生物谷報道：HIF1α是最重要的低氧調(diào)節(jié)因子之一。在常氧下它迅速被降解，是因為氧氣存在情況下，會活化泛素化酶,，從而使HIF1α迅速降解。但是調(diào)控它降解的蛋白了解并不是很多,。最新發(fā)現(xiàn)Siah2是其中關(guān)鍵蛋白之一,。

Introduction
Physiologic systems that regulate oxygen homeostasis are among the most extensively developed, reflecting the body's constant and absolute requirement for oxygen. Changes in oxygen tension are tightly monitored and addressed through a complex and rapidly implemented series of posttranslational modifications that affect the activity and expression of genes involved in control of cellular metabolism, growth, and death. HIF1, hypoxia-inducible factor, is a master regulator of oxygen homeostasis (reviewed in Safran and Kaelin, 2003). HIF1 target genes play key roles in development as well as physiological processes such as angiogenesis, vascular remodeling, erythropoiesis, glucose transport, glycolysis, iron transport, cell proliferation, and cell survival (reviewed in Semenza 2002 and Pugh and Ratcliffe 2003). HIF1 is a heterodimer composed of a hypoxia-inducible  subunit and a constitutively expressed  subunit (Wang et al., 1995).

Cellular O2 concentration is a central determinant of HIF1 expression. In nonhypoxic conditions, HIF1 is constantly degraded through ubiquitination and proteasomal destruction, whereas these processes are inhibited under hypoxic conditions, resulting in rapid increase in HIF1 levels (Huang et al. 1998; Kallio et al. 1999; Salceda and Caro 1997 and Jiang et al. 1996). The constitutive degradation of HIF1 in normoxia requires O2-dependent hydroxylation of proline residues 402 and 564 in HIF1 by prolyl 4-hydroxylases (PHD 1, 2, and 3 for prolyl hydroxylase domain containing; Bruick and McKnight 2001; Yu et al. 2001 and Epstein et al. 2001). Hydroxylation of HIF1 is prerequisite for binding of the von Hippel-Lindau protein (pVHL), which forms an E3 ubiquitin ligase complex with elongin B, C, and Cullin 2, resulting in targeting of HIF1 for proteasomal degradation (Kamura et al. 1999; Bruick and McKnight 2001; Ivan et al. 2001; Jaakkola et al. 2001 and Maxwell et al. 1999). Loss of pVHL function in renal carcinoma cells or ES cells results in constitutive expression of HIF1 and induction of downstream target genes such as VEGF (Maxwell et al. 1997; Mazure et al. 1997 and Richard et al. 1999). SM20, the rat homolog of PHD3, was originally identified in smooth muscle cells as a growth inhibitor-inducible gene (Wax et al., 1994). PHD3 exhibits high affinity and kinetics for hydroxylation of HIF1 (Bruick and McKnight, 2001). Further, inhibition of PHD enzymatic activities during hypoxia is thought to be important for stabilization of HIF1 (Ivan et al. 2001; Jaakkola et al. 2001 and Epstein et al. 2001). Yet, mechanisms underlying the functional differences among the three PHDs are not well understood.

RING finger proteins consist of a characteristic cysteine-rich zinc binding domain defined by a pattern of conserved cysteine and histidine residues that can catalyze polyubiquitination, thereby functioning as E3 ubiquitin ligases (Borden 2000). Siah proteins are highly conserved mammalian homologs of Drosophila Seven in Absentia, which possesses potent RING finger E3 ubiquitin ligase activities implicated in the degradation of diverse proteins including DCC, -catenin, N-CoR, c-myb, Numb, and TRAF2 (Hu et al. 1997; Tiedt et al. 2001; Matsuzawa and Reed 2001; Matsuzawa et al. 1998; Zhang et al. 1998; Susini et al. 2001 and Habelhah et al. 2002). In searching for novel targets of Siah2, we performed mass spectrometry analysis of Siah2-associated proteins and identified several enzymes that mediate oxidation and hydroxylation. Here, we identify PHD1 and 3 as new substrates of Siah2 and demonstrate that Siah2 limits PHD1 and PHD3 availability during hypoxia, thereby revealing a novel mechanism underlying the increase in HIF1 expression during hypoxic conditions.

Results
Siah1a/2 Target PHD1/3 for a Proteasome-Mediated Degradation
To test the possibility that Siah2 may affect the stability of prolyl hydroxylases, we first examined the association of Siah2 with each of the 3 PHDs. PHD1, 2, and 3 were cotransfected with RING mutant Siah2 (Siah2Rm), which lacks E3 ligase activity and is more highly expressed than wild-type Siah2 due to the absence of self-ubiquitination (Hu et al. 1997 and Habelhah et al. 2002). Immunoprecipitation of each PHD revealed that PHD3 exhibited the highest degree of binding to Siah2 and that PHD1 also exhibited strong association. A substantially weaker association was found for PHD2 (Figure 1A). In light of these findings, further analysis was primarily carried out using PHD3.

