The parkinsonian neurotoxin, MPP+ induces phosphorylated c-Jun in dopaminergric neurons of mesencephalic cultures.

The stress-activated protein kinase (SAPK) cascade serves a critical role in the apoptotic death of neuronal cells in response to a variety of cellular stresses. Recent in vitro and in vivo evidence has directly implicated this kinase in the death of dopaminergic nigral neurons in the MPTP model of Parkinson's disease (PD). To assess the involvement of c-Jun, a key transcription factor substrate of SAPK (also known as c-Jun N-terminal kinase, or JNK) in the MPTP-induced death of dopaminergic nigral neurons, we determined the ability of MPP+, the active toxin metabolite of MPTP, to induce the phosphorylated form of c-Jun in dopaminergic neurons in nigral (ventral mesencephalon) cultures. At a dose of MPP+ that specifically induces apoptotic changes in nuclear morphology in tyrosine hydroxylase-positive (dopaminergic) cells in these cultures, MPP+ induces nuclear phospho-c-Jun immunoreactivity (IR). The peak induction of phospho-c-Jun IR was observed 16h after beginning MPP+ exposure, and preceded the maximal induction of apoptotic nuclear changes by approximately 8h. These data support an important role for the SAPK/JNK pathway including its c-Jun transcriptional target in the apoptotic death of dopaminergic nigral neurons in the MPTP model of PD.

Leukemia inhibitory factor and NGF regulate signal transducers and activators of transcription activation in sympathetic ganglia: convergence of cytokine- and neurotrophin-signaling pathways.

We have used the response of the superior cervical ganglia (SCG) to axotomy to investigate interactions between neuropoietic cytokines and neurotrophins. Postganglionic sympathetic axotomy leads to a prolonged leukemia inhibitory factor (LIF)-dependent activation of signal transducers and activators of transcription (STAT) factors. To study regulation of LIF-dependent activation of STAT proteins and to mimic the loss of target-derived NGF resulting from postganglionic axotomy in vivo, SCG were explanted into media lacking NGF and activation of STAT proteins was assessed by electrophoretic mobility shift assay. Like postganglionic axotomy in vivo. STAT proteins were activated for up to 8 days after explantation of SCG in vitro. SCG cultured in the presence of NGF showed decreased STAT binding when compared to ganglia cultured in NGF-free media. This inhibition of STAT activation by NGF was only present in ganglia cultured for more than 5 days and was mimicked by brain-derived neurotrophic factor (BDNF). The serine kinase inhibitor H7 augmented the increase of STAT binding produced by explantation, suggesting the presence of a labile repressor of STAT activation in the SCG. These data indicated that the neuropoietic cytokine-signaling pathway interacts with neurotrophin and H7-sensitive-signaling pathways to regulate activation of STAT proteins in sympathetic neurons. Moreover, these data suggest that one of the mechanisms leading to prolonged activation of STAT proteins after postganglionic axotomy in vivo is loss of target-derived neurotrophins.

NFAT interactions with the vasoactive intestinal peptide cytokine response element.

The vasoactive intestinal peptide cytokine response element (VIP CyRE) is responsible for mediating the transcriptional induction of the VIP gene to the neuropoietic cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF). In investigating the sequence and function of the CyRE, we found a region of DNA with homology to the distal NFAT site in the IL-2 promoter. In this paper we characterize this sequence and show that the VIP NFAT site recognizes T cell NFAT with similar affinity to the previously characterized IL-2 NFAT site. However, despite its location in the middle of the CyRE, we find no CNTF/LIF induced binding to it. Instead we show that in NBFL neuroblastoma cells, the calcium ionophore A23187 induces a protein to bind to the VIP NFAT site. This A23187-mediated induction of nuclear protein binding to an NFAT oligonucleotide is dependent on extracellular calcium but not dependent on de novo protein synthesis. Thus, this protein has the characteristics of an NFAT-like protein and is recognized by an NFAT3-specific antiserum suggesting that it is indeed an NFAT protein. The location of the NFAT site in the VIP CyRE suggests that this may be one mechanism through which different signaling pathways engage in cross talk to alter VIP gene transcription.

The protein tyrosine phosphatase SHP-2 negatively regulates ciliary neurotrophic factor induction of gene expression.

Ciliary neurotrophic factor, along with other neuropoietic cytokines, signals through the shared receptor subunit gp130 [1-3], leading to the tyrosine phosphorylation of a number of substrates [4,5], including the transcription factors STAT1 and STAT3 and the protein tyrosine phosphatase SHP-2 [6,7] [8]. SHP-2 (also known as PTP1D, SHPTP2, Syp and PTP2C) is a positive regulatory molecule required for the activation of the mitogen-activated protein kinase pathway and the stimulation of gene expression in response to epidermal growth factor, insulin and platelet-derived growth factor stimulation [9-11]. We have previously shown that cytokines that signal via the gp130 receptor subunit activate transcription of the vasoactive intestinal peptide (VIP) gene through a 180 bp cytokine response element (CyRE) [12,13]. To characterize the role of SHP-2 in the regulation of gp130-stimulated gene expression, we examined the regulation of the VIP CyRE in two systems that prevented ligand-dependent SHP-2 phosphorylation. Inhibition of SHP-2, either by mutating the tyrosine residue in gp130 that mediates the SHP-2 interaction, or by expression of dominant-negative SHP-2, resulted in dramatic increases in gp130-dependent gene expression, through the VIP CyRE and more specifically through multimerized STAT-binding sites. These data suggest that SHP-2 has a negative role in gp130 signaling by modulating STAT-mediated transcriptional activation.

Integration of Jak-Stat and AP-1 signaling pathways at the vasoactive intestinal peptide cytokine response element regulates ciliary neurotrophic factor-dependent transcription.

Ciliary neurotrophic factor (CNTF)-dependent induction of expression of the neuropeptide vasoactive intestinal peptide (VIP) gene is mediated by a 180-base pair cytokine response element (CyRE) in the VIP promoter. To elucidate the molecular mechanisms mediating the transcriptional activation by CNTF, intracellular signaling to the CyRE has been studied in a neuroblastoma cell line. It has been shown previously that CNTF induces Stat proteins to bind to a site within the CyRE. CNTF also induces a second protein to bind to a C/EBP-like site within the CyRE. In this report, we show that this inducible CyRE binding protein is composed of the AP-1 proteins c-Fos, JunB, and JunD. These proteins bind to a non-canonical AP-1 site located near the previously characterized C/EBP site. The serine/threonine kinase inhibitor H7 prevents CNTF-dependent induction of AP-1 binding and CyRE-mediated transcription, suggesting that an H7-sensitive kinase is important to mediating CNTF effects on VIP transcription. The integration at the VIP CyRE of the Jak-Stat and AP-1 signaling pathways with other pre-existing proteins provides a cellular mechanism for cell- and cytokine-specific signaling.