Differential expression of adrenomedullin and its receptor component, receptor activity modifying protein (RAMP) 2 during hypoxia in cultured human neuroblastoma cells.

Adrenomedullin is a potent vasodilator peptide originally isolated from a pheochromocytoma. Recently, a novel adrenomedullin receptor has been identified as a complex consisting of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein (RAMP) 2. To explore possible pathophysiological roles of adrenomedullin and its receptor component RAMP2 in hypoxic tissues, we studied effects of hypoxia on expression of adrenomedullin and RAMP2 in two human neuroblastoma cell lines, IMR-32 and NB69, by radioimmunoassay and Northern blot analysis. Expression levels of adrenomedullin were increased by hypoxia in both cell lines. Treatment with cobalt chloride or desferrioxamine mesylate also increased expression levels of adrenomedullin mRNA. On the other hand, expression levels of RAMP2 mRNA were decreased in IMR-32 cells and were not changed in NB69 cells by hypoxia. Treatment with cobalt chloride or desferrioxamine mesylate decreased expression levels of RAMP2 mRNA in both IMR-32 and NB69 cells. These findings indicate that adrenomedullin expression is induced during hypoxia in IMR-32 and NB69 neuroblastoma cells, but RAMP2 expression is rather suppressed under the same conditions. The decreased expression of RAMP2 and the ADM expression induction under hypoxia may constitute one mechanism of cellular adaptation to hypoxic stress.     

Suppression of cytokine-induced expression of adrenomedullin and endothelin-1 by dexamethasone in T98G human glioblastoma cells

There is accumulating evidence showing that glial cells and gliomas secrete some neuropeptides and vasoactive peptides, such as adrenomedullin and endothelin-1. We have previously shown that expression of these two peptides is induced by inflammatory cytokines in T98G human glioblastoma cells. Glucocorticoids are frequently used for the treatment of inflammatory diseases and glioblastomas. We therefore studied effects of dexamethasone on expression of adrenomedullin and endothelin-1 in T98G human glioblastoma cells. Dexamethasone dose-dependently increased adrenomedullin mRNA levels and immunoreactive-adrenomedullin levels in the medium in T98G cells, whereas it decreased immunoreactive-endothelin levels in the medium. A combination of three cytokines, interferon-gamma (100 U/ml), tumor necrosis factor-alpha (20 ng/ml) and interleukin-1beta (10 ng/ml) induced expression of adrenomedullin and endothelin-1 in T98G cells. Dexamethasone (10(-8) mol/l) suppressed increases in expression of both adrenomedullin and endothelin-1 induced by these three cytokines. Thus, dexamethasone alone increased adrenomedullin expression whereas it suppressed the cytokine-induced expression of adrenomedullin in T98G cells. These findings raised the possibility that effects of dexamethasone on brain inflammation and glioblastomas may be partly mediated or modulated by its effects on expression of adrenomedullin and endothelin-1.     

Increased secretion of adrenomedullin from cultured human astrocytes by cytokines

Abstract: Adrenomedullin, originally discovered from pheochromocytoma, is a member of the calcitonin gene-related peptide family. The production and secretion of adrenomedullin by cultured human astrocytes were studied by northern blot analysis and radioimmunoassay. Northern blot analysis showed the expression of adrenomedullin mRNA in cultured human astrocytes. Immunoreactive adrenomedullin concentrations in the culture medium were 29.6 ± 1.2 fmol/10⁵ cells/24 h (mean ± SEM, n = 4). Treatment with interferon-γ (100 U/ml), tumor necrosis factor-α (1 and 10 ng/ml), or interleukin-1β (1 and 10 ng/ml) for 24 h caused >20-fold increases in immunoreactive adrenomedullin levels in the culture medium of human astrocytes. On the other hand, northern blot analysis showed only small increases (∼40%) in the adrenomedullin mRNA expression of human astrocytes with either 100 U/ml interferon-γ or 10 ng/ml interleukin-1β and no noticeable change with tumor necrosis factor-α. Reverse phase HPLC of the medium extracts of human astrocytes treated with interferon-γ, tumor necrosis factor-α, or interleukin-1β showed that most of immunoreactive adrenomedullin was eluted in the position of adrenomedullin-(1-52). On the other hand, immunoreactive adrenomedullin in the medium of human astrocytes without cytokine treatment was eluted earlier than the adrenomedullin standard, suggesting that this immunoreactive adrenomedullin represents adrenomedullin with some modifications or fragments of the adrenomedullin precursor. The present study has shown the production and secretion of adrenomedullin by human astrocytes and increased secretion of adrenomedullin by cytokines.     

