Abstract
Following injury to the peripheral and central nervous systems, tissue levels of transforming growth factor (TGF)-β1 often increase, which is key for wound healing and scarring. However, active wound regions and scars appear to inhibit process outgrowth by regenerating neurons. We recently showed that corneal wound myofibroblasts block corneal nerve regeneration in vivo, and sensory neurite outgrowth in vitro in a manner that relies critically on TGF-β1. In turn, delayed, abnormal re-innervation contributes to long-term sensory dysfunctions of the ocular surface. Here, we exposed morphologically and biochemically-differentiated sensory neurons from the ND7/23 cell line to TGF-β1 to identify the intracellular signals regulating these anti-neuritogenic effects, contrasting them with those of Semaphorin(Sema)3A, a known inhibitor of neurite outgrowth. Neuronal morphology was quantified using phase-contrast imaging. Western blotting and specific inhibitors were then used to identify key molecular mediators. Differentiated ND7/23 cells expressed neuron-specific markers, including those involved in neurite extension and polarization. TGF-β1 increased phosphorylation of collapsin response mediator protein-2 (CRMP2), a molecule that is key for neurite extension. We now show that both glycogen synthase kinase (GSK)-3β and Smad3 modulate phosphorylation of CRMP2 after treatment with TGF-β1. GSK-3β appeared to exert a particularly strong effect, which could be explained by its ability to phosphorylate not only CRMP2, but also Smad3. In conclusion, TGF-β1's inhibition of neurite outgrowth in sensory neurons appears to be regulated through a highly-conserved signaling pathway, which involves the GSK-3β/CRMP-2 loop via both canonical and non-canonical mechanisms. It is hoped that by defining the signaling pathways that control neurite outgrowth in wound environments, it will become possible to identify optimal molecular targets to promote re-innervation following injury.
Highlights
We previously reported that transforming growth factor (TGF)-β1-induced reduction in neurite outgrowth by ND7/23 cells is associated with increased phosphorylation of collapsin response mediator protein-2 (CRMP2) [16]
To investigate the effects of TGF-β1 on neuritogenesis, we examined the morphology of ND7/ 23 cells after 1 day in culture, and measured their relative expression of pGSK-3β/tGSK-3α/β and pCRMP2/tCRMP2 using western blots
Compared to undifferentiated ND7/23 cells, culture in serum free medium (SFM) for 1 day caused an upregulation of growth associated protein-43 (GAP-43), Ac-Tub and calcitonin gene-related peptide (CGRP)
Summary
Nerve injuries pose many challenges to patients, ranging from mild discomfort to life-long impairment due to pain, loss of sensitivity, motor function and/or autonomic control [1]. Corneal nerves arise from the trigeminal ganglion, and as schematically represented, are distributed through the anterior half of the stroma and the epithelium They are predominantly mechano-sensory and nociceptive, and serve to protect the eye from outside elements [20,21,22]. Key to the present experiments, this effect was reproduced in vitro, when corneal myofibroblasts were co-cultured with neurons derived from the ND7/23 cell line, they inhibited neurite outgrowth [16]. TGF-β1 was shown to be both necessary and sufficient for the anti-neuritogenic effect of myofibroblasts on differentiated ND7/23 cells [16], allowing us to use this growth factor as a surrogate [in place of myofibroblasts] to define molecular pathways that inhibit neurite outgrowth in pure neuronal cultures (i.e., cellautonomously).
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