 
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Figure 1. Interaction of PHDs with Siah2 and Their Degradation(A) The HA-tagged RING mutant form of Siah2 (Siah2Rm) and Flag-tagged PHD1, 2, and 3 were transfected into 293T cells to detect the association between Siah2 and PHDs. Cell lysates were immunoprecipitated with anti-Flag antibody and subjected to SDS-PAGE. Three PHDs coprecipitated HA-Siah2Rm (top image). Expression of PHDs (middle image) and loading control -actin (lower image) are shown. Asterisk indicates nonspecific band detected by Flag polyclonal antibody.(B) HA-Siah2 or Siah2Rm and Flag-PHD3 were transfected into 293T cells. Transfected cells, treated with or without MG132 (20 M), were lysed and 1 mg of lysate was used for immunoprecipitation with anti-Flag antibody. Coprecipitation of Siah2Rm as well as Siah2 in the MG132-treated condition was detected using anti-HA antibody (top image). Expressions of PHD3, Siah2, and -actin are shown (three lower images).(C) In vitro-translated 35S-labeled PHD3 was incubated with bacterially purified GST-Siah2 or GST-Siah2Rm fusion protein attached to glutathione beads in phosphate-buffered saline (PBS). GST and unrelated GST-ATF2 were used as negative controls. After incubation, the beads were extensively washed and subjected to SDS-PAGE. Association of 35S-PHD3 and Siah2 in vitro was detected by autoradiography. Input of GST fusion proteins is shown as Coomassie blue-stained gel (lower image).(D) Flag-Siah1 or HA-Siah2 with Flag-PHD1, 2, and 3 were transfected into 293T cells. The total cell lysate was subjected to Western blotting using anti-Flag tag antibody (top two images). -actin was probed for the loading control (bottom image).(E) Effect of various inhibitors was tested in 293T cells transfected with PHD3 and Siah2. Cells were treated with MG132 (20 M), lactacystin (10 g/ml), epoxomicin (2 M), MG101 (10 M), E64 (100 g/ml), and chloroquine (1 M) for 5 hr and harvested. Cell lysates were subjected to Western blotting and the membrane was probed with anti-Flag tag antibody (upper image). -actin was probed for the loading control (lower image).(F) Siah2-dependent ubiquitination of PHD3. Flag-PHD3 (4 g), Siah2 expression vectors and pSup-PHD3 were cotransfected into 293T cells. After 48 hr, cells were harvested and lysed in 1% SDS/TBS followed by immediate boiling. Cell lysate was subjected to immunoprecipitation using anti-Flag antibody followed by separation on SDS-PAGE and immunoblot analysis with antiubiquitin- (upper image) or anti-Flag-antibody (lower image). Nonspecific (ns) and the IgG bands are indicated.

Associations of wild-type Siah2 and PHD3 could be detected only in cells treated with a proteasome inhibitor (Figure 1B). The potent self-ubiquitination of Siah2 renders very low expression levels of endogenous Siah2 preventing detection of the association between endogenous proteins. Expression of Siah2 also resulted in decreased PHD3 steady-state level, which could be attenuated upon treatment with proteasome inhibitor (Figure 1B). These observations suggest that Siah2 and PHD3 associate in vivo and that this association subjects PHD3 to active degradation.

To further characterize the association between Siah2 and PHD3, we divided PHD3 into three fragments whose binding to Siah2Rm in vivo was assessed. This analysis revealed that Siah2 binds to the carboxy-terminal domain of PHD3 (Supplemental Figure S1 available at http://www.cell.com/cgi/content/full/117/7/941/DC1). In vitro binding assays using bacterially expressed and purified GST-Siah2 or GST-Siah2Rm and in vitro-translated 35S-labeled PHD3 revealed that both wild-type and RING mutant Siah2 associate with PHD3 (Figure 1C). Incubation of immunopurified 35S-labeled PHD3 with GST-Siah2 also confirmed their association (Supplemental Figure S2 available on Cell website).

Comparing Siah1a and Siah2's ability to affect the steady-state level of the PHDs revealed that both decreased abundance of PHD1 and PHD3, but not PHD2, and that Siah2 exhibited a stronger effect on both PHDs than Siah1a (Figure 1D). These findings show that both Siah1a and Siah2 (Siah1/2) reduce the steady-state levels of PHD1/3. Since Siah2's activity was more potent than Siah1a's, we focused on Siah2 in subsequent studies.

