Calpain mediates a von Hippel-Lindau protein-independent destruction of hypoxia-inducible factor-1alpha

Hypoxia-inducible factor 1 (HIF-1) is controlled through stability regulation of its alpha subunit, which is expressed under hypoxia but degraded under normoxia. Degradation of HIF-1alpha requires association of the von Hippel Lindau protein (pVHL) to provoke ubiquitination followed by proteasomal digestion. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha under normoxia but destabilizes the protein under hypoxia. To understand the role of NO under hypoxia we made use of pVHL-deficient renal carcinoma cells (RCC4) that show a high steady state HIF-1alpha expression under normoxia. Exposing RCC4 cells to hypoxia in combination with the NO donor DETA-NO (2,2'-(hydroxynitrosohydrazono) bis-ethanimine), but not hypoxia or DETA-NO alone, decreased HIF-1alpha protein and attenuated HIF-1 transactivation. Mechanistically, we noticed a role of calpain because calpain inhibitors reversed HIF-1alpha degradation. Furthermore, chelating intracellular calcium attenuated HIF-1alpha destruction by hypoxia/DETA-NO, whereas a calcium increase was sufficient to lower the amount of HIF-1alpha even under normoxia. An active role of calpain in lowering HIF-1alpha amount was also evident in pVHL-containing human embryonic kidney cells when the calcium pump inhibitor thapsigargin reduced HIF-1alpha that was stabilized by the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). We conclude that calcium contributes to HIF-1alpha destruction involving the calpain system.     

Thioredoxin 1 and thioredoxin 2 have opposed regulatory functions on hypoxia-inducible factor-1alpha

Hypoxia inducible factor 1 (HIF-1), a key regulator for adaptation to hypoxia, is composed of HIF-1alpha and HIF-1beta. In this study, we present evidence that overexpression of mitochondria-located thioredoxin 2 (Trx2) attenuated hypoxia-evoked HIF-1alpha accumulation, whereas cytosolic thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount. Transactivation of HIF-1 is decreased by overexpression of Trx2 but stimulated by Trx1. Inhibition of proteasomal degradation of HIF-1alpha in Trx2-overexpressing cells did not fully restore HIF-1alpha protein levels, while HIF-1alpha accumulation was enhanced in Trx1-overexpressing cells. Reporter assays showed that cap-dependent translation is increased by Trx1 and decreased by Trx2, whereas HIF-1alpha mRNA levels remained unaltered. These data suggest that thioredoxins affect the synthesis of HIF-1alpha. Trx1 facilitated synthesis of HIF-1alpha by activating Akt, p70S6K, and eIF-4E, known to control cap-dependent translation. In contrast, Trx2 attenuated activities of Akt, p70S6K, and eIF-4E and provoked an increase in mitochondrial reactive oxygen species production. MitoQ, a mitochondria specific antioxidant, reversed HIF-1alpha accumulation as well as Akt activation under hypoxia in Trx2 cells, supporting the notion of translation control mechanisms in affecting HIF-1alpha protein accumulation.     

The zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, increases the level of nonfunctional HIF-1alpha protein in normoxic cells

The hypoxia-inducible factor-1alpha (HIF-1alpha) subunit is activated in response to lack of oxygen. HIF-1alpha-specific prolyl hydroxylase and factor inhibiting HIF-1alpha (FIH-1) catalyze hydroxylation of the proline and asparagine residues of HIF-1alpha, respectively. The hydroxyproline then interacts with ubiquitin E3 ligase, the von Hippel-Lindau protein, leading to degradation of HIF-1alpha by ubiquitin-dependent proteasomes, while the hydroxylation of the asparagine residue prevents recruitment of the coactivator, cAMP-response element-binding protein (CBP), thereby decreasing the transactivation ability of HIF-1alpha. We found that the Zn-specific chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), enhances the activity of HIF-1alpha-proline hydroxylase 2 but the level of HIF-1alpha protein does not fall because TPEN also inhibits ubiquitination. Since the Zn chelator does not prevent FIH-1 from hydroxylating the asparagine residue of HIF-1alpha, its presence leads to the accumulation of HIF-1alpha that is both prolyl and asparaginyl hydroxylated and is therefore nonfunctional. In hypoxic cells, TPEN also prevents HIF-1alpha from interacting with CBP, so reducing expression of HIF-1alpha target genes. As a result, Zn chelation causes the accumulation of nonfunctional HIF-1alpha protein in both normoxia and hypoxia.     

VHL protein-interacting deubiquitinating enzyme 2 deubiquitinates and stabilizes HIF-1alpha

Hypoxia-inducible factor (HIF)-1alpha is a short-lived protein and is ubiquitinated and degraded through the von Hippel-Lindau protein (pVHL)-E3 ubiquitin ligase pathway at normoxia. Deubiquitination, by reversing ubiquitination, has been recognized as an important regulatory step in ubiquitination-related processes. Here, we show that pVHL-interacting deubiquitinating enzyme 2, VDU2, but not VDU1, interacts with HIF-1alpha. VDU2 can specifically deubiquitinate and stabilize HIF-1alpha and, therefore, increase expression of HIF-1alpha targeted genes, such as vascular endothelial growth factor (VEGF). These findings suggest that ubiquitination of HIF-1alpha is a dynamic process and that ubiquitinated HIF-1alpha might be rescued from degradation by VDU2 through deubiquitination. Although pVHL functions as a master control for HIF-1alpha stabilization, as pVHL-E3 ligase mediates the ubiquitination of both HIF-1alpha and VDU2, the balance between the pVHL-mediated ubiquitination and VDU2-mediated deubiquitination of HIF-1alpha provides another level of control for HIF-1alpha stabilization.     

