Ciliary Neurotrophic Factor Stimulates the Phosphorylation of Two Forms of STAT3 in Chick Ciliary Ganglion Neurons

Abstract

Ciliary neurotrophic factor (CNTF) is a neuropoietic cytokine that was identified, purified, and cloned based on its neurotrophic activity on cultured chick ciliary ganglion neurons. The molecular mechanisms by which CNTF elicits its effects on these neurons are unknown. We have previously identified functional receptors for CNTF on ciliary ganglion neurons and demonstrated the CNTF-specific tyrosine phosphorylation of an approximately 90-kDa protein. Here we show that CNTF induced the rapid tyrosine phosphorylation and nuclear accumulation of this protein and identify it as an avian form of the transcription factor, STAT3. Identification was confirmed by its recognition with two distinct anti-STAT3 antibodies and the lack of binding to antibodies against STAT1, -2, -4, -5, or -6. The phosphorylation was stable for up to 2 h but required the continued presence of CNTF. CNTF also induced the tyrosine phosphorylation of a similar protein in cultured chick dorsal root ganglion and retinal neurons. In addition, we identify a second, 100-kDa form of STAT3 that appears in response to CNTF. Unlike previous reports, utilizing mammalian cell lines that detected a slower migrating form of STAT3 resulting from H7-sensitive protein phosphorylation, H7 did not prevent the appearance of the 100-kDa form in ciliary neurons. Thus, the 100-kDa avian protein may represent a novel form of CNTF-inducible STAT3. Ciliary neurotrophic factor (CNTF)1 is a cytokine that shows activity toward a variety of cell types in the nervous system. However, the molecular mechanisms for these effects have not been clearly elucidated in defined neuronal populations. CNTF belongs to a family of neuropoietic cytokines, which display a high degree of redundancy in their biological activities, have structural similarity, share receptor subunits and signal transduction pathways, and yet are capable of generating unique cellular responses as well (1, 2). The known receptor complexes for CNTF, leukemia inhibitory factor, oncostatin M, cardiotrophin-1, and interleukin-6 and -11, include gp130 as a ␤-receptor component, and all except interleukin-6 and -11 share the leukemia inhibitory factor ␤-receptor component (3-7). CNTF and interleukin-6 and -11 also utilize unique ligand-binding ␣ subunits (1,8,9). Receptor activation by CNTF-related cytokines results in dimerization of the ␤-receptor components and activation of receptor-associated tyrosine kinases of the Jak family. Jaks or other cytoplasmic kinases phosphorylate tyrosine on the cytokine receptor components that selectively bind cytoplasmic transcription factors of the STAT (signal transducers and activators of transcription) family (10). In this model, established primarily from cell lines, STAT proteins are directly tyrosine-phosphorylated by Jaks and then dimerize and translocate to the cell nucleus and regulate gene transcription (11-13). There are at least six members of the STAT family, and the selection and activation of a particular STAT is dependent on receptor substrate-specifying motifs (1...

Direct effects of ciliary neurotrophic factor on brown adipocytes: evidence for a role in peripheral regulation of energy homeostasis

“…CNTF causes a robust increase in STAT3 phosphorylation in brown adipocytes which is consistent with findings in a variety of neuronal cell-populations (Rajan 1996, Wishingrad et al 1997. Furthermore, while there is no effect on p38 MAP kinase activation, CNTF selectively activates p42/44 MAP kinase.…”

Reciprocal Regulation of Ciliary Neurotrophic Factor Receptors and Acetylcholine Receptors during Synaptogenesis in Embryonic Chick Atria

“…Soluble cytoplasmic proteins were collected after centrif ugation to remove particulate material and were dissolved in Laemmli sample buffer. Protein samples were subjected to SDS-PAGE (7.5% acrylamide) and transferred to nitrocellulose membranes as described previously (Koshlukova et al, 1996;Wishingrad et al, 1997). Immunoblots were assayed first with a polyclonal antibody directed against tyrosine-phosphorylated (Y705) STAT3 (1:1000) and then were stripped and assayed with a monoclonal antibody to STAT3 for normalization.…”

“…Jaks induce phosphorylation of tyrosine residues on the receptor that then selectively bind cytoplasmic proteins including signal transducers and activators of transcription, STATs (Ihle, 1996). In chick ciliary ganglia, CNTF induces the tyrosine phosphorylation primarily of STAT3 (Wishingrad et al, 1997).…”

Original article Neuroprotective properties of ciliary neurotrophic factor on retinoic acid (RA)-predifferentiated SH-SY5Y neuroblastoma cells

“…Binding of CNTF to CNTFRα triggers heterodimerization of gp130 and LIFR, forming an active trimeric receptor complex and activates the downstream signalling pathway [25]. It has been established that JAK2/STAT3 pathway is mainly involved in survival of neurons in response to CNTF [11,12,26]. Phosphorylated STAT3 dimerizes and translocates to the nucleus to regulate target gene transcription [27].…”

“…In particular, CNTF is responsible for neurotransmitter synthesis and neurite outgrowth in certain neuronal populations [22,26]. CNTF is a 25-kDa polypeptide that was originally isolated as a target-derived survival factor of parasympathetic ciliary ganglion neurons [19].…”