Glucocorticoids Flashcards

Question

how are GCCs implicated in osteoporosis?

Answer 1

Glucocorticoid-induced osteoporosis is caused by impairment of this remodelling process. - leads to loss of trabecular lattice and loss of structural strength - Effects of GCs on osteoblasts most important - GCs inhibit osteoblast function and proliferation, increase their apoptosis (programmed cell death)

Answer 2

Turnover of bone is continual – remodelling - osteoblast lays down bone - Osteoclast (releated to macrophages) destry bone – refines bone - Work together as functional unit – communicate with one another via soluble mediators made by osteoblasts - RANKL increases osteoclast activity, OPG which inhibits osteoclast activity with - osteoblast produce these mediators to instruct osteoclast feedback and activation – tightly regulated

Answer 3

GCCs cause breakdown communication between bone cells, where osteoclasts dominate and cause net loss of bone - GCCs increase RANKL production and decrease OPG - indirectly increases osteoclast activity - GCCs also drive osteoblast apoptosis - increases resorption of bone and decreases formation of bone This process is most active at trabechular bone – so this is where most GCC effects are seen

Answer 4

Fragility fractures occur in 30-50% of patients taking long term systemic GCs. Fracture risk increases markedly in first 3 months after initiation of GC Hip fracture risk increases up to 7-fold, vertebral fracture risk up to 17-fold with > 3 months prednisone > 10 mg/d. This is dangerous for elderly people and those with joint problems - significant morbidity, mortality and health-care costs

Answer 5

Can detect markers in blood of increased bone destruction - Monitoring bone mineral density, serum osteocalcin, collagen telopeptides (marker of increased bone turnover). Bisphosphonates can treat osteoporosis - inhibits increased bone turnover Can reduce GCCs to minimal needed dose - tapering

Answer 6

GR is a TF, ubiquitously expressed with modular structure: - DBD – binding to DNA, responsible for dimerisation of GR - LBD – ligand-activated TF, this is where GCC binds on the GR - AF1/2 (activation domains) switches on transcription to increase gene expression

Answer 7

1. Guidance system to bind DNA at the right gene - mediated by sequence-specific DBD which recognises palindromic DNA (AGAACA) - upon binding to the palindromic motif, the GR dimerises 2. Payload = transcriptional activation domains switch genes on - changes gene expression

Answer 8

In abensce of GCC, GR is in cytoplasm, inactive, held by heat shock protein chaperone complex

Answer 9

GCC binds and causes allosteroic modification in GR to release it from chaperones and enter nucleus - GCCs are lipophilic so pass through plasma membrane to bind GR - GR dissociates from heat-shock proteins and enters nucleus

Answer 10

1. GR dimerises, binds palindromic DNA sequences and drives transcription of genes - transactivation 2. Without dimerisation, GR becomes tethered and indirectly bound to DNA by NF-KB – GR stops NF-KB TF complex forming - This stops NF-KB-driven inflammatory responses – transrepression

Answer 11

GRdim mouse: - K/I mouse: point mutation of a single amino acid in the DNA binding domain of mouse GR gene - This impaired the ability of GR to dimerise and bind to palindromic GREs and activate transcription. - The mutant form of GR could still inhibit the function of NF-kB – could still transrepress inflammatory gene expression

Answer 12

Mice expressing only GRdim failed to upregulate gluconeogenic genes such as TAT and PEPCK in response to GCs, but still showed anti-inflammatory responses to GCs in a simple model of irritant-induced skin inflammation Model of inflammation via toxin added to ears of mice Treat mouse with GCC – reduces inflammation When mice have Grdim, they couldn’t activate gluconeogenesis, but was anti-inflammatory

Answer 13

GR, when activated by GCC e.g. dex, enters nucleus and can either: 1. transrepression - switch off gene expression as a monomer - assumes this pathway has all the good effects - desirable anti-inflammatory effects of GCC 2. transactivation - switch on gene expression as a dimer - assumes this pathway has all the side effects - responsible for harmful effects of GCCs like on metabolism assumes 2 distinct biological activities

Answer 14

Suggested to tweak GCC ligand to generate compound that only transrepresses and doesn't transactivate These are called: Dissociated glucocorticoids Selective glucocorticoid receptor agonists (SEGRA) Selective glucocorticoid receptor modulators (SGRM) Dissociated agonists of the glucocorticoid receptor (DAGR) - these bind GR, but have different structure to cortisol

Answer 15

2 reporters: - One for transactivation – couple of palindromic GRE binding sites upstream of reporter - see how well the compound activates gene expression. add fluorescent protein - One for transrepression – couple of kB binding sites upstream of another reporter - reports on anti-inflammatory function of GCC. add fluorescent protein can add both reporters into same cell to assay in one step and test many compounds - measure ratio of one reporter compared to another Apply inflammatory stimulus to switch repressor on for NF-KB to be active

Answer 16

Its too simple - It requires the glucocorticoid receptor to display a convenient mechanistic separation between actions that are all physiological and “on-target”, but which the physician considers as desirable or undesirable. - these separate functions are increasingly at odds with experimental observation. - No SEGRA/SGRM/DAGR has yet made it through clinical trial (demonstrated at least equivalent efficacy as classical GCs, but improved safety profile).