Knockdown of hypoxia-inducible factor-1alpha by siRNA inhibits C2C12 myoblast differentiation

We analyzed the role of Hypoxia-inducible factor (HIF)-1alpha in myoblast differentiation by examining the expression and regulation of HIF-1alpha in proliferating and differentiating C2C12 myoblast, and by knocking down HIF-1alpha of C2C12 myoblasts with small interfering RNA (siRNA), given that HIF-1alpha has been shown to be involved in differentiative process in non-muscle tissues. Although HIF-1alpha mRNA was constantly expressed in C2C12 myoblasts both under growth and differentiating phase, HIF-1alpha protein was hardly detectable in the growth phase but became detectable only during myogenic differentiation even under normoxia. During early stage of C2C12 myogenesis, HIF-1alpha accumulated in the nuclei of myogenin-positive myoblasts. The inhibition of proteasome in the growth phase led to HIF-1alpha protein accumulation, whereas in the differentiation phase the inhibition of Hsp90, which stabilizes HIF-1alpha, suppressed HIF-1alpha accumulation. Therefore, we suggest that the level of HIF-1alpha protein expression is regulated by a proteasome-and chaperon-dependent pathway in C2C12 myoblast. Knockdown of HIF-1alpha effectively blocked myotube formation and myosin heavy chain (MHC) expression. Finally, HIF-1alpha expression in vivo was confirmed in the regenerative muscle tissue of mice after eccentric exercise. We conclude that HIF-1alpha is required for C2C12 myogenesis in vitro, and suggest that HIF-1alpha may have an essential role in regenerative muscle tissue in vivo.     

Hypoxic but not anoxic stabilization of HIF-1alpha requires mitochondrial reactive oxygen species

The molecular mechanisms by which cells detect hypoxia (1.5% O2), resulting in the stabilization of hypoxia-inducible factor 1alpha (HIF-1alpha) protein remain unclear. One model proposes that mitochondrial generation of reactive oxygen species is required to stabilize HIF-1alpha protein. Primary evidence for this model comes from the observation that cells treated with complex I inhibitors, such as rotenone, or cells that lack mitochondrial DNA (rho(0)-cells) fail to generate reactive oxygen species or stabilize HIF-1alpha protein in response to hypoxia. In the present study, we investigated the role of mitochondria in regulating HIF-1alpha protein stabilization under anoxia (0% O2). Wild-type A549 and HT1080 cells stabilized HIF-1alpha protein in response to hypoxia and anoxia. The rho(0)-A549 cells and rho(0)-HT1080 cells failed to accumulate HIF-1alpha protein in response to hypoxia. However, both rho(0)-A549 and rho(0)-HT1080 were able to stabilize HIF-1alpha protein levels in response to anoxia. Rotenone inhibited hypoxic, but not anoxic, stabilization of HIF-1alpha protein. These results indicate that a functional electron transport chain is required for hypoxic but not anoxic stabilization of HIF-1alpha protein.     

The mitochondrial thioredoxin system regulates nitric oxide-induced HIF-1alpha protein

Hypoxia-inducible factor-1 (HIF-1), consisting of two subunits, HIF-1alpha and HIF-1beta, is a key regulator for adaptation to low oxygen availability, i.e., hypoxia. Compared to the constitutively expressed HIF-1beta, HIF-1alpha is regulated by hypoxia but also under normoxia (21% O(2)) by several stimuli, including nitric oxide (NO). In this study, we present evidence that overexpression of mitochondrial-located thioredoxin 2 (Trx2) or thioredoxin reductase 2 (TrxR2) attenuated NO-evoked HIF-1alpha accumulation and transactivation of HIF-1 in HEK293 cells. In contrast, cytosolic-located thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount and activity under NO treatments. Taking into consideration that thioredoxins affect the synthesis of HIF-1alpha by altering Akt/mTOR signaling, we herein show that p42/44 mitogen-activated protein kinase and p70S6 kinase are involved. Moreover, intracellular ATP was increased in Trx1-overexpressing cells but reduced in cells overexpressing Trx2 or TrxR2, providing thus an understanding of how protein synthesis is regulated by thioredoxins.     

Nitric oxide reverses desferrioxamine- and hypoxia-evoked HIF-1alpha accumulation--implications for prolyl hydroxylase activity and iron

Hypoxia inducible factor 1 (HIF-1) senses and coordinates cellular responses towards hypoxia. HIF-1 activity is primarily determined by stability regulation of its alpha subunit that is degraded by the 26S proteasome under normoxia due to hydroxylation by prolyl hydroxylases (PHDs) but is stabilized under hypoxia. Besides hypoxia, nitric oxide (NO) stabilizes HIF-1alpha and promotes hypoxia-responsive target gene expression under normoxia. However, in hypoxia, NO attenuates HIF-1alpha stabilization and gene activation. It was our intention to explain the contrasting behavior of NO under hypoxia. We used the iron chelator desferrioxamine (DFX) or hypoxia to accumulate HIF-1alpha in HEK293 cells. Once the protein accumulated, we supplied NO donors and followed HIF-1alpha disappearance. NO-evoked HIF-1alpha destabilization was reversed by proteasomal inhibition or by blocking PHD activity. By using the von Hippel Lindau (pVHL)-HIF-1alpha capture assay, we went on to demonstrate binding of pVHL to HIF-1alpha under DFX/NO but not DFX alone. Showing increased intracellular free iron under conditions of hypoxia/NO compared to hypoxia alone, we assume that increased free iron contributes to regain PHD activity. Variables that allow efficient PHD activation such as oxygen availability, iron content, or cofactor accessibility at that end allow NO to modulate HIF-1alpha accumulation.