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Takashi Matsui

Publications and source records attributed to Takashi Matsui.

At least 19 recordsLinked to original sources
PubMed2026

2-Mercaptoethanol/DMSO Workflow Enables Highly Reproducible Quantitative Proteomics.

Proteomics provides a systematic and high-throughput approach to comprehensively characterize protein networks, enabling insights into cellular functions and disease mechanisms. Carbamidomethylation using iodoacetamide (IAA), a common method for cysteine alkylation, is known to cause nonspecific modifications that increase spectral complexity in mass spectrometry and reduce quantitative accuracy. Here, we established a reproducibility-focused 2-mercaptoethanol (2-ME)/dimethyl sulfoxide (DMSO) workflow and systematically evaluated its quantitative performance at the proteome-wide level. Mouse liver proteomes were processed using either 2-ME/DMSO or conventional IAA treatment, followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The optimized 2-ME treatment increased the number of cysteine-modified peptides by 1.6- to 1.9-fold. Although total protein identifications were comparable, 77% of proteins exhibited improved sequence coverage with the optimized 2-ME treatment. Quantitative reproducibility was also enhanced, with the peptide quantified CV ≤ 20% increasing from 61.4% with IAA treatment to 86.1% with 2-ME treatment, and protein quantified CV ≤ 20% increasing from 80.6% with IAA treatment to 93.5% with 2-ME treatment. Application of this new workflow to ovarian clear cell carcinoma reliably detected cisplatin-induced alterations. The 2-ME/DMSO workflow offers a simple and highly reproducible proteomics strategy for accurate quantitative proteomics.

Animals↗
PubMed2007

RNA recognition mechanism of the minimal active domain of the human immunodeficiency virus type-2 nucleocapsid protein.

NCp8 of HIV-2 contains two CCHC-type zinc fingers connected by a linker, and is involved in many critical steps of the virus life cycle. It was previously shown that the first zinc finger flanked by the linker is the minimal active domain for specific binding to viral RNA. In our previous study, we determined the three-dimensional structure of NCp8-f1, including the minimal active domain, and found that a hydrogen bond between Asn(11) N(delta)H and Arg(27) O stabilized the conformation of the linker in the vicinity of the zinc finger [Kodera et al. (1998) Biochemistry 37, 17704-17713]. In this study, RNA binding activities of NCp8-f1 and three types of its mutant peptides were analysed by native PAGE assay. The activity and three-dimensional structure of NCp8-f1/N11A, in which alanine is substituted for Asn(11) thereby affecting the conformation of the linker, was analyzed and compared with those of NCp8-f1. We demonstrated that the existence of Arg(4) and/or Lys(5) and Arg(26) and/or Arg(27) were necessary for binding RNA. Furthermore, the linker's flexible orientation, which is controlled by the hydrogen bond between Asn(11) N(delta)H and Arg(27) O, appears to be a structural basis for NCp8 existing as a multi-functional protein.

Capsid Proteins↗
PubMed2007

Two von Recklinghausen's disease cases with pheochromocytomas and gastrointestinal stromal tumors (GIST) in combination.

We here document two cases of von Recklinghausen's disease with both pheochromocytomas and gastrointestinal stromal tumors (GIST). It is very rare that these three disorders are found to occur simultaneously, although the fact that preoperative detection of GIST is difficult except with large tumors may be important in this respect. In the present cases, GIST were not evident on preoperative computed tomography and magnetic resonance imaging, and were identified unexpectedly during surgery. Our findings indicate that investigation of the intraperitoneal space should be performed during adrenalectomy for pheochromocytomas with von Recklinghausen's disease.

Adrenal Gland Neoplasms↗
PubMed2006

Myostatin regulates cardiomyocyte growth through modulation of Akt signaling.

Myostatin is a highly conserved, potent negative regulator of skeletal muscle hypertrophy in many species, from rodents to humans, although its mechanisms of action are incompletely understood. Transcript profiling of hearts from a genetic model of cardiac hypertrophy revealed dramatic upregulation of myostatin, not previously recognized to play a role in the heart. Here we show that myostatin abrogates the cardiomyocyte growth response to phenylephrine in vitro through inhibition of p38 and the serine-threonine kinase Akt, a critical determinant of cell size in many species from drosophila to mammals. Evaluation of male myostatin-null mice revealed that their cardiomyocytes and hearts overall were slightly smaller at baseline than littermate controls but exhibited more exuberant growth in response to chronic phenylephrine infusion. The increased cardiac growth in myostatin-null mice corresponded with increased p38 phosphorylation and Akt activation in vivo after phenylephrine treatment. Together, these data demonstrate that myostatin is dynamically regulated in the heart and acts more broadly than previously appreciated to regulate growth of multiple types of striated muscle.