To identify the degradation pathway by which Siah2 decreases PHD3 stability, we tested the ability of different protease inhibitors to block degradation. Whereas the proteasome inhibitors MG101, MG132, lactacystin, and epoxomicin inhibited the degradation of PHD3 by Siah2, the lysosomal protease inhibitors E-64 or chloroquine failed to do so (Figure 1E).

We next assessed Siah2 effect on ubiquitination of PHD3. In vitro ubiquitination using bacterially produced and purified GST-Siah2 and 35S-labeled PHD3 revealed Siah2-dependent ubiquitination of PHD3. This reaction required the presence of reticulocyte lysates, suggesting that further modification (i.e., posttranslational) or the presence of an adaptor protein is required for Siah2-mediated ubiquitination of PHD3 in vitro (Supplemental Figure S3 available on Cell website). In vivo analysis revealed a low basal ubiquitination of PHD3 (Figure 1F, lane 2), which was also seen in Siah2−/− cells and is likely to be noncanonical ubiquitination that does not affect PHD3 stability (data not shown). Exogenous expression of Siah2 resulted in a dose-dependent increase in PHD3 ubiquitination (Figure 1F, lanes 3 and 4). Higher amounts of Siah2 did not increase further the degree of PHD3 ubiquitination as it also reduced stability of PHD3 ( Figure 1F, lanes 4 and 5). This ubiquitination can be inhibited by reducing PHD3 expression upon transfection of PHD3 RNAi, supporting the specificity of this ubiquitination. These results suggest that Siah2 targets the ubiquitination and degradation of PHD3 in a proteasome-dependent manner, as reported for other Siah2 substrates (Hu et al. 1997 and Habelhah et al. 2002).

PHD3 Is More Stable in Siah2−/− Cells
We next elucidated changes in PHD3 stability in immortalized wild-type (Siah2+/+) and Siah2 null (Siah2−/−) mouse embryonic fibroblasts (Habelhah et al., 2002). Initial analysis of steady-state levels of exogenously expressed PHD3 revealed almost undetectable expression in Siah2+/+ cells, whereas it was clearly detected in Siah2−/− cells (Figure 2C, compare lanes 1 and 7). Pulse chase analysis of PHD3 revealed a half-life of about 1 hr in Siah2+/+cells compared with over 2 hr in Siah2−/− cells (Figure 2A). Parallel analysis revealed that the half-life of PHD3 was shortened in Siah2+/+ cells maintained under hypoxia compared to normoxia conditions (Figure 2B). A similar decrease in PHD3 half-life was also observed in Siah2−/− cells under hypoxia, compared with Siah2+/+ cells, suggesting that other E3 ligases (e.g., Siah1) may also contribute to degradation of PHD3 (Figure 2B). To further confirm that PHD3 stabilization could be attributed to loss of Siah2 in Siah2−/− cells, we reintroduced Siah2 into these cultures. Reexpression of Siah2, but not Siah2Rm, in Siah2−/− cells markedly decreased PHD3 stability and this effect was blocked by addition of proteasome inhibitor (Figure 2C, compare lanes 7, 8, 9, and 11).

 
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Figure 2. PHD3 Is Stabilized in Siah2−/− MEFs(A and B) The half-life of PHD3 protein was monitored by a pulse-chase experiment. Five micrograms of Flag-PHD3 expression vector were transfected into Siah2+/+ and Siah2−/− MEFs. 48 hr after transfection, cells were metabolically labeled with 35S-Met for 2 hr followed by chase carried out during normoxia (A) or hypoxia (B) for the indicated time points. Flag-PHD3 was immunoprecipitated from the cell lysate with anti-Flag antibody and signals were detected by autoradiography.(C) The effect of Siah2 on PHD3 was tested by introducing Siah2 into Siah2−/− cells. HA-Siah2 or Siah2Rm and Flag-PHD3 were transfected into both Siah2+/+ and Siah2−/− cells. Forty-eight hours after transfection, cells were mock treated or treated with MG132 (20M) for 5 hr and harvested. Immunoprecipitates with anti-Flag antibody were subjected to SDS-PAGE followed by Western blotting using anti-Flag antibody. Expressions of HA-Siah2 (middle image) and endogenous PHD3 (bottom image) were also detected.(D) Expression level of PHD3 in normoxia and hypoxia was detected in both Siah2+/+ and Siah2−/− cells. Cells were treated by exposure to hypoxia (1% O2) or normoxia for 5 hr. Total cell lysates were subjected to SDS-PAGE and detected with PHD3 antibody. -actin was probed for the internal control.(E and F) Half-life measurement of PHD3 in Siah1a/2 double-null MEFs was carried out under normoxia (E) or hypoxia (F) conditions as detailed in (A and B) above.

