Michael J. Pellegrino

Sustained activation of extracellular signal-regulated kinase by nerve growth factor regulates c-fos protein stabilization and transactivation in PC12 cells

The duration of intracellular signaling is thought to be a critical component in effecting specific biological responses. This paradigm is demonstrated by growth factor activation of the extracellular signal‐regulated kinase (ERK) signaling cascade in the rat pheochromocytoma cell line (PC12 cells). In this model, sustained ERK activation induced by nerve growth factor (NGF) results in differentiation, whereas transient ERK activation induced by epidermal growth factor (EGF) results in proliferation in these cells. Recently, the immediate early gene product c‐fos has been proposed to be a sensor for ERK signaling duration in fibroblasts. In this study, we ask whether this is true for NGF and EGF stimulation of PC12 cells. We show that NGF, but not EGF, can regulate both c‐fos stability and activation in an ERK‐dependent manner in PC12 cells. This is achieved through ERK‐dependent phosphorylation of c‐fos. Interestingly, distinct sites regulate enhanced stability and transactivation of c‐fos. Phosphorylation of Thr325 and Thr331 are required for maximal NGF‐dependent transactivation of c‐fos. In addition, a consensus ERK binding site (DEF domain) is also required for c‐fos transactivation. However, stability is controlled by ERK‐dependent phosphorylation of Ser374, while phosphorylation of Ser362 can induce conformational changes in protein structure. We also provide evidence that sustained ERK activation is required for proper post‐translational regulation of c‐fos following NGF treatment of PC12 cells. Because these ERK‐dependent phosphorylations are required for proper c‐fos function, and occur sequentially, we propose that c‐fos is a sensor for ERK signaling duration in the neuronal‐like cell line PC12.

STAT3 integrates cytokine and neurotrophin signals to promote sympathetic axon regeneration

The transcription factor STAT3 has been implicated in axon regeneration. Here we investigate a role for STAT3 in sympathetic nerve sprouting after myocardial infarction (MI) - a common injury in humans. We show that NGF stimulates serine phosphorylation (S727) of STAT3 in sympathetic neurons via ERK1/2, in contrast to cytokine phosphorylation of Y705. Maximal sympathetic axon regeneration in vitro requires phosphorylation of both S727 and Y705. Furthermore, cytokine signaling is necessary for NGF-induced sympathetic nerve sprouting in the heart after MI. Transfection studies in neurons lacking STAT3 suggest two independent pools of STAT3, phosphorylated on either S727 or Y705, that regulate sympathetic regeneration via both transcriptional and non-transcriptional means. Additional data identify STAT3-microtubule interactions that may complement the well-characterized role of STAT3 stimulating regeneration associated genes. These data show that STAT3 is critical for sympathetic axon regeneration in vitro and in vivo, and identify a novel non-transcriptional mode of action.

Cytokines inhibit norepinephrine transporter expression by decreasing Hand2

Functional noradrenergic transmission requires the coordinate expression of enzymes involved in norepinephrine (NE) synthesis, as well as the norepinephrine transporter (NET) which removes NE from the synapse. Inflammatory cytokines acting through gp130 can suppress the noradrenergic phenotype in sympathetic neurons. This occurs in a subset of sympathetic neurons during development and also occurs in adult neurons after injury. For example, cytokines suppress noradrenergic function in sympathetic neurons after axotomy and during heart failure. The molecular basis for suppression of noradrenergic genes is not well understood, but previous studies implicated a reduction of Phox2a in cytokine suppression of dopamine beta hydroxylase. We used sympathetic neurons and neuroblastoma cells to investigate the role of Phox2a in cytokine suppression of NET transcription. Chromatin immunoprecipitation experiments revealed that Phox2a did not bind the NET promoter, and overexpression of Phox2a did not prevent cytokine suppression of NET transcription. Hand2 and Gata3 are transcription factors that induce noradrenergic genes during development and are present in mature sympathetic neurons. Both Hand2 and Gata3 were decreased by cytokines in sympathetic neurons and neuroblastoma cells. Overexpression of either Hand2 or Gata3 was sufficient to rescue NET transcription following suppression by cytokines. We examined expression of these genes following axotomy to determine if their expression was altered following nerve injury. NET and Hand2 mRNAs decreased significantly in sympathetic neurons 48 h after axotomy, but Gata3 mRNA was unchanged. These data suggest that cytokines can inhibit NET expression through downregulation of Hand2 or Gata3 in cultured sympathetic neurons, but axotomy in adult animals selectively suppresses Hand2 expression.