Adrenergic alpha-Agonists↗
PubMed2006

[Survey of factors underlying treatment choice for patients with localized prostate cancer (radical prostatectomy vs extrabeam radiotherapy)].

PURPOSE: Little is known regarding factors for decision-making on treatment by localized prostate cancer patients. We therefore conducted a survey series of cases for influence on treatment decision making, and also satisfaction after therapy. MATERIALS AND METHODS: A total of 51 patients with localized prostate cancer treated with radical prostatectomy (RP) or external beam radiation therapy (EBRT) were mailed original questionnaires about their treatment decision-making factors and satisfaction and the results compared between the two groups. RESULT: Some 48 (94.1%) patients responded to the questionnaire, 38 (79.2%) and 10 (20.8%) after RP and EBRT, respectively. The major factor determining the decision as to treatment approach was the physician in both groups (more than 90%). Excluding physicians, family or others were more important in the RP group than the EBRT group (p = 0.023). RP group patients desired removal of their prostate for cancer control, while, EBRT group patients favored the less invasive approach in consideration of side effects. Over 80% patients indicated they would definitely or probably choose the same treatment again, although some of the RP group would switch to watchful-waiting because of sexual dysfunction, urinary incontinence and the invasive nature of the procedure. CONCLUSION: Physicians are in a most important position to help patients understand prostate cancer and treatment, outcomes, and need to help them make their best choice, with appropriate follow up including mental care.

Aged↗
PubMed2006

Evidence that myocardial Na/I symporter gene imaging does not perturb cardiac function.

UNLABELLED: Adenoviral Na/I symporter (NIS) gene transfer has emerged as a promising method for myocardial gene imaging but concern over possible perturbation of cardiac function persists. In this study, we addressed this issue with cultured cardiac cells and serial echocardiography, creatine kinase (CK) measurements, and histologic examination of rats intramyocardially injected with an adenovirus that expresses both NIS and enhanced green fluorescent protein (EGFP) (Ad.EGFP.NIS) or a control virus (Ad.EGFP). METHODS: H9C2 cardiac myoblasts differentiated into cardiomyocytes were evaluated for the effect of Ad.EGFP.NIS and Ad.EGFP infection on viable cell number and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Rats injected intramyocardially with 3 x 10(8) plaque-forming units of Ad.EGFP.NIS (n = 9) or Ad.EGFP (n = 8) underwent serial echocardiographic measurements of heart rate, left ventricular (LV) dimensions, ejection fraction (EF), and fractional shortening (FS) on the day before and on days 4 and 9 after gene transfer. Five Ad.EGFP.NIS rats also underwent repeated (123)I imaging from which ratios of cardiac to mediastinal counts (C/M ratios) were obtained. Separate rats underwent serial measurements of serum CK, myocardial myeloperoxidase assays, and microscopic assessment of inflammation. RESULTS: Cultured cardiac cells showed no change in cell viability or proliferation at 4 and 9 d after Ad.EGFP.NIS or Ad.EGFP infection compared with controls. (123)I scintigraphy demonstrated high cardiac radiouptake at Ad.EGFP.NIS injection sites by days 2 and 4 (C/M ratios, 5.0 +/- 0.6 and 5.1 +/- 1.0, respectively), followed by a complete loss of uptake by day 9 (C/M ratio, 1.4 +/- 0.0). Serial echocardiography revealed no difference in heart rate, LV dimensions, or functional parameters between Ad.EGFP.NIS and Ad.EGFP groups at any given time. Mild reductions in LVEF and LVFS by day 9 compared with baseline were similar for both Ad.EGFP (88.2% +/- 6.4% vs. 79.6% +/- 5.0% for LVEF and 0.55 +/- 0.10 vs. 0.44 +/- 0.05 for LVFS) and Ad.EGFP.NIS groups (88.0% +/- 5.4% vs. 78.7% +/- 4.6% for LVEF and 0.54 +/- 0.09 vs. 0.42 +/- 0.05 for LVFS). Serial serum CK and myocardial myeloperoxidase activities were not elevated in either group, in contrast to substantial increases found after ischemia-reperfusion injury. Histology revealed similar mild inflammatory cell infiltration restricted to the injection site for both groups. CONCLUSION: The results of this study demonstrate that myocardial NIS gene imaging does not cause significant myocardial injury or perturbed cardiac function, other than mild effects likely due to adenoviral vector-associated host response. Thus, this practical and convenient reporter gene strategy can be used safely for noninvasive myocardial gene imaging in living subjects.

