotechnology Master’s Programmesat theUniversity of Pécs and at the University of Debrecen Identification number: TÁMOP-4.1.2-08/1/A-2009-0011 Tímea Berki and Ferenc Boldizsár Signaltransduction Non-genomic steroid receptor signaling pathways

3. Glucocorticoidaction • 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 • Cortisol • Dexamethasone

4. 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

5. GR signaling pathways Hormone mReceptor Plasmamembrane Cytoplasm ? Othercytoplasmicsignalingproteins HSP90 cReceptor Geneexpression RE Mitochondrion Nucleus

6. GR signaling pathways • Genomic (“classical”) • Direct membrane effect • Membrane GR • Interaction with cytoplasmic signaling proteins • Mitochondrial translocation

7. Genomicsteroidactions Levels of regulation Milliseconds (?) Hours-days Seconds-minutes (?) G-ptotein coupled receptor Ion channel Ionotropic receptor Ion pump CBG binding in blood MDR in the membrane ? Molecular assembly Binding Metabolism and nuclear receptor fate ? TFs Proteins Multipleco-regulators RNAs Dimerization Nucleus Nucleus RE GRE GRE TFs Transcription Steroid MR/GR G-protein Neurotransmitter

8. Genomic and non-genomic GC effects Nongenomic GC effects Genomic GC effects Glucocorticoid mGR Plasmamembrane Cytoplasm Specific mGR dependent effects TF Nonspecific GC effects cGR Specific cGR dependent effects Transmembranecurrents Phosphorylationevents Calciumlevels pGRE GRE nGRE Transactivation Transrepression Slow Medium slow Rapid

9. Evidences • Binding of corticosteroidstotheglucocorticoid receptor (GR) stimulates PIP3K and protein kinase AKT, leadingtoeNOSactivation and vasorelaxation • Membraneassociated GR has beenshowntomediatelymphocytolysis • Inaddition, someglucocorticoidshavebeenshowntorapidlyinhibittherelease of theinflammatoryprostaglandin PGE2 • A multi-protein complexcomposed of theunligandedglucocorticoid receptor, Hsp90, and thetyrosinekinases LCK and FYN is recruitedtotheantigenactivated T cell receptor (TCR). This GR complex is necessaryfor TCR signalling. Onbinding of glucocorticoidsto GR, this multi-protein complexdissociatesblocking TCR signalling

10. 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 Ca2+, and increased the H+ uptake into the mitochondria • In canine kidney epithel cell systemthere is a direct effect of DX on tight junction formation • 20 min cortisol treatment caused changes in the excitability of principal basolateralamygdala neurons • GCs, especially at high doses, could change plasma membrane physico-chemical properties due to their lipid soluble nature.

11. 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 ankylosingspondylitis patients • What pathways are activated by the mGR???

12. Evidencesfor mitochondrial GR • Uponligand 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

13. Mitochondrial GR actions • In the mitochondria, the GR might act through diversemechanisms: • 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

14. Ligandinduced mitochondrial GRtranslocationin DP thymocytes Kontroll DX CMX-Ros GR+ CMX- Ros CMX-Ros GR+ CMX- Ros CD4 CD8 CD4 CD8 DIC GR DIC GR Number of mitochondria-GR colocalisedpixels * 500 Ctrl 400 DX 300 200 100 0

15. Mitochondrial GRtranslocation HSP90 cReceptor Membrane potential↓ Other proteins DNA Bcl-2 family Transcription factors Mitochondrion APOPTOSIS

16. Summary of genomic and non-genomicglucocorticoideffects Hormone mReceptor Plasmamembrane Cytoplasm ? Othercytoplasmicsignalingproteins HSP90 cReceptor Geneexpression RE Mitochondrion Nucleus