Maria F. Czyzyk-Krzeska

Regulation of Tyrosine Hydroxylase Gene Expression in the Rat Carotid Body by Hypoxia

Abstract: The activity (Vmax) of tyrosine hydroxylase (TH; EC 1.14.16.2), the rate limiting enzyme in the synthesis of catecholamines, is increased in carotid body, superior cervical ganglion, and the adrenal medulla during hypoxia (i.e., reduced Pao2). The present study was undertaken to determine if the increase in TH activity in these tissues during hypoxia is regulated at the level of TH mRNA. Adult rats were exposed to hypoxia (10% O2) or room air for periods lasting from 1 to 48 h. The carotid bodies, superior cervical ganglia, and adrenals were removed and processed for in situ hybridization using 35S‐labeled oligonucleotide probes. The concentration of TH mRNA was increased by hypoxia at all time points in carotid body type I cells, but not in cells of either superior cervical ganglion or adrenal medulla. The increase in TH mRNA in carotid body during hypoxia did not require innervation of the carotid body or intact adrenal glands. In addition, hypercapnia, another physiological stimulus of carotid body activity, failed to induce an increase in TH mRNA in type I cells. Our findings suggest that hypoxia stimulates TH gene expression in the carotid body by a mechanism that is intrinsic to type I cells.

von Hippel-Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination

The transition from transcription initiation to elongation involves phosphorylation of the large subunit (Rpb1) of RNA polymerase II on the repetitive carboxyl-terminal domain. The elongating hyperphosphorylated Rpb1 is subject to ubiquitination, particularly in response to UV radiation and DNA-damaging agents. By using computer modeling, we identified regions of Rpb1 and the adjacent subunit 6 of RNA polymerase II (Rpb6) that share sequence and structural similarity with the domain of hypoxia-inducible transcription factor 1α (HIF-1α) that binds von Hippel–Lindau tumor suppressor protein (pVHL). pVHL confers substrate specificity to the E3 ligase complex, which ubiquitinates HIF-α and targets it for proteasomal degradation. In agreement with the computational model, we show biochemical evidence that pVHL specifically binds the hyperphosphorylated Rpb1 in a proline-hydroxylation-dependent manner, targeting it for ubiquitination. This interaction is regulated by UV radiation.

VHL-Regulated MiR-204 Suppresses Tumor Growth through Inhibition of LC3B-Mediated Autophagy in Renal Clear Cell Carcinoma

The von Hippel-Lindau tumor-suppressor gene (VHL) is lost in most clear cell renal cell carcinomas (ccRCC). Here, using human ccRCC specimens, VHL-deficient cells, and xenograft models, we show that miR-204 is a VHL-regulated tumor suppressor acting by inhibiting macroautophagy, with MAP1LC3B (LC3B) as a direct and functional target. Of note, higher tumor grade of human ccRCC was correlated with a concomitant decrease in miR-204 and increase in LC3B levels, indicating that LC3B-mediated macroautophagy is necessary for RCC progression. VHL, in addition to inducing endogenous miR-204, triggered the expression of LC3C, an HIF-regulated LC3B paralog, that suppressed tumor growth. These data reveal a function of VHL as a tumor-suppressing regulator of autophagic programs.

Induction of Vascular Endothelial Growth Factor by IGF-I in Osteoblast-Like Cells Is Mediated by the PI3K Signaling Pathway through the Hypoxia-Inducible Factor-2α

IGF-I is known to stimulate the expression of oxygen- and nutrient-sensitive genes in several cell types. In this study we investigated the signaling pathways and transcriptional mechanisms that mediate IGF-I induction of vascular endothelial growth factor (VEGF) expression in human osteoblast-like cells. IGF-I (50 ng/ml) induced a rapid increase (3-fold) in VEGF mRNA in osteoblasts that was accompanied by an increase in the level of hypoxia-inducible factor-2α (HIF-2α) protein without changes in HIF-2α mRNA expression. These effects were mimicked by chemical inhibition of proteosomal degradation of HIF-2α. Transcriptional activation of a proximal VEGF promoter-luciferase construct was greatly enhanced by cotransfection with an HIF-2α, but not an HIF-1α, 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-2α and blunted IGF-I-induced VEG...

