Inhibition of a novel specific neuroglial integrin signaling pathway increases STAT3-mediated CNTF expression
Cell Communication and Signaling
Inhibition of a novel specific neuroglial integrin signaling pathway increases STAT3-mediated CNTF expression
Matthew P Keasey 1
Seong Su Kang 1
Chiharu Lovins 1
Theo Hagg 0 1
0 Pharmacology and Toxicology, University of Louisville , Louisville, KY 40292 , USA
1 Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville , Louisville, KY 40292 , USA
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 v and 5, but not 6 or 1, integrin induced CNTF. Together, the ligand and antibody specificity suggests that CNTF is repressed by v5 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.
Astrocyte; Ciliary neurotrophic factor; Focal adhesion kinase; Integrin; Intercellular communication; Mice; Neurogenesis; Neuron; Pharmacological; Transcriptional gene regulation
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Background
Endogenous CNTF regulates the development of
oligodendrocytes [1] and some neurons [2], synaptic function
[3], and adult CNS neurogenesis [4,5]. CNTF treatment is
neuroprotective in many animal models [6-10], and
promotes retinal ganglion cell regeneration [11,12] and
remyelination [13]. Even so, clinical trials failed due to low
penetration of CNTF into the CNS and systemic side
effects after subcutaneous injections [14]. CNTF is almost
exclusively expressed in the nervous system, suggesting
that its pharmacological induction might solve these
problems. In the CNS, CNTF is produced at very low levels
primarily by astrocytes [15] but little is known about
mechanisms that regulate its expression. We found that a
cAMP-reducing dopamine D2 agonist induces CNTF in
the brain but not the spinal cord [5], indicating the need
to find more universal regulation mechanisms.
The expression of CNTF is rapidly and robustly
induced in astrocytes upon brain injury [16] and stroke
[17], where it serves a neuroprotective role [18], as it
does in an experimental autoimmune encephalomyelitis
(EAE) model [19] and the retina [20]. We found that glial
CNTF is repressed by integrins and, conversely, that loss
of neuron-astroglial interaction increases CNTF in vitro
and in the mouse striatum after ischemic or excitotoxic
neuronal loss [18].
Integrins are a group of 24 heterodimer receptors with
alpha and beta subunits binding extracellular matrix
(ECM) proteins as adhesion partners [21]. The neuronal
ligands that bind astroglial integrins to regulate CNTF are
unknown. Neurons do not make most of the classical
ECM molecules although they express laminin isoforms
[22,23]. Thy-1, whose function is unknown, is highly
expressed by adult neurons [24] and is a ligand of v3 [25]
and v5 integrins [26] which are expressed by astrocytes
and astroglioma cells [27,28]. Integrins signal through focal
adhesion kinase (FAK) which can signal downstream to
the ERK, p38 and JNK pathways [29]. The intracellular
signaling pathways that regulate CNTF are unknown. The
transcription factor Sox-10 regulates CNTF expression in
Schwann cells [30] but is not present in astrocytes [31].
IL6 and CNTF itself induce CNTF expression [12,18],
suggesting a potential role of STAT3, which is downstream
of their gp130 receptor [32,33].
We set out to identify the CNTF-repressing signaling
pathway from neuronal ligand to astroglial transcription
factor, and whether its pharmacological inhibition would
increase functional CNTF using adult SVZ neurogenesis
as an outcome measure.
Results
Glial CNTF is repressed through v5 integrin
To ide (...truncated)