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. 2013 May 21:11:35.
doi: 10.1186/1478-811X-11-35.

Inhibition of a novel specific neuroglial integrin signaling pathway increases STAT3-mediated CNTF expression

Matthew P Keasey, Seong Su Kang, Chiharu Lovins, Theo Hagg

Inhibition of a novel specific neuroglial integrin signaling pathway increases STAT3-mediated CNTF expression

Matthew P Keasey et al. Cell Commun Signal. .

Abstract

Background: Ciliary neurotrophic factor (CNTF) expression is repressed in astrocytes by neuronal contact in the CNS and is rapidly induced by injury. Here, we defined an inhibitory integrin signaling pathway.

Results: The integrin substrates laminin, fibronectin and vitronectin, but not collagen, thrombospondin or fibrinogen, reduced CNTF expression in C6 astroglioma cells. Antibodies against αv and β5, but not α6 or β1, integrin induced CNTF. Together, the ligand and antibody specificity suggests that CNTF is repressed by αvβ5 integrin. Antibodies against Thy1, an abundant neuronal surface protein whose function is unclear, induced CNTF in neuron-astrocyte co-cultures indicating that it is a neuroglial CNTF repressor. Inhibition of the integrin signaling molecule Focal Adhesion Kinase (FAK) or the downstream c-Jun N-terminal kinase (JNK), but not extracellular regulated kinase (ERK) or p38 MAPK, greatly induced CNTF mRNA and protein expression within 4 hours. This selective inhibitory pathway phosphorylated STAT3 on its inhibitory ser-727 residue interfering with activity of the pro-transcription Tyr-705 residue. STAT3 can activate CNTF transcription because it bound to its promoter and FAK antagonist-induced CNTF was reduced by blocking STAT3. Microinjection of FAK inhibitor directly into the brain or spinal cord in adult mice rapidly induced CNTF mRNA and protein expression. Importantly, systemic treatment with FAK inhibitors over 3 days induced CNTF in the subventricular zone and increased neurogenesis.

Conclusions: Neuron-astroglia contact mediated by integrins serves as a sensor to enable rapid neurotrophic responses and provides a new pharmacological avenue to exploit the neuroprotective properties of endogenous CNTF.