To monitor the expression level of endogenous PHD3, we generated an affinity-purified rabbit polyclonal antibody against PHD3, which did not crossreact with ectopically expressed PHD1/2 (Supplemental Figure S4 available on Cell website). As expected, endogenous expression of PHD3 was higher in Siah2−/− than in Siah2+/+ cells grown under normoxic conditions (Figure 2C, lower image). Higher level of PHD3 in Siah2−/− cells could be reduced upon exogenous expression of Siah2 (Figure 2C, lower image). Moreover, hypoxia treatment for 5 hr decreased steady-state levels of endogenous PHD3 (Figure 2D). This finding implicates Siah2's role in the degradation of endogenous PHD3 and also implies that Siah2 activity toward PHD3 may be enhanced during hypoxia (see below). The residual decrease in PHD3 levels following hypoxia in Siah2−/− cells is likely to be mediated by Siah1.

To test this possibility, given that Siah1 also affects PHD3 stability, we monitored changes in PHD3 half-life in MEFs obtained from Siah1a/2 double-null mice. Indeed, half-life of PHD3 in Siah1a/2 DKO MEFs was longer compared with wild-type MEF under normoxia conditions (>4 hr compared with 1 hr; Figure 2E). The half-life of PHD3 under hypoxic conditions was also longer in Siah1a/2 DKO cells, compared with wild-type cells (100 min compared with 50 min; Figure 2F), although some degradation of PHD3 was still noted and could be attributed to other E3 ligase. These findings reveal that Siah1a/2 DKO cells further extended the already prolonged half-life of PHD3 seen in the Siah2−/− cells (compare images E and F with A and B in Figure 2). These results provide strong genetic evidence to support the role of Siah proteins in regulation of PHD3 stability and suggest that the decrease in PHD3 stability seen in the Siah2−/− cells can be attributed to Siah1a. Expression of Siah1a is not altered in Siah2−/− cells (Supplemental Figure S5 available on Cell website).

Siah2−/− Cells Exhibit Impaired Induction of HIF1 under Hypoxia
In light of the role identified for Siah2 in regulating PHD3 stability, we investigated the physiological implications of such regulation by monitoring levels of HIF1 in Siah2+/+ and Siah2−/− cells grown under normoxic and hypoxic conditions. Under normoxia, HIF1 expression was comparable between Siah2+/+ cells and Siah2−/− cells. In contrast, after hypoxia treatment, the amount of HIF1 in Siah2+/+ cells markedly increased compared with Siah2−/− cells (Figure 3A). These data are consistent with the notion that Siah2−/− cells are impaired in their ability to efficiently degrade PHD3, resulting in a concomitant decrease in the level of HIF1 expression under hypoxia.

 
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Figure 3. Siah2−/− Exhibit Impaired Induction of HIF1 in Response to Hypoxia(A) Expression level of HIF1 in normoxia and hypoxia was detected in both Siah2+/+ and Siah2−/− cells. Cells were treated by exposure to hypoxia (1% O2) or normoxia for 5 hr. Total cell lysates were subjected to SDS-PAGE and detected with HIF1 antibody. -actin was probed for the internal control.(B) Expression level of HIF1 under hypoxia was compared among Siah2+/+, Siah2−/−, and Siah1a/2 DKO MEFs. After hypoxia treatment (1% O2), cells were harvested and subjected to SDS-PAGE. Blot was probed with HIF1 antibody (upper image) or -actin antibody (lower image).(C) Expression of VEGF mRNA in Siah2+/+, Siah2−/−, and Siah1a/2 DKO MEFs was detected in normoxia and hypoxia. cDNA synthesized from the cells was subjected to a semiquantitative RT-PCR reaction using specific primer sets for VEGF and GAPDH genes. After PCR labeling with (-32P)dCTP, samples were separated on acrylamide gel and amplified bands were detected by autoradiography.(D) Inhibition of PHD3 by RNAi increases HIF1 expression under hypoxia. pSuper control (empty vector) or pSuper carrying oligonucleotide designed for specific inhibition of PHD3 were transfected (6 g each) into Siah2+/+ and Siah1a/2 DKO MEFs. 72 hr after transfection cells were subjected to normoxia or hypoxia for 5 hr. Proteins were analyzed for HIF1 expression by Western blotting. RNA extracted from the same cultures (Siah2+/+ cells are shown) was analyzed for the expression of PHD2, PHD3, and GAPDH by RT-PCR followed by autoradiography.

