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GROWTH FACTORS-CYTOKINES-ONCOGENES |
Departments of Medicine (N.A., M.Z., T.L.C.), Obstetrics and Gynecology (J.R.), and Molecular and Cellular Physiology (M.F.C.-K., T.L.C.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: Thomas L. Clemens, Ph.D., Division of Endocrinology and Metabolism, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, Ohio 45267-0547. E-mail: clementl{at}UC.edu
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Abstract |
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(HIF-2) protein without changes in HIF-2 mRNA expression. These effects were mimicked by chemical inhibition of proteosomal degradation of HIF-2. Transcriptional activation of a proximal VEGF promoter-luciferase construct was greatly enhanced by cotransfection with an HIF-2, but not an HIF-1, construct. IGF-I acutely stimulated Akt phosphorylation, which was abolished by pretreatment of cells with the PI3K inhibitor LY294002. Pretreatment of the cells with LY294002 also greatly attenuated IGF-I induction of HIF-2 and blunted IGF-I-induced VEGF promoter activity. Finally, forced expression of a constitutively active PI3K expression construct induced VEGF promoter to levels similar to those observed with IGF-I alone. These data indicate that IGF-I, by activation of the PI3K pathway, induces VEGF expression in osteoblasts through a transcriptional control mechanism common to those that activate VEGF and other hypoxia response genes. |
Introduction |
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In addition to its well recognized growth-promoting effects, IGF-I is an important survival factor in a number of cell types, including bone osteoblasts (7). Recent studies suggest that IGF-I may also play a role in tissue response to hypoxic or nutrient stress. For example, IGF-I induces the expression of factors that facilitate the delivery of nutrients and metabolic energy to hypoxic sites (8). Among these factors, vascular endothelial growth factor (VEGF) is an essential cytokine involved in the regulation of angiogenesis. VEGF shares homology with platelet-derived growth factor and functions as both a permeability and an angiogenic factor (9). The VEGF gene is subject to alternative splicing, resulting in distinct protein isoforms that signal through a family of related type III tyrosine kinase receptors (10).
In skeletal tissue, osteoblasts are believed to participate in the regulation of angiogenesis in bone under normal physiological conditions, and in response to pathological signals (11). Several VEGF splice variants and their receptors are expressed in osteoblasts (12), and the cytokine has been shown to induce alkaline phosphatase activity and enhance osteoblast responsiveness to PTH (13). We have recently shown that hypoxia transcriptionally activates VEGF mRNA expression in human osteoblast-like cells by elevating the level of the basic helix-loop-helix-periodic acid-Schiff transcription factor, hypoxia-inducible factor-2 (HIF-2) (14). The ability of IGF-I to induce VEGF and other genes that are also regulated by hypoxia suggested a common mechanism for transcriptional control by these two stimuli. In the present study we show that IGF-I induces VEGF expression in human osteoblast-like cells through transcriptional activation involving the HIF-2 /aryl hydrocarbon nuclear translocator complex. These events appear to occur secondary to IGF-I activation of the PI3K pathway.
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Materials and Methods |
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were purchased from NeoMarkers (Freemont, CA). Antibodies to HIF-1ß and HIF-2 were purchased from Novus Biologicals (Littleton, CO). Phosphorylated Akt antibody was purchased from New England Biolabs, Inc. (Beverly, MA). The proteosome inhibitor N-CBZ-LEU-LEU-NORVALINAL (CBZLLN) and the PI3K inhibitor LY294002 were purchased from Sigma (St. Louis, MO).
Cell culture
Human MG63 osteoblast-like cells and human SaOS-2 osteoblast-like cells were obtained from American Type Tissue Culture Collection (Manassas, VA). MG63 cells were maintained in MEM containing 10% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). SaOS-2 cells were maintained in DMEM containing 15% FBS, penicillin (100 U/ml), and streptomycin (100 µg/ml). Both cell types were cultured in a water-jacketed incubator with a humidified atmosphere (5% CO2/air) at 37 C.
RNA extraction and Northern blot analysis
MG63 cells and SaOS-2 cells were grown in 100-mm tissue culture plates until 90% confluence was reached and were starved for 24 h. Cells were then incubated with 50 ng/ml IGF-I or were left untreated for the times indicated. Total cellular RNA was extracted using TRIzol reagent (Life Technologies, Inc., Gaithersburg, MD), and 20 µg RNA samples were resolved by formaldehyde agarose gel electrophoresis in 3-[N-morpholino]propanesulfonic acid buffer, transferred to a nylon membrane (QIAGEN), and cross-linked to the membrane by UV irradiation. Blots were then hybridized for 2 d at 42 C to a rat VEGF cDNA or a human HIF-2 cDNA probe labeled by the random priming method using the Prime II kit (Stratagene, La Jolla, CA). After hybridization, membranes were washed in 2x SSC/0.1% SDS at 65 C and in 1x SSC/0.1% SDS at 65 C. The final wash was with 0.2x SSC/0.1% SDS at room temperature. For standardization, blots were rehybridized with a cyclophilin cDNA probe. Membrane signal intensity was quantitated by phosphorimaging and analyzed using ImageQuant (Molecular Dynamics, Inc., Sunnyvale, CA) image analysis software.
