Figure 10.

Tis21 associates to the Cxcl3 promoter and induces Cxcl3 transcription. A, B, Real-time RT-PCR analysis of Tis21 and Cxcl3 mRNAs in primary cultures of cerebellar granule neurons. Cells, obtained from P7 rats, were plated and infected with recombinant adenoviruses adeno-Tis21 or control adeno-β-Gal the same day of plating (DIV 0), and harvested after 24 h. The primers amplifying Tis21 (rat sequence) detected both the endogenous and exogenous mRNA. Shown are mean ± SEM values are from five separate experiments. TATA-binding protein mRNA was used as endogenous control for normalization. *p < 0.05 versus control, Student's t test (performed on data normalized to the endogenous controls but not yet relativized as fold expression). C, Cxcl3 promoter activity in cerebellar granule neurons from P7 wild-type rats transfected with an expression vector for Tis21 (pSCT-Tis21) and in PC12 cells expressing inducible Tis21 (rat sequence) increased significantly, relative to the corresponding controls expressing endogenous levels of Tis21. The Cxcl3 promoter construct comprises 628 nt 5′ to the putative transcription start, placed upstream of a luciferase reporter (construct pGL3-Cxcl3-prom/-628). TNF-α, known inducer of Cxcl3 promoter activity, is used in PC12 cells as positive control. Luciferase activity is represented as mean ± SEM fold increase from five separate experiments. *p < 0.05 versus the corresponding control without exogenous or ectopic Tis21, Student's t test. C′, Parallel cultures were analyzed for Tis21 protein expression by Western blot. D, ChIP analysis of Tis21 binding to the Cxcl3 promoter and to the muscle creatine kinase promoter (MCK) (negative control) either in cerebellar granule neurons (graph on the left), infected with adeno-Tis21 or adeno-β-Gal as in A and B, or in GCPs (graph on the right), both isolated from P7 wild-type rats. The amount of Cxcl3 or MCK promoters present in immunoprecipitates, obtained using anti-Tis21 antibody A3H, is quantified by real-time PCR and is expressed as fold enrichment (ratio of the percentage of the Tis21-immunoprecipitated amount of Cxcl3 or MCK promoter detected in the input cell lysates to the percentage of the normal serum-immunoprecipitated amount detected in the input cell lysates). The binding of Tis21 protein to the Cxcl3 promoter (darker gray columns) significantly increases above basal levels (i.e., above the binding levels to the MCK negative control promoter) in correlation with the overexpression of Tis21 mRNA (compare also A). Shown are mean ± SEM values are from five separate experiments performed with cerebellar granule neurons and from three separate experiments with GCPs. *p < 0.05 versus control, Student's t test. E, F, Expression of Tis21 and Cxcl3 mRNAs, respectively, as detected by in situ hybridization in cerebellar midsagittal sections from P7 wild-type mice; their expression colocalizes and is very high in GCPs, throughout the whole EGL width. Black box, Area at higher magnification. Scale bars: 100 μm; enlargement, 20 μm. G, Expression of Cxcr2, receptor of Cxcl3, immunostained by a specific antibody in a representative confocal image of a cerebellar midsagittal section from P7 wild-type mice. Nuclei are visualized by Hoechst 33258 staining. Clusters of receptors are clearly detectable in GCPs (white arrowhead in EGL) and to a lower extent in the pial membrane (PM) and in Purkinje cells (black-white arrowhead); staining of putative Bergmann's glia is indicated by white arrows. Scale bar, 25 μm.