We next examined changes in expression levels of HIF1 in Siah1a/2 DKO cells. The lower levels of HIF1 expression seen in Siah2−/− cells was even more pronounced in DKO cells, where HIF1 expression was barely detectable following hypoxia treatment (Figure 3B). These data point to the role of Siah1a in complementing Siah2-targeted PHD3 degradation, which concomitantly affects HIF1 expression.

Lower HIF1 expression under hypoxia in Siah2−/− and Siah1a/2 DKO cells is expected to cause a corresponding change in the expression of HIF1 target genes. Thus, we investigated the expression of VEGF mRNA, a representative HIF1-inducible gene, by semiquantitative RT-PCR. Consistent with the HIF1 expression levels, the relative increase of VEGF mRNA in hypoxia-treated Siah2+/+ cells was greater than that in Siah2−/− cells and barely detectable in Siah1a/2 DKO cells (Figure 3C).

Inhibition of PHD3 Recovers HIF1 Expression in Siah-Deficient Mouse Fibroblasts
Given the effect of Siah1a and Siah2 on PHD3, but not on PHD2, we have assessed the changes in HIF1 expression in Siah2+/+ and Siah1a/2 DKO cells that were maintained in either normoxia or hypoxia, in which PHD3 expression was inhibited by a specific RNAi. Significantly, lack of HIF1 expression in Siah1a/2 DKO cells subjected to hypoxia could be rescued upon inhibition of PHD3 expression via RNAi (Figure 3D). This finding provides direct support for the role of Siah1a/2 in the regulation of PHD3 expression under hypoxic conditions. Unlike the changes seen under hypoxia, there was no significant increase in the level of HIF1 under normoxia in cells in which PHD3 expression was inhibited (Figure 3D), as was observed upon inhibition of PHD2 expression (Berra et al., 2003). These findings suggest that whereas PHD2 primarily limits HIF1 under normoxia, PHD3, which is regulated by Siah2, determines HIF1 availability under hypoxic conditions.

Alteration of HIF1 Stability in Siah2−/− Cells Is Dependent on PHD3 Abundance
Since we have shown that PHD3 protein levels are higher in Siah2−/− cells (Figure 2C) and given the changes in HIF1 levels upon inhibition of PHD3 expression in cells that lack Siah, we tested whether altering the levels of PHD3 during hypoxia is sufficient to modulate HIF1 levels. Transfection of PHD3 expression vector into HeLa cells decreased, in a dose-dependent manner, basal as well as hypoxia-induced expression of HIF1 (Figure 4A). Similarly, exogenous expression of PHD3 decreased the HIF1 level in Siah2+/+ cells in both normoxia and hypoxia to levels seen in Siah2−/− cells (Figure 4B). Thus, expression of PHD3 mimics the changes of HIF1 expression seen in Siah2−/− cells.

 
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Figure 4. Expression of HIF1 Depends on PHD3 Abundance(A) Different amounts of PHD3 expression plasmid were transfected into HeLa cells as indicated in the image. Thirty-six hours after transfection, cells were maintained under hypoxia (1% O2) or normoxia, and harvested 5 hr after treatment. The total cell lysate was probed for HIF1 (upper image), Flag-PHD3 (middle image), and -actin (lower image). PHD3 expression inhibited the induction of HIF1 in hypoxia in a dose-dependent manner.(B) The effect of exogenously expressed PHD3 in Siah2+/+ and Siah2−/− cells was tested. Six micrograms of PHD3 expression plasmid were transfected into cells of both types, which were either maintained under normoxia or hypoxia (1% O2) for 5 hr. The total cell lysate was probed for HIF1 (upper image), Flag-PHD3 (middle image), and -actin (lower image).(C) The effect of MG132 treatment on HIF1 expression was examined in Siah2+/+ and Siah2−/− cells. Cells were mock treated or treated with MG132 (20M) for 5 hr and harvested. The total cell lysate was subjected to Western blotting with anti-HIF1 antibody.

If the lower abundance of HIF1 in Siah2−/− cells under hypoxia results entirely from improper degradation of PHD3, then inhibition of PHD3-dependent (VHL-mediated) degradation should result in comparable levels of expression of HIF1 in mutant and wild-type cells. Treatment with proteasome inhibitor increased HIF1 abundance to similar levels in both genotypes of MEF (Figure 4C). Treatment with DFO, an iron chelator known to inhibit the activity of PHDs, increased HIF1 abundance to similar levels in both Siah2+/+ cells and Siah2−/− cells under normoxia (Supplemental Figure S6 available on Cell website). Collectively, these results suggest that the relative abundance of PHD3 is one of the key determinants that regulate HIF1 expression under hypoxic conditions

