Immunocytochemical localization and expression of heme oxygenase-1 in primary astroglial cell cultures during differentiation: effect of glutamate

doi:10.1016/j.bbrc.2004.01.090

Biochemical and Biophysical Research Communications

Volume 315, Issue 2, 5 March 2004, Pages 517-524

https://doi.org/10.1016/j.bbrc.2004.01.090 Get rights and content

Abstract

Heme oxygenase-1 (HO-1) catalyzes the rate-limiting step in heme degradation releasing iron, carbon monoxide (CO), and biliverdin. We investigated subcellular localization of HO-1 using confocal laser scanning microscopy (CLSM) and the expression by Western blot in primary astroglial cells during differentiation and after exposure to glutamate (100 μM). CLSM analysis of immunostained HO-1 in cultured astroglial cells during differentiation showed an increase of fluorescence between 7 and 14 days and a decrease between 14 and 21, although HO-1 peaked at 14 days it remained at high levels. The distribution of HO-1 protein undergoes modification in the various cellular compartments. Furthermore, localization of the protein in untreated astrocytes at 7 days appeared prevalently localized in the cytosol and in the perinuclear region. In contrast, at 14 and 21 days, fluorescence detection suggests that HO-1 was present also in the nucleus, and in the nucleoli. Fluorescence intensity significantly increased in glutamate-treated astrocytes during all development stages and the protein appeared in the cytosol, in the nucleus and in the nucleoli. The involvement of AMPA/Ka receptors was studied in glutamate-treated astroglial cells at 14 days by the preincubation of the cells with GYKI 52466, a specific receptor inhibitor, of AMPA/Ka receptor demonstrating the involvement of these receptors. Western blot analysis of HO-1 confirmed the CLSM results. Our results demonstrate that changes in HO-1 protein expression and localization in primary cultured astroglial cells may be part of the underlying mechanisms involved in brain development as well as in neurodegenerative diseases.

Section snippets

Materials and methods

Materials. Cell culture medium and sera were obtained from Life Technologies (Milano, Italy). GYKI 52466 was from Research Biochemicals (Natick, MA, USA). Rabbit monoclonal antibody to Glial Fibrillary Acidic Protein (GFAP) was from Chemicon (Milano, Italy). Polyclonal anti-goat IgG conjugated to tetramethylrhodamine-isothiocyanate (TRITC) and polyclonal anti-mouse IgG conjugated to fluoresceinisothiocyanate (FITC) were from Sigma (Milano, Italy). TO-PRO-3,a fluorescent probe was purchased from

Results

Astroglial cell cultures during development were characterized, using CLSM, by immunocytochemical staining of a specific marker such as GFAP [18]. More than 90% of cells were GFAP-positive, indicating that cultures were enriched in astrocytes (Fig. 1A). The levels of cell maturation and differentiation were also tested by measuring GS activity as an enzyme marker. GFAP was also determined by CLSM analysis in glutamate-treated astroglial cell cultures at 7, 14, and 21 days in culture (DIV) (Fig.

Discussion

A growing body of evidence shows that receptor ligand-gated channels are expressed in both neurons and glial cells [20], [21]. The activation of these receptors is followed by biological events, including neurotransmitter release and growth factors, suggesting an active role in neurotoxic damage and repair. Therefore, glial cells are sensitive and vulnerable to excitotoxic injury [22]. In mammalian cells, HO-1 plays an important role in iron homeostasis and confers protection against various

Acknowledgements

This work was supported by NIH Grants RO1 HL54138 and PO1 HL34300 and Catania University.

References (44)

