Regulation of Hypoxia-Inducible Factor (HIF)-1 Activity and Expression of HIF Hydroxylases in Response to Insulin-Like Growth Factor I

Caroline Treins, Sophie Giorgetti-Peraldi, Joseph Murdaca, Marie-Noëlle Monthouël-Kartmann and Emmanuel Van Obberghen

Institut National de la Santé et de la Recherche Médicale, Unité 145, Institut Fédératif de Recherche 50, Faculté de Médecine, 06107 Nice Cedex 2, France

Address all correspondence and requests for reprints to: S. Giorgetti-Peraldi, Institut National de la Santé et de la Recherche Médicale, Unité 145, Institut Fédératif de Recherche 50, Faculté de Médecine, Avenue de Valombrose, 06107 Nice Cedex 2, France. E-mail: peraldis{at}unice.fr.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Hypoxia-inducible factor-1 (HIF-1), a transcription factor composed of two subunits (HIF-1{alpha} and HIF-1ß), initially described as a mediator of adaptive responses to changes in tissue oxygenation, has been shown to be activated in an oxygen-independent manner. In this report, we studied the action of IGF-I on the regulation of HIF-1 in human retinal epithelial cells. We show that IGF-I stimulates HIF-1{alpha} accumulation, HIF-1{alpha} nuclear translocation, and HIF-1 activity by regulation of HIF-1{alpha} expression through a posttranscriptional mechanism. In addition, we demonstrate that IGF-I stimulates HIF-1 activity through phosphatidylinositol-3-kinase/ mammalian target of rapamycin and MAPK-dependent signaling pathways leading to VEGF (vascular endothelial growth factor) mRNA expression. Three human prolyl-hydroxylases PHD-1, -2, and -3 (PHD-containing protein) and an asparaginyl-hydroxylase factor inhibiting HIF-1, which regulate HIF-1{alpha} stability and HIF-1 activity in response to hypoxia, have been described. Our analysis of their mRNA expression showed a different magnitude and time course of expression pattern in response to insulin and IGF-I compared with CoCl2. Taken together, our data reveal that growth factors and CoCl2, which mimics hypoxia, lead to HIF-1 activation and ensuing VEGF expression by different mechanisms. Their joined actions are likely to lead to an important and sustained increase in VEGF action on retinal blood vessels, and hence to have devastating effects on the development of diabetic retinopathy.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HYPOXIA-INDUCIBLE FACTOR-1 (HIF-1) is a transcription factor that mediates adaptive responses to changes in tissue oxygenation. It regulates the transcription of numerous genes involved in vascular development, in glucose and energy metabolism, in iron metabolism, and in cell proliferation and viability. HIF-1 is a basic helix-loop-helix transcription factor that is composed of two subunits, HIF-1{alpha} and HIF-1ß. HIF-1ß, also known as the arylhydrocarbon nuclear translocator, is constitutively expressed, whereas the level of HIF-1{alpha} is regulated. In normoxia, HIF-1{alpha} is hydroxylated by PHD-1, -2, and -3 (prolyl-hydroxylase domain-containing protein) on two conserved proline residues, Pro402 and Pro564, within the oxygen-dependent degradation domain (1). These prolyl-hydroxylases, which belong to the iron- and 2-oxoglutarate-dependent dioxygenase superfamily, are the regulators of HIF-1{alpha} degradation (1, 2, 3, 4, 5). Indeed, this posttranslational modification of HIF-1{alpha} induces binding to the Von Hippel-Lindau protein, which is an E3-ubiquitin ligase, and targets HIF-1{alpha} for subsequent proteasomal degradation (3). Under hypoxic conditions, the prolyl-hydroxylases are inactive; HIF-1{alpha} degradation is inhibited leading to its accumulation, and its association with HIF-1ß resulting in a functional heterodimeric transcription factor. In addition, hydroxylation of a specific asparagine residue, Asn803, on the C-terminal activation domain of HIF-1-{alpha}, by an asparaginyl-hydroxylase [factor inhibiting HIF-1 (FIH-1)], decreases the transcriptional activity of HIF-1 (6, 7, 8). Prolyl- and asparaginyl-hydroxylases show an ubiquitous pattern of expression but differ in the relative abundance of their mRNA and in their cellular localization (9, 10, 11). More recently, it has been described that PHD-2 mRNA is induced by hypoxia through a HIF-1-dependent signaling pathway (12). However, to the best of our knowledge the effect of oxygen-independent regulators of HIF-1{alpha} levels on prolyl- and asparaginyl-hydroxylases expression has not been investigated. Indeed, HIF-1 has been shown to be activated in response to growth factors leading to the expression of several genes, including vascular endothelial growth factor (VEGF) (13, 14, 15).

VEGF is a key angiogenic factor, involved in a wide variety of biological processes including embryonic development and tumor progression. In addition, VEGF is a major mediator of intraocular neovascularization and plays a key role in the etiology of diabetic retinopathy (16). Clinical observations support a pathogenic link between IGF-I and intraocular neovascularization (17, 18). Indeed, IGF-I has been reported to play a role in the development of diabetic retinopathy (19, 20). Furthermore, IGF-I stimulates the expression of VEGF (21, 22). IGF-I is homologous to insulin and acts through its own, but closely related tyrosine kinase receptor, that shares many signaling components and cellular responses with the insulin receptor. After ligand binding, the IGF-I receptor is activated, leading to its autophosphorylation and the subsequent phosphorylation of intracellular proteins including insulin receptor substrate proteins, Grb2-associated binding protein 1 and SH2-collagen (Shc). These initial events stimulate multiple signaling cascades, including the phosphatidylinositol-3-kinase (PI-3-kinase) pathway and the Ras-Raf-MAPK pathway, which mediate cellular responses to IGF-I. Although both IGF-I and insulin receptors target the same array of intracellular substrates and activate the same signaling pathways, they are able to lead to distinct final biological programs. In contrast to insulin, which regulates anabolic metabolism in animals and humans, IGF-I stimulates cell proliferation and differentiation and is a potent inhibitor of apoptosis.

