Calcineurin-nuclear factor of activated T cells regulation of Krox-20 expression in Schwann cells requires elevation of intracellular cyclic AMP.

The transcription factor Krox-20 (Egr2) is a master regulator of Schwann cell myelination. In mice from which calcineurin B had been excised in cells of the neural crest lineage, calcineurin-nuclear factor of activated T cells (NFAT) signaling was required for neuregulin-related Schwann cell myelination (Kao et al. [2009] Immunity 12:359-372). Whether NFAT signaling required simultaneous elevation of intracellular cAMP levels was not explored. In vivo, Krox-20 expression requires continuous axon-Schwann cell signaling that in Schwann cell cultures can be mimicked by elevation of intracellular cAMP. We have investigated the role of the calcineurin-NFAT pathway in Krox-20 induction in purified rat Schwann cell cultures. Activation of this pathway requires elevation of intracellular Ca(2+) levels. The calcium ionophore A23187 or ionomycin was used to increase intracellular Ca(2+) levels in Schwann cell cultures that had been treated with dibutyryl cAMP to induce Krox-20. Increase in Ca(2+) levels significantly potentiated Krox-20 induction, determined by Krox-20 immunolabeling of individual cells and Western blotting. Levels of the myelin proteins periaxin and P(0) were also elevated. The potentiating effect was blocked by cyclosporin A, a specific blocker of the calcineurin-NFAT pathway. We found that, in the absence of cAMP elevation, treatment with A23187 alone failed to induce Krox-20 expression, indicating that NFAT upregulation of Krox-20 requires elevation of cAMP levels in Schwann cells. P-VIVIT, another specific inhibitor of calcineurin-NFAT interaction, blocked Krox-20 induction in response to dibutyryl cAMP and ionophore. HA-NFAT1 (1-460)-GFP translocated to the nucleus on treatment with dibutyryl cAMP with or without added ionophore. NFAT isoforms 1-4 were detected in purified Schwann cells by quantitative RT-PCR.

c-Jun reprograms Schwann cells of injured nerves to generate a repair cell essential for regeneration.

The radical response of peripheral nerves to injury (Wallerian degeneration) is the cornerstone of nerve repair. We show that activation of the transcription factor c-Jun in Schwann cells is a global regulator of Wallerian degeneration. c-Jun governs major aspects of the injury response, determines the expression of trophic factors, adhesion molecules, the formation of regeneration tracks and myelin clearance and controls the distinctive regenerative potential of peripheral nerves. A key function of c-Jun is the activation of a repair program in Schwann cells and the creation of a cell specialized to support regeneration. We show that absence of c-Jun results in the formation of a dysfunctional repair cell, striking failure of functional recovery, and neuronal death. We conclude that a single glial transcription factor is essential for restoration of damaged nerves, acting to control the transdifferentiation of myelin and Remak Schwann cells to dedicated repair cells in damaged tissue.

Identification and characterization of ZFP-57, a novel zinc finger transcription factor in the mammalian peripheral nervous system.

To isolate new zinc finger genes expressed at early stages of peripheral nerve development, we have used PCR to amplify conserved zinc finger sequences. RNA from rat embryonic day 12 and 13 sciatic nerves, a stage when nerves contain Schwann cell precursors, was used to identify several genes not previously described in Schwann cells. One of them, zinc finger protein (ZFP)-57, proved to be the homologue of a mouse gene found in F9 teratocarcinoma cells. Its mRNA expression profile within embryonic and adult normal and transected peripheral nerves, and its distribution in the rest of the nervous system is described. High levels of expression are seen in embryonic nerves and spinal cord. These drop rapidly during the first few weeks after birth, a pattern mirrored in other parts of the nervous system. ZFP-57 localizes to the nucleus of Schwann and other cells. The sequence contains an N-terminal Krüppel-associated box (KRAB) domain and ZFP-57 constructs containing green fluorescent protein reveal that the protein colocalizes with heterochromatin protein 1alpha to centromeric heterochromatin in a characteristic speckled pattern in NIH3T3 cells. The KRAB domain is required for this localization, because constructs lacking it target the protein to the nucleus but not to the centromeric heterochromatin. When fused to a heterologous DNA binding domain, the KRAB domain of ZFP-57 represses transcription, and full-length ZFP-57 represses Schwann cell transcription from myelin basic protein and P(0) promoters in co-transfection assays. Zfp-57 mRNA is up-regulated in Schwann cells in response to leukemia inhibitory factor and fibroblast growth factor 2.

Sensitization by extracellular Ca(2+) of rat P2X(5) receptor and its pharmacological properties compared with rat P2X(1).

The recombinant rat P2X(5) (rP2X(5)) receptor, a poorly understood ATP-gated ion channel, was studied under voltage-clamp conditions and compared with the better understood homomeric rP2X(1) receptor with which it may coexist in vivo. Expressed in defolliculated Xenopus laevis oocytes, rP2X(5) responded to ATP with slowly desensitizing inward currents that, for successive responses, ran down in the presence of extracellular Ca(2+) (1.8 mM). Replacement of Ca(2+) with either Ba(2+) or Mg(2+) prevented rundown, although agonist responses were very small, whereas reintroduction of Ca(2+) for short periods of time (<300 s) before and during agonist application yielded consistently larger responses. Using this Ca(2+)-pulse conditioning, rP2X(5) responded to ATP and other nucleotides (ATP, 2-methylthio-ATP, adenosine-5'-O-(thiotriphosphate), 2'-&-3'-O-(4-benzoylbenzoyl)-ATP, alpha,beta-methylene-ATP, P(1)-P((4))-diadenosine-5'-phosphate, and more) with pEC(50) values within 1 log unit of respective determinations for rP2X(1). Only GTP was selective for rP2X(5), although 60-fold less potent than ATP. At rP2X(5), lowering extracellular pH reduced the potency and efficacy of ATP, whereas extracellular Zn(2+) ions (0.1-1000 microM) potentiated then inhibited ATP responses in a concentration-dependent manner. However, these modulators affected rP2X(1) receptors in subtly different ways-with increasing H(+) and Zn(2+) ion concentrations reducing agonist potency. For P2 receptor antagonists, the potency order at rP2X(5) was pyridoxal-5-phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) > 2',3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) > suramin > reactive blue 2 (RB-2) > diinosine pentaphosphate (Ip(5)I). In contrast, the potency order at rP2X(1) was TNP-ATP = Ip(5)I > PPADS > suramin = RB-2. Thus, the Ca(2+)-sensitized homomeric rP2X(5) receptor is similar in agonist profile to homomeric rP2X(1)-although it can be distinguished from the latter by GTP agonism, antagonist profile, and the modulatory effects of H(+) and Zn(2+) ions.