N.G. Abraham et al.
The physiological significance of heme oxygenase
Int. J. Biochem.
(1988)
W.K. McCoubrey et al.
Human heme oxygenase-2: characterization and expression of a full-length cDNA and evidence suggesting that the two HO-2 transcripts may differ by choice of polyadenylation signal
Arch. Biochem. Biophys.
(1992)
T. Kushida et al.
TNF-alpha-mediated cell death is attenuated by retrovirus delivery of human heme oxygenase-1 gene into human microvessel endothelial cells
Transplant. Proc.
(2002)
Y. Lavrovsky et al.
Novel regulatory sites of the human heme oxygenase-1 promoter region
Biochem. Biophys. Res. Commun.
(1993)
A. Campisi et al.
Glutamate-induced increases in transglutaminase activity in primary cultures of astroglial cells
Brain Res.
(2003)
M. Bergeron et al.
Developmental expression of heme oxygenase-1 (HSP32) in rat brain: an immunocytochemical study
Brain Res. Dev. Brain Res.
(1998)
G. Scapagnini et al.
Gene expression profiles of heme oxygenase isoforms in the rat brain
Brain Res.
(2002)
A. Bignami et al.
Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence
Brain Res.
(1972)
G. Li Volti et al.
Differential effect of heme oxygenase-1 in endothelial and smooth muscle cell cycle progression
Biochem. Biophys. Res. Commun.
(2002)
C. Matute et al.
Excitotoxicity in glial cells
Eur. J. Pharmacol.
(2002)

K. Nagata et al.

Microglia-derived plasminogen enhances neurite outgrowth from explant cultures of rat brain

Int. J. Dev. Neurosci.

(1993)

J.A. Nathanson et al.

The cellular Na⁺ pump as a site of action for carbon monoxide and glutamate: a mechanism for long-term modulation of cellular activity

Neuron

(1995)

T. Shinomura et al.

Reduction of depolarization-induced glutamate release by heme oxygenase inhibitor: possible role of carbon monoxide in synaptic transmission

Neurosci. Lett.

(1994)

A.F. McDonagh

Is bilirubin good for you?

Clin. Perinatol.

(1990)

J.Y. Kwak et al.

Bilirubin inhibits the activation of superoxide-producing NADPH oxidase in a neutrophil cell-free system

Biochim. Biophys. Acta

(1991)

T. Hon et al.

The yeast heme-responsive transcriptional activator Hap1 is a preexisting dimer in the absence of heme

J. Biol. Chem.

(1999)

C. Colombrita et al.

Heme oxygenase-1 expression levels are cell cycle dependent

Biochem. Biophys. Res. Commun.

(2003)

H. Fung et al.

Key role of a downstream specificity protein 1 site in cell cycle-regulated transcription of the AP endonuclease gene APE1/APEX in NIH3T3 cells

J. Biol. Chem.

(2001)

J.M. Segui-Simarro et al.

Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L

J. Struct. Biol.

(2003)

K. Nakaso et al.

Induction of heme oxygenase-1 in the rat brain by kainic acid-mediated excitotoxicity: the dissociation of mRNA and protein expression in hippocampus

Biochem. Biophys. Res. Commun.

(1999)

Y. Matsuoka et al.

Kainic acid induction of heme oxygenase in vivo and in vitro

Neuroscience

(1998)

D.W. Choi

Glutamate neurotoxicity and diseases of the nervous system

Neuron

(1988)

Cited by (56)