Diabetic retinopathy is the major cause of acquired blindness in adults in Western countries. It corresponds to a microvascular complication of diabetes that is characterized by occlusion of retinal vessels, leading to an increase in angiogenesis at the level of the retina. IGF-I seems to play an important role in the development and the progression of this pathology. Indeed, transgenic mice overexpressing IGF-I in the retina developed most of the alterations seen in the human diabetic eye disease (23).

In the present manuscript, we studied the action of IGF-I on the regulation of the couple HIF-1/VEGF in human retinal epithelial cells. We demonstrate that IGF-I stimulates HIF-1{alpha} subunit accumulation, HIF-1{alpha} nuclear localization, HIF-1 activation, and VEGF expression through a PI-3-kinase/ mammalian target of rapamycin (mTOR) and MAPK-dependent pathways. In addition, our results show that IGF-I regulates HIF-1{alpha} expression through a posttranscriptional mechanism. Finally, our study of prolyl- and asparaginyl-hydroxylases mRNA expression showed a regulation pattern in response to insulin and IGF-I, which differs from that seen with CoCl2 and deferoxamine (DFO) treatment.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IGF-I Stimulates HIF-1{alpha} Accumulation and VEGF mRNA Expression in Human Retinal Epithelial Cells
We have investigated the effect of IGF-I on HIF-1{alpha} protein expression in human retinal epithelial cells. Arising retinal pigment epithelial (ARPE)-19 cells were treated with IGF-I or with CoCl2 for the indicated period of time. Whole cell lysates were analyzed by Western blotting using antibodies to HIF-1{alpha} and Shc (Fig. 1AGo). We used cobalt chloride (CoCl2) as a positive control because it is known to induce HIF-1{alpha} expression and transactivation of HIF-1 target genes (24, 25, 26). We observe that IGF-I stimulates HIF-1{alpha} expression within 90 min of stimulation. CoCl2 effect is more rapid because HIF-1{alpha} is detectable within 60 min of treatment. Moreover, we find that CoCl2 is a more potent inducer of HIF-1{alpha} expression compared with IGF-I. These observations suggest that IGF-I and CoCl2 use distinct signaling pathways to increase the HIF-1{alpha} protein amount.



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Fig. 1. IGF-I Stimulates HIF-1{alpha} Accumulation and VEGF mRNA Expression in ARPE-19 Cells

A, ARPE-19 cells were stimulated with IGF-I (100 nM) or CoCl2 (200 µM) for indicated periods of time. Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1{alpha} and Shc. B, ARPE-19 cells were stimulated with insulin (100 nM), IGF-I (100 nM), CoCl2 (200 µM) or DFO (0.1 µM) for 6 or 16 h. mRNA levels for VEGF were determined by real-time quantitative PCR as detailed in Materials and Methods. Results shown represent the mean ± SE of three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001). C, ARPE-19 cells were stimulated with IGF-I (100 nM) for 6 h. RNA was extracted and analyzed by Northern blotting using a VEGF165 cDNA probe. The membrane was subsequently hybridized with a probe for 18S rRNA as control.

 
Because HIF-1 regulates transcription of several genes, we studied the effect of IGF-I on VEGF expression. ARPE-19 cells were stimulated with insulin, IGF-I, CoCl2 or DFO for 6 or 16 h. DFO is an iron chelator, which has been shown to stimulate HIF-1{alpha} accumulation and VEGF mRNA expression (24, 27, 28). VEGF mRNA level was determined by real-time quantitative PCR (Fig. 1BGo). As expected, insulin, CoCl2 and DFO stimulate VEGF mRNA expression. IGF-I induces a 2.7 (±0.35)-fold increase in VEGF mRNA expression within 6 h of treatment. Elevated VEGF mRNA level is found within 16 h of IGF-I treatment. The same result was obtained by northern blot analysis using a VEGF165 cDNA probe (Fig. 1CGo).

To summarize, IGF-I stimulates HIF-1{alpha} protein accumulation and VEGF mRNA expression in ARPE-19 cells.

IGF-I Stimulates Nuclear Translocation of HIF-1{alpha}
Nuclear entry of HIF-1{alpha} subunit is a necessary step for its association with HIF-1ß, which is constitutively localized in the nucleus (29, 30). To study the effect of IGF-I and insulin on the intracellular localization of HIF-1{alpha}, ARPE-19 cells were stimulated for 4 h with insulin, IGF-I or CoCl2, nuclear and cytoplasmic fractions were prepared, and were analyzed by Western blot using antibodies to HIF-1{alpha}, HIF-1ß, Shc, and cAMP response element binding protein (CREB) (Fig. 2Go). The analysis of Shc and CREB expression was used to confirm the separation of cytoplasmic vs. nuclear fractions. We observe that HIF-1{alpha} protein induced by IGF-I or insulin was localized exclusively in the nuclear fraction. In cells exposed to CoCl2, HIF-1{alpha} is accumulated predominantly in the nuclear fraction, whereas some HIF-1{alpha} protein is also found in the cytoplasmic fraction. As expected, HIF-1ß is constitutively expressed in the nuclear fraction of human retinal epithelial cells, and insulin, IGF-I and CoCl2 do not modulate its expression and cellular localization. These observations indicate that IGF-I stimulates nuclear expression of HIF-1{alpha}. However, to confirm that IGF-I stimulates nuclear translocation of HIF-1{alpha}, we have expressed a HIF-1{alpha}-green fluorescent protein (GFP) fusion protein in HEK-293 cells. Transfected HEK-293 cells were stimulated with IGF-I or CoCl2 and cellular localization of HIF-1{alpha} was examined by confocal microscopy (Fig. 3Go). We find that in absence of stimulation, the HIF-1{alpha}-GFP fusion protein is localized exclusively in the cytosol (Fig. 3Go, A and D). After stimulation with IGF-I (Fig. 3Go, B and C) or CoCl2 (Fig. 3EGo), a marked accumulation of HIF-1{alpha}-GFP in the nucleus is detected. This increase in the nucleus is accompanied with a slight decrease in the labeling in the cytosol of the cells. Taken together, our results show that IGF-I stimulates HIF-1{alpha} expression in the nucleus, which is likely to be due to IGF-I-induced nuclear translocation.