Production and Secretion of Adrenomedullin by Cultured Choroid Plexus Carcinoma Cells

Abstract: Adrenomedullin is a potent vasodilator peptide that was originally isolated from pheochromocytoma. The production and secretion of adrenomedullin by cultured choroid plexus carcinoma cells were studied by radioimmunoassay and northern blot hybridization. Choroid plexus carcinoma is a rare malignant tumor derived from the epithelium of the choroid plexus. Immunoreactive adrenomedullin was detected in the conditioned medium of choroid plexus carcinoma cells (40.8 ± 7.5 fmol/10⁵ cells/24 h; mean ± SEM, n = 5). Reverse-phase HPLC of the conditioned medium showed one major peak of the immunoreactive peptide eluting in the position of synthetic human adrenomedullin and two smaller peaks eluting earlier. Addition of interleukin-1β (10 ng/ml) alone or in combination with three cytokines, interferon-γ (100 U/ml), tumor necrosis factor-α (20 ng/ml), and interleukin-1β (10 ng/ml), caused significant increases in the immunoreactive adrenomedullin concentrations in the medium (∼175 and 293% of the control level, respectively). Northern blot analysis showed the expression of 1.6-kb adrenomedullin mRNA in the total RNA sample prepared from cultured choroid plexus carcinoma cells. Treatment with either interleukin-1β or the combination of three cytokines caused significant increases in levels of adrenomedullin mRNA in parallel with those in immunoreactive adrenomedullin concentrations in the conditioned medium. These findings raise a possibility that adrenomedullin is secreted from the choroid plexus and has physiological roles in the CNS via the CSF. In addition, adrenomedullin secreted from choroid plexus carcinoma may be related to the pathophysiology of the tumor.     

Adrenomedullin in the eye

Adrenomedullin (AM) is a multifunctional regulatory peptide that is produced and secreted by various types of cells. We showed the presence of high concentrations of adrenomedullin-immunoreactivity in the vitreous fluid, and the levels were elevated in patients with proliferative vitreoretinopathy. Furthermore, adrenomedullin mRNA expression levels were elevated in the tissues of intraocular tumors and orbital tumors. Adrenomedullin is produced and secreted by cultured human retinal pigment epithelial (RPE) cells. Inflammatory cytokines and hypoxia are strong stimulators for the adrenomedullin expression in retinal pigment epithelial cells. Adrenomedullin stimulated the proliferation of retinal pigment epithelial cells both under normoxia and hypoxia. Dexamethasone (DEX) increased the adrenomedullin expression in two cultured cell lines of human retinal pigment epithelial cells; ARPE-19 cells and D407 cells, while it had no noticeable effects on the cytokine-induced adrenomedullin expression. These findings suggest that adrenomedullin is involved in the pathophysiology of inflammatory and neoplastic eye diseases as an autocrine or paracrine growth stimulator. The findings on glucocorticoid-induced AM expression raise the possibility that it may be related to the pathogenesis of some eye diseases, such as central serous chorioretinopathy and multifocal posterior pigment epitheliopathy, which are frequently seen in patients treated with high doses of glucocorticoids.     

