Neonatal hypoxic ischemic brain injury is a significant cause of mortality and lifelong
neurological morbidity. Despite its public health significance, little is know about
the underlying molecular pathways linking hypoxia to neuronal ischemia and cell death.
In this issue of Neurotoxicity Research, Li et al. show that the integrin β8 is upregulated
in cultured astrocytes in response to hypoxia, which subsequently activates a TGFβ-dependant
neuroprotective pathway. These findings support a novel function for astrocyte-derived
integrin β8 and sheds light on the molecular mechanisms underlying hypoxic ischemic
brain injury.
TGFβ-mediated Cytoprotection
TGFβ is a pleotropic cytokine with well-characterized cytoprotective and apoptotic
effects (Annes et al. 2003; Flanders et al. 1998; Unsicker and Krieglstein 2002).
Whether TGFβ promotes cell survival or induces apoptosis depends largely on the cellular
and environmental context, as well as the specificity of TGFβ activation and signaling.
This specificity is achieved through various mechanisms including pericellular extracellular
matrix localization, regulated liberation of active TGFβ from its latent form, and
a vast array of intracellular signaling molecules integrating TGFβ effects on target
tissues. The integrin αvβ8 is a critical activator of TGFβ in vitro and in vivo. Using
cell culture assays, Cambier et al. (2005) showed that astrocyte-derived αvβ8 binds
to and activates TGFβ, which then transactivates vascular endothelial cell TGFβ dependent
signaling. Conditional deletion of β8 from dendritic cells abrogates TGFβ-mediated
activation of regulatory T cells and results in an immunophenotype identical to that
found in TGFβ knockout mice (Travis et al. 2007). Mice with a mutated form of TGFβ1
blocking integrin-mediated activation develop a phenotype identical to that of TGFβ1
null mice (Yang et al. 2007) and similar to that of αv or β8 deficient mice (Bader
et al. 1998; Zhu et al. 2002a, b). Finally, genetic loss of TGFβ in mice results in
apoptotic neuronal loss accompanied by diffuse astrogliosis, and increased neuronal
susceptibility to kainic acid-induced excitotoxic injury (Brionne et al. 2003). Moreover,
in vivo TGF-β1 administration in mice protects against ischemic brain injury (McNeill
et al. 1994; Zhu et al. 2002a, b), and overexpression of TGFβ1 from astrocytes protects
against excitotoxic neuronal injury (Brionne et al. 2003). While these experiments
support β8’s role in the activation of TGFβ, and TGFβ’s role in neuroprotection, there
has been little direct evidence demonstrating transcellular activation of neuroprotective
signaling pathways by β8. Li et al. (2009) provide such evidence. They show that in
the presence of β8 expressing astrocytes, TGFβ protects against hypoxia-induced apoptotic
cell death, in part by upregulating canonical antiapoptotic proteins BCL2 and BCLxl.
Importantly, BCLxl is induced by TGFβ1 via TGFβ receptor, ALK1 activation of NF-kappaB,
promoting neuronal survival after injury (König et al. 2005). It will be interesting
to see if this same β8–TGFβ–ALK1 pathway is important for neuronal maintenance and
for protection against hypoxic ischemic injury in vivo.
TGFβ Activation
As was previously shown by other groups (Cambier et al. 2005; Mobley et al. 2009),
Li et al. (2009) demonstrate that astrocyte-derived β8 activates TGFβ. Different from
other studies (Cambier et al. 2005), however, they found that neither matrix metaloprotease
(MMP) inhibition nor β8 knockdown could completely abrogate TGFβ activation. While
the authors note this may be due to ineffective MMP inhibition, or due to incomplete
β8 knockdown, it is also possible that alternative activators of TGFβ are upregulated
in response to hypoxia, such as other integrins or receptors. For example, the VEGF
co-receptor, neuropilin 1 (Nrp1) is highly expressed on neurons, regulates neuroprotection
in response to hypoxia (Oosthuyse et al. 2001), and was recently found to bind to
and activate TGFβ (Glinka and Prud’homme 2008). Interestingly, there are numerous
parallels between Nrp1 and β8. For instance, Nrp1 and β8 knockouts have similar cerebrovascular
phenotypes. Also, the adult neurological phenotype of αv or β8 deficient mice (McCarty
et al. 2005; Proctor et al. 2005; Mobley et al. 2009) is strikingly similar to that
of mice with deletion of the hypoxia-response element in the VEGF promoter (Oosthuyse
et al. 2001), where hypoxia-induced VEGF was found to have a neuroprotective role
mediated in part through neuronal Nrp1. Considering these parallels, it will be interesting
to determine whether neuro-glial Nrp1 can specifically activate TGFβ in response to
hypoxia, and whether this occurs in the context of the β8–TGFβ interaction.
Hypoxia-Induced β8 Expression
Li et al. (2009) show that astocytic β8 is upregulated in response to hypoxemia and
that the timing of hypoxia-induced TGFβ activation mirrors peak expression levels
of β8. Why might β8 expression be responsive to hypoxia/ischemia? During development,
β8 plays an essential role in vascular ingression and remodeling in the brain (Zhu
et al. 2002a, b; Proctor et al. 2005). Here, it is plausible that glial-derived β8
regulates neovascularization, coupling the metabolic needs of developing neuroepithelial
cells to the vasculature that supplies oxygen and nutrients. It is tempting to speculate
that β8 plays a similar dual role in regulating neo-vascularization and neuronal survival
in response to hypoxic damage. How does hypoxia signal astrocytes to upregulate β8?
Based on the observations of Li et al. (2009), β8 may have direct or indirect autocrine
signaling effects through TGFβ. TGFβ and hypoxia cooperatively signal through hypoxia
inducible factor (HIF)-1α to regulate transcription of endothelial-derived VEGF, and
control angiogenesis and endothelial apoptosis (Ferrari et al. 2006; Sánchez-Elsner
et al. 2001). HIF1α and its major target gene, VEGF, are upregulated in response to
hypoxia/ischemia, and may protect against neuronal cell death in this setting (Sheldon
et al. 2009). Considering these recent reports, it will be important to determine
whether β8 is involved in regulation of HIF1α and VEGF, and alternatively how hypoxia,
HIF1α and VEGF may regulate expression of β8. This line of study may more fully elucidate
the pathophysiology of hypoxic ischemic brain injury and could help identify novel
treatment targets. Taken together the findings of Li et al. (2009), one may speculate
that β8 is critically important for neuronal maintenance and protection against hypoxic
insult in vivo. Testing of this hypothesis will require astrocyte specific deletion
of β8, and in vivo characterization of these mice in various injury paradigms including
hypoxia/ischemia brain injury.