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Fig. 2. IGF-I and Insulin Stimulate Nuclear Expression of HIF-1{alpha}

ARPE-19 cells were stimulated with insulin (100 nM), IGF-I (100 nM) or CoCl2 (200 µM) for 4 h. Nuclear and cytoplasmic fractions were prepared and analyzed by Western blotting using antibodies to HIF-1{alpha}, HIF-1ß, Shc, and CREB.

 


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Fig. 3. IGF-I Stimulates Nuclear Translocation of HIF-1{alpha}

HEK-293 cells were transfected with pEGFP-HIF-1{alpha}, and were stimulated with IGF-I (100 nM) or CoCl2 (200 µM) for indicated periods of time. HIF-1{alpha}-GFP fusion protein was detected by confocal microscopy.

 
IGF-I Activates the Transcription Factor HIF-1
Next, we determined whether IGF-I-induced HIF-1{alpha} accumulation was correlated with activation of the transcription factor HIF-1. We measured the transcriptional activity of HIF-1 using a simian virus 40 promoter-luciferase unit downstream of four hypoxia-response element sequences [pGL2-HRE(4X)WT] (Fig. 4AGo). After IGF-I or CoCl2 treatment, luciferase activity in cell extracts was determined, and normalized to the ß-galactosidase activity. IGF-I and CoCl2 induced a statistically significant 1.373 (±0.046)- and 3.583 (±1.036)-fold increase in luciferase activity, respectively. In contrast, IGF-I or CoCl2 did not activate the mutated hypoxia response element pGL2-HRE(4X)MUT (Fig. 4AGo).



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Fig. 4. IGF-I Activates the Transcription Factor HIF-1

ARPE-19 cells were transfected with the pGL2-HRE(4X)WT-luciferase reporter construct (HRE-WT), pGL2-HRE(4X)MUT-luciferase reporter construct (HRE-MUT) (A) or pGL2-basic P12 VEGF-luciferase reporter construct (VEGF promoter) (B) and an expression vector encoding for the ß-galactosidase gene under the control of the Rous sarcoma virus promoter. ARPE-19 cells were serum-starved for 24 h and incubated with IGF-I (100 nM) or CoCl2 (200 µM) for 16 h. Luciferase activity was measured and results are expressed as a ratio of luciferase activity over ß-galactosidase activity. Results shown represent the mean ± SE of three independent experiments performed in triplicate (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
To determine the specific action of HIF-1 on the transactivation of the VEGF promoter observed in response to IGF-I, we measured the ability of IGF-I to activate pGL2-basic P12 VEGF promoter that contains a fragment (–1005 to –906) of the VEGF promoter (Fig. 4BGo). IGF-I and CoCl2 induced a statistically significant fold increase in luciferase activity, 1.566 (±0.135) and 3.864 (±1.074), respectively.

In conclusion, our data show that IGF-I stimulates the activity of the transcription factor HIF-1.

IGF-I Induces HIF-1{alpha} Expression through Posttranscriptional Mechanisms
To obtain a better understanding of the processes involved in HIF-1{alpha} accumulation in response to IGF-I, we investigated the effect of IGF-I on the amount of HIF-1{alpha} mRNA. ARPE-19 cells were stimulated with IGF-I, RNA was extracted and Northern blot analysis was performed using a VEGF165 and a HIF-1{alpha} cDNA as probe (Fig. 5AGo). Despite its stimulatory action on VEGF mRNA expression, IGF-I did not modify HIF-1{alpha} mRNA expression, showing that IGF-I is unlikely to regulate HIF-1{alpha} mRNA transcription.



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Fig. 5. IGF-I Induces HIF-1{alpha} through a Posttranscriptional Mechanism

A, ARPE-19 cells were stimulated with IGF-I (100 nM) for 6 h. RNA was extracted and analyzed by Northern blotting using HIF-1{alpha} cDNA or VEGF165 cDNA probe. The blot was subsequently hybridized with 18S rRNA as control. B, ARPE-19 cells were stimulated with IGF-I (100 nM) or CoCl2 (200 µM) for 4 h to induce HIF-1{alpha}. Cells are maintained in presence of IGF-I or CoCl2 (upper panels) or cycloheximide (CHX) was added to a final concentration of 10 µg/ml (lower panels). Cells were harvested after being incubated for the indicated times. Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1{alpha} and to Shc. C, A quantification of three independent experiments ± SE are shown (*, P < 0.05; **, P < 0.01). Results are expressed as percent of IGF-I or CoCl2-induced HIF-1{alpha} protein level.

 
To analyze the possible effect of IGF-I on HIF-1{alpha} protein synthesis, we performed a time-course of HIF-1{alpha} turnover in presence of the protein translation inhibitor, cycloheximide (Fig. 5BGo). ARPE-19 cells were treated for 4 h with IGF-I or CoCl2 to induce HIF-1{alpha} accumulation, and cells were maintained in presence of inducers in absence (upper panels) or in presence of cycloheximide (lower panels). After 15–60 min of incubation, HIF-1{alpha} protein level was analyzed by Western blot with antibodies to HIF-1{alpha} or to Shc. As expected, CoCl2 stimulates HIF-1{alpha} accumulation, and the CoCl2-induced HIF-1{alpha} protein level remained unchanged despite the blockade of protein synthesis by cycloheximide. Indeed, after 60 min of cycloheximide treatment, the HIF-1{alpha} protein level was approximately 90% of the level seen before cycloheximide addition (Fig. 5CGo). This observation is consistent with previous studies showing that CoCl2 had no effect on HIF-1{alpha} synthesis but blocked its degradation.

After 4 h of IGF-I stimulation, addition of cycloheximide led within 15 min to a decrease in the HIF-1{alpha} protein level. Within 60 min of protein synthesis inhibition, 66% of the amount of IGF-I-induced HIF-1{alpha} protein has disappeared (Fig. 5CGo).