Adrenomedullin protects neurons against oxygen glucose deprivation stress in an autocrine and paracrine manner

The understanding of mechanisms involved in ischaemic brain tolerance may provide new therapeutical targets for stroke. In vivo genomic studies revealed an up-regulation of adrenomedullin expression by hypoxic pre-conditioning. Furthermore, adrenomedullin reduced ischaemia-induced brain damage in rodents. However, whether adrenomedullin is involved in hypoxic pre-conditioning-induced tolerance and whether adrenomedullin protects directly neurons against ischaemia remain unknown. Using a neuronal model of hypoxic pre-conditioning and oxygen glucose deprivation (OGD), we showed that 0.1% or 0.5% of O₂ pre-conditioning reduced the OGD-induced neuronal death, whereas 1% or 2% of O₂ pre-treatment did not induce neuroprotection. Adrenomedullin expression increased following the hypoxic period, and following OGD only in pre-conditioned (0.1% or 0.5% of O₂) neurons. Adrenomedullin pre-treatment and post-treatment reduced the OGD-induced neuronal death, partly through PI3kinase-dependent pathway. However, adrenomedullin antagonism during hypoxic pre-conditioning failed to inhibit the neuroprotection whereas adrenomedullin antagonism following OGD abolished the hypoxic pre-conditioning-induced neuroprotection. Finally, we showed that adrenomedullin is involved in neuroprotection induced by endothelial cells and microglia. In contrast, neuroprotection induced by astrocytes occurred through adrenomedullin-independent mechanisms. Altogether, our results suggest that adrenomedullin is an effector of the hypoxic pre-conditioning-induced neuronal tolerance and a potent autocrine and paracrine neuroprotective factor during cerebral ischaemia.     

Hypoxia induces expression of a GPI-anchorless splice variant of the prion protein

The human prion protein (PrP) is a glycoprotein with a glycosylphosphatidylinositol (GPI) anchor at its C-terminus. Here we report alternative splicing within exon 2 of the PrP gene (PRNP) in the human glioblastoma cell line T98G. The open reading frame of the alternatively spliced mRNA lacked the GPI anchor signal sequence and encoded a 230 amino acid polypeptide. Its product, GPI-anchorless PrP (GPI(-) PrPSV), was unglycosylated and soluble in non-ionic detergent, and was found in the cytosolic fraction. We also detected low levels of alternatively spliced mRNA in human brain and non-neuronal tissues. When long-term passaged T98G cells were placed in a low-oxygen environment, alternatively spliced mRNA expression increased and expression of normally spliced PrP mRNA decreased. These findings imply that oxygen tension regulates GPI(-) PrPSV expression in T98G cells.     

Effects of adipokines on expression of adrenomedullin and endothelin-1 in cultured vascular endothelial cells

Obesity is a major risk factor for the development of hypertension. Adipokines may cause hypertension by acting both centrally and directly on the vascular vessels. We wished to clarify whether three adipokines, leptin, resistin and tumor necrosis factor-alpha, affect expression of adrenomedullin and endothelin-1 in vascular endothelial cells. Human umbilical vein endothelial cells were cultured for 24 h with leptin (1-10 nmol/l), resistin (1-10 nmol/l) or tumor necrosis factor-alpha (1-10 ng/ml). Expression of adrenomedullin and endothelin-1 was examined by radioimmunoassay and northern blot analysis. Immunoreactive-adrenomedullin in the medium and adrenomedullin mRNA expression levels were decreased by treatment of tumor necrosis factor-alpha time- and dose-dependently, whereas endothelin-1 secretion was not significantly changed by it. Leptin or resistin had no significant effects on expression of adrenomedullin or endothelin-1 in human umbilical vein endothelial cells. Under hypoxic conditions (1% O2), expression of both adrenomedullin and endothelin-1 was induced in these cells. Immunoreactive-adrenomedullin levels in the medium were decreased by treatment of tumor necrosis factor-alpha under hypoxia. Leptin or resistin had no significant effects on adrenomedullin or endothelin-1 expression also in hypoxia. These findings have raised the possibility that decreased expression of adrenomedullin by tumor necrosis factor-alpha may be related to the increased risk of hypertension and other cardiovascular diseases in obese subjects.     

