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Small ubiquitin-related modifier-1 modification mediates resolution of CREB-dependent responses to hypoxia
Katrina M Comerford1, Martin O Leonard, Jorn Karhausen
1The Conway Institute for Biomolecular and Biomedical Research, University College, Dublin, D4 Ireland.
Abstract:
Phosphorylation-dependent ubiquitination combined with proteasomal degradation of transcriptional regulators is a recently appreciated mechanism for control of a number of inflammatory genes. Far less is known about the counterregulatory mechanisms that repress transcriptional activity in these pathways during resolution. Here, we investigated the transient nature of hypoxia-induced tumor necrosis factor (TNF)alpha in T84 cells, a process we have previously shown to involve phosphorylation-dependent degradation of the cAMP-response element-binding protein (CREB). Initial studies indicate hypoxia-induced TNFalpha to be a transient event, the resolution of which is associated with the appearance of a higher molecular weight modified form of CREB. Gene array analysis of mRNA derived from hypoxic cells identified a time-dependent induction of small ubiquitin-related modifier (SUMO)-1 mRNA. In prolonged hypoxia, CREB is posttranslationally modified by SUMO-1. Furthermore, SUMO-1 overexpression stabilizes CREB in hypoxia and enhances CREB-dependent reporter gene activity. Site-directed mutagenesis of lysine residues K285 and K304 identifies them as SUMO acceptors in vivo and in vitro. Mutation of K304 also results in loss of CREB nuclear localization, implying a role for SUMO-1 modification at this site in the subcellular localization of CREB. Thus, in prolonged hypoxia, CREB is modified by association with SUMO-1. Furthermore, we hypothesize that such an event stabilizes and promotes nuclear localization of CREB and thus complements an endogenous resolution phase for hypoxia-induced inflammatory processes.
Insights
Hypoxia triggers tumor necrosis factor-alpha (TNFα) via CREB degradation. SUMO-1 modification of CREB stabilizes it, promoting resolution of inflammatory responses during prolonged hypoxia.
Area of Science:
- Cellular biology
- Molecular mechanisms of inflammation
- Transcriptional regulation
Background:
- Phosphorylation-dependent ubiquitination and proteasomal degradation control inflammatory genes.
- Counterregulatory mechanisms for repressing inflammatory gene activity during resolution are poorly understood.
- Hypoxia-induced tumor necrosis factor-alpha (TNFα) in T84 cells involves cAMP-response element-binding protein (CREB) degradation.
Purpose of the Study:
- Investigate the transient nature of hypoxia-induced TNFα.
- Identify mechanisms resolving hypoxia-induced inflammatory gene expression.
- Elucidate the role of CREB modification in inflammatory gene resolution.
Main Methods:
- Gene array analysis to identify differentially expressed genes under hypoxia.
- Western blotting to detect modified forms of CREB.
- SUMO-1 overexpression and site-directed mutagenesis of CREB.
- Reporter gene assays to assess CREB transcriptional activity.
Main Results:
- Hypoxia-induced TNFα is transient, with resolution linked to a higher molecular weight CREB form.
- Small ubiquitin-related modifier (SUMO)-1 mRNA is upregulated during prolonged hypoxia.
- CREB is post-translationally modified by SUMO-1 in prolonged hypoxia.
- SUMO-1 overexpression stabilizes CREB and enhances its transcriptional activity.
- Lysine residues K285 and K304 are SUMOylation sites on CREB.
- Mutation of K304 disrupts CREB nuclear localization.
Conclusions:
- CREB is modified by SUMO-1 in prolonged hypoxia, stabilizing the protein.
- SUMO-1 modification of CREB promotes its nuclear localization.
- SUMO-1 modification of CREB likely complements endogenous resolution of hypoxia-induced inflammatory processes.