Degradation of PHD3 Is Enhanced under Hypoxia
As the differences in HIF1 abundance between Siah2+/+ cells and Siah2−/− cells are more striking under hypoxia than normoxia (Figure 3A), we hypothesized that Siah2 targeting of PHD3 may be increased by hypoxia. Consistent with this idea, we showed that hypoxia enhanced the ability of Siah2 to induce degradation of exogenously and endogenously expressed PHD3 (Figure 5A). Increase in Siah2 activity under hypoxia conditions was seen for both PHD3 and PHD1 (Figure 5B). Moreover, this effect was observed even under mild hypoxic conditions (5% and 10% oxygen concentration; Figure 5C compare lanes 3, 6, 9, and 12 and lanes 1, 4, 7, and 10), demonstrating that the regulation of PHD3 stability is sensitive to oxygen tension. Monitoring GFP driven from the same promoter as PHD3 revealed that neither Siah nor hypoxia affect transcription or translation of GFP (Supplemental Figure S7 available on Cell website). However, although hypoxia alone decreased PHD3 abundance and enhanced the ability of exogenous Siah2 to degrade PHD3, the degree of association between exogenously expressed PHD3 and Siah2Rm was not altered by hypoxia under conditions where PHD3 was not the limiting factor (Figure 5A compare lanes 6 and 12). Thus, enhancement of interaction between PHD3 and Siah2 does not appear to account for increased degradation of PHD3 during hypoxia.

 
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Figure 5. Siah2 Decreases Abundance of PHD1/3 also Under Mild Hypoxic Conditions(A) Flag-PHD3 and HA-Siah2 or Siah2Rm were transfected into 293T cells. Forty-eight hours later, cells were maintained under normoxia or hypoxia (1% O2) for 5 hr and harvested. Cell lysates were immunoprecipitated with anti-Flag antibody. Total cell lysate or immunoprecipitates were subjected to Western blotting and probed for HA-Siah2 (two upper images), Flag-PHD3 (middle image), and -actin (lower image). Expression of endogenous PHD3 was also detected with the increase amount of Siah2 transfection (bottom two images).(B) Degradation of PHD1 and PHD3 by Siah2 increases under hypoxia. Expression vectors of Flag-PHD1, 2, 3, and HA-Siah2 were transfected into 293T cells. Forty-eight hours after the transfection, cell were maintained under normoxia or hypoxia (1% O2) for 5 hr and harvested. Cell lysate was subjected to SDS-PAGE followed by probing with anti-Flag antibody (upper two images) or anti--actin antibody (lower image).(C) Activity of Siah2 to target PHD3 was tested in 10%, 5%, and 1% O2 concentrations. Cells transfected with different amount of Siah2 and PHD3 were treated under different hypoxic conditions for 5 hr. Cell lysates were subjected to Western blotting probed with anti-Flag tag antibody (upper image). The membrane was also probed with -actin for internal control (lower image).

Siah2 Transcription Is Induced under Hypoxia
Since our biochemical and genetic experiments suggested that Siah2 expression levels are an important determinant of PHD3 abundance, we reasoned that hypoxia might increase Siah2 expression and hence potentiate PHD3 degradation. To test this possibility, semiquantitative RT-PCR was used to monitor levels of Siah2 mRNA following exposure to hypoxia. This analysis revealed a marked increase in the amount of Siah2 transcripts as early as 2 hr after exposure to hypoxia. Further increase in the level of Siah2 mRNA was observed within 5 hr after exposure to hypoxia, followed by a decrease after 14 hr to levels somewhat higher than that seen in normoxia (Figure 6A). Moreover, consistent with our findings of decreased PHD3 stability under even mild hypoxia (Figure 5C), we found that Siah2 expression is induced to a similar extent by 1%, 5% or 10% oxygen concentration (Figure 6B). Under the same conditions HIF1 expression is elevated as early as 2 hr following hypoxia and in mild hypoxic conditions (10% or 5%; Figures 6C and 6D). Since the increase in Siah2 transcripts precedes the increase in HIF1 expression (at 10% hypoxia Siah2 transcripts increase substantially, whereas HIF1 levels are minimally elevated), it is likely that factors other than HIF1 induce Siah2 expression during hypoxia. Collectively, these findings lead us to propose a model in which transcriptional upregulation of Siah2 during hypoxia induces degradation of PHD3, providing a positive feed-forward mechanism to enhance HIF1 stability.