Induction of adrenomedullin by hypoxia in cultured human coronary artery endothelial cells.

To explore the role of adrenomedullin (ADM) in pathophysiology of ischemic heart disease, we investigated the effects of hypoxia on the production and secretion of ADM in cultured human coronary artery endothelial cells. Treatment with hypoxia (5% CO2/94% N2/1% O2) for 6 and 12 h increased expression levels of ADM mRNA 2.2-fold and fivefold compared with the normoxia control, respectively. The levels of immunoreactive ADM in the media were increased by 12-h hypoxia about fivefold compared with the control (39.0+/-1.1 fmol/10(5) cells per 12 h under hypoxia and 7.9+/-0.4 fmol/10(5) cells per 12 h under normoxia; P<0.01, n = 4, mean +/- SEM). Reverse-phase high-performance liquid chromatography of the extracts of culture media under normoxia and hypoxia showed one major peak eluting in the position of human ADM standard. The production and secretion of ADM were increased in cultured human coronary artery endothelial cells under hypoxia. ADM may therefore play an important pathophysiological role in ischemic heart disease.     

Expression of Heme Oxygenase Isozyme mRNAs in the Human Brain and Induction of Heme Oxygenase-1 by Nitric Oxide Donors

Abstract: Heme oxygenase is an essential enzyme in the heme catabolism that produces carbon monoxide (CO). This study was designed to examine the expression of two heme oxygenase isozyme mRNAs in the human brain and to explore the involvement of nitric oxide (NO) and various neuropeptides in the regulation of their expression. Northern blot analysis showed the expression of heme oxygenase-1 and heme oxygenase-2 mRNAs in every region of the brain examined, with the highest levels found in the frontal cortex, temporal cortex, occipital cortex, and hypothalamus. In a human glioblastoma cell line, T98G, treatment with any of three types of NO donors—sodium nitroprusside, 3-morpholinosydnonimine, and S -nitroso- l -glutathione—caused a significant increase in the levels of heme oxygenase-1 mRNA but not in the levels of heme oxygenase-2 and heat-shock protein 70 mRNAs. Sodium nitroprusside increased the levels of heme oxygenase-1 protein but not the levels of heat-shock protein 70 in T98G cells. The increase in content of heme oxygenase-1 mRNA caused by sodium nitroprusside was completely abolished by the treatment with actinomycin D. On the other hand, the levels of heme oxygenase isozyme mRNAs were not noticeably changed in T98G cells following the treatment with 8-bromo cyclic GMP, sodium nitrite, or various neuropeptides, such as calcitonin gene-related peptide, endothelin-1, and corticotropin-releasing hormone. The present study has shown the expression profiles of heme oxygenase-1 and -2 mRNAs in the human brain and the induction of heme oxygenase-1 mRNA caused by NO donors in T98G cells. These findings raise a possibility that the CO/heme oxygenase system may function in concert with the NO/NO synthase system in the brain.     

Hypoxia‐induced apoptosis of astrocytes is mediated by reduction of Dicer and activation of caspase‐1

