A1 and A3 Adenosine Receptors Inhibit LPS-Induced Hypoxia-Inducible Factor-1 Accumulation in Murine Astrocytes

Abstract

Adenosine (Ado) exerts neuroprotective and anti-inflammatory functions by acting through four receptor subtypes: A1, A2A, A2B, and A3. Astrocytes are one of its targets in the central nervous system. Hypoxia-inducible factor-1 (HIF-1), a master regulator of oxygen homeostasis, is induced after hypoxia, ischemia, and inflammation and plays an important role in brain injury. Although HIF-1 is expressed by astrocytes, the regulatory role played by adenosine on HIF-1α modulation induced by inflammatory and hypoxic conditions has not been investigated.

Primary murine astrocytes were activated with lipopolysaccharide (LPS) with or without adenosine, adenosine receptor agonists, antagonists, and receptor silencing, before exposure to normoxia or hypoxia. HIF-1α accumulation and downstream gene regulation were determined.

Adenosine inhibited LPS-increased HIF-1α accumulation under both normoxic and hypoxic conditions, through activation of A1 and A3 receptors. In cells incubated with blockers of p44/42 MAPK and Akt, LPS-induced HIF-1α accumulation was significantly decreased in normoxia and hypoxia, suggesting the involvement of p44/42 MAPK and Akt in this effect. Adenosine inhibited kinases phosphorylation. A series of angiogenesis and metabolism-related genes were modulated by hypoxia in an HIF-1 dependent way, but not further increased by LPS, except for GLUT-1 and hexokinase II, which were elevated by LPS only in normoxia and inhibited by adenosine receptors. Instead, genes involved in inflammation, like inducible nitric-oxide synthase (iNOS) and A2B receptors, were increased by LPS in normoxia, strongly stimulated by LPS in concert with hypoxia, and inhibited by adenosine through A1 and A3 receptor subtypes. In conclusion, A1 and A3 receptors reduce the LPS-mediated HIF-1α accumulation in murine astrocytes, resulting in a downregulation of genes involved in inflammation and hypoxic injury, such as iNOS and A2B receptors, in both normoxic and hypoxic conditions.

Introduction

Astrocytes are the predominant cell type in the central nervous system (CNS), maintaining neuronal homeostasis and plasticity. Upon CNS injury, astrocytes become reactive and modulate inflammation, which is critical in many CNS disorders such as stroke, ischemia, trauma, and neurodegenerative diseases. Hypoxia-inducible factor-1 (HIF-1) is a key transcription factor activated during hypoxia, ischemia, and inflammation, regulating genes involved in oxygen homeostasis and brain injury. While HIF-1 is expressed by astrocytes, its regulation by adenosine under inflammatory and hypoxic conditions was previously unknown.

Adenosine, a signaling molecule derived from ATP breakdown, mediates physiological effects via four G-protein-coupled receptors: A1, A2A, A2B, and A3. In glial cells, adenosine receptor activation mediates neuroprotection after hypoxic insult. Specifically, A1 and A3 receptor activation protects astrocytes from hypoxic damage and inhibits inflammatory cell migration. Adenosine is known to affect HIF-1 induction in peripheral inflammatory cells, but its role in primary astrocytes was unclear.

This study examined whether adenosine and its analogs modulate HIF-1α accumulation and transcription in primary astrocytes activated by LPS under normoxic and hypoxic conditions.

Materials and Methods

Primary Astrocyte Cultures: Astrocytes were isolated from newborn Balb/c mice and cultured in DMEM with FBS and antibiotics. Cultures were characterized by GFAP immunostaining.

Treatments: Cells were treated with LPS (1 μg/mL) and/or adenosine, adenosine receptor agonists/antagonists, or siRNAs targeting A1, A2B, or A3 receptors, then exposed to normoxia or hypoxia (1% O₂).

Immunofluorescence and Western Blot: HIF-1α and other proteins were detected by immunofluorescence or immunoblotting.Real-Time RT-PCR: Quantified mRNA levels of HIF-1α, VEGF, GLUT1, GLUT3, HK2, GAPDH, iNOS, and A2B.

Nitrite Assay: Measured NO₂⁻ as a readout of NOS activity.

Statistical Analysis: Data were analyzed by ANOVA and Dunnett’s test; p < 0.05 was considered significant.

Results

LPS Increases HIF-1α Expression

LPS significantly increased HIF-1α accumulation in murine astrocytes under both normoxic and hypoxic conditions, in a time- and dose-dependent manner. Hypoxia alone also induced HIF-1α, but LPS further enhanced this effect.

Adenosine Inhibits LPS-Induced HIF-1α Accumulation

Adenosine reduced LPS-induced HIF-1α accumulation in a dose-dependent manner under both normoxia and hypoxia. This effect was mimicked by A1 agonist (CHA) and A3 agonist (Cl-IB-MECA), and blocked by their respective antagonists (PSB 36 for A1, MRS 1523 for A3), confirming receptor specificity. Silencing A1 or A3 receptors by siRNA partially reversed the adenosine effect.

Mechanisms of HIF-1α Regulation

LPS-induced HIF-1α accumulation was transcriptionally regulated, as actinomycin D blocked the increase. LPS increased HIF-1α mRNA under both normoxic and hypoxic conditions; adenosine reduced this effect. LPS and adenosine did not affect HIF-1α protein degradation after reoxygenation.

Involvement of MAPK and Akt Pathways

LPS-induced HIF-1α accumulation was significantly reduced by inhibitors of MEK1/2 (U0126) and Akt (SH5), implicating ERK1/2 and Akt pathways. Adenosine inhibited LPS-induced phosphorylation of these kinases.

Downstream Gene Regulation

Metabolic/Angiogenic Genes: Hypoxia upregulated VEGF, GLUT1, GLUT3, HK2, and GAPDH in an HIF-1-dependent manner. LPS did not further increase these genes under hypoxia, but did increase GLUT1 and HK2 in normoxia, which was inhibited by A1 and A3 activation.

Inflammatory Genes: LPS increased iNOS and A2B receptor expression in normoxia and even more so with hypoxia. Adenosine, via A1 and A3 receptors, inhibited this upregulation. These effects were dependent on HIF-1α and involved ERK1/2 and Akt pathways.

Protein Expression

LPS increased iNOS and A2B protein levels, especially under hypoxia; adenosine reduced these increases. Nitrite assays confirmed reduced NO production with adenosine treatment.

Discussion

This study demonstrates for the first time that adenosine, via A1 and A3 receptor activation, inhibits LPS-induced HIF-1α accumulation and expression in murine astrocytes under both normoxic and hypoxic conditions. This effect is mediated by inhibition of ERK1/2 and Akt phosphorylation, leading to downregulation of genes involved in inflammation and hypoxic injury, such as iNOS and A2B receptors. Adenosine did not affect genes related to angiogenesis and metabolism, except for GLUT1 and HK2, which were stimulated by LPS in normoxia and reduced by A1 and A3 activation. These findings reveal a new molecular pathway by which adenosine reduces inflammatory and hypoxic gene expression in astrocytes, potentially offering neuroprotection in stroke, ischemia, and other CNS disorders.

Conclusion

Adenosine, through A1 and A3 receptor activation, reduces LPS-stimulated HIF-1α mRNA expression and protein accumulation by inhibiting LPS-triggered ERK1/2 and Akt phosphorylation in both normoxic and hypoxic conditions. This leads to inhibition of inflammation-related genes, such as iNOS and A2B receptors, providing a potential CGS 21680 neuroprotective mechanism in CNS inflammatory and hypoxic injury.