Characterization of the Hypoxia-inducible Protein Binding Site within the Pyrimidine-rich Tract in the 3*-Untranslated Region of the Tyrosine Hydroxylase mRNA*

Reduced tension of O slows the degradation rate of mRNA for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis, in the pheochromocytoma (PC12) clonal cell line. The observed increase in half-life (30 h versus 10 h) correlates with enhanced binding of a 66-kDa protein (hypoxia inducible protein) to the pyrimidine-rich tract located between bases 1552-1578 in the 3′-untranslated region of TH mRNA (hypoxia-inducible protein binding site (HIPBS)). The present study investigates the protein binding site within the 27-base HIPBS, first by using specific cleavages of HIPBS and its flanking sequences with antisense oligodeoxynucleotides and RNase H and then by using mutational analysis of the binding properties. We found that the 27-base HIPBS oligoribonucleotide was sufficient to bind the protein in vitro in a hypoxia-stimulated manner. We further identified the optimal hypoxia-inducible protein binding site that is represented by the motif (U/C)(C/U)CCCU, where the core binding site is indicated by the underlined cytidines. Substitutions of either one of the cytidines with purine or uridine abolished the protein binding. The mutations within HIPBS, which partially reduced binding, did not prevent stimulation of protein binding for extracts from hypoxic cells. The hypoxia-induced increase in complex formation was proportional to the strength of binding using proteins from normoxic cells. The HIPBS element is conserved in TH mRNAs derived from different species.

The von Hippel-Lindau Tumor Suppressor Protein and Egl-9-Type Proline Hydroxylases Regulate the Large Subunit of RNA Polymerase II in Response to Oxidative Stress

ABSTRACT Human renal clear cell carcinoma (RCC) is frequently associated with loss of the von Hippel-Lindau (VHL) tumor suppressor (pVHL), which inhibits ubiquitylation and degradation of the alpha subunits of hypoxia-inducible transcription factor. pVHL also ubiquitylates the large subunit of RNA polymerase II, Rpb1, phosphorylated on serine 5 (Ser5) within the C-terminal domain (CTD). A hydroxylated proline 1465 within an LXXLAP motif located N-terminal to the CTD allows the interaction of Rpb1 with pVHL. Here we report that in RCC cells, pVHL regulates expression of Rpb1 and is necessary for low-grade oxidative-stress-induced recruitment of Rpb1 to the DNA-engaged fraction and for its P1465 hydroxylation, phosphorylation, and nondegradative ubiquitylation. Egln-9-type prolyl hydroxylases, PHD1 and PHD2, coimmunoprecipitated with Rpb1 in the chromatin fraction of VHL+ RCC cells in response to oxidative stress, and PHD1 was necessary for P1465 hydroxylation while PHD2 had an inhibitory effect. P1465 hydroxylation was required for oxidative-stress-induced Ser5 phosphorylation of Rpb1. Importantly, overexpression of wild-type Rpb1 stimulated formation of kidney tumors by VHL+ cells, and this effect was abolished by P1465A mutation of Rpb1. These data indicate that through this novel pathway involving P1465 hydroxylation and Ser5 phosphorylation of Rbp1, pVHL may regulate tumor growth.

Calcium signaling stimulates translation of ΗΙF-α during hypoxia

Hypoxia‐inducible factors (HIFs) are ubiquitous transcription factors that mediate adaptation to hypoxia by inducing specific sets of target genes. It is well accepted that hypoxia induces accumulation and activity of HIFs by causing stabilization of their α subunits. We have demonstrated that hypoxia stimulates translation of HIF‐1α and ‐2α proteins by distributing HIF‐α mRNAs to larger polysome fractions. This requires influx of extracellular calcium, stimulation of classical protein kinase C‐α (cPKC‐α), and the activity of mammalian target of rapamycin, mTOR. The translational component contributes to α40–50% of HIF‐α proteins accumulation after3hof1%O2. Hypoxia also inhibits general protein synthesis and mTOR activity; however, cPKC‐α inhibitors or rapamycin reduce mTOR activity and total protein synthesis beyond the effects of hypoxia alone. These data show that during general inhibition of protein synthesis by hypoxia, cap‐mediated translation of selected mRNAs is induced through the mTOR pathway. We propose that calcium‐induced activation of cPKC‐α hypoxia partially protects an activity of mTOR from hypoxic inhibition. These results provide an important physiologic insight into the mechanism by which hypoxia‐stimulated influx of calcium selectively induces the translation of mRNAs necessary for adaptation to hypoxia under conditions repressing general protein synthesis.—Hui, A. S., Bauer, A. L., Striet, J. B., Schnell, P. O., Czyzyk‐Krzeska, M. F. Calcium signaling stimulates translation of HIF‐α during hypoxia. FASEB J. 20, 466–475 (2006)