Hypoxia is a condition in which the whole body or a region of the body is deprived of oxygen supply. The brain is very sensitive to the lack of oxygen and cerebral hypoxia can rapidly cause severe brain damage. Astrocytes are essential for the survival and function of neurons. Therefore, protecting astrocytes against cell death is one of the main therapeutic strategies for treating hypoxia. Hence, the mechanism of hypoxia‐induced astrocytic cell death should be fully elucidated. In this study, astrocytes were exposed to hypoxic conditions using a hypoxia work station or the hypoxia mimetic agent cobalt chloride (CoCl₂). Both the hypoxic gas mixture (1% O₂) and chemical hypoxia‐induced apoptotic cell death in T98G glioblastoma cells and mouse primary astrocytes. Reactive oxygen species were generated in response to the hypoxia‐mediated activation of caspase‐1. Active caspase‐1 induced the classical caspase‐dependent apoptosis of astrocytes. In addition, the microRNA processing enzyme Dicer was cleaved by caspase‐3 during hypoxia. Knockdown of Dicer using antisense oligonucleotides induced apoptosis of T98G cells. Taken together, these results suggest that astrocytic cell death during hypoxia is mediated by the reactive oxygen species/caspase‐1/classical caspase‐dependent apoptotic pathway. In addition, the decrease in Dicer levels by active caspase‐3 amplifies this apoptotic pathway via a positive feedback loop. These findings may provide a new target for therapeutic interventions in cerebral hypoxia.     

Growth regulation of human glioblastoma t98g cells by insulin-like growth factor-1 and its receptor

The interaction of insulin-like growth factors (IGFs) with the IGF-1 receptor is an important step in the control of cell proliferation and development. In particular, IGF-1 and IGF-2 are key regulators of central nervous system development, and may modulate the growth of glial tumors. We have investigated the growth factor regulation of the human glioblastoma cell line T98G. These cells growth arrested in serum-free medium at 34°C, despite their secretion of substantial amounts of bioactive IGF-1. To be stimulated to divide, growth-arrested cells required the addition of platelet-derived growth factor (PDGF) or its equivalent, 1% serum. Cell proliferation in serum-free medium could also be obtained by shifting the cells to a temperature of 39.6°C. Treatment of growth-arrested cells with PDGF or temperature shift was accompanied by a transient increase in the expression of the mRNA for the IGF-1 receptor. Transfection with a plasmid constitutively expressing the full cDNA for the human IGF-1 receptor allowed autonomous growth in serum-free medium at 34°C. By contrast, growth induction by growth factors or temperature shift was abrogated by transfection of the cells with a plasmid expressing a 300 bp segment of mRNA antisense to the IGF-1 receptor mRNA. Cloning in soft agar was also inhibited by expression of antisense IGF-1 receptor mRNA. These results demonstrate that the IGF-1 receptor is strictly required for the growth of T98G glioblastoma cells. Moreover, the autocrine interaction of IGF-1 with its receptor regulates both autonomous and anchorage-independent growth of these cells. © 1994 wiley-Liss, Inc.     

Adrenomedullin promotes cell cycle transit and up-regulates cyclin D1 protein level in human glioblastoma cells through the activation of c-Jun/JNK/AP-1 signal transduction pathway

Adrenomedullin is a secreted peptide hormone with multiple functions. Although a number of reports have indicated that adrenomedullin may be involved in tumor progression, its mechanism of action remains obscure. In this study, we have analysed the signal transduction pathway activated by adrenomedullin in human glioma cells. Our results revealed that adrenomedullin induced the phosphorylation of both c-Jun and JNK in glioblastoma cells. Silencing JNK expression with siRNA reversed the phosphorylation of c-Jun induced by adrenomedullin, indicating that JNK is responsible of c-Jun activation. In addition, electrophoretic mobility-shift assays showed that the increase in phosphorylation of c-Jun was associated with increased AP-1 DNA binding activity. Supershift assays and co-immunoprecipitation demonstrated that c-Jun and JunD are part of the AP-1 complex, indicating that activated c-Jun is dimerized with JunD in response to adrenomedullin. Furthermore, adrenomedullin was shown to promote cell transit beyond cell cycle phases with a concomittant increase in cyclin D1 protein level, suggesting that adrenomedullin effect cell proliferation through up-regulation of cyclin D1. The inhibition of JNK activation or the suppression of c-Jun or JunD expression with siRNA impaired the effects of adrenomedullin on cell proliferation and on cyclin D1. Taken together, these data demonstrate that activation of cJun/JNK pathway is involved in the growth regulatory activity of adrenomedullin in glioblastoma cells.