Angiogenesis and G-protein-coupled receptors: signals that bridge the gap.

Angiogenesis is a mechanism that has repercussions in a number of physiological and pathological situations. Vascular endothelial growth factor and basic fibroblast growth factor have understandably received enormous research coverage for being the major mediators of new blood vessel growth, often overshadowing other agonist that also have strong angiogenic potential. We wish to put the spotlight on GPCR agonists that undoubtedly have their word to say on the subject of angiogenesis. In this short review, we will discuss our findings along with the work from other groups on the mechanisms by which GPCR agonists, like thrombin and angiotensin II, control a number of angiogenic signals. A complete understanding of these mechanisms could, by the design of new therapeutic strategies, have a strong impact in clinical oncology.

Short-term desensitization of the angiotensin II receptor of bovine adrenal glomerulosa cells corresponds to a shift from a high to a low affinity state.

Angiotensin II (Ang II) is an important regulator of aldosterone production by bovine adrenal glomerulosa (BAG) cells. Ang II interacts with a specific receptor coupled to a guanyl nucleotide-binding protein (G protein) that controls the activity of phospholipase C. A primary culture of BAG cells was used to study short-term desensitization of the Ang II receptor. After short exposures to Ang II, BAG cells lost some [125I]Ang II binding capacity. This loss was dependent on the duration of the pretreatment and on the concentration of Ang II used. A maximal loss of [125I]Ang II binding of 55 +/- 10% was observed after a pretreatment of 30 min with 30 nM Ang II. The EC50 was 1.3 +/- 0.6 nM (mean +/- SD of three experiments). The desensitization was readily reversible, since most of the binding capacity (higher than 90%) was recovered after a 60-min incubation, at 37 C, in the absence of Ang II. Scatchard studies revealed that the Ang II receptor of BAG cells exists under two affinity states with one dissociation constant of 0.2 nM and another dissociation constant of 1.5 nM. After a 30-min exposure of BAG cells to 10 nM Ang II, an important decrease of high affinity binding sites was observed. The maximal amount of binding sites was similar on control and desensitized cells (around 52,000 receptors per cell). GTP gamma S, a potent activator of G proteins, decreased [125I]Ang II binding to permeabilized BAG cells. This GTP gamma S effect was not observed on permeabilized BAG cells that had previously been desensitized with 10 nM Ang II. These results suggested that, similarly to GTP gamma S, short exposure to 10 nM Ang II caused the uncoupling of Ang II receptor from its G protein. DuP-753 (a selective AT1 angiotensin II type 1 receptor antagonist) markedly unhibited, whereas PD-123319 (a selective AT2 angioten II type 2 receptor antagonist) had no effect on Ang II receptor desensitization, indicating that the AT1 receptor subtype was responsible for the observed phenomenon. Pretreatment of BAG cells with staurosporine (a protein kinase C inhibitor) and R24571 (a calmodulin inhibitor) did not modify Ang II-induced desensitization of AT1 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)

Desensitization of AT1 receptor-mediated cellular responses requires long term receptor down-regulation in bovine adrenal glomerulosa cells.

Angiotensin II (Ang II) regulates aldosterone production in bovine adrenal glomerulosa cells by interacting with the AT1 receptor. This receptor is coupled to a G protein that controls the activity of phospholipase C. With a primary culture of bovine adrenal glomerulosa cells, we evaluated the desensitization of cellular responses after pretreatment with Ang II. When cells were pretreated for 30 min with 1 microM Ang II at 37 C, we observed a 48% loss of [125I]Ang II-binding activity. Scatchard analysis revealed that this decreased binding activity corresponded to a 53% loss of the total number of binding sites. This phenomenon was time dependent, with a t(1/2) of 20 min, and a maximal loss of 76% of the total binding sites was observed after 14 h. A time-dependent decrease in AT1 receptor messenger RNA levels was also observed after pretreatment with 1 microM Ang II for 12-24 h. Taken together, these results are interpreted as a down-regulation of the AT1 receptor. Desensitization of phospholipase C activity under similar conditions was, however, a slower process, with a t(1/2) of 9 h and a maximal response reduction of 83% observed after 24 h. Dose-response experiments indicated that maximal phospholipase C desensitization was obtained in the presence of 1 microM Ang II, with an EC50 of 90 nM. The desensitization was of a homologous nature, as a 24-h pretreatment with Ang II did not affect bradykinin-induced inositol phosphate production. A 24-h pretreatment with 1 microM Ang II also significantly desensitized the steroidogenic effect of Ang II and the potentiating effect of Ang II on ACTH-induced cAMP production. Lower concentrations of Ang II (10 nM) did not produce any desensitizing effect on these two parameters. This study provides evidence that glomerulosa cells are functionally resistant to short term desensitization of the AT1 receptor and that long term down-regulation with high concentrations of Ang II is needed to desensitize AT1-mediated cellular responses.

