Hypoxia-induced Protein Binding to O2-responsive Sequences on the Tyrosine Hydroxylase Gene

Abstract

Cells in mammalian tissues are primarily aerobic and therefore highly dependent upon a continuous supply of oxygen. A primary mechanisms by which mammals respond to reduced O 2 (hypoxia) is hyperventilation, which enhances the delivery of O 2 to cells by increasing arterial O 2 tension. The hyperventilation that occurs during hypoxia is mediated by the O 2 -sensitive (type I) cells in the carotid body. The carotid body is located bilaterally at the bifurcation of the carotid artery and releases dopamine in response to a reduction in arterial O 2 tension. The activity of tyrosine hydroxylase (EC 1.14.16.2, TH), 1 the rate-limiting enzyme in the biosynthesis of dopamine, is enhanced in the carotid body (3) and adrenal gland (4) during hypoxia. We reported recently that hypoxia enhances TH gene expression in the rat carotid body (1) and stimulates both the rate of TH gene transcription and TH mRNA stability in pheochromocytoma (PC12) cells (2). We use the PC12 clonal cell line as a model system to study the molecular genetic mechanisms that regulate TH gene expression during hypoxia. Transcriptional studies using nested deletions of the proximal promoter region of the TH gene linked to a reporter gene revealed that the O 2 -responsive sequences reside within a fragment that extends from Ϫ272 to ϩ 27 bases, relative to transcription start site (2).In the present study experiments were undertaken to further characterize the sequences and trans-acting protein factors that regulate the rate of transcription of TH gene during hypoxia. We report here that the O 2 responsiveness of the TH gene is mediated by a short fragment of the proximal promoter that contains the AP1 and HIF-1 (hypoxia-induced factor binding site) sequences. Findings from immunological studies revealed that c-Fos and JunB bind to the AP1 element during hypoxia. In addition, we report that mutation of the AP1 element abolished the transcription response to hypoxia. EXPERIMENTAL PROCEDURES Materials[␣-32 P]dATP (Ͼ3000 Ci/mmol) and [ 14 C]chloramphenicol (50 -60 mCi/mmol) were purchased from Amersham Corp. Klenow, G-25, and G-50 purification spin columns were purchased from Boehringer Mannheim. pCAT plasmids were originally obtained from Promega. Lipofectin reagent was purchased from Bio-Rad, and all cell culture reagents were obtained from Life Technologies, Inc. Antibodies for supershift assays, shift-Western analysis, and immunoblotting assays were purchased from Santa Cruz Biotechnology. Immunoblotting assays were performed using the ECL kit from Amersham. MethodsCell Culture-Rat pheochromocytoma (PC12) cells were maintained in Dulbecco's modified Eagle's medium/F-12 containing 15 mM Hepes buffer, L-glutamine, 10% fetal calf serum, penicillin (100 units/ml), and streptomycin (100 g/ml). Cells were grown on culture dishes to 90% confluence in a strict environment (21% O 2 , 5% CO 2 , remainder N 2 at 37°C). Medium was changed twice weekly. Hypoxia exposures were carried out in an O 2 regulated incubator by exposing cells to 5% or 10% O 2 , 5% CO 2 , r...

Regulation of Transcription by Hypoxia Requires a Multiprotein Complex That Includes Hypoxia-Inducible Factor 1, an Adjacent Transcription Factor, and p300/CREB Binding Protein

“…The dependence of p300 binding on occupancy of the HRE is consistent with finding that nuclear hormone receptors interact with p300 (7,26). The tyrosine hydroxylase gene, which encodes the rate-limiting enzyme in dopamine synthesis, is regulated by hypoxia and has adjacent HIF-1 and AP-1 binding sites, both of which play a role in oxygen-dependent expression of tyrosine hydroxylase (34). AP-1 proteins have been shown to interact with p300 to activate transcription (3), and so it is likely that HIF-1, p300, and AP-1 form a complex at the tyrosine hydroxylase promoter.…”

Defective carotid body function and impaired ventilatory responses to chronic hypoxia in mice partially deficient for hypoxia-inducible factor 1α

“…HIF-1␣ dimerizes with HIF-1␤, which is constitutively expressed, binds to the core DNA sequence 5Ј-RCGTG-3Ј present within hypoxia response elements of target genes, and activates their transcription. Over three dozen genes are targets for transactivation by HIF-1 (21), and several of these may be involved in O 2 sensing͞signal transduction by the carotid body, including endothelin-1 (22,23) and tyrosine hydroxylase (24). Expression of endothelin-1 is induced by hypoxia in the carotid body and augments chemoreceptor responses by enhancing intracellular Ca 2ϩ levels and stimulating proliferation of glomus cells (25,26).…”

Biology of hypoxia-inducible factor-2α in development and disease

“…Indeed, HIF-2a is expressed in neural crest derivatives that synthesize catecholamines and tyrosine hydroxylase, a critical enzyme in catecholamine synthesis that is hypoxia inducible. 41 A fraction of these embryos could be rescued to birth by exogenous administration of D,L-threo-3,4-dihydroxyphenylserine, a metabolic intermediate that can be directly converted to noradrenaline. 15 Nevertheless, homozygous pups died soon after birth.…”