Reciprocal regulation of nuclear factor kappa B and its inhibitor ZAS3 after peripheral nerve injury

Abstract

Background: NF-kappaB binds to the kappaB motif to regulate transcription of genes involved in growth, immunity and inflammation, and plays a pivotal role in the production of pro-inflammatory cytokines after nerve injuries. The zinc finger protein ZAS3 also binds to the kappaB or similar motif. In addition to competition for common DNA sites, in vitro experiments have shown that ZAS3 can inhibit NF-kappaB via the association with TRAF2 to inhibit the nuclear translocation of NF-kappaB. However, the physiological significance of the ZAS3-mediated inhibition of NF-kappaB has not been demonstrated. The purpose of this study is to characterize ZAS3 proteins in nervous tissues and to use spinal nerve ligation, a neuropathic pain model, to demonstrate a functional relationship between ZAS3 and NF-kappaB.

Results: Immunohistochemical experiments show that ZAS3 is expressed in specific regions of the central and peripheral nervous system. Abundant ZAS3 expression is found in the trigeminal ganglion, hippocampal formation, dorsal root ganglia, and motoneurons. Low levels of ZAS3 expressions are also found in the cerebral cortex and in the grey matter of the spinal cord. In those nervous tissues, ZAS3 is expressed mainly in the cell bodies of neurons and astrocytes. Together with results of Western blot analyses, the data suggest that ZAS3 protein isoforms with differential cellular distribution are produced in a cell-specific manner. Further, neuropathic pain confirmed by persistent mechanical allodynia was manifested in rats seven days after L5 and L6 lumbar spinal nerve ligation. Changes in gene expression, including a decrease in ZAS3 and an increase in the p65 subunit of NF-kappaB were observed in dorsal root ganglion ipsilateral to the ligation when compared to the contralateral side.

Conclusion: ZAS3 is expressed in nervous tissues involved in cognitive function and pain modulation. The down-regulation of ZAS3 after peripheral nerve injury may lead to activation of NF-kappaB, allowing Wallerian regeneration and induction of NF-kappaB-dependent gene expression, including pro-inflammatory cytokines. We propose that reciprocal changes in the expression of ZAS3 and NF-kappaB might generate neuropathic pain after peripheral nerve injury.

Figure 1

Primary structure of ZAS3 and expression of ZAS3 in the trigeminal ganglion and dorsal root ganglia. (A) Schema showing the domain structure of the ZAS3 protein and fusion proteins used to raise polyclonal antisera, AbN and AbC. Black boxes, zinc finger(s); grey box, middle conserved region; ZAS, zinc finger, acidic- and serine/threonine-rich regions; S, S protein; and mbp, maltose binding protein. (B) and (C) Immunohistochemical staining of sections of naïve rat trigeminal ganglion using antisera AbN and AbC, respectively. Inserts are higher magnifications showing uniform cellular staining of AbN and prominent nuclear staining with AbC. Bars represent 200 μM. (D) and (E) Longitudinal sections of DRG immunostained with AbN and AbC, respectively. Arrows highlight differential nuclear staining of AbN and AbC. Bars represent 100 μM.

Figure 2

Expression of ZAS3 in neuronal cell bodies and astrocytes at the hippocampal formation. (A) Rat coronal brain section (Bregma -3.80) stained with cresyl violet; (B) immunostaining of a similar brain sections with AbC; (C) CA2 region stained with AbC; and (D) CA3 region stained with AbN. Star-like AbN-positive cells are highlighted with arrows. pcl, pyramidal cell layer; sl, stratum lucidum; PL, GL and ML, polymorphic, granule and molecular cell layer, respectively, of the dentate gyrus. Scale bars represent 2 mm (A and B); 0.2 mm (C); 0.1 mm (D).

Figure 3

Expression of ZAS3 in astrocytes and neurons in the brain cortex. (A) Images of sections of brain cortex stained with AbN (left) and GFAP antibodies (middle). The images were superimposed in the right. Most AbN-IR cells were also stained by GFAP antibodies. Whereas AbN stained the cell bodies and the cylindrical processes, GFAP also stained the more elongated branching processes. An example of a cell stained with both AbN and GFAP antibodies is shown in a circle. (B) and (C) are high magnification of neurons stained with AbN, and AbC, respectively and DAPI. While AbC stained the nucleus, AbN yielded intense cytoplasmic staining.

Figure 4

Expression of ZAS3 in the spinal cord. Cross section of rat L3 spinal cord stained with (A) pre-immune sera, (B) AbC, and (C) AbN. The expression of ZAS3 was higher in the grey matter than white matter.

Figure 5

Expression of ZAS3 in motoneurons of lumbar spinal cord. Triple fluorescent ZAS3, GFAP, and DAPI staining of lumber spinal cord sections. Many cell nuclei were devoid of ZAS3 (circled), suggesting that the antisera, AbC and AbN, were specific. While AbC stained the nuclei of motoneurons, AbN stained both the nuclei and cytoplasm of motoneuorns and astrocytes. When the images of AbN and GFAP are superimposed, cells that expressed both proteins are yellow (highlighted with a white arrow in A, C, and E). When the images of AbC and DAPI are superimposed, nuclei expressing ZAS3 appear purple (highlighted with a yellow arrow), whereas nuclei that did not express ZAS3 are blue (circled).

Figure 6

Immunoblot analysis of ZAS3 in brain and DRG. Proteins (T), cytoplasmic extracts (C), and nuclear extracts (N) resolved by SDS-PAGE was sequentially incubated with antibodies AbC (A), p65 (B), and AbN (C). T, total protein lysate; C, cytoplasmic extracts; and N, nuclear extracts.

Figure 7

The rat ZAS3 gene and gene products. (A) A diagonal blot of the rat ZAS3 cDNA against mouse ZAS3 cDNA. (B) Schema of the rat ZAS3 gene (top panel), the complete protein product (middle panel), and a product generated by alternative splicing of exon 13 (bottom panel). White boxes represent exons; vertical arrows indicate two additional exons corresponded to a cDNA sequence present in GenBank databases (XM_233464); asterisk highlights exon 13 that is involved in alternative splicing; horizontal arrows mark the positions of primers used in RT-PCR; and black boxes represent zinc finger pairs. The 5' end of the rat ZAS3 gene has not been elucidated. Therefore, exon numberings, referring to the mouse gene, are shown on top of exons.

Figure 8

Downregulation of ZAS3 in SpNL. (A) Upper panels, RT-PCR transcript analyses of ZAS1, ZAS2, ZAS3, NGF and actin of DRG seven days after SpNL nerves (top panel). The experiments were carried out as described in the text (n = 4 per group) and a representative result is shown. (Bottom panel) Bar charts showing relative levels of each PCR products adjusted with actin in the same cDNA sample. Significance (*) is p < 0.05. (B) Immunoblot analyses of total protein lysates in ipsilateral (I) DRG and contralateral (Con) DRG prepare from rats seven days after surgery using AbC (a), AbN (b), and p65 antibodies (c). Asterisk indicates the 200 kDa species observed only in contralateral DRG but not in ipsilateral DRG. (C) Immunoblot analysis of cytoplasmic and nuclear extracts with p65 and histone H1 antibodies.