Together, these results are compatible with the idea that IGF-I increases HIF-1{alpha} protein level through a posttranscriptional mechanism without modulating HIF-1{alpha} mRNA transcription.

IGF-I Stimulates HIF-1{alpha} Accumulation and VEGF mRNA Expression through Pathways Dependent on PI-3-Kinase/mTOR and MAPK
To evaluate the contribution of the MAPK and the PI-3-kinase/mTOR pathways in regulation of the HIF-1{alpha} protein level and VEGF mRNA expression induced by IGF-I, we used pharmacological inhibitors of MAPK kinase (MEK) 1/2, PI-3-kinase, and mTOR. ARPE-19 cells were treated with IGF-I in absence or in presence of increasing concentrations of inhibitors of PI-3-kinase (25, 50, and 75 µM of LY 294002), MEK 1/2 (5, 10, and 15 µM of U0126) or mTOR (25, 50, and 75 nM of rapamycin). Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1{alpha} or to Shc (Fig. 6AGo). We observe a dose-dependent inhibition for each drug. To determine the effect of these inhibitors on IGF-I-induced HIF-1{alpha} and VEGF expression, the inhibitors were used at the following concentrations: 50 µM LY294002, 50 nM rapamycin, and 10 µM U0126. Inhibition of PI-3-kinase activation by LY294002 decreases by 63% the expression of HIF-1{alpha} in response to IGF-I. In addition, U0126 and rapamycin treatments decrease by 31% and 56%, respectively, the IGF-I-induced HIF-1{alpha} protein level (Fig. 6BGo).



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Fig. 6. IGF-I Stimulates HIF-1{alpha} Accumulation and VEGF mRNA Expression through a PI-3-Kinase/mTOR and a MAPK-Dependent Pathway

A, ARPE-19 cells were pretreated with LY294002 (25, 50, and 75 µM), rapamycin (Rapa, 25, 50, 75 nM) or U0126 (5, 10, 15 µM) for 30 min before being stimulated with IGF-I (100 nM) for 4 h. Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1{alpha} or to Shc. B, ARPE-19 cells were stimulated with IGF-I (100 nM) with or without pretreatment for 30 min with LY294002 (50 µM), U0126 (10 µM), or rapamycin (50 nM). Whole cell lysates were prepared and analyzed by Western blotting using antibodies to HIF-1{alpha} or to Shc. A representative experiment (left) and a quantification of seven independent experiments ± SE (right) are shown (***, P < 0.001). C, ARPE-19 cells were stimulated for 6 h with IGF-I (100 nM) with or without pretreatment for 30 min with LY294002 (50 µM), U0126 (10 µM), or rapamycin (50 nM). RNA was extracted and analyzed by Northern blotting using a VEGF165 cDNA probe. The blot was subsequently probed with a probe for 18S rRNA as control.

 
Finally, we show that these inhibitors modulate VEGF mRNA expression in response to IGF-I. Indeed, ARPE-19 cells were treated with IGF-I in absence or in presence of LY294002, U0126, or rapamycin. RNA was extracted and Northern blot analysis was performed using a VEGF165 cDNA probe (Fig. 6CGo). IGF-I induced a 2.5-fold increase in VEGF mRNA expression, and LY294002, U0126 and rapamycin exposure resulted in 40, 38, and 35% inhibition, respectively, of IGF-I-induced VEGF mRNA.

In summary, IGF-I-induced accumulation of HIF-1{alpha} protein and VEGF mRNA are dependent on both the PI-3-kinase/mTOR and the MAPK pathway.

Phosphorylation Level of Ribosomal S6 Kinase-1 (S6K-1) and eIF-4E-Binding Protein-1 (4E-BP1) in Response to IGF-I and Insulin
The PI-3-kinase/mTOR signaling cascade has been shown to regulate protein translation via phosphorylation of S6K-1 and 4E-BP1 (31). Because we found that IGF-I stimulates HIF-1{alpha} expression through a translation-dependent mechanism, we evaluated the phosphorylation state of S6K-1 and 4E-BP1 in response to IGF-I and to insulin.

ARPE-19 cells were treated with IGF-I or insulin in absence or presence of inhibitors of PI-3-kinase (LY 294002), MEK 1/2 (U0126), or mTOR (rapamycin). Whole cell lysates were prepared and analyzed by Western blotting using antibodies to phospho-S6K-1 (Thr389), phospho-4E-BP1 (Thr36/45), phospho-MAPK (Thr202/Tyr204), phospho-PKB (Ser473) and Shc. IGF-I and insulin induce phosphorylation of S6K-1 and 4E-BP1, as revealed by a decrease in the mobility of the two proteins on immunoblot analysis and with phosphospecific antibodies. In addition, phosphorylation of S6K-1 and 4E-BP1 induced by IGF-I treatment is decreased by inhibition of PI-3-kinase and mTOR. However, blockade of MEK/MAPKs by the MEK 1/2 inhibitor (U0126) does not prevent phosphorylation of S6K-1 and 4E-BP1 induced by IGF-I (Fig. 7Go). We obtain the same result in ARPE-19 cells treated with insulin (Fig. 7Go). Note that IGF-I- and insulin-induced phosphorylation of PKB and MAPK were indeed blocked by their respective inhibitors.



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Fig. 7. Phosphorylation Level of S6K-1 and 4E-BP1 in Response to IGF-I and to Insulin

ARPE-19 cells were stimulated for 4 h with IGF-I (100 nM), or with insulin (100 nM) with or without pretreatment for 30 min with LY294002 (50 µM), U0126 (10 µM), or rapamycin (50 nM). Whole cell lysates were prepared and analyzed by Western blotting using antibodies to phosphoS6K-1, S6K-1, phospho4E-BP1, 4E-BP1, phospho-MAPK, phospho-PKB (protein kinase B) and Shc.