 
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Figure 6. Siah2 Transcripts Are Induced under Hypoxic Conditions(A and C) Expression of Siah2 mRNA and HIF1 in Siah2+/+ cells was detected at different time points during hypoxia (1% O2). cDNA synthesized from the cells was subjected to a semiquantitative RT-PCR reaction using specific primer sets for Siah2 and GAPDH genes. PCR product labeled with (-32P)dCTP was separated on acrylamide gel and detected by autoradiography (A). Cell lysates prepared from those cells were subjected to SDS-PAGE and probed with anti-HIF1 antibody (C).(B and D) Expression level of Siah2 mRNA and HIF1 was detected in Siah2+/+ cells treated with different hypoxic conditions for 5 hr. cDNA synthesized from the cells was subjected to a semiquantitative RT-PCR reaction using specific primer sets for Siah2 and GAPDH genes. Amplified bands labeled with (-32P)dCTP were separated on acrylamide gel and detected by autoradiography (B). Cell lysates prepared from those cells were subjected to SDS-PAGE and probed with anti-HIF1 antibody (D).

Defective Physiological Responses to Hypoxia in Siah2 Null Mice
The role of Siah2 in the regulation of PHD3 stability and HIF1 abundance under hypoxic conditions suggested that Siah2 null animals may exhibit altered physiological responses to hypoxia. Exposure of mice to chronic hypoxia induces an increase in red blood cell production (polycythemia), attributable to increased expression of the HIF1 target gene erythropoietin (Wenger, 2000). HIF1 heterozygous mice exhibit a delayed polycythemic response following chronic hypoxia (Yu et al., 1999), demonstrating the importance of HIF1 expression levels for this response. Consistent with our biochemical data showing that Siah2 is a positive regulator of HIF1 protein abundance, we found that Siah2−/− mice also display an impaired increase in red blood cell production, as measured by hemoglobin levels, following continuous treatment with hypoxia (10% oxygen) for 1–2 weeks (Figure 7A). This finding provides important physiological evidence demonstrating that Siah2 modulates HIF1 function in vivo.

 
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Figure 7. Defective Response to Hypoxia in Siah2 Null Mice(A) The effect of hypoxia (10% O2) on hemoglobin concentration in wild-type (+/+) and Siah2 null mice (−/−). Bars are mean ± 1 SE, N = 7. *, Significant difference for same strain from day 0 (normoxia); †, significant difference between strains on the same day.(B) The relationship between ventilation and rate of oxygen consumption in normoxia (position 1) and on exposure to acute hypoxia (10% O2, position 2) for wild-type mice (•) and Siah2 null mice (). Closed symbols are normoxic values; open symbols are hypoxic values. Dashed lines represent E/o2 isopleths (values given). Symbols are mean values ± 1 SE, N = 7. A significant difference exists between cohorts in  E and  O2 at 2, no significant difference exists in either variable in normoxia.

Since mammals regulate ventilation and metabolism in response to available oxygen, we investigated whether Siah2 also regulates physiological oxygen-sensitive systems. Analysis of Siah2+/+ and Siah2−/− mice under normoxia (Figure 7B; position 1) revealed identical rates of ventilation ( E) and metabolism ( O2, rate of oxygen consumption) between genotypes. The average convective requirement determined for both genotypes ( E/ O2) was close to 41, a value similar to that previously reported for mammals in general (Frappell et al., 1992). On exposure to acute hypoxia ( Figure 7B, position 2), wild-type mice hyperventilate (shift to a new  E/ O2 ratio of 82), achieved both by an increase in ventilation (hyperpnea) and a decrease in rate of oxygen consumption (hypometabolism). In contrast, Siah2−/− mice display the same hyperventilatory response ( E/ O2 ratio of 82) but achieve it solely through a dramatic hypometabolic response and completely lack a hyperpneic response. Thus, although the absence of Siah2 does not prevent hyperventilation appropriate to the level of hypoxia, Siah2 is necessary for ventilatory changes (hyperpnea) in response to acute hypoxia. However, Siah2−/− mice display similar ventilatory and metabolic responses to those of wild-type mice during and following chronic hypoxia (data not shown).

Discussion
The present study identifies a novel layer in the regulation of HIF1 hydroxylation by PHD1/3. We demonstrate that PHD1 and PHD3 are regulated by members of the E3 ubiquitin ligase family, Siah2 and Siah1a, and that such regulation determines the level of HIF1 expression under hypoxic conditions. The biological significance of our findings is best illustrated by the finding that Siah2−/− cells exhibit lower HIF1 abundance than wild-type cells during hypoxia, and that Siah1a/2 DKO cells fail to exhibit hypoxia-induced increase in HIF1 abundance. Consequently, there are correspondingly lower levels of expression of the HIF1 target gene VEGF, in Siah2 null cells and Siah1a/2 DKO cells during hypoxia.

