Notch1-STAT3-ETBR signaling in brain injury and cancer.
Based on their relative distance from sites of CNS injury or disease and the severity of insult, astrocytes and other glial cells characteristically become ‘‘reactive”. Reactive astrocytes are phenotypically different from mature protoplasmic or fibrous astrocytes that reside in normal, healthy tissue. During the process of reactive astrogliosis, mature astrocytes undergo dramatic changes in gene and protein expression that beget changes in morphology (hypertrophy) and cell cycle status (Fig. 1). Proliferating reactive astrocytes in close proximity to damaged/dying cells carry out key functions that include neuroprotection, preservation/ re-establishment of the blood–brain barrier (BBB), regulation of immune cell responses, and glial scar formation [1]. Importantly, mechanistic understanding and mapping of the signaling network that controls reactive astrogliosis has great potential to elucidate therapeutic targets and inform the development of new treatments to promote recovery and improve outcomes in patients with CNS injury or disease. Multiple proteins/peptides commonly released during CNS injury have been shown to stimulate reactive astrocyte proliferation such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and endothelin (ET-1). Other cytokines/growth factors, molecules, and conditions known to trigger reactive astrogliosis include (but are not limited to): IL-1, IL-6, IL-10, LIF, CNTF, BMP, TGF-b, TNF-a, INF-c, lipopolysaccharide, glutamate, adenosine triphosphate, nitric oxide, reactive oxygen species, and hypoxia and glucose deprivation during tissue ischemia [1]. Although numerous signals released or produced during CNS injury have been shown to stimulate reactive astrocytes in culture, the complex signaling network that regulates reactive astrocyte proliferation and function(s) in vivo remains poorly understood. Reactive astrocytes commonly exhibit increased expression of several intermediate filament proteins such as Glial Fibrillary Acidic Protein (GFAP), Nestin, and RC2, a specialized isoform of Nestin with post-translational modifications (Fig. 1). Previously, our group demonstrated Notch1 signaling was required for the proliferation of reactive astrocytes after stroke. Using an inducible transgenic mouse model with Cre–loxP-mediated gene excision (GFAP-CreER-Notch1-cKO), we reported that conditional knockout (cKO) of Notch1 in reactive astrocytes significantly reduced the number of RC2-positive cells immediately adjacent to the infarct core and increased immune cell invasion after stroke [2]. Recently, we demonstrated that the RC2 antigen marks specifically the subpopulation of proliferating reactive astrocytes after stroke and traumatic brain injury and reported a method to prospectively isolate the subpopulation of proliferating reactive astrocytes from injured CNS tissue based on expression of the glutamate–aspartate transporter (Glast) and Jagged1, a Notch1 ligand [3]. In the peri-infarct area after stroke, RC2-positive reactive astrocytes expressed high levels of ETBR, prompting us to investigate whether Notch1 signaling interacted with ETBR. We found that Jagged1/Notch1 signaling increased ETBR levels in an indirect manner, by stabilizing phosphorylated STAT3 (p-STAT3), a transcriptional activator for EDNRB (ETBR gene). Subsequent experiments with GFAP-CreER-ETBR-cKOmice demonstrated that ETBR was necessary for reactive astrocyte proliferation after stroke. Notably, the deficit observed in GFAP-CreER-ETBR-cKO mice closely resembled that of GFAP-CreER-Notch1-cKO mice. Notch1–STAT3–ETBR signaling may affect numerous cell types that proliferate during development, tissue repair, and cancer. In addition to primary adult astrocytes, we observed that Notch1–STAT3–ETBR signaling regulated proliferation and/or survival of primary neonatal astrocytes as well as human tumor cell lines derived from kidney (HEK 293) or brain (U87; glioma, astrocytoma) [3]. Whereas we found that Jagged1/Notch1 signaling promoted increased ETBR levels in postnatal astrocytes and adult reactive astrocytes, ET-1 was recently shown to induce Jagged1 expression in cultured astrocytes and to alter Notch signaling in vivo during the first week after lysolecithin-induced focal demyelination [4]. In the same injury model, reactive astrogliosis was previously shown to result in ET-1 signaling through ETBR, ERK and JNK pathways, and activation of the transcription factor c-jun [5]. Importantly, c-jun can bind to cis-acting AP-1 DNA elements in the Jag1 promoter to drive Jagged1 expression [6]. In light of these observations, our recent data suggest a feed-forward loop whereby ET-1 signaling through ETBR increases Jagged1 levels, which then can activate Notch1 signaling in adjacent astrocytes. Notch1 signaling increases the expression of ETBR by stabilizing p-STAT3, and in turn enhances expression of Jagged1 by amplifying ET-1 signaling through ETBR. Accordingly, we expect that Jagged1/Notch1, STAT3, and ET-1/ETBR signaling all contribute to driving reactive astrocyte proliferation after CNS injury through this feed-forward loop. Similar feed-forward signaling loops promoted by STAT3