HIF-1-dependent transcriptional activity is required for oxygen-mediated HIF-1alpha degradation.

Hypoxia-inducible factor-1alpha (HIF-1alpha) plays a central role in oxygen homeostasis. In normoxia, HIF-1alpha is a short lived protein, whereas hypoxia rapidly increases HIF-1alpha protein levels by relaxing its ubiquitin-proteasome-dependent degradation. In this study, we show that the p42/p44 MAP kinase cascade, known to phosphorylate HIF-1alpha, does not modulate the degradation/stabilization profile of HIF-1alpha. However, we present evidence that the rate of HIF-1alpha degradation depends on the duration of hypoxic stress. We demonstrate that degradation of HIF-1alpha is suppressed by: (i) inhibiting general transcription with actinomycin D or (ii) specifically blocking HIF-1-dependent transcriptional activity. In keeping with these findings, we postulate that HIF-1alpha is targetted to the proteasome via a HIF-1alpha proteasome targetting factor (HPTF) which expression is directly under the control of HIF-1-mediated transcriptional activity. Although HPTF is not yet molecularly identified, it is clearly distinct from the von Hippel-Lindau protein (pVHL).

Novel cyclic analogs of angiotensin II with cyclization between positions 5 and 7: conformational and biological implications.

To study the conformational features of molecular recognition of angiotensin II (Asp1-Arg2-Val3-Tyr4-Val/IIe5-His6-Pro7-Phe8, AII), the synthesis and biological testing of several cyclic analogs of AII cyclized between positions 5 and 7 have been performed. The synthesized analogs were Sar1-Arg2-Val3-Tyr4-cyclo(Cys5-His6-Pen7)-Phe8 (3), Sar1-Arg2-Val3-Tyr4-cyclo(Asp5-His6-Apt7)-Phe8 (4), Sar1-Arg2-Val3-Tyr4-cyclo(Glu5-His6-Apt7)-Phe8 (5), Sar1-Arg2-Val3-Tyr4-cyclo-(Cys5-His6-Mpt7)-Phe8 (6), Sar1-Arg2-Val3-Tyr4-cyclo(Cys5-His6-Mpc7)-Phe8 (7), Sar1-Arg2-Val3-Tyr4-cyclo(Hcy5-His6-Mpt7)-Phe8 (8), and Sar1-Arg2-Val3-Tyr4-cyclo(Hcy5-His6-Mpc7)-Phe8 (9), where Apt stands for 4-amino-trans-proline, and Mpt and Mpc for 4-mercapto-trans- and -cis-prolines, respectively. Compound (9) showed good affinity at AT-1 receptors, namely a KD = 20 nM. In functional assays, it showed the characteristics of a weak partial agonist with a relative affinity of 0.26% of that for AII and an intrinsic efficacy, alpha E, of 0.42. Molecular modeling suggested a possible explanation for this finding: the relatively strong binding and the weak partial agonistic activity of compound 9 are due to interaction with AT-1 receptor of only two functionally important groups, namely, the side chains of the His6 and Phe8 residues.

Bovine adrenal glomerulosa cells express such a low level of functional B2 receptors that bradykinin does not significantly increase their aldosterone production.