 
As a whole, our experiments show that IGF-I and insulin induce phosphorylation of S6K-1 and 4E-BP1 through a PI-3-kinase/mTOR-dependent pathway.

IGF-I and Insulin Do Not Regulate Prolyl- and Asparaginyl-Hydroxylases mRNA Expression
Next, we investigated IGF-I and insulin effects on prolyl- and asparaginyl-hydroxylases mRNA expression. ARPE-19 cells were treated for the indicated times with insulin or IGF-I and with CoCl2 or DFO as positive controls. We verified that all compounds stimulated VEGF mRNA expression (data not shown), and we analyzed PHD-1, PHD-2, PHD-3, PH-4, and FIH-1 mRNA levels by quantitative real-time PCR (Fig. 8Go).



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Fig. 8. PHD-1, PHD-2, PH-4, and FIH-1 mRNA Expression

ARPE-19 cells were stimulated with insulin (100 nM), IGF-I (100 nM), CoCl2 (200 µM), or DFO (0.1 µM) for the indicated time. Level of PHD-1 (A), PHD-2 (B), PH-4 (C), and FIH-1 (D) mRNA expression was analyzed by quantitative real-time PCR as detailed in Materials and Methods. Results shown represent the mean ± SE of three independent experiments (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

 
We found that insulin and IGF-I do not modulate PHD-1, PH-4, and FIH-1 mRNA expression (Fig. 8Go, A, C, and D). However, insulin and IGF-I induce a slight but significant increase in the PHD-2 mRNA level after 6 h of treatment [1.33 (±0.123)- and 1.47 (±0.088)-fold increase, respectively]. This induction is not detectable after long-term treatment because 16 and 24 h of stimulation by insulin or IGF-I do not stimulate PHD-2 mRNA expression (Fig. 8BGo). As expected, compounds that mimic hypoxia, CoCl2 and DFO, stimulate PHD-2 mRNA expression by 2 (±0.15)- and 1.9 (±0.04)-fold increase respectively after 6 h of treatment. The PHD-2 mRNA level remains elevated after 24 h of treatment with CoCl2 and DFO [1.8 (±0.083)- and 2.45 (±0.3)-fold increase, respectively] (Fig. 8BGo). PHD-3, another target of hypoxia, is not detected in our cells (data not shown).

Unexpectedly, CoCl2 decreases PHD-1, PH-4, and FIH-1 mRNA expression (Fig. 8Go, A, C, and D). CoCl2 decreases FIH-1 mRNA expression after 6 h of treatment (23% ± 5.4). The maximal inhibition is reached within 24 h of CoCl2 stimulation (50% ± 6.9) (Fig. 8DGo). The decrease in PHD-1 and PH-4 mRNA level is not detectable after 6 h. However, after 16 h of CoCl2 treatment we observed a decrease in PHD-1 and PH-4 mRNA level (27% ± 6.6 and 40% ±7.4, respectively) (Fig. 8Go, A and C).

In conclusion, we show that IGF-I and insulin stimulate transiently PHD-2 mRNA levels in ARPE-19 cells, but do not appear to regulate PHD-1, PH-4, and FIH-1 mRNA expression. In addition, we found that CoCl2 and DFO stimulate PHD-2 mRNA expression. However, only CoCl2 decreases PHD-1, PH-4, and FIH-1 mRNA expression in these retinal cells.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
HIF-1 is a transcription factor involved in cellular adaptation in response to decreased oxygen availability. HIF-1 regulates expression of numerous genes such as angiogenic factors, glucose transporters, and glycolytic enzymes, survival and invasion factors (32). One of HIF-1 target gene encodes for VEGF, which is a key player in angiogenesis. Angiogenesis is involved in pathological situations such as tumor development and proliferative diabetic retinopathy. During proliferative diabetic retinopathy, it has been demonstrated that VEGF expression is increased in the vitreous of patients with and proliferative diabetic retinopathy, and that VEGF plays an important role in the development of this pathology. VEGF expression is mainly induced by hypoxia. In addition, we and others, have shown that oxygen-independent pathways, such as stimulation by growth factors, hormones, or advanced glycation end products, stimulate HIF-1 activity and VEGF expression (13, 14, 15, 33, 34). The aim of our present work was to identify the molecular mechanisms involved in the regulation of HIF-1 activation and VEGF expression in response to IGF-I, and to compare the mechanisms used by IGF-I and insulin.

HIF-1 is composed of two subunits, HIF-1{alpha} and HIF-1ß. HIF-1ß subunit expression is not regulated by insulin, IGF-I or CoCl2 and is strictly localized in the nucleus (29, 30). In contrast, we demonstrate that IGF-I stimulates nuclear translocation of HIF-1{alpha}. Indeed, in ARPE-19 cells, we have observed that IGF-I stimulates nuclear expression of HIF-1{alpha} because it has been observed in the tumor cell line HCT116 cells (35). To follow the changes in cellular localization of HIF-1{alpha}, we have expressed ectopic HIF-1{alpha}-GFP fusion protein in HEK-293 cells. Stimulation with IGF-I appears to stimulate nuclear translocation of HIF-1{alpha}. A similar observation was made with insulin (data not shown). Several studies have suggested that changes in nuclear vs. cytoplasmic localization of HIF-1{alpha} can be an additional mode of HIF-1 regulation. Indeed, it has been shown that hypoxia induces the nuclear localization of HIF-1{alpha}, and that it could be regulated by Fe(II)- and 2-oxoglutarate-dependent oxygenases (36, 37, 38, 39, 40). In response to growth factors such as IGF-I, mechanisms involved in the translocation of HIF-1{alpha} remains to be elucidated. It is possible that IGF-I stimulates an active nuclear export or inhibits cytoplasmic retention. Because HIF-1{alpha} contains a bipartite nuclear localization signal in the C-terminal portion of the molecule, which induces constitutive nuclear import irrespective of oxygen, mechanisms involved in cytoplasmic retention remain to elucidated.