Adenoviridae↗
PubMed2005

Effects of chronic Akt activation on glucose uptake in the heart.

Acute activation of the serine-threonine kinase Akt is cardioprotective and increases glucose uptake, at least in part, through enhanced expression of GLUT4 on the sarcolemma. The effects of chronic Akt activation on glucose uptake in the heart remain unclear. To address this issue, we examined the effects of chronic Akt activation on glucose uptake, glycogen storage, and relevant glucose transporters in the hearts of transgenic mice. We found that chronic cardiac activation of Akt led to a substantial increase in the rate of basal glucose uptake (P < 0.05) but blunted the response to insulin (1.9 vs. 18.1-fold increase compared with baseline) using NMR in ex vivo perfused heart. Basal glucose uptake was also increased in Akt transgenic mice in vivo (P < 0.005). These changes were associated with an increase on glycogen deposition, examined with histochemical staining, biochemical (>6-fold, P < 0.001) and in vivo radioactive (5-fold, P < 0.01) assays. Studies in chimeric hearts of female X-linked transgenic Akt mice suggested that increased glycogen deposition occurred as a cell autonomous effect of transgene expression. Interestingly, although sarcolemmal GLUT1 was not significantly altered, chronic Akt activation actually decreased plasma membrane GLUT4. Moreover, intracellular pools of GLUT1 were modestly reduced, whereas intracellular GLUT4 was substantially reduced. It seems likely that neither GLUT1 nor GLUT4 explains the increase in basal glucose uptake but that these reductions contribute to the loss of insulin responsiveness that we observed. These data demonstrate that chronic Akt activation increases basal glucose uptake and glycogen deposition while inhibiting the response to insulin.

Animals↗
PubMed2005

PI3K rescues the detrimental effects of chronic Akt activation in the heart during ischemia/reperfusion injury.

Acute activation of the serine-threonine kinase Akt is cardioprotective and reduces both infarction and dysfunction after ischemia/reperfusion injury (IRI). However, less is known about the chronic effects of Akt activation in the heart, and, paradoxically, Akt is activated in samples from patients with chronic heart failure. We generated Tg mice with cardiac-specific expression of either activated (myristoylated [myr]) or dominant-negative (dn) Akt and assessed their response to IRI in an ex vivo model. While dn-Akt hearts demonstrated a moderate reduction in functional recovery after IRI, no function was restored in any of the myr-Akt-Tg hearts. Moreover, infarcts were dramatically larger in myr-Akt-Tg hearts. Biochemical analyses demonstrated that chronic Akt activation induces feedback inhibition of PI3K activity through both proteasome-dependent degradation of insulin receptor substrate-1 (IRS-1) and inhibition of transcription of IRS-1 as well as that of IRS-2. To test the functional significance of these signaling changes, we performed in vivo cardiac gene transfer with constitutively active PI3K in myr-Akt-Tg mice. Restoration of PI3K rescued function and reduced injury after IRI. These data demonstrate that PI3K-dependent but Akt-independent effectors are required for full cardioprotection and suggest a mechanism by which chronic Akt activation can become maladaptive.

Animals↗
PubMed2005

Serum and glucocorticoid-responsive kinase-1 regulates cardiomyocyte survival and hypertrophic response.

BACKGROUND: Serum- and glucocorticoid-responsive kinase-1 (SGK1), a serine-threonine kinase that is highly expressed in the heart, has been previously reported to regulate sodium channels. Because SGK1 is a PI 3-kinase-dependent kinase with structural homology to Akt, we examined its regulation in the heart and its effects on cardiomyocyte (CM) apoptosis and hypertrophy in vitro. METHODS AND RESULTS: Rats were subjected to aortic banding, and expression of total and phosphorylated SGK1 was examined. Both phospho- and total SGK1 increased 2 to 7 days after banding. Phospho-SGK1 was also upregulated in CMs stimulated in vitro with IGF-I or phenylephrine. Infection of CMs with an adenoviral vector encoding constitutively active SGK1 (Ad.SGK1.CA) inhibited apoptosis after serum-deprivation or hypoxia (P<0.05), whereas expression of kinase-dead SGK1 (Ad.SGK1.KD) increased it and partially mitigated the protective effects of IGF-I (P<0.05). SGK1 activation was also sufficient to increase cell size, protein synthesis, sarcomere organization, and ANF expression both at baseline and in response to phenylephrine but was not necessary for the hypertrophic response to phenylephrine. Evaluation of potential downstream signaling pathways demonstrated that SGK1 induces phosphorylation of tuberin, p70s6kinase, and GSK3beta in CMs, which may contribute to its effects. CONCLUSIONS: SGK1 is dynamically regulated during acute biomechanical stress in the heart and inhibits CM apoptosis while enhancing the hypertrophic response.

