Glucocorticoids promote Von Hippel Lindau degradation and Hif-1α stabilization

Abstract

Glucocorticoid (GC) and hypoxic transcriptional responses play a central role in tissue homeostasis and regulate the cellular response to stress and inflammation, highlighting the potential for cross-talk between these two signaling pathways. We present results from an unbiased in vivo chemical screen in zebrafish that identifies GCs as activators of hypoxia-inducible factors (HIFs) in the liver. GCs activated consensus hypoxia response element (HRE) reporters in a glucocorticoid receptor (GR)-dependent manner. Importantly, GCs activated HIF transcriptional responses in a zebrafish mutant line harboring a point mutation in the GR DNA-binding domain, suggesting a nontranscriptional route for GR to activate HIF signaling. We noted that GCs increase the transcription of several key regulators of glucose metabolism that contain HREs, suggesting a role for GC/HIF cross-talk in regulating glucose homeostasis. Importantly, we show that GCs stabilize HIF protein in intact human liver tissue and isolated hepatocytes. We find that GCs limit the expression of Von Hippel Lindau protein (pVHL), a negative regulator of HIF, and that treatment with the c-src inhibitor PP2 rescued this effect, suggesting a role for GCs in promoting c-src-mediated proteosomal degradation of pVHL. Our data support a model for GCs to stabilize HIF through activation of c-src and subsequent destabilization of pVHL.hypoxia-inducible factor | glucocorticoid signaling | Von Hippel Lindau | metabolism | liver G lucocorticoids (GCs) are steroid hormones secreted from the adrenal glands that regulate carbohydrate, lipid, and protein metabolism. GCs are widely used as anti-inflammatory agents for treating pathological conditions where hypoxia plays a role in disease progression such as rheumatoid arthritis and chronic obstructive pulmonary disease. GCs and hypoxia pathways have a close interplay in physiology and disease (1-3); however, recent studies report conflicting results on the cross-talk between GC action and hypoxia (4, 5). Hypoxia-inducible factors (HIFs) are oxygen-sensitive transcriptional complexes constituted by α-and β-subunits that activate diverse pathways regulating cellular glucose and lipid metabolism and proliferation (6, 7). Under normoxic conditions, the HIF-1α transcriptional subunit is recognized by prolyl hydroxylases and targeted for degradation via the Von Hippel Lindau (VHL)-mediated ubiquitin proteasome pathway; however, under hypoxic conditions HIF-1α is stabilized and translocates to the nucleus to exert its transcriptional activity. HIFs play a central role in many disease processes and provide a therapeutic target for treating pathological conditions including cancer, ischemia, stroke, inflammation, and chronic anemia (8-11). Screens to identify agents that stabilize HIFs have identified numerous agents, with the majority acting either via iron chelation or as 2-oxyglutarate analogs (12). In vitro HIFreporter screening methods, although extremely valuable, do not provide physiological information and may overlook tissue-s...

Bidirectional crosstalk between Hypoxia-Inducible Factor and glucocorticoid signalling in zebrafish larvae

“…Together, these data allow us to show the presence of a HIF-induced negative feedback, aimed to blunt steroidogenesis in order to regulate HIF activity itself. By the way, HIF-mediated negative feedback seems to be a logic homeostatic response aimed to avoid a further GCs-induced upregulation of HIF pathway, since hypoxia has been shown to trigger glucocorticoid response (Leonard et al, 2005) and consequently, cortisol was shown to increase HIF expression (Vettori et al, 2017).…”

“…In our previous work, we identified new activators of the HIF pathway, e.g. betamethasone, a synthetic glucocorticoid drug (Vettori et al, 2017). Counterintuitively, GR loss of function was shown by Facchinello and colleagues to hamper the transcriptional activity linked to immune-response (i.e of cytokines Il1β, Il8 and Il6 and of the metalloproteinase Mmp-13) (Facchinello et al, 2017).…”

“…vhl hu2117/2117 ;phd3::EGFP i144/i144 hypoxia reporter line (this line will be called gr +/-;vhl +/hereafter). We created this line because the existing gr s357 allele may still have some activity via non-genomic pathways or tethering, promoting HIF activation upon GC treatment(Griffiths et al, 2012;Ziv et al, 2012;Vettori et al, 2017). Of note, gr mutants are hypercortisolemic(Facchinello et al, 2017;Faught and Vijayan, 2018).…”

Modulation of macrophage phenotype via phagocytosis of drug‐loaded microparticles

“…Administration of the corticosteroid dexamethasone (Dex) affects macrophage phenotype by inhibiting the expression of genes related to inflammation and promoting genes related to homing, phagocytosis, and iron metabolism, functions that promote tissue regeneration. However, systemic Dex administration has failed in a number of clinical trials and cannot be tolerated by patients over long treatment regimens because of adverse side effects .…”

“…However, attempts to overcome chronic inflammation by locally administering anti-inflammatory macrophages into the damaged tissue have been unsuccessful because macrophages rapidly respond to their environment and quickly revert to a detrimental, inflammatory state. 9,10 Administration of the corticosteroid dexamethasone (Dex) affects macrophage phenotype by inhibiting the expression of genes related to inflammation [11][12][13] and promoting genes related to homing, 14 phagocytosis, [15][16][17][18][19][20] and iron metabolism, 21 functions that promote tissue regeneration. However, systemic Dex administration has failed in a number of clinical trials and cannot be tolerated by patients over long treatment regimens because of adverse side effects.…”