| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,1
* Laboratory of Oxygen Metabolism, University Hospital, University of Buenos Aires, Argentina; and
Institut National de la Santé et de la Recherche Médicale (INSERM U700), Université Paris 7, Faculté X. Bichat, Paris, France
1Correspondence: INSERM U700 Faculté X. Bichat BP416, Paris 75870, Cedex 18, France. E-mail: jbb2{at}bichat.inserm.fr
SPECIFIC AIMS
This study is devoted to the investigation of the inducible isoform of heme oxygenase (HO). We explore the role of HO-1 (which is located in liver mitochondria) on mitochondria heme content and metabolism. The rationale is that HO-1 location in mitochondria could be involved in control of mitochondrial heme protein expression and activity via modulation of mitochondrial heme content.
PRINCIPAL FINDINGS
The most significant and novel findings of this study are the following:
1. Western-blot and immune-electron microscopy in whole purified and fractionated organelles showed basal expression of HO-1 protein in both microsomes and mitochondria (Fig. 1
A), accompanied by a parallel HO activity.
Inducers of HO-1 increased HO-1 targeting to the inner mitochondrial membrane, which also contained biliverdin reductase. By contrast, HO-2 was only expressed in microsomes.
|
2. Induction of mitochondrial HO-1 was associated with a decrease of mitochondrial heme content and with a selective reduction in protein expression of cytochrome oxidase (COX) subunit I (Fig. 1 B), which is coded by the mitochondrial genome and synthesized in the mitochondria depending on heme availability.
These changes resulted in decreased COX spectrum and activity. Mitochondrial HO-1 induction was also associated with down-regulation of mitochondrial-targeted NO synthase expression and activity, resulting in a reduction of NO-dependent mitochondrial oxidants production; inhibition of HO-1 activity reverted these phenomena (Fig. 2
).
|
CONCLUSIONS AND SIGNIFICANCE
In this study, we show for the first time that, in addition to microsomes, HO-1 protein is located in liver mitochondria, operates on redox components through heme availability, and as a part of its antioxidant properties, modulates mitochondrial O2 uptake and ROS production.
The modulation of mitochondrial HO-1 by inducers (hemin or LPS) closely followed the variations in the microsomal fraction. In purified mitochondria with negligible microsomal contamination, mitochondrial HO-1 activity and content were within 
10–20% of that of microsomes. This percentage indicates that only a fraction of induced HO-1 is targeted to mitochondria, suggesting that tightly controlled HO-1 import could be limited by the intrinsic translocon activity. HO-1 lacks N-termini targeting presequence to mitochondria and thus, as transporters colocalized in the inner membrane, probably requires internal hydrophobic targeting domains, and a specific import machinery, like the Tim 23 transporter.
HO-1 targeting to mitochondria was associated with significant variations of mitochondrial heme pool. In fact, in hemin-treated animals, a different subcellular distribution of total heme content exclusively resulted from 50% decrease of mitochondrial heme, without variations in cytosol or microsomes. The reduction in mitochondrial heme content resulted in a decreased expression and activity of COX, the terminal acceptor of the electron transfer chain. Indeed, we found that a decrease of activity and spectrum of COX isolated from hemin-treated animals was associated with a selective decrease in protein expression of heme-bound subunit I, whereas expression of the heme-independent subunit VIc was unchanged. Because mARN expression of subunit I was unchanged in hemin-treated animals, the effect of HO-1 is likely to be posttranslational and secondary to the decreased mitochondrial heme content. In accord, all processing of subunit I occurs in the mitochondria: subunit I is coded on mitochondrial DNA, translated on mitochondrial ribosomes, and heme incorporation takes place locally. A critical role of heme incorporation on subunit I expression and COX stability and function was demonstrated in human fibroblasts in which heme deficiency secondary to inhibition of ferrochelatase decreased protein expression of subunit I and COX activity, whereas independency of heme pool and protein expression, subunit VIc was reported in mouse liver.
Similarly to COX subunit I, HO-1 could affect mitochondrial-targeted NO (mtNOS) content because NOS protein dimerization and stability depends on heme availability. Because in hemin-treated animals, liver nNOS mRNA expression and a small amount of microsomal neuronal NOS did not change, the in situ effect of targeted HO-1 on mtNOS protein is emphasized. Considering that CO binds heme and inhibits electron transfer, some contribution of CO released by HO-1 activity to COX or mtNOS inhibition remains to be elucidated.