It was recently demonstrated that bradykinin (BK) stimulates aldosterone secretion in bovine adrenal glomerulosa (BAG) cells. The aim of the present study was to characterize the mechanism of action of BK on these cells. Binding experiments with the radioligand 125I-[Tyr8]BK revealed the presence of a relatively small amount (Bmax = 180 +/- 55 fmol/mg of protein) of high affinity (Kd = 0.65 +/- 0.17 nM) binding sites. BK induced a time- and concentration-dependent increase of [3H]inositol trisphosphate ([3H]IP3) in myo-[3H]inositol-labeled BAG cells. A maximal response was obtained with 10 nM BK and the EC50 value was 1.0 +/- 0.5 nM. 125I-[Tyr8]BK binding and BK-induced IP3 production were inhibited by the selective B2 receptor antagonist Icatibant (1 microM) and unaffected by the selective B1 receptor antagonist [DesArg9, Leu8]BK (1 microM). In fura-2 loaded BAG cells, BK (100 nM) induced a typical biphasic Ca2+ response composed of a rapid and transient increase of intracellular Ca2+ concentration [Ca2+]i which slowly declined to a level that remained above basal level for about 5 min. In the presence of EGTA (2 mM), the rapid and transient calcium increase was unaffected whereas the plateau phase was abolished. Angiotensin II (Ang II, 100 nM) also elicited a typical biphasic response in BAG cells. However the rapid and transient elevation of [Ca2+]i was followed by a sustained plateau phase which remained above the basal level for more than 10 min. Although BAG cells express functional B2 receptors, no secretion of aldosterone was observed after stimulation with 100 nM BK for 120 min. Under the same conditions Ang II increased by about 10-fold the basal level of aldosterone. The lack of effect of BK is probably attributable to its very transient effect on IP3 production. Pretreatment of BAG cells with 100 nM BK for 20 min reduced by 70 +/- 10% their total binding capacity. These results suggest a rapid and very efficient desensitization process. We conclude that BAG cells express functional B2 receptors. The weak production of second messengers and the rapid desensitization process could explain why BK fails to increase aldosterone production in these cells. Since functional B2 receptors are expressed in BAG cells it is likely that under some specific physiological or pathological conditions these receptors may play a significant role in aldosterone secretion. However these conditions remain to be determined.

Signaling angiogenesis via p42/p44 MAP kinase and hypoxia.

Angiogenesis is associated with a number of pathological situations. In this study, we have focused our attention on the role of p42/p44 MAP (mitogen-activated protein) kinases and hypoxia in the control of angiogenesis. We demonstrate that p42/p44 MAP kinases play a pivotal role in angiogenesis by exerting a determinant action at three levels: i) persistent activation of p42/p44 MAP kinases abrogates apoptosis; ii) p42/p44 MAP kinase activity is critical for controlling proliferation and growth arrest of confluent endothelial cells; and iii) p42/p44 MAP kinases promote VEGF (vascular endothelial growth factor) expression by activating its transcription via recruitment of the AP-2/Sp1 (activator protein-2) complex on the proximal region (-88/-66) of the VEGF promoter and by direct phosphorylation of hypoxia-inducible factor 1 alpha (HIF-1 alpha). HIF-1 alpha plays a crucial role in the control of HIF-1 activity, which mediates hypoxia-induced VEGF expression. We show that oxygen-regulated HIF-1 alpha protein levels are not affected by intracellular localization (nucleus versus cytoplasm). Finally, we propose a model which suggests an autoregulatory feedback mechanism controlling HIF-1 alpha and therefore HIF-1-dependent gene expression.

Stimulation of the angiotensin II type I receptor on bovine adrenal glomerulosa cells activates a temperature-sensitive internalization-recycling pathway.

Angiotensin II (Ang II) is an important regulator of aldosterone production by bovine adrenal glomerulosa cells. On these cells Ang II interacts with the AT1 receptor that is coupled to a G protein controlling the activity of phospholipase C. A primary culture of bovine adrenal glomerulosa cells was used to study the internalization-recycling mechanism of the AT1 receptor after stimulation with Ang II. When cells were pretreated with 10 nM Ang II for 30 min at 37 degrees C and binding studies were performed at 12 degrees C we observed a 48% loss in [125I]Ang II binding. Scatchard analysis revealed that this loss in binding translated into a decreased affinity of the AT1 receptor without any loss in the total amount of binding sites. Under the same conditions an important internalization of [125I]Ang II was invariably observed. These observations suggest that a mechanism was at work to recycle the internalized receptors to the cell surface during the binding studies. Following internalization we indeed observed an externalization of [125I]Ang II. This phenomenon relatively rapid at 37 degrees C was much slower at 12 degrees C and completely inhibited at 4 degrees C. When cells were pretreated with 10 nM Ang II for 30 min at 37 degrees C binding assays at 4 degrees C no longer revealed a loss of binding affinity but rather a 54% reduction in the total amount of binding sites. The maximal binding capacity could be recovered during incubations at 12 degrees C. These results reveal the existence of a dynamic recycling process for the AT1 receptor. In accordance with this interpretation the phenomenon was blocked by monensin, a known inhibitor of receptor recycling. These studies suggest that the stimulation of the AT1 receptor sets in motion an internalization-recycling process that seems to be a fundamental aspect of the AT1 receptor transduction mechanism.