Western blot analysis
MG63 cells were cultured until 90% confluent in 150-mm dishes for nuclear extract preparation and in 100-mm dishes for cell lysate preparation. After fasting for 24 h, cells were incubated with 50 ng/ml IGF-I or were left untreated in serum-free medium. For the LY294002 treatment group, cells were treated with IGF-I (50 ng/ml) and LY294002 (25 µM) for 6 h. To determine the protein levels of HIFs, cells were washed twice with ice-cold PBS, and nuclei were prepared as described previously (15). Nuclear extracts (50 µg) were boiled for 5 min in Laemmli buffer [62.5 mM Tris (pH 6.8), 1% SDS, 20% glycerol, 0.01% bromophenol blue, and 100 mM dithiothreitol] and separated by 6% SDS-PAGE. Gels were then transferred to 0.2-µm-pore-size nitrocellulose membranes. After blocking with Tris-buffered saline (pH 7.4) and 0.1% Tween-20 (TBS-T) containing 5% low fat milk, the membranes were incubated with a primary antibody for HIF-1 or HIF-2 in TBS-T containing 5% BSA at 4 C overnight. After three washes with TBS-T, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 2 h and developed by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ). The membranes were then stripped in a buffer containing 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 100 mM ß-mercaptoethanol for 30 min at 50 C and reprobed with primary antibody for HIF-1ß by a similar procedure. Quantitation of band intensities was performed using a Kodak Image Station 440 and Kodak 1D software (Rochester, NY). The levels of HIF-1 and HIF-2 were expressed as a ratio of the density obtained for the constitutive HIF-1ß band.
To determine the concentration of Akt, cells were washed twice with cold PBS, and then cell lysates were prepared with a buffer containing 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ß-glycerophosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin. The proteins (40 µg) were boiled for 5 min in Laemmli buffer, resolved by 8% SDS-PAGE, and transferred onto a 0.2 µm pore size nitrocellulose membrane. The membranes were blocked with TBS-T containing 5% low fat milk and incubated with primary antibody for phospho-Akt (Ser473) in TBS-T containing 5% BSA at 4 C overnight. After three washes with TBS-T, the membranes were incubated with antirabbit IgG conjugated to peroxidase for 2 h at room temperature. After three additional washes in TBS-T, the membranes were developed by enhanced chemiluminescence. Membranes were then stripped as described above, and total Akt was determined by reprobing the membranes with primary antibody for Akt. Bands were quantified using the Kodak Image Station. The extent of Akt phosphorylation was measured by calculating the ratio of phospho-Akt and Akt bands.
Reporter and expression constructs
The KpnI-NheI fragment comprising -2273 to 51 bp of VEGF promoter was cloned upstream of the luciferase reporter vector in the pGL3-Basic reporter plasmid (gift from Dr. J. Abraham, Scios, Inc., Sunnyvale, CA). The HIF-1 cDNA was cloned in the pEBP expression vector under control of human elongation factor-1 promoter (Novus Biologicals). The HIF-2 cDNA was cloned in the pcDNA3 expression vector (Invitrogen, San Diego, CA) under control of the cytomegalovirus promoter (gift from Dr. S. McKnight). The p11 wild-type and mutant VEGF promoter constructs were provided by Dr. G. Semenza. Each contains a 53-bp sequence encoding the hypoxia-responsive element (HRE) cloned into pGL2-promoter reporter vector (Promega Corp.). p11m contains a 3-nucleotide substitution in the HRE element (16). The p110* constitutively active PI3K expression vector (17) was provided by Dr. M. Waterfield.