In the present study, we show that IGF-I increases HIF-1{alpha} expression through a posttranscriptional mechanism. The underlying process is distinct from that observed during hypoxia. Indeed, it has been shown that hypoxia inhibits HIF-1{alpha} proteasomal degradation, leading to its accumulation (41). It is tempting to propose that IGF-I regulates HIF-1{alpha} expression through a translation-dependent mechanism, even if we cannot exclude that IGF-I regulates the translation of a protein that is involved in HIF-1{alpha} degradation. It has been previously shown that heregulin, which activates HER-2 tyrosine kinase receptor, stimulates HIF-1{alpha} translation (14). The stimulatory action on HIF-1{alpha} translation seems to be a general mechanism used by tyrosine kinase receptors, such as insulin and IGF-I receptors, HER2, and by cytosolic tyrosine kinases such as Src (14, 15, 42). Moreover, we cannot exclude the possibility that IGF-I regulates the stability of HIF-1{alpha}.

We have observed that, although IGF-I and CoCl2 induces HIF-1{alpha} and VEGF expression to the same extent, IGF-I is less potent than CoCl2 to activate reporter genes, containing only hypoxia response element sequence from the VEGF promoter. This observation suggests that other transcriptions factors activated by IGF-I could be involved in VEGF mRNA expression.

In contrast with what we found for insulin-induced HIF-1{alpha} expression (15), IGF-I regulates HIF-1{alpha} and VEGF expression through both the PI-3-kinase and the MAPK cascades. Moreover, we show that IGF-I and insulin induce phosphorylation of S6K-1 and 4E-BP1 through a PI-3-kinase/mTOR-dependent pathway. The phosphorylation of 4E-BP1 leads to the release of the eukaryotic initiator factor 4E (eIF4E), and the phosphorylation of S6K-1 results in the phosphorylation and activation of the 40S ribosomal protein S6. The ultimate outcome of all these events is the stimulation of protein synthesis. We found that MAPK is not involved in the phosphorylation of 4E-BP1 and S6K-1 in response to insulin and IGF-I. This is in contrast with a previous study in which IGF-I stimulates phosphorylation of S6K-1 and 4E-BP1 through both PI-3-kinase and MAPK-dependent pathways (43). We propose that in retinal epithelial cells, MAPK regulates a step downstream of 4E-BP1. Indeed, it has been shown that MnK1 (MAPK signal integrating kinase-1) phosphorylates eIF4E leading to increased protein synthesis (44).

Hypoxia modulates HIF-1{alpha} protein level and HIF-1 activity by regulating its hydroxylation level. Three human prolyl-hydroxylases PHD-1, -2, and -3 (prolyl hydroxylase domain-containing protein) and an asparaginyl-hydroxylase FIH-1, that catalyze, respectively, the oxygen-dependent prolyl- and asparaginyl-hydroxylation of HIF-1{alpha}, have been described (1, 5, 6, 7, 8). In addition, by sequence homology a novel HIF prolyl-hydroxylase, termed PH-4, has been cloned (10). Recently, it has been shown that PHD-2 and PHD-3 mRNA expression is stimulated by hypoxia (1, 11). In addition, PHD-2 expression is itself regulated by HIF-1 (12). In agreement with these studies, we find here that CoCl2 and DFO stimulate PHD-2 mRNA expression in human retinal epithelial cells. To the best of our knowledge, this is the first demonstration that growth factors stimulate prolyl-hydroxylases expression. We observe the same level of stimulation of VEGF expression in response to IGF-I, insulin and CoCl2. In contrast, compared with CoCl2, insulin and IGF-I have a moderate effect on PHD-2 mRNA level. Therefore, it is likely that HIF-1, which has been involved in the regulation of PHD-2 mRNA level, needs additional cofactors to activate PHD-2 transcription, which are not recruited by growth factors. In normoxia, insulin and IGF-I stimulate HIF-1{alpha} accumulation, in contrast PHD-2 increases HIF-1{alpha} ubiquitination and degradation, suggesting that they have an opposite effect. This counter action could explain that insulin and IGF-I have only a slight effect on PHD-2 mRNA level. The level of the other hydroxylases, PHD-1, PH-4, and FIH-1, does not appear to be modulated by insulin, IGF-I or DFO. This is in agreement with studies showing that hypoxia and DFO do not modify PHD-1, PH-4, and FIH-1 mRNA levels (1, 11). Surprisingly, our results reveal that CoCl2 decreases PHD-1, PH-4, and FIH-1 mRNA levels. The effects of CoCl2 and DFO on the regulation of the mRNA level of these hydroxylases, suggest that divalent metal and iron chelator mimic hypoxia by distinct mechanisms. In addition, the different expression pattern, observed in response to insulin and IGF-I compared with CoCl2 and DFO treatments, add support to the hypothesis that growth factors and hypoxia use distinct signaling pathways to modulate HIF-1 activity and VEGF expression in cells.

To conclude, pathological retinal neovascularization is a debilitating complication of diabetes. Exaggerated VEGF expression is the chief event involved in this process. Several factors, i.e. hypoxia due to micro-occlusions, increased production of advanced glycation end products and high levels of insulin/IGF-I, will lead in concert to increased VEGF expression. Because the mechanisms used by these factors are distinct, identification of specific molecules targeted at the machinery going from the HIF-1 message to the VEGF protein will lead to important insights at the cell biology level, but more importantly, they will provide new therapeutic approaches for the diabetic eye.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Insulin was a kind gift from Novo-Nordisk (Copenhagen, Denmark). Recombinant human IGF-I was purchased from PromoKine (PromoCell, Heidelberg, Germany). Antibody to HIF-1{alpha} (clone H1{alpha}67) and HIF-1ß/ARNT were purchased from Novus Biologicals, Inc. (Littleton, CO). Antibody to Shc was purchased from BD Transduction Laboratories (Franklin Lakes, NJ). Antibody to CREB was a gift from M. Montminy (Salk Institute for Biological Studies, La Jolla, CA). Antibody to phospho-PKB (Ser473) was purchased from Sigma-Aldrich (St. Louis, MO). Antibodies to phospho-p42/44 MAPK (Thr202/Tyr204) E10, and to phospho-S6K-1 were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Antibody to S6K-1 (C-18) was purchased from Santa Cruz Biotechnology, Inc. (Tebu, France). Antibodies to 4E-BP1 and to phospho-4E-BP1 (Thr36/45) were a gift from J. C. Lawrence Jr. (University of Virginia School of Medicine, Charlottesville, VA).