Signaling angiogenesis via p42/p44 MAP kinase cascade.

Vascular endothelial growth factor (VEGF), a potent agonist secreted by virtually all cells, controls migration and division of vascular endothelial cells. Disruption of one VEGF allele in mice has revealed a dramatic lethal effect in early embryogenesis, suggesting a key role in vasculogenesis. We analyzed the regulation of VEGF mRNA in normal and transformed CCL39 fibroblasts and then dissected the VEGF promoter to identify the signaling pathway(s) controlling the activation of this promoter in response to growth factors, oncogenes, and hypoxic stress. We demonstrated that the p42/p44 MAP kinase signaling cascade controls VEGF expression at least at two levels. In normoxic conditions, MAPKs activate the VEGF promoter at the proximal (-88/-66) region where Sp-1/AP-2 factors bind. Activation of p42/p44 MAPKs is sufficient to turn on VEGF mRNA. At low O2 tension, hypoxia inducible factor-1 alpha (HIF-1 alpha), a limiting factor rapidly stabilized and phosphorylated, plays a key role in the expression of several genes including VEGF. We demonstrated that p42/p44MAPKs stoichiometrically phosphorylate HIF-1 alpha in vitro and that HIF-1-dependent VEGF gene expression is strongly enhanced by the exclusive activation of p42/p44MAPKs. Finally, we demonstrated that the regulation of p42/p44MAPK activity is critical for controlling proliferation and growth arrest of vascular endothelial cells at confluency. These results point to at least three major targets of angiogenesis where p42/p44 MAP kinases exert a determinant action.

Aerated biofiltration for simultaneous removal of iron and polycyclic aromatic hydrocarbons from groundwater.

Filters incorporating the principles of biological accumulation have been used in Europe to remove iron from drinking water for many years. The authors of this study hypothesized that a modified biological iron-removing filter could simultaneously degrade polycyclic aromatic hydrocarbons (PAHs) in groundwater impacted by former manufactured gas plant (MGP) operations. The MGP-impacted groundwater obtained for this study had an average total iron concentration of 3.8 mg/L and an average total dissolved PAH concentration of 3.0 mg/L. Naphthalene was the primary PAH, with an average concentration of 2.8 mg/L. The groundwater was passed intermittently through duplicate gravel-media columns at 15 to 30 cm/d for 2 months while filtered air was delivered countercurrently at 4 mL/min. The columns remained partially saturated throughout the study. Flooding, which would have indicated plugging of the columns, was not observed, and the pressure needed to aerate the columns remained constant. Total iron in the effluent was below the detection limit of 0.1 mg/L (97% removal) for 40 days of operation. Removal of total PAHs, primarily two- and three-ring compounds, averaged 99%. This single-stage treatment process represents an economical alternative to biological treatment systems currently available for MGP-impacted groundwater that require pretreatment to remove iron.

Selective interaction of chemical dyes with inositol 1,4,5-trisphosphate recognition sites.