Transient expression assays
Plasmid DNA was prepared using commercial kits (QIAGEN, Chatsworth, CA). MG63 cells and SaOS-2 cells were cultured to 60–80% confluence in 12-well plates. For each well, 0.75 µg plasmid DNA and 2.5 µl LipofectAMINE reagent (Life Technologies, Inc.) were used. DNA and LipofectAMINE reagents were diluted separately in 50 µl Opti-MEM I reduced serum medium (Life Technologies, Inc.), mixed together, and incubated at room temperature for 30 min. Plates were then washed with serum-free medium, 0.4 ml Opti-MEM I reduced serum medium was added, and the diluted solution was added to the cells. Plates were incubated at 37 C for 5 h, after which time growth medium containing 20% serum was added, and cells were incubated at 37 C for approximately 19 h. Medium was then replaced with serum-free medium, and cells were maintained for an additional 24 h. Cells were then left untreated or were treated with 50 ng/ml IGF-I for 24 h. For the experiments using LY294002, cells were pretreated with 10 µM LY294002 for 1 h and then treated with IGF-I (50 ng/ml) for 6 h. Luciferase assays were carried out using the Steady-Glo luciferase assay system (Promega Corp.). The relative luciferase activity (mean ± SEM) was calculated as light units per µg protein. All experiments were repeated at least three times with two different batches of purified DNA. The protein concentration was measured using the Coomassie Plus protein assay reagent (Pierce Chemical Co., Rockford, IL).
Statistical analysis
Data were compared by t test. Significance was defined as P < 0.05.
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, IGF-I induced VEGF mRNA by approximately 3-fold over basal by 3 h (Fig. 1). This time course of VEGF mRNA induction by IGF-I is similar to that previously shown for hypoxia (14, 18). Similar results were obtained in SaOS-2 cells (not shown).
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and HIF-2 protein in nuclear extracts from IGF-I-treated MG63 cells. IGF-I treatment elevated HIF-2 levels by 2-fold over basal levels by 3 h. Levels continued to rise over a 24-h period to 10-fold the basal level (Fig. 2B). There was no change in the level of HIF-1. By contrast, treatment of MG63 cells with IGF-I had no effect on HIF-2 mRNA levels (Fig. 3). Treatment with the proteosome inhibitor CBZLLN increased the levels of both HIF-1 and HIF-2 subunits, demonstrating the specificity of the antibodies for each protein. Furthermore, treatment of cells with IGF-I together with the proteosome inhibitor caused no further accumulation over that observed with the proteosome inhibitor alone (data not shown). The increases in HIF-1 and HIF-2 protein levels in cells treated with CBZLLN (Fig. 2A) were associated with increased VEGF mRNA expression, further supporting the role of the HIF proteins in the regulation of VEGF gene transcription (data not shown).
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or HIF-2. The cells were then treated with serum-free medium, followed by 50 ng/ml IGF-I. IGF-I treatment resulted in a 5-fold increase in VEGF promoter activity compared with that in untreated cells. Cotransfection with the HIF-2 construct further increased VEGF promoter activity to 40-fold over basal. In contrast, cotransfection with the HIF-1 construct had no effect on VEGF promoter activity (Fig. 4A). Transfection experiments with SaOS-2 cells yielded similar results (Fig. 4B).
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expression vector. Cotransfection with HIF-2 significantly induced the activity of the p11w construct, and this was further enhanced by treatment with IGF-I (Fig. 5). By contrast, IGF-I had no significant effect on the activity of the p11m construct (Fig. 5).
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). Phospho-Akt levels were increased 7-fold over basal by 2 h and remained elevated at 6 h. Treatment of cells with the PI3K inhibitor LY294002 blocked IGF-I-induced phosphorylation of Akt in a dose-dependent fashion (Fig. 6B). LY294002 treatment also attenuated the IGF-I-induced elevation of HIF-2 (Fig. 7) and significantly reduced the IGF-I activation of pVEGF activity (Fig. 8A). These results suggested that activation of the PI3K pathway by IGF-I was required for transcriptional activation of VEGF. To test this more directly, cells were transfected with the pVEGF construct together with an expression plasmid encoding a constitutively active PI3K (p110*). As shown in Fig. 8B, p110* dose-dependently induced the pVEGF to levels comparable to those observed in cells treated with IGF-I alone.
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Discussion |
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mediates the transcriptional activation of VEGF by IGF-I. In support of this conclusion, the IGF-I induction of VEGF mRNA occurred concurrently with the increased levels of HIF-2. Moreover, both basal and IGF-I-induced VEGF promoter activities were increased in cells cotransfected with a HIF-2 expression construct. Finally, a VEGF promoter construct containing a mutation in HRE was not induced by IGF-I when transfected into MG63 cells.
Previous studies in Hep-G2 hepatoma cells showed that both IGF-I and hypoxia stimulated VEGF gene expression (8). In addition, HIF-1 expression was shown to be required for expression of genes encoding IGF-II, IGF-binding protein-2 and IGF-binding protein-3 (23). Moreover, hypoxia has been shown to induce IGF-I production in osteoblasts (20), suggesting that it might mediate the effects of hypoxia on VEGF expression. However, mouse embryonic fibroblasts lacking the IGF-I receptor are still capable of elevating HIF levels in response to hypoxia. It is possible, therefore, that hypoxia stimulates the production of IGF-I, which functions in an autocrine/paracrine mode to reinforce the cells’ ability to respond to hypoxic or nutrient stress.