Hypoxia Induces Vascular Endothelial Growth Factor Gene Transcription in Human Osteoblast-Like Cells through the Hypoxia-Inducible Factor-2α

VEGF is produced by osteoblasts and has been postulated to function as an angiogenic stimulus during normal skeletal development and in fracture repair. In this study, we characterized the molecular mechanisms by which experimental hypoxia increases VEGF mRNA in human MG63 osteoblast-like cells. Exposure of MG63 cells to 1% O2 for 24 h resulted in a four-fold increase in VEGF mRNA. Immunoblotting of nuclear extracts demonstrated a time-dependent increase in the level of the Hif-2α protein, which preceded the rise in VEGF mRNA. To determine the effect of hypoxia on VEGF gene transcription, MG63 cells were transiently transfected with a segment of the VEGF promoter construct fused to luciferase and then exposed to 1% O2. Hypoxia induced VEGF promoter activity five-fold by 24 h. Forced expression of Hif-2α, but not Hif-1α, increased both basal and hypoxia induced VEGF promoter activity. By contrast, the ability of the VEGF reporter to respond to hypoxia or recombinant Hif-2α was abolished in cells transfecte...

Regulation of tyrosine hydroxylase promoter activity by the von Hippel-Lindau tumor suppressor protein and hypoxia-inducible transcription factors.

Tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamine biosynthesis, is induced by hypoxia in oxygen‐sensitive cells of the carotid body and pheochromocytoma‐derived PC12 cells. TH is also regulated by the von Hippel–Lindau tumor suppressor protein (pVHL). Here, we report that induction of TH gene expression involves activation of the hypoxia‐inducible transcription factors (HIFs) that interact with a specific hypoxia‐responsive element (HRE) in the proximal region of the TH promoter. We also show that some of the effects of pVHL on activity of the TH promoter are mediated through HIFs. Low levels of pVHL are associated with decreased HIFα ubiquitination, increased accumulation of HIFα proteins, increased binding of HIFs to the HRE within the TH promoter, and increased activity of a TH promoter–reporter construct. In contrast, high levels of pVHL repress HIF accumulation and inhibit its activity in hypoxic cells. These results indicate that HIFs may play an important role in regulation of TH gene expression in oxygen‐sensitive cells and also in the development of hypercatecholaminemia in pheochromocytoma tumors.

Regulation of Catecholamines by Sustained and Intermittent Hypoxia in Neuroendocrine Cells and Sympathetic Neurons

Abstract—Chronic intermittent hypoxia, a characteristic feature of sleep-disordered breathing, induces hypertension through augmented sympathetic nerve activity and requires the presence of functional carotid body arterial chemoreceptors. In contrast, chronic sustained hypoxia does not alter blood pressure. We therefore analyzed the biosynthetic pathways of catecholamines in peripheral nervous system structures involved in the pathogenesis of intermittent hypoxia-induced hypertension, namely, carotid bodies, superior cervical ganglia, and adrenal glands. Rats were exposed to either intermittent hypoxia (90 seconds of room air alternating with 90 seconds of 10% O2) or to sustained hypoxia (10% O2) for 1 to 30 days. Dopamine, norepinephrine, epinephrine, dihydroxyphenylacetic acid, and 5-hydroxytyptamine contents were measured by high-performance liquid chromatography. Expression of tyrosine hydroxylase and its phosphorylated forms, dopamine &bgr;-hydroxylase, phenylethanolamine N-methyltransferase, and GTP cyclohydrolase-1 were determined by Western blot analyses. Both sustained and intermittent hypoxia significantly increased dopamine and norepinephrine content in carotid bodies but not in sympathetic ganglia or adrenal glands. In carotid bodies, both types of hypoxia augmented total levels of tyrosine hydroxylase protein and its phosphorylation on serines 19, 31, 40, as well as levels of GTP cyclohydrolase-1. However, the effects of intermittent hypoxia on catecholaminergic pathways were significantly smaller and delayed than those induced by sustained hypoxia. Thus, attenuated induction of catecholaminergic phenotype by intermittent hypoxia in carotid body may play a role in development of hypertension associated with sleep-disordered breathing. The effects of both types of hypoxia on expression of catecholaminergic enzymes in superior cervical neurons and adrenal glands were transient and small.