Importantly, our findings demonstrate the effect of Siah1a/2 on PHD1/3, but not on PHD2, thereby pointing to a functional difference among the three PHD enzymes in the regulation of HIF1. RNAi for PHD2 increases HIF1 expression under normoxia (Berra et al., 2003). In contrast, elevated expression of PHD3 suffices to decrease expression of HIF1 under hypoxia conditions in wild-type cells and RNAi-mediated inhibition of PHD3 rescues hypoxia-induced HIF1 expression in Siah1a/2 DKO cells. We therefore conclude that whereas PHD2 primarily limits HIF1 expression under normoxia, PHD3 regulates HIF1 availability under moderate hypoxic conditions, under which sufficient oxygen molecules are available for its activity.

Since severe hypoxia or anoxia deprive PHDs of the oxygen molecules that are required for their enzymatic activities (Bruick and McKnight 2001; Jaakkola et al. 2001 and Epstein et al. 2001), it is necessary to highlight the physiological significance of Siah2-mediated degradation of PHD3 under hypoxia. The enzymatic activity of PHDs is inhibited in a graded manner with respect to decreasing oxygen concentration, with only partial inhibition in vitro at 10% oxygen (Epstein et al., 2001). Consistent with functional PHD under mild hypoxia conditions, both that level (mRNA) and activity (toward degradation of PHD3) of Siah2 are elevated upon exposure to even mild hypoxia (5% and 10% oxygen; Figure 5 and Figure 6). We therefore propose that Siah2-regulation of PHD3 stability is of physiological importance within the range of moderate hypoxia where it is likely that there remains sufficient oxygen for PHD3 activity.

Increased expression of PHD3 mRNA, but not PHD1/2, was observed in cardiac myocytes, smooth muscle cells, and endothelial cells subjected to hypoxia (Cioffi et al., 2003), while induced expression of both PHD3 and PHD2 mRNA under hypoxia conditions has been reported for certain cell lines, pointing to a tissue-specific regulation (Epstein et al. 2001 and Metzen et al. 2003). As Siah2 mRNA induction and enhancement of Siah2-mediated PHD3 degradation were as efficient at 10% and 1% oxygen, it is possible that Siah2-mediated PHD3 degradation may represent an oxygen-sensing mechanism that responds to even mild hypoxic conditions, thereby constituting a primary response to hypoxia.

Variations in oxygen concentration (or oxygen partial pressure) in different tissues have both physiological and pathophysiological implications. For example, oxygen tension is an important determinant of proliferation and differentiation of hematopoietic cells; whereas the formation of mature erythrocytes and megakaryocytes is more extensive under 20% O2 (LaIuppa et al., 1998), greater production of differentiated granulocytes is observed under 5% O2 (Hevehan et al., 2000). Further, regulation of human placental development, lung branching morphogenesis, and angiogenesis are affected by altering oxygen tension (Genbacev et al. 1997; Adelman et al. 2000 and Gebb and Jones 2003).

Our analyses of physiological responses to hypoxia in Siah2 null mice provide strong evidence supporting our proposal that Siah2 is a component of oxygen-sensing systems. First, we showed that Siah2 null mice exhibit a defect similar to that of HIF1 heterozygous mutant mice in induction of polycythemia following exposure to chronic hypoxia. Second, we observed that Siah2 null mice lack a hyperpneic response to acute hypoxia and instead achieve hyperventilation solely through a dramatic decrease in metabolic rate. Interestingly, isolated carotid bodies of HIF1 heterozygous mutant mice displayed impaired neural activity on exposure to hypoxia (Kline et al., 2002). The normal ventilatory response to acute hypoxia in these mice was attributed to increased utilization of chemoreceptors other than the carotid body (Kline et al., 2002). In this context, it is particularly interesting that Siah2 null mice lack a hyperpneic response to acute hypoxia, suggesting that Siah2 is important for the ventilatory response. Presumably the absence of Siah2 changes the expression of various genes encoding receptors or neurotransmitting enzymes in the chemoreceptors involved in transduction of the hypoxic signal to the central nervous system.

In summary, we propose that induction of Siah2 by hypoxia serves to enhance the degradation of PHD1/3 and consequently increase the abundance of HIF1. Accordingly, Siah2 is part of a feed-forward mechanism that regulates PHD1/3 availability in hypoxia.

原文：

Siah2 Regulates Stability of Prolyl-Hydroxylases, Controls HIF1α Abundance, and Modulates Physiological Responses to Hypoxia
Koh Nakayama, Ian J. Frew, Mette Hagensen, Marianne Skals, Hasem Habelhah, Anindita Bhoumik, Takayuki Kadoya, Hediye Erdjument-Bromage, Paul Tempst, Peter B. Frappell, David D. Bowtell, and Ze'ev Ronai
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