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Figures

Figure 1
Figure 1
CNTF is repressed by ECM and specific αvβ5 integrin and neuronal Thy-1 ligand. A) C6 astroglioma cells grown for 4 h on laminin (LAM), fibronectin (FN1) and vitronectin (VTN) had lower levels of CNTF mRNA compared to poly-d-lysine control (Ctrl, set at 1.00) as quantified by qRT-PCR. Fibrinogen (FG), thrombospondin (TSP), and collagen (COL) had no significant effect. Because of the binding specificity this suggests that a limited number of integrins repress CNTF. B) Neutralizing antibodies against αv or β5, but not α6 or β1 induce CNTF mRNA expression relative to IgG (Ctrl) in C6 cells after 4 hours. Antibody selection was guided by elimination of unlikely candidates (Table 1). The β5 subunit is only found in αvβ5. C) Neutralizing antibody against Thy-1, an αvβ5 ligand, induces CNTF mRNA expression in primary astrocyte-neuron co-cultures compared to IgG (Ctrl). Data represent means (± SEM) of 3–4 (A) or 6 (B, C) independent experiments and are fold changes expressed relative to controls.
Figure 2
Figure 2
FAK inhibition induces CNTF expression in vitro. A) C6 cells incubated for 4 hours with FAK inhibitor PF573228 have increased levels of CNTF mRNA compared to vehicle-only cultures (Ctrl). B) The inhibitor reduced FAK activity as shown in Western blots by the reduced levels of phosphorylated FAK (pFAK tyr-397). C) CNTF protein was also increased in the same protein extracts, showing the rapid and robust nature of the disinhibition. Blots are representative of 4 independent observations. D) C6 cell injury by mechanical dissociation robustly induced CNTF mRNA within 2 hours which was lost by 6 hours. E) FAK inhibition did not augment the dissociation-induced CNTF mRNA up-regulation. Data are fold change compared to controls and are shown as average +/− SEM. In A, n = 6, D and E, n = 4.
Figure 3
Figure 3
A specific intracellular FAK-JNK signaling pathway represses CNTF. A) Incubation of C6 cells with inhibitors of JNK, but not p38 or ERK, for 4 hours, increases CNTF mRNA compared to vehicle controls (Ctrl). Data are means +/− SEM and are from 4 independent experiments. B) Incubation with FAK inhibitor reduced JNK activation as shown by reduced JNK phosphorylation in western blots, which are representative of 4 independent observations. Antibodies against total JNK were used as internal controls.
Figure 4
Figure 4
STAT3 inhibition mediates CNTF repression by FAK. A) FAK inhibition reduces STAT3 phosphorylation on its inhibitory residue (s727) as shown in western blots of C6 cells incubated for 4 hours. Total STAT3 antibodies were used as internal controls. B) Pre-incubation with a STAT3 antagonist reduced the CNTF-inducing effects of FAK inhibitor in C6 cells. Thus, FAK represses CNTF by inhibiting STAT3. An AP1 transcription factor antagonist was without effect. Data are fold compared to control (not shown) and means +/−SEM. C) STAT3 binds the CNTF promoter region of C6 cells as shown by ChIP analysis. STAT3 antibody immunoprecipitates (IP) CNTF promoter DNA as shown by the PCR amplification products of two promoter-specific primer sets. Normal rabbit IgG was used as control for non-specific binding. Histone H3 was a positive control. D) Sequencing of the amplification products of the STAT3 immunoprecipitate gave the predicted DNA regions. Key: Consensus STAT3 binding sites; formula image: CNTF initiation site; CNTF1 primer set (FWD, REV); formula image; < > denote DNA regions excluded from this panel for presentation purposes. E) IL-6 treatment of C6 cells for 15 minutes robustly increased phosphorylation of STAT3 at the Tyr-705 residue (y705) with modest increases after CNTF and LIF as shown by western blot. Ser-727 phosphorylation (s727) or total STAT3 (tSTAT3) was not affected. Similar results were seen at 4 hours. The blot is representative of 4 independent experiments. F) IL-6 induced only an ~10% increase in CNTF mRNA expression in C6 cells after 4 hours and did not augment FAKi-induced CNTF expression (n = 3-4 each, p < 0.05). G) FAK inhibition reduced phosphorylation of STAT3 (y705) in C6 cells most notably under IL-6 treated conditions. Antibodies against total STAT3 were used as internal controls for western blots. Results were repeatable in independent experiments.
Figure 5
Figure 5
FAK inhibition induces CNTF expression in the adult CNS in vivo. A) Intracerebral injection of the FAK inhibitor PF573228 directly in the striatum of mice results in increased CNTF protein 4 hours later as shown in western blots. FAK activity was greatly decreased as shown by lack of phosphorylation. (pFAK). α-tubulin = internal control. Lanes are from individual animals. B) Similar results were obtained after direct injection into the spinal cord. C) Systemic intraperitoneal injections of the FAK inhibitors PF573228 or FAK14 induce CNTF mRNA in the spinal cord or SVZ relative to vehicle only injected mice (Ctrl). Drugs were given 3 times every 24 hours and tissues collected 2 hours after the last injection. Data are fold change compared to control (Ctrl) and are means +/− SEM from 3–4 mice per group.
Figure 6
Figure 6
Systemic FAK inhibitor treatment increases adult CNS neurogenesis. A) mRNA for the proliferation marker Ki67 and the marker for proliferating neural C cell progenitors, EGFR, increased in the SVZ after systemic FAK inhibitor PF573228 treatment in mice. CNTF mRNA was increased in the same extracts (Figure 6C). Drugs were given 3 times every 24 hours and tissues collected 2 hours after the last injection. Data are mean fold change compared to vehicle treated mice +/− SEM, n = 3–4 mice. B) In other mice, the systemic injections of FAK inhibitor PF573228 (n = 6) increased the number of DCX + neuroblasts in the SVZ. Data are expressed as a mean percentage of vehicle (n = 4) treated mice +/− SEM. C) Representative confocal images of the dorsal SVZ showing increased numbers of nuclei (Hoechst) and neuroblasts (dcx) in an FAK inhibitor-injected mouse compared to a vehicle-injected mouse. Scale bar = 20 μm. CC = corpus callosum, LV = lateral ventricle, STR = striatum.
Figure 7
Figure 7
Schematic representation of the proposed pathways that regulate astroglial CNTF. This study identifies an inhibitory pathway (Red) where ligands such neuronal Thy-1 bind to astrocyte αvβ5 integrin resulting in FAK phosphorylation. Subsequently, JNK is activated and phosphorylates STAT3 at an inhibitory serine-727 (s727) residue potently repressing transcription of CNTF. Conversely, the CNTF-stimulatory pathway is activated by neural cytokines such as CNTF, LIF and IL-6 which bind to their respective gp130 (gp) receptor complexes (CNTF: CNTFαR/LIFβR/gp130; LIF: LIFβR/gp130; IL-6: IL-6αR/gp130/gp130; Lβ indicates LIFβR), triggering recruitment of JAK to activate STAT3 by phosphorylation at the pro-transcription residue tyrosine-705, y705; Green). CNTF is known to activate STAT3 in astrocytes in vitro [9,87-90] and in vivo [91]. FAK may simultaneously stimulate the activating pathway, potentially representing another component of the tightly regulated CNTF gene expression under physiological conditions.
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