Adaptation, Physiological↗
PubMed2005

Magnetic resonance imaging of cardiomyocyte apoptosis with a novel magneto-optical nanoparticle.

The ability to image cardiomyocyte apoptosis in vivo with high-resolution MRI could facilitate the development of novel cardioprotective therapies. The sensitivity of the novel nanoparticle AnxCLIO-Cy5.5 for cardiomyocyte apoptosis was thus compared in vitro to that of annexin V-FITC and showed a high degree of colocalization. MRI was then performed, following transient coronary artery (LAD) occlusion, in five mice given AnxCLIO-Cy5.5 and in four mice given an identical dose (2 mg Fe/kg) of CLIO-Cy5.5. MR signal intensity and myocardial T2* were evaluated, in vivo, in hypokinetic regions of myocardium in the LAD distribution. Ex vivo fluorescence imaging was performed to confirm the in vivo findings. Myocardial T2* was significantly lower in the mice given AnxCLIO-Cy5.5 (8.1 versus 13.2 ms, P<0.01), and fluorescence target to background ratio was significantly higher (2.1 versus 1.1, P<0.01). This study thus demonstrates the feasibility of obtaining high-resolution MR images of cardiomyocyte apoptosis in vivo with the novel nanoparticle, AnxCLIO-Cy5.5.

Analysis of Variance↗
PubMed2005

Transcriptional coactivator PGC-1 alpha controls the energy state and contractile function of cardiac muscle.

Skeletal and cardiac muscle depend on high turnover of ATP made by mitochondria in order to contract efficiently. The transcriptional coactivator PGC-1alpha has been shown to function as a major regulator of mitochondrial biogenesis and respiration in both skeletal and cardiac muscle, but this has been based only on gain-of-function studies. Using genetic knockout mice, we show here that, while PGC-1alpha KO mice appear to retain normal mitochondrial volume in both muscle beds, expression of genes of oxidative phosphorylation is markedly blunted. Hearts from these mice have reduced mitochondrial enzymatic activities and decreased levels of ATP. Importantly, isolated hearts lacking PGC-1alpha have a diminished ability to increase work output in response to chemical or electrical stimulation. As mice lacking PGC-1alpha age, cardiac dysfunction becomes evident in vivo. These data indicate that PGC-1alpha is vital for the heart to meet increased demands for ATP and work in response to physiological stimuli.

Animals↗
PubMed2005

Effect of pet removal on pet allergic asthma.

BACKGROUND: Allergen avoidance has been recommended in the management of allergic asthma. Very few studies have assessed the effect of pet removal on pet allergic asthma. OBJECTIVE: We examined the effect of pet removal from homes on pulmonary function testing, airway hyperresponsiveness, and medication use. DESIGN: Prospective, nonrandomized, nonblinded observational study. PATIENTS AND METHODS: Subjects included 20 symptomatic patients with newly diagnosed pet allergic asthma who were keeping domestic animals, including hamsters, cats, dogs, and ferrets, and were sensitized to the animals. They were treated with inhaled corticosteroids or other medications according to recommendations by level of severity of the Global Initiative for Asthma. Methacholine inhalation tests were performed regularly before and after starting medication. Clinical features were compared between the patients who gave away their pets according to recommendations by the clinician (removal group) and the patients who refused to give away their pets (keeping group). RESULTS: There were 10 patients in both the removal group and the keeping group. After >or= 1 year of follow-up with or without pet removal, a 5.9-fold increase in the provocative concentration of methacholine causing a 20% fall in FEV(1) was observed in the removal group compared with a 2.3-fold increase in the keeping group (p = 0.04). There were no significant differences in the changes in FEV(1) and peak flow variability. Finally, no patient received inhaled corticosteroids in the removal group, whereas all but one of the patients needed beclomethasone dipropionate (mean dose, 600 mug/d) in the keeping group. CONCLUSION: This study indicates that removal of pets from homes reduces airway responsiveness in patients with pet allergic asthma more than optimal pharmacotherapy alone, thereby enabling a decrease in inhaled corticosteroid doses.

