Research Article: The Chaperone-Dependent Ubiquitin Ligase CHIP Targets HIF-1α for Degradation in the Presence of Methylglyoxal

Date Published: November 29, 2010

Publisher: Public Library of Science

Author(s): Carla Figueira Bento, Rosa Fernandes, José Ramalho, Carla Marques, Fu Shang, Allen Taylor, Paulo Pereira, Ben C. B. Ko.

Abstract: Hypoxia-inducible factor-1 (HIF-1) plays a key role in cell adaptation to low oxygen and stabilization of HIF-1 is vital to ensure cell survival under hypoxia. Diabetes has been associated with impairment of the cell response to hypoxia and downregulation of HIF-1 is most likely the event that transduces hyperglycemia into increased cell death in diabetes-associated hypoxia. In this study, we aimed at identifying the molecular mechanism implicated in destabilization of HIF-1 by high glucose. In this work, we identified a new molecular mechanism whereby methylglyoxal (MGO), which accumulates in high-glucose conditions, led to a rapid proteasome-dependent degradation of HIF-1α under hypoxia. Significantly, MGO-induced degradation of HIF-1α did not require the recruitment of the ubiquitin ligase pVHL nor did it require hydroxylation of the proline residues P402/P564 of HIF-1α. Moreover, we identified CHIP (Carboxy terminus of Hsp70-Interacting Protein) as the E3 ligase that ubiquitinated HIF-1α in the presence of MGO. Consistently, silencing of endogenous CHIP and overexpression of glyoxalase I both stabilized HIF-1α under hypoxia in the presence of MGO. Data shows that increased association of Hsp40/70 with HIF-1α led to recruitment of CHIP, which promoted polyubiquitination and degradation of HIF-1α. Moreover, MGO-induced destabilization of HIF-1α led to a dramatic decrease in HIF-1 transcriptional activity. Altogether, data is consistent with a new pathway for degradation of HIF-1α in response to intracellular accumulation of MGO. Moreover, we suggest that accumulation of MGO is likely to be the link between high glucose and the loss of cell response to hypoxia in diabetes.

Partial Text: Cell response to ischemia is primarily regulated by the transcription factor HIF-1 (hypoxia-inducible factor-1) [1] that triggers protective and adaptive mechanisms, promoting cell survival under hypoxia. Thus, any mechanism that destabilizes HIF-1 has a negative impact on cell adaptation to hypoxia. HIF-1 is a heterodimer composed of two subunits: a labile HIF-1α subunit and a stable HIF-1β subunit. Under normoxia, HIF-1α is hydroxylated on prolines 402 and 564 in the oxygen dependent degradation domain (ODD) by specific prolyl hydroxylases. Once hydroxylated, HIF-1α binds to the von Hippel Lindau protein (pVHL), which is part of an E3 ligase complex, resulting in HIF-1α polyubiquitination and subsequent proteasomal degradation [2], [3], [4]. In addition, asparagine 803 is also hydroxylated inhibiting the interaction of HIF-1α with the co-activator p300, leading to further repression of HIF-1 transcriptional activity [5]. When oxygen becomes limiting, the proline residues are not hydroxylated and HIF-1α escapes degradation, accumulating in the cell. HIF-1α is imported into the nucleus, dimerizes with HIF-1β and binds to hypoxia responsive elements (HREs), enabling transcriptional activation of more than 70 genes that help cells to cope and survive under hypoxia [1], [6], such as the vascular endothelial growth factor (VEGF).

In this study we elucidated a new pathway for degradation of HIF-1α. Data presented in this paper is consistent with a molecular mechanism in which HIF-1α-modification by MGO leads to increased association with the molecular chaperones Hsp40/70. This, in turn, recruits CHIP, leading to the ubiquitination and proteasome-dependent degradation of HIF-1α (Figure 8). We further showed that this mechanism is independent on the recruitment of pVHL and does not require hydroxylation of HIF-1α. This new mechanism is triggered by accumulation of MGO and is likely to have a significant physiological impact in conditions with increased availability of glucose, such as diabetes. Indeed, we showed that exposure of cells to high glucose leads to a sustained increase in the intracellular levels of MGO, which were sufficient to activate the degradation of HIF-1α. Moreover, scavenging of intracellular MGO by overexpressing glyoxalase I prevented degradation of HIF-1α under high glucose. Additionally, MGO-induced degradation of HIF-1α led to decreased transcriptional activity of HIF-1 and decreased expression of VEGF under hypoxia. Significantly, we also observed that data obtained in cell culture systems is consistent with observations in retinas of diabetic animals. Indeed, increased availability of MGO in the retina of Goto-Kakizaki rats was accompanied by decreased levels of HIF-1α and VEGF, as well as increased levels of apoptotic markers and enhanced vascular permeability [34]. The reduction of VEGF expression under high glucose and in response to hypoxia was also described before [22], [35]. The authors suggested that increased levels of MGO under hyperglycemia induce HIF-1α [22] and p300 modifications [35], which were sufficient to disrupt the interaction between HIF-1α/HIF-1β and HIF-1α/p300, leading to loss of HIF-1 transcriptional activity and poor response to hypoxia. Although this and other modifications induced by MGO cannot be excluded and are likely to account for the plethora of noxious effects of MGO in cells, our study provides robust data that elucidates an independent molecular mechanism whereby MGO can lead to proteasomal degradation of HIF-1α mediated by CHIP. It is perhaps interesting to note that the two observations are not inconsistent. Indeed, it is plausible that the decreased interaction between HIF-1α/p300 and HIF-1α/HIF-1β in the presence of MGO increases the amount of modified and monomeric HIF-1α that is available to undergo degradation through a CHIP-mediated pathway.