Inositol 1,4,5-trisphosphate (InsP3) is an important second messenger that interacts with a specific intracellular receptor and triggers a release of Ca2+ from intracellular stores. InsP3 is preferentially metabolized by two enzymes. A specific 5-phosphatase (InsP3 phosphatase) produces an inactive metabolite, inositol 1,4-bisphosphate, while a specific 3-kinase (InsP3 kinase) produces an active metabolite, inositol 1,3,4,5-tetrakisphosphate. With the goal of developing selective ligands of the diverse InsP3 recognition sites, we have studied the effects of some chemical dyes on the binding of InsP3 to its receptor and on the activity of its metabolic enzymes. Although these dyes possess similar chemical structures, they showed varied selectivities towards the three recognition sites. Thymol Blue was the most potent inhibitor of InsP3 binding activity, with an IC50 of 105 microM. Phenol Red demonstrated a higher selectivity for InsP3 phosphatase inhibition, with an IC50 of 100 microM. 3',3",5',5"-Tetraiodophenolsulfonephthalein showed its most potent inhibitory effect on InsP3 kinase activity, with an IC50 of 35 microM. Tetrabromophenol Blue potently inhibited InsP3 phosphatase and InsP3 kinase activities, with respective IC50 values of 25 and 12 microM. Phenolphthalein Diphosphate and Phenolphthalein Carbinol Disulfate demonstrated weak inhibitory effects towards the three recognition sites for InsP3. These results reveal certain structural clues that should help in the development of more selective inhibitors.

Nonhypoxic pathway mediates the induction of hypoxia-inducible factor 1alpha in vascular smooth muscle cells.

Hypoxia-inducible factor-1 (HIF-1) controls the expression of a number of genes such as vascular endothelial growth factor (VEGF) and Erythropoietin in low oxygen conditions (hypoxia). VEGF is strongly induced at both the mRNA and protein expression level by a number of hormones and growth factors in vascular smooth muscle cells (VSMC) independently of the oxygen environment. However, the role of HIF-1alpha in this induction has not been studied. We report here that HIF-1alpha protein levels are strongly increased by fetal calf serum in quiescent VSMC. More interestingly, Angiotensin II (Ang II), thrombin, platelet-derived growth factor, and other hormones can also increase HIF-1alpha in VSMC to levels that are substantially more elevated than the hypoxic treatment. HIF-1alpha induced by Ang II is located in the nucleus, binds to the hypoxic response element, and is transcriptionally active. The induction of HIF-1alpha by hormones is mediated through the production of reactive oxygen species (ROS), since it can be blocked by the ROS inhibitors, diphenyleneiodonium and catalase. Finally, strong induction of VEGF mRNA by Ang II can also be inhibited by these ROS inhibitors. These results implicate HIF-1alpha and HIF-1-dependent transcriptional activity in the induction of VEGF expression after agonist stimulation and define novel hypoxia-independent mechanisms that should play a major role in vascular remodeling.

Angiogenesis: how a tumor adapts to hypoxia.

The growth of new blood vessels from the preexisting vascular tree, also known as angiogenesis, occurs in situations such as wound and fracture healing, arthritis, cardiovascular and cerebral ischemia, and nearly every type of cancer known. Vascular endothelial growth factor (VEGF) has been shown to play a crucial role in these events. Hypoxia-dependent VEGF induction is mediated by hypoxia-inducible factor-1 (HIF-1). HIF-1 is a heterodimeric transcription factor tightly regulated by oxygen concentration. In this short review, we summarize recent data concerning the control of HIF-1 activity and notably the regulation of HIF-1alpha subunit by phosphorylation and the ubiquitin proteasomal degradation system. A complete knowledge of this mechanism could, by the design of new antiangiogenic strategies, have a strong impact in clinical oncology.

Hypoxia-inducible factor-1 alpha (HIF-1 alpha) escapes O(2)-driven proteasomal degradation irrespective of its subcellular localization: nucleus or cytoplasm.

Eukaryotic cells sense oxygen and adapt to hypoxia by regulating a number of genes. Hypoxia-inducible factor 1 (HIF-1) is the 'master' in this pleiotypic response. HIF-1 comprises two members of the basic helix--loop--helix transcription factor family, HIF-1 alpha and HIF-1 beta. The HIF-1 alpha protein is subject to drastic O(2)-dependent proteasomal control. However, the signalling components regulating the 'switch' for 'escaping' proteasomal degradation under hypoxia are still largely unknown. The rapid nuclear translocation of HIF-1 alpha could represent an efficient way to escape from this degradation. We therefore asked, where in the cell is HIF-1 alpha degraded? To address this question, we trapped HIF-1 alpha either in the cytoplasm, by fusing HIF-1 alpha to the cytoplasmic domain of the Na(+)-H(+) exchanger (NHE-1), or in the nucleus, by treatment with leptomycin B. Surprisingly, we found that HIF-1 alpha is stabilized by hypoxia and undergoes O(2)-dependent proteasomal degradation with an identical half-life (5--8 min) in both cellular compartments. Therefore, HIF-1 alpha entry into the nucleus is not, as proposed, a key event that controls its stability. This result markedly contrasts with the mechanism that controls p53 degradation via MDM2.