Adult↗
PubMed2005

Accuracy of myocardial sodium/iodide symporter gene expression imaging with radioiodide: evaluation with a dual-gene adenovirus vector.

UNLABELLED: The sodium/iodide symporter (NIS) gene allows convenient reporter imaging with gamma-cameras and free radioiodide. In this study, we investigated the accuracy of this technique for assessing myocardial gene expression in living rats by using a dual-gene adenovirus that expresses both NIS protein and enhanced green fluorescent protein (EGFP). METHODS: Rats underwent myocardial injection with the dual-gene adenovirus (Ad.EGFP.NIS) or a control virus (Ad.EGFP). 123I scintigraphy was performed at day 4, and ratios of cardiac counts to mediastinal count were measured. The site of radiouptake was localized by use of dual-isotope 201Tl images, and radioiodide efflux rates were evaluated by dynamic imaging. The dependence of cardiac radiouptake on viral titers and the sensitivity of detection of cardiac radiouptake were investigated at various viral titers. The radioactivity, EGFP, and NIS protein expression sites were compared microscopically. The correlation of image-based uptake with fluorometric measurements of EGFP concentrations was assessed by regression analysis. RESULTS: 123I scintigraphy demonstrated clear focal myocardial uptake at the Ad.EGFP.NIS injection site, with relatively slow washout rates (3.3% +/- 0.8%/h), despite the absence of organification. Image-based cardiac uptake increased in a viral titer-dependent manner, with a detection threshold of between 3 x 10(6) and 1 x 10(7) plaque-forming units. Western blotting showed titer-dependent increases in myocardial NIS protein expression. Tissue radioactivity levels approached 12.5-fold control levels and correlated closely with image-based uptake (r = 0.91; P < 0.0001). Histologic analysis confirmed the colocalization of radiouptake, EGFP fluorescence, and NIS staining. Image-based uptake showed a strong correlation with fluorometric measurements of myocardial EGFP concentrations (r = 0.81; P < 0.0001). CONCLUSION: Our results demonstrate that convenient myocardial gene imaging with gamma-cameras and radioiodide is feasible with the NIS gene. Moreover, the use of this technique with a dual-gene vector appears to allow accurate assessment of the level of myocardial expression of a second gene of interest.

Adenoviridae↗
PubMed2004

Convergent signal transduction pathways controlling cardiomyocyte survival and function: the role of PI 3-kinase and Akt.

Effective cardioprotection in clinical settings requires not only cardiomyocyte survival but also preservation of function. Multiple growth factors protect the heart from ischemic and other injury. While the downstream signaling pathways of these cardioprotective factors are complex, activation of phosphoinositide 3-kinase (PI 3-kinase) and its downstream effector, the serine-threonine kinase Akt (or Protein Kinase B), is a common feature in many cases. Genetic manipulations in cardiomyocytes both in vitro and in vivo suggest that acute activation of this pathway can promote both cardiomyocyte survival and function. Here, we review PI 3-kinase and Akt signaling, with a focus on their role in cardiomyocyte growth, survival, and function. Finally, the clinical implications of these studies will be considered.

Animals↗
PubMed2004

Inhibition of ErbB2 causes mitochondrial dysfunction in cardiomyocytes: implications for herceptin-induced cardiomyopathy.

