STAT3 integrates cytokine and neurotrophin signals to promote sympathetic axon regeneration
Introduction
Sympathetic neurons, like other peripheral neurons, can regenerate following injury although the mechanisms are not completely understood. Nerve growth factor (NGF) is required for sympathetic neuron survival and enhances sympathetic axon outgrowth during development (Glebova and Ginty, 2005), but its role in adult nerve regeneration is less clear. Mature sympathetic neurons no longer require NGF for their survival (Ruit et al., 1990, Sofroniew et al., 2001), and regeneration of adult sympathetic axons to the skin after nerve transection does not require NGF (Gloster and Diamond, 1995). However, collateral sprouting of sympathetic axons into injured skin (Gloster and Diamond, 1992, Gloster and Diamond, 1995), arthritic joints (Ghilardi et al., 2012), or damaged myocardium (Hasan et al., 2006, Wernli et al., 2009) is stimulated by NGF in the target tissue and can be blocked by NGF antibodies.
The milieu produced by tissue damage is complex and includes many factors in addition to NGF, including the inflammatory cytokines CNTF (Ciliary Neurotrophic Factor) and LIF (Leukemia Inhibitory Factor) (Adler, 1993, Rao et al., 1993). These cytokines act via the gp130 receptor (Ip et al., 1992, Taga and Kishimoto, 1997) to promote axon regeneration in the central and peripheral nervous systems (Cafferty et al., 2001, Ekstrom et al., 2000, Homs et al., 2011, Leibinger et al., 2009). In sympathetic neurons cytokines are involved in the “conditioning lesion” response whereby prior injury enhances the subsequent regeneration (Hyatt Sachs et al., 2010, McQuarrie and Grafstein, 1973, Navarro and Kennedy, 1990, Shoemaker et al., 2005). Cytokines are thought to enhance nerve regeneration after injury through stimulating transcription of regeneration associated genes via tyrosine phosphorylation of Signal Transducer and Activator of Transcription 3 (STAT3) (Ben-Yaakov et al., 2012, Habecker et al., 2009, Lee et al., 2004, Liu and Snider, 2001, O'Brien and Nathanson, 2007, Qiu et al., 2005, Smith and Skene, 1997). A second subcellular locus of STAT3 action was identified recently in embryonic motor neurons, where tyrosine-phosphorylated STAT3 enhanced microtubule stability in motor axons from pmn (progression motor neuronopathy) mice, which contain unstable microtubules (Selvaraj et al., 2012).
NGF acting through the TrkA receptor can also stimulate phosphorylation of STAT3, but NGF triggers phosphorylation of STAT3 on serine (Ng et al., 2006b, Zhou and Too, 2011) rather than tyrosine. Likewise, activation of TrkB by Brain Derived Neurotrophic Factor (BDNF) leads to serine 727 (S727) phosphorylation of STAT3 in hippocampal (Y.P. Ng et al., 2006) and cortical (Zhou and Too, 2011) neurons. Thus, STAT3 serves as a downstream mediator for neurotrophin signaling as well as cytokine signaling. Interestingly, serine phosphorylation of STAT3 is required for neurotrophin-stimulated neurite extension in PC12 cells and axon regeneration in hippocampal and cortical neurons (Ng et al., 2006b, Zhou and Too, 2011). NGF stimulates transcription of regeneration associated genes in PC12 cells via STAT3 (Y.P. Ng et al., 2006), but serine phosphorylated STAT3 is also found in axons and growth cones (Ng et al., 2006b, Zhou and Too, 2011), suggesting that it may play a non-transcriptional role.
We have investigated the role of NGF and gp130 cytokines on sympathetic nerve sprouting after myocardial infarction (MI), which is a common source of nerve damage in humans. Over 1 million people in the U.S. suffer an MI each year (Roger et al., 2012), resulting in the loss of sympathetic nerve terminals in undamaged peri-infarct myocardium (Barber et al., 1983, Inoue and Zipes, 1988, Vaseghi et al., 2012). NGF is elevated in the heart following MI (Abe et al., 1997, Hiltunen et al., 2001, Meloni et al., 2010, Zhou et al., 2004), and blocking NGF with antibodies or preventing infiltration of NGF-producing immune cells into the heart inhibits post-infarct sympathetic nerve sprouting (Hasan et al., 2006, Wernli et al., 2009). However, LIF and several other gp130 cytokines are also elevated in the heart following myocardial infarction (Aoyama et al., 2000, Fischer and Hilfiker-Kleiner, 2007, Frangogiannis et al., 2002, Gwechenberger et al., 1999, Hilfiker-Kleiner et al., 2010), and their role in sympathetic regeneration has not been examined in vivo. In this study, we identify STAT3 as an integrator of neurotrophin and inflammatory cytokine signaling. We show that gp130 cytokine signaling is necessary for the NGF-induced sympathetic nerve sprouting in the heart after MI, and provide evidence that STAT3 plays a non-transcriptional role in sympathetic axons that complements its role in stimulating regeneration associated genes.
Section snippets
STAT3 enhances sympathetic axon regeneration
STAT3 is involved in axon regeneration in several types of neurons (Miao et al., 2006, Qiu et al., 2005, Smith et al., 2009), and we asked if STAT3 was required for sympathetic axon regeneration. To address this question, we cultured SCG explants from wild-type mice and mice whose sympathetic neurons lack STAT3 (Fig. 1A–B). We compared growth rates from STAT3 KO and wild-type ganglia and discovered that axon growth was impaired in neurons lacking STAT3 (Fig. 1C).
NGF stimulates serine phosphorylation of STAT3 in sympathetic axons via ERK1/2 pathway
Ng and colleagues made the
gp130 signaling is required for NGF-dependent nerve sprouting after myocardial infarction
Sympathetic nerve regeneration has been characterized in many peripheral tissues, and two general categories of regeneration have been identified: NGF-independent, as seen in the regrowth of transected nerves back to skin (Gloster and Diamond, 1995), and NGF-dependent, as seen in the collateral sprouting of sympathetic axons within injured skin (Gloster and Diamond, 1992, Gloster and Diamond, 1995) or damaged myocardium (Gardner and Habecker, in press, Hasan et al., 2006). Neurotrophins and
Materials
Matrigel™ was purchased from BD Biosciences (San Jose, CA). Fetal bovine serum was purchased from ATCC (Manassas, VA). Goat serum was from Jackson Immunoresearch Laboratories (West Grove, PA). CNTF was from PreproTech (Rocky Hills, NJ). Nerve growth factor (NGF) was purchased from Austral Biologicals (San Ramon, CA). Dispase was purchased from Boehringer Mannheim (Indianapolis, IN). Collagenase type II was purchased from Worthington Biochemicals (Freehold, NJ). Nitrocellulose membranes were
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgments
This work was supported by R01 HL068231 & HL093056 (BAH) and NINDS P30 Center Grant (Jungers Center-OHSU). The authors thank Diana Parrish, Eric Alston, and Dr. Xiao Shi for technical assistance, and thank Dr. Richard Zigmond and Jon Niemi for comments on the manuscript. After this work was completed, we were informed by Addgene (Cambridge, MA) that STAT3-WT (pcDNA3), a construct we purchased from them, had a mutation in the open reading frame resulting in an amino acid change (E16K). We
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