In general, exposure of various cells types to hypoxia is associated with increases in both HIF-1 and HIF-2. This has led to the speculation that these two transcription factors perform overlapping or redundant functions. However, in the case of osteoblasts, IGF-I (current report) and hypoxia (14) appear to selectively elevate HIF-2. Thus, our data indicate the existence of cell type-specific mechanisms in the regulation of HIF-2 subunits by IGF-I and hypoxia, possibly at the level of inhibition of HIF’s ubiquitination. This idea is supported by the observation that HIF-2 expression is augmented in endothelial cells and cells from the sympathoadrenal origin from the embryonic lethal HIF-1-null mice (24).
The current results also implicate the PI3K pathway and its downstream target, Akt, in the signaling pathway through which IGF-I elevates HIF-2. Thus, IGF-I treatment of MG63 cells induced a rapid phosphorylation of Akt, a Ser/Thr kinase that preceded the increase in HIF-2. Furthermore, chemical inhibition of PI3K with LY294002 virtually eliminated the IGF-I-induced accumulation of HIF-2 and blunted IGF-I activation of pVEGF activity. Moreover, forced expression of a constitutively active PI3K induced the pVEGF to levels comparable to those seen with IGF-I treatment. Signaling through the PI3K pathway has also been implicated in activation of oxygen-sensing genes in other cell types. Thus, exposure of PC12 cells to hypoxia was also associated with increased phosphorylation of Akt. Active Akt translocates to the nucleus, where it phosphorylates a number of proteins involved in regulating metabolic functions, such as glycogen synthesis, glucose uptake, and glycolysis. These targets include glycogen synthase kinase-3 (25), glucose transporter-4 (26), and proteins involved in cell fate, including Bad (27) and the forkhead family of transcription factors (28). Additionally, Akt has been reported to mediate VEGF induction under hypoxia (25). However, the precise mechanisms that link Akt phosphorylation to HIF-2 stabilization in osteoblasts remain to be determined.
The existence of a common transcriptional mechanism through which IGF-I and hypoxia regulate VEGF gene expression in osteoblasts is notable. As mentioned above, hypoxia initiates the transcription of gene products that help to sustain the supply of O2 to tissues and to enhance cell survival during severe O2 deprivation. There are a number of physiological and pathophysiological situations that require cells of the osteoblast lineage to respond to both oxygen and nutrient deprivation. For example, intramembraneous and endochondral ossification occur in close association and proximity to capillary in-growth and angiogenesis (29). Moreover, after fracture, which disrupts normal afferent blood supply to bone, compensatory flow through small periosteal arterioles is elevated via an endothelial cell- mediated process (30). The initial hematoma is filled with growth factors that enhance cell recruitment and differentiation (11). VEGF mRNA is highly expressed during fracture healing and is believed to promote angiogenesis during skeletal development (31). Moreover, treatment of mouse osteoblast-like cells with recombinant VEGF-A stimulates differentiation and nodule formation (12). In addition, VEGF has recently been shown to mimic macrophage colony-stimulating factor in supporting osteoclastogenesis, suggesting that it might also contribute to the formation and/or recruitment of osteoclasts to the fracture site (32). Therefore, it is reasonable to propose that IGF-I participates in a regulatory pathway to enable osteoblasts to respond to fluctuations oxygen or nutrient supply during endochondral bone formation and fracture healing.
In conclusion, we have shown that IGF-I induces VEGF mRNA in osteoblast-like cells through transcriptional mechanisms involving HIF-2 and that these events occur secondary to IGF-I activation of the PI3K signaling cascade. We postulate that the HIF-2/ARNT transcriptional pathway affords bone cells an efficient means of responding to changes in oxygen or nutrient availability during osteogenesis. A better definition of the molecular targets that stabilize HIF-2 could be useful in the design of drugs to promote angiogenesis and relieve tissue ischemia in conditions such as avascular necrosis of bone.
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Acknowledgments |
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Footnotes |
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Abbreviations: HIF-2, Hypoxia-inducible factor-2; HRE, hypoxia-responsive element; IRS, insulin receptor substrate; pVEGF, vascular endothelial growth factor promoter; TBS-T, Tris-buffered saline (pH 7.4) and 0.1% Tween-20; VEGF, vascular endothelial growth factor.
Received August 10, 2001.
Accepted for publication October 22, 2001.
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/ARNT. EMBO J 17:5085–5094[CrossRef][Medline]
. Endocrinology 142:959–962 and insulin-like growth factor 2. Cancer Res 59:3915–3918This article has been cited by other articles:
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) or were left untreated (
), and total RNA was analyzed by Northern blotting at the indicated times (A). Blots were imaged to calculate the normalized values presented in B. The bars represent the relative abundance of HIF-2