Exploring the pathophysiological influence of heme oxygenase-1 on neuroinflammation and depression: A study of phytotherapeutic-based modulation
2024, Phytomedicine
The heme oxygenase (HO) system plays a significant role in neuroprotection and reduction of neuroinflammation and neurodegeneration. The system, via isoforms HO-1 and HO-2, regulates cellular redox balance. HO-1, an antioxidant defense enzyme, is highlighted due to its association with depression, characterized by heightened neuroinflammation and impaired oxidative stress responses.
We observed the pathophysiology of HO-1 and phytochemicals as its modulator. We explored Science Direct, Scopus, and PubMed for a comprehensive literature review. Bibliometric and temporal trend analysis were done using VOSviewer.
Several phytochemicals can potentially alleviate neuroinflammation and oxidative stress-induced depressive symptoms. These effects result from inhibiting the MAPK and NK-κB pathways - both implicated in the overproduction of pro-inflammatory factors - and from the upregulation of HO-1 expression mediated by Nrf2. Bibliometric and temporal trend analysis further validates these associations.
In summary, our findings suggest that antidepressant agents can mitigate neuroinflammation and depressive disorder pathogenesis via the upregulation of HO-1 expression. These agents suppress pro-inflammatory mediators and depressive-like symptoms, demonstrating that HO-1 plays a significant role in the neuroinflammatory process and the development of depression.
The non-canonical effects of heme oxygenase-1, a classical fighter against oxidative stress
2021, Redox Biology
The role of heme oxygenase-1 in resisting oxidative stress and cell protection has always been a hot research topic. With the continuous deepening of research, in addition to directly regulating redox by catalyzing the degradation of heme, HO-1 protein also participates in the gene expression level in a great diversity of methods, thereby initiating cell defense. Particularly the non-canonical nuclear-localized HO-1 and HO-1 protein interactions play the role of a warrior against oxidative stress. Besides, HO-1 may be a promising marker for disease prediction and detection in many clinical trials. Especially for malignant diseases, there may be new advances in the treatment of HO-1 by regulating abnormal ROS and metabolic signaling. The purpose of this review is to systematically sort out and describe several aspects of research to facilitate further detailed mechanism research and clinical application promotion in the future.
Temporal evolution of heme oxygenase-1 expression in reactive astrocytes and microglia in response to traumatic brain injury
2020, Brain Hemorrhages
Citation Excerpt :
HO-3 is a protein related to HO-2 but product of a different gene, is expressed in the brain but has poor heme catalyst activity.39 HO is primarily an endoplasmic reticulum-resident protein but has also been located in the nucleus of cultured fibroblast cells exposed to heme or hypoxia,35 in the nucleus of astroglial cells exposed to glutamate,34 and in the nucleus of endothelial cells and smooth muscle cells.33 The products of HO activation are known to have diverse consequences: biliverdin is converted through biliverdin reductase to bilirubin,54 which has antioxidant properties by scavenging peroxyl radicals formed from oxidation of lipids.53
Heme oxygenase-1 (HO-1) is an inducible enzyme that catabolizes heme into biliverdin (which is converted to bilirubin), carbon monoxide, and free iron. HO-1 and its downstream molecules have antioxidant and anti-inflammatory functions, making the effects of HO-1 difficult to predict. It is unknown if HO-1 expression has neuroprotective or neurodegenerative sequelae after traumatic brain injury (TBI). In adult male mice, we quantitatively investigated HO-1 expression in reactive astrocytes and microglia in a controlled cortical impact (CCI) model of TBI at 1, 7, 14, and 30 days post-injury (dpi). Immunoglobulin G (IgG) staining for blood-brain barrier (BBB) permeability was significantly increased at 1 and 7 dpi in TBI mice compared to controls. HO-1 expression in astrocytes was significantly increased acutely and sub-acutely (1, 7, 14 dpi) compared to controls. Significantly elevated expression of HO-1 in microglia was only observed at 14 and 30 dpi relative to controls. HO-1 expression remained elevated at 30 dpi following TBI relative to controls. This study for the first time demonstrates that HO-1 is highly expressed in perilesional tissues after TBI, but primarily in cells that contribute to the neuroinflammatory response. Modulating HO-1 expression may provide a path to therapeutic intervention by enhancing the neuroprotective aspects of HO-1.
Heme oxygenase-1 expression after traumatic brain injury in adult male mice occurs first in reactive astrocytes followed by dramatic increases in microglia adjacent to the injury site.
The sinister face of heme oxygenase-1 in brain aging and disease
2019, Progress in Neurobiology
Citation Excerpt :