Sympathetic denervation of peri-infarct myocardium requires the p75 neurotrophin receptor.

Development of cardiac sympathetic heterogeneity after myocardial infarction contributes to ventricular arrhythmias and sudden cardiac death. Regions of sympathetic hyperinnervation and denervation appear in the viable myocardium beyond the infarcted area. While elevated nerve growth factor (NGF) is implicated in sympathetic hyperinnervation, the mechanisms underlying denervation are unknown. Recent studies show that selective activation of the p75 neurotrophin receptor (p75(NTR)) in sympathetic neurons causes axon degeneration. We used mice that lack p75(NTR) to test the hypothesis that activation of p75(NTR) causes peri-infarct sympathetic denervation after cardiac ischemia-reperfusion. Wild type hearts exhibited sympathetic denervation adjacent to the infarct 24h and 3 days after ischemia-reperfusion, but no peri-infarct sympathetic denervation occurred in p75(NTR)-/- mice. Sympathetic hyperinnervation was found in the distal peri-infarct myocardium in both genotypes 3 days after MI, and hyperinnervation was increased in the p75(NTR)-/- mice. By 7 days after ischemia-reperfusion, cardiac sympathetic innervation density returned back to sham-operated levels in both genotypes, indicating that axonal pruning did not require p75(NTR). Prior studies revealed that proNGF is elevated in the damaged left ventricle after ischemia-reperfusion, as is mRNA encoding brain-derived neurotrophic factor (BDNF). ProNGF and BDNF preferentially bind p75(NTR) rather than TrkA on sympathetic neurons. Immunohistochemistry using Bdnf-HA mice confirmed the presence of BDNF or proBDNF in the infarct after ischemia-reperfusion. Thus, at least two p75(NTR) ligands are elevated in the left ventricle after ischemia-reperfusion where they may stimulate p75(NTR)-dependent denervation of peri-infarct myocardium. In contrast, NGF-induced sympathetic hyperinnervation in the distal peri-infarct ventricle is attenuated by p75(NTR).

Cyclic AMP Analogs Induce Synthesis, Processing, and Secretion of Prepro Nociceptin/Orphanin FQ-Derived Peptides by NS20Y Neuroblastoma Cells

Recent studies have shown that cAMP analogs can induce expression of prepro (pp) orphanin FA (OFQ)/nociceptin-related gene products in NS20Y mouse neuroblastoma cells (Saito et al. [1996]. J Biol Chem 271, 15615-15622). Additionally, exposure of NS20Y cells to cAMP analogs promoted neurite outgrowth and large dense-core vesicle formation. Even though an OFQ-like precursor (called 27K) was identified in NS20Y cell extracts, no secretion of OFQ-related peptides was detected. We have used reversed-phase high-performance liquid chromatography combined with a specific radioimmunoassay for OFQ(1-17) to determine if NS20Y cells secrete ppOFQ-derived peptides when stimulated by the cAMP analog ctp-cAMP. We found that NS20Y cells secreted abundant amounts of OFQ-derived products when stimulated by cAMP analogs. We also have determined that secretion of OFQ peptides was both time and concentration dependent and reversible on removal of cAMP analogs from the culture medium. In addition, the opioid agonist D-Pen2-D-Pen5-enkephalin inhibited forskolin-stimulated OFQ peptide secretion. Further, the synthetic glucocorticoid dexamethasone virtually abolished ctp-cAMP-stimulated OFQ peptide secretion. These results suggest that the biosynthesis, processing, and secretion of the OFQ neuropeptide transmitter system can be modulated through intracellular cAMP levels and that these functions are regulated by opioids and molecules involved in mediating the stress response. The NS20Y cell system will be extremely valuable for studying the regulation of OFQ-derived peptides by a variety of intra-cellular and extracellular signaling pathways.

Carboxyl terminal peptides derived from prepro-orphanin FQ/nociceptin (ppOFQ/N) are produced in the hypothalamus and possess analgesic bioactivities