All chemical reagents were purchased from Sigma-Aldrich. U0126 was purchased from Promega Inc. (Madison, WI). Oligonucleotides were purchased from Invitrogen (Gaithersburg, MD). Culture media were purchased from Cambrex (East Rutherford, NJ).

DNA Plasmids
pGL2-basic HRE(4X)WT corresponds to four hypoxia response element (HRE) of the VEGF promoter cloned upstream of a luciferase coding sequence, and pGL2-basic HRE(4X)MUT corresponds to four HRE of the VEGF promoter cloned upstream of a luciferase coding sequence, in the opposite sense (45). These two constructs were obtained from F. Kapner (Mayer Cancer Biology Research Laboratory, Stanford University, CA). pGL2-basic P12 VEGF promoter corresponds to nucleotides –1005 to –906 of the human VEGF promoter, containing the hypoxia response element, cloned upstream of a luciferase coding sequence (46). This construct was obtained from G. L. Semenza (The Johns Hopkins University School of Medicine, Baltimore, MD).

HIF-1{alpha}-GFP construct was generated by subcloning HIF-1{alpha} cDNA into BamH1 sites of pEGFP-C1 vector (CLONTECH, BD Biosciences, Palo Alto, CA).

Cell Culture
ARPE-19 cells (ATCC no. CRL-2302) were grown in F12/DMEM containing 10% (vol/vol) fetal calf serum (Cambrex) at 37 C with 5% CO2. Human embryonic kidney cells (HEK-293 EBNA) were maintained in culture in DMEM containing 10% (vol/vol) fetal calf serum (Cambrex) and 500 µg/ml geneticin at 37 C with 5% CO2.

Transfection
ARPE-19 cells were transiently transfected using the FuGENE 6 transfection reagent (Roche Diagnostics Corp., Indianapolis, IN), according to manufacturer’s instructions. Sixteen hours after addition of DNA, the medium was changed, and the cells were incubated for an additional 16 h with F12/DMEM containing 0.2% (wt/vol) BSA.

Transfection of human embryonic kidney cells (HEK-293 EBNA) was performed by calcium phosphate precipitation (10 µg of DNA/9.5-cm2 dish). Sixteen hours after transfection, the calcium phosphate-DNA precipitates were removed, and cells were incubated in DMEM containing 5% (vol/vol) fetal calf serum. Before use, cells were serum starved for 16 h in DMEM containing 0.2% (wt/vol) BSA.

Western Blot Analysis
Serum-starved cells were treated with the indicated compounds, chilled to 4 C and washed with ice-cold PBS [140 mM NaCl, 3 mM KCl, 6 mM Na2HPO4, 1 mM KH2PO4 (pH 7.4)], and solubilized with lysis buffer [50 mM HEPES, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 100 mM NaF, 2 mM vanadate, 1 mM 4-(2-aminoethyl)-benzene-sulfonylfluoride hydrochloride (AEBSF), 10 µg/ml aprotinin, 10 µg/ml leupeptin (pH 7.4), 1% Triton X-100] for 20 min at 4 C. Proteins were separated by SDS-PAGE and transferred by electroblotting to nitrocellulose membranes (Hybond C; Amersham Pharmacia Biotech, Uppsala, Sweden). The membranes were soaked first in blocking buffer [20 mM Tris (pH 7.4), 137 mM NaCl, 0.5% (vol/vol) Tween 20] containing 5% (wt/vol) BSA or nonfat milk, and second in blocking buffer containing antibodies. After washes, proteins were detected using horseradish peroxidase-linked secondary antibodies and enhanced chemiluminescence according to the manufacturer’s instructions (Amersham Pharmacia Biotech).

RNA Isolation and Northern Blot Analysis
Trizol reagent (Invitrogen Life Technologies Inc., Gaithersburg, MD) was used to extract total cellular RNA from confluent cells grown in 100-mm tissue culture plates according to the manufacturer’s instructions. Cells were serum-deprived overnight in medium containing 0.2% (wt/vol) BSA and were pretreated or not with inhibitors for 30 min before being stimulated for indicated lengths of time. RNA was extracted, and 10 µg of total RNA was denatured in formamide and formaldehyde, and separated by electrophoresis in formaldehyde-containing agarose gels. RNA was transferred to Immobilon-Ny+ membranes (Millipore Corp., Bedford, MA), and cross-linked to the membrane by heating to 80 C. Human VEGF165 cDNA or a PCR product encoding nucleotides 990-1584 of human HIF-1{alpha} mRNA were used as probe (14). Probes were labeled with [{gamma}-32P] deoxy-CTP by random priming using the Rediprime kit (Amersham Pharmacia Biotech) and purified with the Probequant kit (Amersham Pharmacia Biotech). Hybridizations were performed at 42 C in NorthernMax Hybridization buffer (Ambion Inc., Austin, TX). Membranes were washed in 1x standard saline citrate, 0.5% (wt/vol) sodium dodecyl sulfate and radioactivity was quantitated using a Storm 840, Molecular Dynamics (Amersham Pharmacia Biotech).

Luciferase Assays
To assay the transcriptional activity of HIF-1, we used pGL2-basic HRE(4X)WT, pGL2-basic HRE(4X)MUT or pGL2-basic P12 VEGF-promoter (45, 46). ARPE-19 cells in 12-well plates were transiently cotransfected with the reporter plasmid and with Rous sarcoma virus-ß-galactosidase as a control for transfection efficiency. Cells were stimulated for 16 h and luciferase assays were performed as described in the Protocols and Applications Guide (Promega Inc.). Luciferase activity was measured using a chemioluminometer Wallac 1420 (PerkinElmer Inc., Courtaboeuf, France). The ß-galactosidase activity was performed as described in the Promega Protocols and Applications Guide. Cells lysates were incubated with a 2x assay buffer (200 mM sodium phosphate buffer (pH 7.8), 2 mM MgCl2, 100 mM ß-mercaptoethanol, 1.33 mg/ml o-nitrophenyl ß-D-galactopyranoside). The absorbance at 420 nm was measured with a spectrophotometer Wallac 1420 (PerkinElmer Inc.).