On the basis of these findings, oxygen uptake and the weight of electron transfer rate would depend on the relative participation of [NO] and [O2], resulting from differential HO-1 effects on the turnover of mitochondrial-targeted NOS and COX. It is noteworthy that despite 40% decreased COX activity, we found no modification in state 4 oxygen uptake rate, and even a significant increase of state 3 oxygen uptake rate was observed in hemin-treated animals. This effect could be due to a threshold effect, since the relative COX capacity is in excess with respect to that required for supporting the endogenous respiration rate. Alternatively, HO-1 could modulate other heme proteins involved in mitochondrial metabolism. A concomitantly decreased mtNOS content and activity contributes to explain the observed variations of O2 uptake in hemin-treated animals. NO produced by mitochondria is vectorially directed to the matrix and reversibly inhibits cytochrome oxidase by binding to heme (nitrosyl-Fe2+). Likewise, hemin-induced HO-1 targeting was associated with decreased mtNOS and thus, O2 uptake increased and was less sensitive to NOS substrate L-arginine.
In addition to effects on oxygen utilization, mitochondrial NO promotes a high production rate of superoxide anions and the dismutation product, hydrogen peroxide. This effect relies on inhibition of electron transfer at transition between cytochromes b and c1 and on direct reactions between ubiquinol and NO, leading to ubisemiquinone formation that transfers electrons to O2 to form superoxide anion. Accordingly, mitochondria from hemin-treated animals with low mtNOS had 50% less NO-dependent H2O2 yield than controls. This effect could contribute to the antioxidant properties of HO-1, especially in conditions associated with increased mtNOS expression, such as endotoxemia.
In conclusion, the present results provide a new insight into the functions and biological implications of HO-1 and, more broadly, into the mechanisms controlling the mitochondrial metabolism. The presence of functional HO-1 in mitochondria, regulating mitochondrial heme pool, heme protein content, and ROS production may have important biological roles. First, HO-1 is suggested as an important in situ modulator of the turnover of respiratory hemoproteins, and of electron transfer rate and O2 uptake. Second, by restricting mitochondrial oxidant and NO yields, it could participate in cell signaling and life and death programs. Finally, by the same token, mitochondrial localization could explain protective properties of HO-1 in conditions such as neurodegenerative diseases, ischemia reperfusion, or sepsis, in which substantially increased mitochondrial NO and ROS production has been implicated.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4204fje
This article has been cited by other articles:
|
|
 |
|
  Z. Zhong, V. K. Ramshesh, H. Rehman, R. T. Currin, V. Sridharan, T. P. Theruvath, I. Kim, G. L. Wright, and J. J. Lemasters Activation of the oxygen-sensing signal cascade prevents mitochondrial injury after mouse liver ischemia-reperfusion Am J Physiol Gastrointest Liver Physiol, October 1, 2008; 295(4): G823 - G832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D Goven, A Boutten, V Lecon-Malas, J Marchal-Somme, N Amara, B Crestani, M Fournier, G Leseche, P Soler, J Boczkowski, et al. Altered Nrf2/Keap1-Bach1 equilibrium in pulmonary emphysema Thorax, October 1, 2008; 63(10): 916 - 924. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Chang, L. Wu, and R. Wang Inhibition of vascular smooth muscle cell proliferation by chronic hemin treatment Am J Physiol Heart Circ Physiol, September 1, 2008; 295(3): H999 - H1007. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. L. Lagan, D. D. Melley, T. W. Evans, and G. J. Quinlan Pathogenesis of the systemic inflammatory syndrome and acute lung injury: role of iron mobilization and decompartmentalization Am J Physiol Lung Cell Mol Physiol, February 1, 2008; 294(2): L161 - L174. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D.-J. Slebos, S. W. Ryter, M. van der Toorn, F. Liu, F. Guo, C. J. Baty, J. M. Karlsson, S. C. Watkins, H. P. Kim, X. Wang, et al. Mitochondrial Localization and Function of Heme Oxygenase-1 in Cigarette Smoke-Induced Cell Death Am. J. Respir. Cell Mol. Biol., April 1, 2007; 36(4): 409 - 417. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