The gliopathy was more pronounced after 6 days of transfection and comprised severe mitochondrial degeneration, numerous multivesicular bodies, elaborate multilamellar whorls, pleomorphic electron-dense granules and spherical concretions resembling human CA. Treatment of the HMOX1-transfected cultures with SnMP obviated these morphological changes, attesting to their dependence on HO enzymatic activity rather than some signalling or other non-canonical behavior of the HO-1 protein (Li Volti et al., 2004; Lin et al., 2007a, 2008). The cytopathological profile of these cells is classical for mitophagy (Klionsky et al., 2016) and indicates that overexpression of HMOX1 promotes macroautophagy in astroglia (Zukor et al., 2009).
Under stressful conditions, cellular heme catabolism to carbon monoxide, iron and biliverdin is mediated by the 32 kDa enzyme, heme oxygenase-1 (HO-1). A wide range of pro-oxidant and inflammatory stimuli act on diverse consensus sequences within the Hmox1 promoter to rapidly induce the gene. There is ample evidence attesting to the beneficial effects of HO-1 upregulation in brain. By converting pro-oxidant heme to the antioxidants, biliverdin and bilirubin, HO-1/biliverdin reductase may help restore a more favorable tissue redox microenvironment. Contrariwise, in some cell types and under certain circumstances, heme-derived carbon monoxide and iron may amplify intracellular oxidative stress and exacerbate the disease process. This inimical side of neural HO-1 has often been ignored in biomedical literature promulgating interventions aimed at boosting central HO-1 expression for the management of diverse CNS conditions and is the focus of the current review. A comprehensive model of astroglial stress is presented wherein sustained Hmox1 induction promotes oxidative mitochondrial membrane damage, iron sequestration and mitophagy (macroautophagy). The HO-1 mediated gliopathy renders nearby neuronal constituents vulnerable to oxidative injury and recapitulates ‘core’ neuropathological features of many aging-related neurodegenerative and some neurodevelopmental brain disorders. A balanced literature should acknowledge that, in a host of chronic human CNS afflictions, the glial HO-1 response may serve as a robust transducer of noxious stimuli, an important driver of relevant neuropathology and a potentially disease-modifying therapeutic target.
Regulation of electrical activity and neuronal excitability in Helisoma trivolvis by carbon monoxide
2015, Neuroscience
Citation Excerpt :
Moreover, the effects of NO on growth cone morphology were dependent on the production of APs and their shape (Estes et al., 2014), linking the modulation of firing activity by gaseous signals to their developmental effects. Given that CO, like NO, is produced in nervous systems (Wu and Wang, 2005), is present during development (Raju et al., 1997; Bergeron et al., 1998; Li Volti et al., 2004), and shares similar cellular targets, we here tested whether CO affected neuronal firing activity of neurons as well. While we have evidence that a CO producing, heme oxygenase (HO)-like enzyme (unpublished data) is present in the CNS of the freshwater snail, Helisoma trivolvis, signaling roles for CO in the gastropod CNS are presently not known.
Carbon monoxide (CO), like other gaseous neuromodulators, has a dual nature as both a toxic gas and a physiologically relevant signaling molecule. In the nervous system, high concentrations of CO can lead to neuronal injury while lower concentrations are found to be neuroprotective. The number of cellular targets affected by physiological concentrations of CO is rapidly growing and includes ion channels in various cell types. The modulation of ion channels by CO in neurons, however, and the effect it has on neural activity are incompletely understood. Here, the well-characterized buccal neurons, B5 and B19, of the freshwater snail, Helisoma trivolvis, were used to investigate the role that CO plays in regulating spontaneous firing activity and neuronal excitability. Neurons were studied in single-cell culture, thereby removing other signals normally present in the intact nervous system and allowing for the optimal characterization of physiological effects of CO. We found that the CO donor molecule, carbon monoxide releasing molecule-2 (CORM-2), hyperpolarized the resting membrane potential of B5 neurons and silenced their spontaneous firing activity. These effects were mediated through the inhibition of a persistent sodium current. CORM-2 also inhibited neuronal excitability. This effect was mediated by the inhibition of voltage-gated calcium channels by CO. The general findings of CO acting as a hyperpolarizing signal and an inhibitor of neuronal excitability extended to B19 neurons. Taken together, these findings suggest that CO is a potent modulator of ion channels with broad implications for the modulation of neural activity in a wide range of neuron-types.
The Nuclear Translocation of Heme Oxygenase-1 in Human Diseases
2022, Frontiers in Cell and Developmental Biology

View all citing articles on Scopus

View full text

Immunocytochemical localization and expression of heme oxygenase-1 in primary astroglial cell cultures during differentiation: effect of glutamate

Abstract

Section snippets

Materials and methods

Results

Discussion

Acknowledgements

Int. J. Biochem.

Arch. Biochem. Biophys.

Transplant. Proc.

Biochem. Biophys. Res. Commun.

Brain Res.

Brain Res. Dev. Brain Res.

Brain Res.

Brain Res.

Biochem. Biophys. Res. Commun.

Eur. J. Pharmacol.

Int. J. Dev. Neurosci.

Neuron

Neurosci. Lett.

Clin. Perinatol.

Biochim. Biophys. Acta

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Struct. Biol.

Biochem. Biophys. Res. Commun.

Neuroscience

Neuron