Nuclear Extract Preparation
Nuclear extracts were prepared as previously described (34). Serum-starved cells were treated with indicated compounds, chilled to 4 C and washed with ice-cold PBS. Cells were scraped into 5 ml for 150-mm dish of PBS and pelleted by centrifugation at 1500 rpm for 10 min at 4 C. Cell pellets were washed with four packed cell volumes of buffer A [10 mM Tris-HCl (pH 7.6), 1.5 mM MgCl2, 10 mM KCl, supplemented with 2 mM dithiothreitol, 1 mM AEBSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 2 µg/ml pepstatine, and 1 mM Na3VO4] resuspended in four packed cell volumes of buffer A, and incubated on ice for 10 min. Cells were lysed by 20 strokes in a glass Dounce homogenizer (VWR, Fontenay sous bois, France) with a type B pestle. Nuclei were pelleted at 3000 rpm for 10 min and resuspended in three packed nuclear volumes of buffer C [0.42 M KCl, 20 mM Tris-HCl (pH 7.8), 20% (vol/vol) glycerol, 1.5 mM MgCl2] supplemented with 2 mM dithiothreitol, 1 mM AEBSF, 2 µg/ml leupeptin, 2 µg/ml aprotinin, 2 µg/ml pepstatine, and 1 mM Na3VO4. Nuclear proteins were extracted by stirring at 4 C for 30 min. After centrifugation at 13,500 rpm for 30 min, the supernatant was dialyzed against buffer Z-100 [25 mM Tris-HCl (pH 7.6), 0.2 mM EDTA, 20% (vol/vol) glycerol, 100 mM KCl] at 4 C. The dialysate was clarified by centrifugation at 13,500 rpm for 30 min at 4 C and designated as crude nuclear extract. The nuclear extracts were aliquoted, frozen in liquid N2, and stored at –80 C.

Immunofluorescence
For immunofluorescence studies, cells were grown on coverslips and used 48 h after transfection. The cells were washed and fixed in 3.7% (wt/vol) paraformaldehyde for 15 min at room temperature. Immunofluorescence was analyzed with a TCS-SP confocal microscope (Leica, Heidelberg, Germany) using a x63 magnification lens. Each picture represents the projection of four serial confocal optical sections.

Reverse Transcription and Quantitative Real-Time PCR
Total RNA was treated with deoxyribonuclease I (Ambion, Inc.). One microgram of total RNA was reverse transcribed using random priming, oligo (deoxythymidine), and avian myeloblastosis virus reverse transcriptase according manufacturer’s instructions (Promega Inc.). Quantitative PCR was performed by monitoring in real-time the increase in fluorescence of SYBR Green on an ABI PRISM 7000 Sequence Detector System (Applied Biosystems, Foster City, CA) according to manufacturer’s instructions. VEGF, PHD-1, PH-4, and SB34 primers were designed using the Primer Express software from Applied Biosystems. The primer sets used for VEGF were (F) 5'-TTGCTGCTCTACCTCCACCAT-3' and (R) 5'-TGATTCTGCCCTCCTCCTTCT-3', for PHD-1 were (F) 5'-ACCAGATTGCCTGGGTGGA-3' and (R) 5'-CACCAATGCTTCGACAGCCT-3', for PH-4 were (F) 5'-AACATGGACCTTCGGGACTTC-3' and (R) 5'-TGTTCCGCACCAGCTCACT-3', and for the housekeeping gene SB34 (F) 5'-ACCAGATTGCCTGGGTGGA-3' and (R) 5'-CACCAATGCTTCGACAGCCT-3'. The primer sets used for PHD-2 (F) 5'-GCACGACACCGGGAAGTT-3' and (R) 5'-CCAGCTTCCCGTTACAGT-3', and for FIH-1 (F) 5'-ACAGTGCCAGCACCCACAA-3' and (R) 5'-GCCCACAGTGTCATTGAGCG-3' have been previously described (11). The amount of cDNA used in each reaction was normalized to the SB34 cDNA and expressed as sample cDNA/SB34 cDNA.


    ACKNOWLEDGMENTS
 
We are grateful to J. Plouet (Toulouse, France) for VEGF165 cDNA and to M. Montminy and J. C. Lawrence, Jr. for the gift of antibodies.


    FOOTNOTES
 
Our research was supported by funds from Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche contre le Cancer (Association pour la Recherche contre le Cancer Grant 3293), University of Nice-Sophia Antipolis, Région Provence-Alpes-Côte-d’Azur. C.T. is recipient of a Ph.D. student fellowship from the Association pour la Recherche contre le Cancer.

First Published Online February 3, 2005

Abbreviations: AEBSF, 4-(2-Aminoethyl)-benzene-sulfonylfluoride hydrochloride; ARPE-19, arising retinal pigment epithelial; CoCl2, cobalt chloride; CREB, cAMP response element binding protein; DFO, deferoxamine; 4E-BP1, eIF-4E-binding protein-1; FIH-1, factor inhibiting HIF-1; GFP, green fluorescent protein; HIF-1, hypoxia-inducible factor 1; HRE, hypoxia response element; MEK, MAPK kinase; mTOR, mammalian target of rapamycin; PHD, prolyl hydroxylase-containing protein; PI-3-kinase, phosphatidylinositol-3-kinase; S6K-1, ribosomal S6 kinase-1; Shc, SH2-collagen domain, VEGF, vascular endothelial growth factor; VHL, von Hippel-Lindau.

Received for publication June 11, 2004. Accepted for publication January 25, 2005.


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