OBJECTIVES: We investigated the effects of erbB2 inhibition by anti-erbB2 antibody on cardiomyocyte survival and mitochondrial function. BACKGROUND: ErbB2 is an important signal integrator for the epidermal growth factor family of receptor tyrosine kinases. Herceptin, an inhibitory antibody to the erbB2 receptor, is a potent chemotherapeutic but causes cardiac toxicity. METHODS: Primary cultures of neonatal rat ventricular myocytes were exposed to anti-erbB2 antibody (Ab) (7.5 mug/ml) for up to 24 h. Cell viability, mitochondrial function, and apoptosis were measured using multiple complementary techniques. RESULTS: ErbB2 inhibition was associated with a dramatic increase in expression of the pro-apoptotic Bcl-2 family protein Bcl-xS and decreased levels of anti-apoptotic Bcl-xL. There was a time-dependent increase in mitochondrial translocation and oligomerization of bcl-associated protein (BAX), as indicated by 1,6-bismaleimidohexane crosslinking. The BAX oligomerization was associated with cytochrome c release and caspase activation. These alterations induced mitochondrial dysfunction, a loss of mitochondrial membrane potential (psi) (76.9 +/- 2.4 vs. 51.7 +/- 0.1; p < 0.05; n = 4), a 35% decline in adenosine triphosphate levels (p < 0.05), and a loss of redox capacity (0.72 +/- 0.04 vs. 0.64 +/- 0.02; p< 0.01). Restoration of Bcl-xL levels through transactivating regulatory protein-mediated protein transduction prevented the decline in psi mitochondrial reductase activity and cytosolic adenosine triphosphate. CONCLUSIONS: Anti-erbB2 activates the mitochondrial apoptosis pathway through a previously undescribed modulation of Bcl-xL and -xS, causing impairment of mitochondrial function and integrity and disruption of cellular energetics.

Animals↗
PubMed2004

Rydberg-valence interactions of CO, and spectroscopic evidence characterizing the C' 1Sigma+ valence state.

Rotationally cold absorption and two-photon ionization spectra of CO in the 90-100 nm region have been recorded at a resolution of 0.3-1.0 cm(-1). The analyses of up to four isotopomers seek to clarify the observations in regions where the Rydberg levels built on the ground state X (2)Sigma(+) of the ion interact with valence states of (1)Sigma(+) and (1)Pi symmetry. Previous observations of the 3ssigma, B (1)Sigma(+) Rydberg state, reviewed by Tchang-Brillet et al. [J. Chem. Phys. 96, 6735 (1992)], have been extended to energies above its avoided crossing with the repulsive part of the D(') (1)Sigma(+) valence state where resonances of varying intensities and widths have been attributed to the fully coupled 3ssigma or 4ssigma and D(') potentials, and where the B state approaches a second avoided crossing with the C(') (1)Sigma(+) valence state [Cooper and Kirby, J. Chem. Phys. 87, 424 (1987); 90, 4895 (1989); Chem. Phys. Lett. 152, 393 (1988)]. Fragments of a progression of weak and mostly diffuse bands, observed for all four isotopomers, have been assigned to the C(')<--X transition. The least-squares modeling of the 4p and 5p complexes reveals the 3ppi, E (1)Pi Rydberg state to be one of the perturbers, violating the Deltav=0 selection rule for Rydberg-Rydberg interactions on account of its rapid transition with increasing v from Rydberg to valence state. A second (1)Pi perturber, very loosely bound and clearly of valence type, contributes to the confusion in the published literature surrounding the 5p, v=0 complex.

Journal Article↗
PubMed2004

Hypoxia-induced activation of the retinoic acid receptor-related orphan receptor alpha4 gene by an interaction between hypoxia-inducible factor-1 and Sp1.

Hypoxia plays a key role in the pathophysiology of many disease states, and expression of the retinoic acid receptor-related orphan receptor alpha (RORalpha) gene increases under hypoxia. We investigated the mechanism for this transient hypoxia-induced increase in RORalpha expression. Reverse transcription-coupled PCR analysis revealed that the steady-state level of mRNA for the RORalpha4 isoform, but not the RORalpha1 isoform, increased in HepG2 cells after 3 h of hypoxia. Transient transfection studies showed that the hypoxia-induced increase in RORalpha4 mRNA occurs at the transcriptional level and is dependent on a hypoxia-responsive element (HRE) located downstream of the promoter. A dominant-negative mutant of hypoxia-inducible factor-1alpha (HIF-1alpha) abrogates the transcription activated by hypoxia as well as the transcription activated by exogenously expressed HIF-1alpha, demonstrating the direct involvement of HIF-1alpha in the transcriptional activation. However, HIF-1 alone was not sufficient to activate transcription in hypoxic conditions but, rather, required Sp1/Sp3, which binds to a cluster of GC-rich sequences adjacent to the HRE. Deletion of one or more of these GC boxes reduced or eliminated the HIF-1-dependent transcription. Together, these results suggest that the hypoxia-responsive region of the RORalpha4 promoter is composed of the HRE and GC-rich sequences and that the transcriptional activation under hypoxia is conferred through the cooperation of HIF-1 with Sp1/Sp3.

Cell Line↗