Answer 17

GCCs can inhibit p38 pathway by switching on gene expression of DUSP1 - transactivation - Feedback mechanism to switch off p38 - Anti-inflammatory effects of GCC require DUSP1 gene – K/O of DUSP1 means GCC cant work Transactivation is crucial: - This can enable anti-inflammatory effects - The hypothesis is too simplified: GCCs can transactivate genes that turn off inflammation disputes transrepression hypothesis that transactivation is always bad

Answer 18

Immune cells undergo metabolic changes upon activation - Resting = TCA, OXPHOS – 32 ATP e.g. M2 macrophages - Active = glycolysis, increased glucose uptake, despite presence of oxygen, driven by HIF1a – rapid response, but inefficient ATP production – only 2 per molecule glucose e.g. M1 macrophages metabolism is coupled to immune effector functions, specifically in cytokine production

Answer 19

GCCs alter metabolism of immune system to conserve glucose - Suppression of inflammation is as a consequence GCC affects metabolic tissues e.g. liver, muscle, adipose - can cause metabolic harm to patients e.g. disturbs glucose homeostasis and lipid metabolism - immune cells could be classed as a metabolic tissue - both harmful and beneficial effects of GCCs may be related to metabolism

Answer 20

Seahorse assay – measure ECAR (glycolysis-mediated lactate excretion): - Unstim = resting cell ECAR - Red = LPS stimulation = increases ECAR, high glucose consumption, increases lactate secretion - Add dex with LPS = reduces acidification as it reduces glycolyisis, reduces glucose uptake, reduces lactate secretion into medium

Answer 21

HIF1a drives metabolic reprogramming - Regulated by oxygen availability - TF that allows cells to metabolically adapt to hypoxia

Answer 22

Resting = low HIF expression LPS stim = upregulated HIF Dex = reduced HIF activation – dex blocks HIF upregulation

Answer 23

HIF K/O macrophage stimulated with LPS compared to WT - See how gene expression changes with RNA seq data - HIF-dependent inflammatory chemokines downregulated when HIF-K/O e.g. CXCLs Another group of genes involved in glycolysis downregulated when HIF K/O: - glut1 (slc2a1) is reduced when HIF is deficient - Slc16a3 (MCT4 – enables lactate excretion) is reduced are these HIF-dependent genes sensitive to dex?

Answer 24

Yes - qPCR measures gene expression - These glycolytic genes e.g. slc2a1 are induced by LPS and inhibited by dex By altering HIF expression/activation in macrophages, GCCs block glycolysis

Answer 25

ibuprofen aspirin naproxen

Answer 26

Treats inflammation and pain - Acetyl-salicylic acid = aspirin (modified salicylic acid) How it works: - Lipids in membrane are processed to form prostaglandins, derived from arachadonic acid (AA), via COX enzymes (COX1 or COX2) - COX convert arachidonic acid to PGG2 and then to PGH2 and then PG synthases make PG products - Aspirin inhibits COX1 and COX2

Answer 27

COX is rate limiting step in prostaglandin synthesis to generate PGH2 - Downstream of PGH2 is prostaglandin synthases to generate different PG products

Answer 28

COX varies in tissues, constitutive or inducible - COX1 is constitutive - COX2 is inducible – LPS stimulation upregulates COX2 in macrophages Synthase enzymes are tissue specific, some constitutive some inducible - They work via different GPCRs - Proteins associated with GPCRs change according to cell type and condition

Answer 29

PGE2 important in inflammatory responses - Causes vasodilation - Increases vascular permeability - Sensitises neurons – increases pain signalling at inflammatory site PGE2 affects immune cells

Answer 30

Inhibits COX1/2 equally to prevent PGE2 synthesis - COX1 also important for gut homeostasis – enables cells to produce PGI2 to maintain gut barrier integrity and prevent leakage - aspirin therefore causes side effects in gut - Aspirin at high doses = ulceration and bleeding of gut - Inhibiting COX1 is harmful, need COX2-specific inhibitor

Answer 31

- Platelets express COX1 which generates PGH2 to thromboxin A2 (TA2) - TA2 is procoagulant and clots blood - This is countered by PGI2 downstream of COX2 in vascular endothelial cells - Selective inhibition of COX2 means there's no block of TA2 by PGI2, so more clotting, thrombosis, vasoconstriction and cardiovascular risk - This is a theory