Hypoxia-inducible factor induces local thyroid hormone inactivation during hypoxic-ischemic disease in rats - PubMed (original) (raw)

. 2008 Mar;118(3):975-83.

doi: 10.1172/JCI32824.

Michelle A Mulcahey, Everaldo M Redout, Alice Muller, Marian J Zuidwijk, Theo J Visser, Frank W J S Wassen, Alessandra Crescenzi, Wagner S da-Silva, John Harney, Felix B Engel, Maria-Jesús Obregon, P Reed Larsen, Antonio C Bianco, Stephen A Huang

Affiliations

Hypoxia-inducible factor induces local thyroid hormone inactivation during hypoxic-ischemic disease in rats

Warner S Simonides et al. J Clin Invest. 2008 Mar.

Abstract

Thyroid hormone is a critical determinant of cellular metabolism and differentiation. Precise tissue-specific regulation of the active ligand 3,5,3'-triiodothyronine (T3) is achieved by the sequential removal of iodine groups from the thyroid hormone molecule, with type 3 deiodinase (D3) comprising the major inactivating pathway that terminates the action of T3 and prevents activation of the prohormone thyroxine. Using cells endogenously expressing D3, we found that hypoxia induced expression of the D3 gene DIO3 by a hypoxia-inducible factor-dependent (HIF-dependent) pathway. D3 activity and mRNA were increased both by hypoxia and by hypoxia mimetics that increase HIF-1. Using ChIP, we found that HIF-1alpha interacted specifically with the DIO3 promoter, indicating that DIO3 may be a direct transcriptional target of HIF-1. Endogenous D3 activity decreased T3-dependent oxygen consumption in both neuronal and hepatocyte cell lines, suggesting that hypoxia-induced D3 may reduce metabolic rate in hypoxic tissues. Using a rat model of cardiac failure due to RV hypertrophy, we found that HIF-1alpha and D3 proteins were induced specifically in the hypertrophic myocardium of the RV, creating an anatomically specific reduction in local T3 content and action. These results suggest a mechanism of metabolic regulation during hypoxic-ischemic injury in which HIF-1 reduces local thyroid hormone signaling through induction of D3.

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Figures

Figure 1

Figure 1. Hypoxia induces D3 activity and mRNA.

(A) Endogenous D3 activity in SK-N-AS neurons, rat neonatal cardiomyocytes, NCLP6E hepatocytes, choriocarcinoma cells (JEG-3 cells), endometrial cells (ECC-1 cells), and AG04526 fibroblasts exposed to normoxia (21% O2) versus hypoxia (1% O2) for 24 h. Values are mean ± SEM of 2 or 3 cell plates; mean of 3 experiments is shown for each cell type. *P < 0.005. (B) Northern blot analysis of total RNA obtained from SK-N-AS or NCLP6E cells exposed to hypoxia versus normoxia for 24 h. Lanes were run on the same gel but were noncontiguous. (C) D3 activity and Northern blotting in NCLP6E cells exposed to continuous normoxia (condition A), continuous hypoxia (condition B), or transient hypoxia for 24 h followed by normoxia (condition C). Representative experiment with mean ± SEM of 2 cell plates is shown; this experiment was reproduced.

Figure 2

Figure 2. The hypoxia mimetics DFO and CoCl2 increase endogenous D3 in NCLP6E hepatocytes and SK-N-AS neurons.

(A) D3 activity and Western blot analysis of HIF-1α protein in NCLP6E hepatocytes 24 h after exposure to the indicated hypoxia mimetics. Hepatocytes that were exposed to hypoxia (without hypoxia mimetics) are included in the HIF-1α Western blot as a positive control. (B) D3 activity and Western blot analysis of HIF-1α protein in SK-N-AS neurons 6 or 24 h after exposure to DFO or CoCl2. Values are mean ± SEM of 3 cell plates. *P < 0.05; **P < 0.005. (C) Western blot analysis of HIF-1α protein in cells that increase endogenous D3 expression in response to hypoxia (JEG-3, SK-N-AS neurons, and NCLP6E hepatocytes) and in cells with undetectable endogenous D3 activity (HepG2, HEK-293 cells, and MSTO cells). (D) ChIP analysis of the DIO3 or the SCN3A promoter in DFO-stimulated SK-N-AS cells using antibodies directed against endogenous HIF-1α. Representative experiments are shown and were reproduced.

Figure 3

Figure 3. Inhibition of endogenous D3 activity with iopanoic acid for 6 h increases metabolic rate in isolated cells.

Oxygen consumption (A and C) and extracellular acidification (B and D) in SK-N-AS neurons and NCLP6E hepatocytes that were propagated in media with (A and B) or without (C and D) a euthyroid concentration of T3 and exposed to iopanoic acid (Iop) versus vehicle control. Oxygen consumption rate (pmol/min/mg protein) and extracellular acidification rate (mpH/min/mg protein) are expressed as relative fold change from vehicle control. Values are mean ± SEM of 10 cell plates. *P = 0.07; **P < 0.05; ***P < 0.005.

Figure 4

Figure 4. HIF-1α and D3 are selectively induced in the RV in a rat model of monocrotaline-induced RV hypertrophy.

Thickening of the RV wall (A) and increased RV weight (B) were observed as CHF developed after monocrotaline administration. Values are mean ± SEM of 11–16 animals per group. (C–F) Western blot analysis of HIF-1α (C) and HIF-2α protein (D), D3 activity (E), and quantitative real-time PCR of D3 mRNA (F) in tissue prepared from the RV and LV of rats administered monocrotaline (CHF) versus saline control (CON). The HIF-2α Western blot in D depicts RV samples from control and CHF animals, with extracts of primary microvascular endothelial cells obtained from human foreskin and cultured under hypoxic conditions (1% O2) serving as a positive control (+). Values are mean ± SEM of 3–9 animals per group. (F) D3 mRNA is expressed as the RV/LV ratio and shown as the relative fold change from the saline control group. *P < 0.05 versus control; **P < 0.05 versus LV; ***P < 0.005 versus control. (G) Myocardial reporter activity after in vivo cardiomyocyte transfection of the pLuc-TRE T3-responsive Firefly luciferase reporter, normalized to the expression of the pRen-C transfection control. Values are mean ± SEM of 5–29 animals per group. LV and RV reporter activity is shown in CHF versus control animals. In control animals with systemic hypothyroidism (Hypo), euthyroidism (Eu), or thyrotoxicosis from T3 treatment (Hyper), reporter levels from pooled LV/RV homogenates are shown. *P < 0.05 versus euthyroidism or hypothyroidism, ANOVA; **P < 0.05 versus control and LV.

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