RCAN1 overexpression exacerbates calcium overloading-induced neuronal apoptosis - PubMed (original) (raw)
RCAN1 overexpression exacerbates calcium overloading-induced neuronal apoptosis
Xiulian Sun et al. PLoS One. 2014.
Abstract
Down Syndrome (DS) patients develop characteristic Alzheimer's Disease (AD) neuropathology after their middle age. Prominent neuronal loss has been observed in the cortical regions of AD brains. However, the underlying mechanism leading to this neuronal loss in both DS and AD remains to be elucidated. Calcium overloading and oxidative stress have been implicated in AD pathogenesis. Two major isoforms of regulator of calcineurin 1 (RCAN1), RCAN1.1 and RCAN1.4, are detected in human brains. In this report we defined the transcriptional regulation of RCAN1.1 and RCAN1.4 by two alternative promoters. Calcium overloading upregulated RCAN1.4 expression by activating RCAN1.4 promoter through calcineurin-NFAT signaling pathway, thus forming a negative feedback loop in isoform 4 regulation. Furthermore, RCAN1.4 overexpression exacerbated calcium overloading-induced neuronal apoptosis, which was mediated by caspase-3 apoptotic pathway. Our results suggest that downregulating RCAN1.4 expression in neurons could be beneficial to AD patients.
Conflict of interest statement
Competing Interests: The authors have declared that no competing interests exist.
Figures
Figure 1. Two alternative promoters distinctly control RCAN1 expression.
(A) Genomic organization of the RCAN1 gene. RCAN1 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 RCAN1 gene, in the 5′UTR of exon 1 and 5′UTR of exon 4 respectively. TSS, transcription start site. E stands for exon. (B) Human RCAN1 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. (C) 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. (D) 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. Renilla luciferase activity was used to normalize transfection efficiency. Values represent means ± SE (n = 4), *P<0.05 by student's t-test. (E) 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. Renilla luciferase activity was used to normalize transfection efficiency. Values represent means ± SE (n = 4), *P<0. 05 by student's t-test.
Figure 2. Calcium overloading upregulates RCAN1 isoform 4 expression through activation of NFAT signaling pathway.
(A) Calcium ionophore A23187 increased RCAN1 isoform 4 mRNA expression in HEK293 cells. A specific set of primers were used to amplify a RCAN1 isoform 4 mRNA through RT-PCR. Samples were analyzed on 1.5% agarose gel. β-actin was used as internal control. (B) Quantification of (A). Values represent means ± SEM. n = 3, *P<0. 05 by student's t-test. (C) Calcium ionophore A23187 increased RCAN1 isoform 4 expression in HEK293 cells. RCAN1.4 protein expression is upregulated by A23187. HEK293 cells were treated with 2.5 µM A23187 for 12 hours. 150 ug cell lysates were separated in a 15% glycine SDS-PAGE gel. RCAN1 was detected with anti-RCAN1 antibody DCT3. β-actin detected with anti-β-actin antibody (Sigma, AC15) served as internal control. (D) Quantification of (C). Values represent means ± SE (n = 3), *P<0. 05 by student's t-test. (E) Calcium ionophore A23187 increased RCAN1 isoform 4 mRNA expression in SH-SY5Y cells. SH-SY5Y cells were treated with 2.5 µM A23187 for 12 hours. A specific set of primers were used to amplify a RCAN1 isoform 4 mRNA by RT-PCR. The samples were analyzed with 1.5% agarose gel. β-actin was used as an internal control. (F) Quantification of (E). Values represent means ± SE (n = 3), *P<0. 05 by student's t-test. (G) Calcium ionophore A23187 increased RCAN1.4 expression in SH-SY5Y cells. RCAN1.4 protein expression was upregulated by the treatment with A23187. SH-SY5Y cells were treated with 2.5 µM A23187 for 12 hours and 150 ug cell lysates were separated in a 15% glycine SDS-PAGE gel. RCAN1.4 was detected with anti-RCAN1 antibody DCT3. β-actin served as an internal control. (H) Quantification of (G). Values represent means ± SE (n = 3), *P<0. 05 by student's t-test. (I) NFAT significantly increased pDE4luc promoter activity. NFAT expression plasmid pHA-NFAT was co-transfected with RCAN1.4 promoter pDE4luc into HEK293 cells. pGL3-Basic was used as a negative control. And pIL2Luc that contains NFAT responsive elements was used as a positive control. Luciferase activity was measured 48 hours after transfection by a luminometer. Values represent means ± SE (n = 4), *P<0. 05 by student's t-test. (J) and (K) RCAN1 exon 4 promoter is activated by NFAT independent of AP1. HEK293 cells co-transfected with pHA-NFAT and pIL2Luc (J) or pDE4luc (K) were exposed to 100 nM tPA for 20 hours. NFAT dominant negative plasmid pDN-NFAT and mutant NFAT plasmid pRIT-NFAT that cannot interact with AP1 were also co-transfected. Luciferase activity was measured with a luminometer 48 hours after transfection. The X axis indicates fold increase of luciferase activity. Values represent means ± SE (n = 4), *P<0. 05 by student's t-test. (L) RCAN1 overexpression decreased RCAN1 exon 4 promoter activity. HEK293 cells were co-transfected with pRCAN1mychis and RCAN1 promoter constructs pDE4Luc. Luciferase activity was measured 48 hours after transfection. Values represent means ± SE (n = 4), *P<0. 05 by student's t-test.
Figure 3. RCAN1.4 overexpression exacerbates calcium overloading-induced neuronal apoptosis.
(A) 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) and (C) 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). (D) Quantification of (B) and (C). Values represent means ± SE (n = 3), *P<0. 05 by ANOVA.
Figure 4. Caspase-3 mediates the neurotoxic effect of RCAN1.4 and calcium overloading.
(A) 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) 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.
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