The tyrosine within the NPXnY motif of the human angiotensin II type 1 receptor is involved in mediating signal transduction but is not essential for internalization.

The NPXnY motif is involved in the internalization process of several types of receptors, including lipoprotein receptors and G protein-coupled receptors. We replaced Tyr302 with either phenylalanine or alanine in the NPLFY site of the human angiotensin II receptor type 1 and determined the pharmacological properties of the resulting mutant receptors. Competitive binding experiments revealed that COS-7 cells transfected with either the wild-type or mutant receptors expressed approximately the same amount of high affinity binding sites (Bmax 70,000 sites/cell and Kd approximately 2 nM). Photoaffinity labeling of both native and mutant receptors revealed apparent molecular masses of 110 kDa. Incubation of transfected cells with 0.2 nM [125I]Ang II at 37 degrees revealed an efficient internalization of the wild-type receptor and the mutant receptors, although the mutant receptors were internalized at a slower rate. Interestingly, however, the transmembrane signaling was severely impaired in transfected cells expressing mutant receptors. No significant production of inositol-1,4,5-trisphosphate was observed when these cells were challenged for 3 min with a concentration of angiotensin II as high as 1 microM. This is in contrast to the dose-dependent stimulation of inositol-1,4,5-trisphosphate production in cells expressing the wild-type receptor. Thus, our results show that the Tyr302 in the NPXnY motif of the human angiotensin II receptor type 1 is not essential for agonist binding properties or for internalization of the receptor but plays an important role in transmembrane signaling.

The high affinity state of inositol 1,4,5-trisphosphate receptor is a functional state.

Inositol 1,4,5-trisphosphate (InsP3) is a second messenger responsible for the rapid and discontinuous release of Ca2+ from intracellular stores. In this study, the effects of the sulfhydryl reagent thimerosal were investigated on Ca2+ mobilization and on InsP3 binding. Thimerosal was shown to release Ca2+, in a dose-dependent manner, with an EC50 of 135.8 +/- 5.2 microM, from bovine adrenal cortex microsomes. Thimerosal-induced Ca2+ release was not prevented by heparin (250 micrograms/ml), ruling out a participation of InsP3 receptor in that effect. The slow rate of thimerosal-induced Ca2+ release rather suggested an inhibition of microsomal Ca2+ ATPase. At submaximal concentration, thimerosal (100 microM) was also shown to potentiate the release of Ca2+ induced by InsP3. Dose-response experiments revealed that thimerosal enhanced the apparent affinity of InsP3 by a factor 2.21 +/- 0.28, without modifying the maximal amount of Ca2+ released by InsP3. Thimerosal also enhanced, in a dose-dependent manner, [3H]InsP3 binding to adrenal cortex microsomes (EC50 = 43.3 +/- 7.6 microM). A similar effect was also observed on [3H]InsP3 binding to solubilized receptors, suggesting a direct modification of the receptor protein by thimerosal. The effects of thimerosal on Ca2+ release and [3H]InsP3 binding were abolished in the presence of the reducing agent dithiothreitol (1 mM), suggesting a modification by thimerosal of specific thiol groups on these microsomal proteins. Scatchard analysis revealed that thimerosal (100 microM) increased InsP3 receptor affinity by 1.87 +/- 0.26-fold. Kinetic analysis indicated that this increased affinity was due to an enhancement of InsP3 association rate constant. The concomitant increases of binding affinity and Ca2+ releasing potency suggest that the high affinity state of InsP3 receptor is a functional state.

Identification of alternative spliced variants of human hypoxia-inducible factor-1alpha.