Orphanin FQ/nociceptin (OFQ/N), the endogenous ligand for the ORL-1/KOR-3 receptor, produces a wide variety of behavioral responses. Its precursor protein, prepro-OFQ/N (ppOFQ/N) contains several series of amino acids bounded by pairs of basic amino acids, raising the possibility that additional functional neuropeptides could be generated by proteolytic posttranslational processing. Several of these processing products have been shown to have pharmacological activity, including the 17 amino acid peptide OFQ/N (mppOFQ/N(140-157)) which is a major product of this precursor in the hypothalamus. Here we have used a newly developed radioimmunoassay and RP-HPLC to detect mppOFQ/N(160-187) in mouse hypothalamic extracts. Murine ppOFQ/N(160-187) has potent analgesic activity supraspinally (3.4 nmol, i.c.v.) and spinally (4.3 nmol, i.t.). This analgesic activity was reversed by the opioid antagonist naloxone (5 mg/kg, s.c.) and kappa(1)-selective opioid antagonist nor-BNI (60 microg, i.c.v.), despite the inability of ppOFQ/N(160-187) to compete binding in mu, delta, kappa(1), kappa(3), or OFQ/N binding assays. These findings suggest that murine ppOFQ/N(160-187) may be a physiologically relevant neuropeptide with a novel mechanism of action.

Leptin stimulates sympathetic axon outgrowth

The neurohormone leptin regulates energy homeostasis. Circulating levels of leptin secreted by adipose tissue act on hypothalamic neurons in the brain leading to decreased appetite and increased energy expenditure. Although leptin signaling in the central nervous system (CNS) is fundamental to its ability to regulate the bodys metabolic balance, leptin also has a variety of effects in many peripheral tissues including the heart, the liver, and the sympathetic nervous system. Leptin stimulation of the hypothalamus can stimulate glucose uptake via the sympathetic nervous system in heart, muscle, and brown adipose tissue. Leptin receptors (Ob-Rb) are also expressed by peripheral sympathetic neurons, but their functional role is not clear. In this study, we found that leptin stimulates axonal growth of both adult and neonatal sympathetic neurons in vitro. Leptin stimulates acute activation of the transcription factor STAT3 via phosphorylation of tyrosine 705. STAT3 phosphorylation is required for leptin-stimulated sympathetic axon outgrowth. Thus, circulating levels of leptin may enhance sympathetic nerve innervation of peripheral tissues.

Unusual Stüve-Wiedemann syndrome with complete maternal chromosome 5 isodisomy

A woman was isozygous for a novel mutation in the leukemia inhibitory factor receptor gene (LIFR) (c.2170C>G; p.Pro724Ala) which disrupts LIFR downstream signaling and results in Stüve‐Wiedemann syndrome (STWS). She inherited two identical chromosomes 5 from her mother, heterozygous for the LIFR mutation. The presentation was typical for STWS, except there was no long bone dysplasia. Prominent cold‐induced sweating and heat intolerance lead to an initial diagnosis of cold‐induced sweating syndrome, excluded by exome sequencing. Skin biopsies provide the first human evidence of failed postnatal cholinergic differentiation of sympathetic neurons innervating sweat glands in cold‐induced sweating, and of a neuropathy.

ERK5 induces ankrd1 for catecholamine biosynthesis and homeostasis in adrenal medullary cells

Extracellular signal-regulated kinases (ERKs) play important roles in proliferation, differentiation and gene expression. In our previous study, we demonstrated that both ERK5 and ERK1/2 were responsible for neurite outgrowth and tyrosine hydroxylase (TH) expression in rat pheochromocytoma cells (PC12) (J Biol Chem 284, 23,564-23,573, 2009). However, the functional differences between ERK5 and ERK1/2 signaling in neural differentiation remain unclear. In the present study, we show that ERK5, but not ERK1/2 regulates TH levels in rat sympathetic neurons. Furthermore, microarray analysis performed in PC12 cells using ERK5 and ERK1/2-specific inhibitors, identified ankyrin repeat domain 1 (ankrd1) as an ERK5-dependent and ERK1/2-independent gene. Here, we report a novel role of the ERK5/ankrd1 signaling in regulating TH levels and catecholamine biosynthesis. Ankrd1 mRNA was induced by nerve growth factor in time- and concentration-dependent manners. TH levels were reduced by ankrd1 knockdown with no changes in the mRNA levels, suggesting that ankrd1 was involved in stabilization of TH protein. Interestingly, ubiquitination of TH was enhanced and catecholamine biosynthesis was reduced by ankrd1 knockdown. Finally, we examined the relationship of ERK5 to TH levels in human adrenal pheochromocytomas. Whereas TH levels were correlated with ERK5 levels in normal adrenal medullas, ERK5 was down-regulated and TH was up-regulated in pheochromocytomas, indicating that TH levels are regulated by alternative mechanisms in tumors. Taken together, ERK5 signaling is required for catecholamine biosynthesis during neural differentiation, in part to induce ankrd1, and to maintain appropriate TH levels. This pathway is disrupted in pathological conditions.