RCAN1.4 overexpression exacerbates calcium overloading-induced neuronal apoptosis.

<p>(<b>A</b>) RCAN1.4 overexpression reduced cell survival. SH-SY5Y cells transfected with empty vector pcDNA4mychisA or pcDNA4-RCAN1.4mychis were treated with 2.5 uM A23187. MTS assay was used to indicate the viability of SH-SY5Y cells. Values represent mean ± SEM, n = 3, *P<0. 05 by ANOVA. (<b>B</b>) and (<b>C</b>) RCAN1.4 overexpression exacerbated cell apoptosis. SH-SY5Y cells transfected with empty vector pcDNA4mychisA and pcDNA4-RCAN1.4mychis were treated with vehicle control solution (B) or 2.5 uM A23187and (C). TUNEL staining was used to indicate cell apoptosis (green color). Nuclei were counterstained with DAPI (blue color). (<b>D</b>) Quantification of (B) and (C). Values represent means ± SE (n = 3), *P<0. 05 by ANOVA.</p

Caspase-3 mediates the neurotoxic effect of RCAN1.4 and calcium overloading.

<p>(<b>A</b>) RCAN1.4 overexpression increased caspase-3 activation. SH-SY5Y cells transfected with empty vector pcDNA4mychisA (Vector) and pcDNA4-RCAN1.4mychis (RCAN1) were treated with 2.5 uM A23187 for 12 hours. 100 µg cell lysates were separated in a 16% tricine SDS-PAGE gel. Procaspase-3 and cleaved caspase-3 were detected with anti-caspase-3 antibody from Sigma. Myc-tagged RCAN1.4 was detected by 9E10 antibody. β-actin served as loading control. (<b>B</b>) Quantification of (A). The ratio of cleaved caspase-3 to procaspase-3 was calculated. Values represent means ± SE (n = 3), *P<0. 05 by ANOVA.</p

Two alternative promoters distinctly control RCAN1 expression.

<p>(<b>A</b>) Genomic organization of the <i>RCAN1</i> gene. <i>RCAN1</i> gene has seven exons and six introns. The first four exons are alternatively spliced and the last three exons are constitutive. There are two promoters and two translation initiation codons in the <i>RCAN1</i> gene, in the 5′UTR of exon 1 and 5′UTR of exon 4 respectively. TSS, transcription start site. E stands for exon. (<b>B</b>) Human <i>RCAN1</i> isoform 4 promoter sequence. A 1200 bp fragment of the 5′ flanking region of human RCAN1 exon 4 was amplified from a human genomic library. Thymine +1 represents the major transcription start site. Positions of some of the unique and common restriction enzymes are indicated in italics and boldface. Putative transcription factor binding sites are underlined in boldface. The codon of the first nine amino acids of exon 4 is indicated. (<b>C</b>) 1200-bp fragment upstream of the exon 4 had significant promoter activity. 1200-bp fragment upstream of the exon 4 was cloned into pGL3-Basic to generate the pDE4Luc luciferase reporter plasmid. pDE4Luc was transfected into HEK293 cells. pGL3-Basic was used as negative control. Luciferase activity was measured 24 hours after transfection. Values represent means ± SE (n = 4), *P<0.05 by student's t-test. (<b>D</b>) Compared to the RCAN1 exon 1 promoter pRCAN1Luc-A, RCAN1 exon 4 promoter pDE4Luc had a higher promoter activity in C6 cells but a lower activity in N2A cells. RCAN1 promoter constructs pDE4Luc and pRCAN1Luc-A were transfected into C6 and N2A cells. pGL3-Basic was used as negative control. Luciferase activity was measured 24 hours after transfection by a luminometer. <i>Renilla</i> luciferase activity was used to normalize transfection efficiency. Values represent means ± SE (n = 4), *P<0.05 by student's t-test. (<b>E</b>) Calcium ionophore A23187 significantly increased pDE4Luc activity. HEK293 cells transfected with pDE4Luc were treated with 2.5 µM A23187 for 12 hours. Luciferase assay was used to measure the promoter activity. pGL3-Basic was used as negative control. <i>Renilla</i> luciferase activity was used to normalize transfection efficiency. Values represent means ± SE (n = 4), *P<0. 05 by student's t-test.</p