Mammalian cells are able to sense oxygen and regulate a number of genes in response to hypoxia. The transcription factor Hypoxia Inducible Factor-1 (HIF-1) was identified as an important key component of the hypoxia signaling pathway. HIF-1 is a heterodimer composed of two members of the basic helix-loop-helix transcription factor superfamily containing a PAS (PER-ARNT-SIM) domain: HIF-1alpha and HIF-1beta/ARNT. During the cloning by reverse transcriptase-polymerase chain reaction of the human HIF-1alpha subunit, we isolated two cDNA clones which corresponded to alternative splicing of the HIF-1alpha gene. Polymerase chain reaction analysis and sequencing revealed that both clones possessed three additional base pairs between exons 1 and 2. Also, one of them lacked 127 base pairs corresponding to exon 14. We demonstrate that the mRNA of this truncated form is expressed in several human cells lines and human skin but apparently not in rodents. When transfected in HEK 293 cells, the corresponding 736 amino acid protein (HIF-1alpha(736)) is regulated by hypoxia in a similar manner as the full-length HIF-1alpha (HIF-1alpha(FL)). In luciferase transfection assays, both recombinant proteins HIF-1alpha(736) and HIF-1alpha(FL) dimerize with HIF-1beta/ARNT and activate the VEGF promoter upon hypoxia. However, the shorter HIF-1alpha isoform is 3-fold less active than HIF-1alpha(FL), a result consistent with the lack of the C-terminal transactivation domain. As expected, this small isoform can compete with the endogenous and transfected full-length HIF-1alpha. Altogether, these results suggest that the HIF-1alpha(736) isoform modulates gene expression upon hypoxia.

Effect of uncoupling agents on AT1 receptor affinity for antagonist analogs of angiotensin II.

AT1 receptor is responsible for most of the physiological effects of Angiotensin II (Ang II). AT1 receptor belongs to the G-protein-coupled receptor (GPCR) family, and it mediates its actions through the coupling of the Gq/11 protein with phospholipase C beta. Classical pharmacology has used the sensitivity of GPCR ligands to uncoupling agents as a criteria to discriminate agonists (which are sensitive) from antagonists (which are insensitive). In this study, the uncoupling agents GTP gamma S and pentosan sulfate (PS) (a low molecular weight polyanion) were used to further characterize the molecular interactions between Ang II analogs and the AT1 receptor. We show that some Ang II antagonists are sensitive to the conformational change of the AT1 receptor induced by uncoupling agents. These results demonstrate that there is no direct relationship between the intrinsic activity of a ligand and its affinity for different conformations of the AT1 receptor and that the sensitivity of GPCR ligands to uncoupling agents can not be used as a criteria to discriminate agonists from antagonists.

p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1alpha (HIF-1alpha) and enhance the transcriptional activity of HIF-1.

Hypoxia-inducible factor-1 (HIF-1) controls the expression of a number of genes such as vascular endothelial growth factor and erythropoietin in low oxygen conditions. However, the molecular mechanisms that underlie the activation of the limiting subunit, HIF-1alpha, are still poorly resolved. Results showing that endogenous HIF-1alpha migrated 12 kDa higher than in vitro translated protein led us to evaluate the possible role of phosphorylation on this phenomenon. We report here that HIF-1alpha is strongly phosphorylated in vivo and that phosphorylation is responsible for the marked differences in the migration pattern of HIF-1alpha. In vitro, HIF-1alpha is phosphorylated by p42 and p44 mitogen-activated protein kinases (MAPKs) and not by p38 MAPK or c-Jun N-terminal kinase. Interestingly, p42/p44 MAPK stoichiometrically phosphorylate HIF-1alpha in vitro, as judged by a complete upper shift of HIF-1alpha. More importantly, we demonstrate that activation of the p42/p44 MAPK pathway in quiescent cells induced the phosphorylation and shift of HIF-1alpha, which was abrogated in presence of the MEK inhibitor, PD 98059. Finally, we found that in a vascular endothelial growth factor promoter mutated at sites previously shown to be MAPK-sensitive (SP1/AP2-88-66 site), p42/p44 MAPK activation is sufficient to promote the transcriptional activity of HIF-1. This interaction between HIF-1alpha and p42/p44 MAPK suggests a cooperation between hypoxic and growth factor signals that ultimately leads to the increase in HIF-1-mediated gene expression.