Yonsei Med J.  2013 May;54(3):772-777. 10.3349/ymj.2013.54.3.772.

Human SNF2L Gene Is Regulated Constitutively and Inducibly in Neural Cells via a cAMP-Response Element

• Xia Y
• Wang L
• Ma C
• Gong Y
• Zhao Y

• KMID: 1727896
• DOI: http://doi.org/10.3349/ymj.2013.54.3.772

Abstract

PURPOSE
SNF2L belongs to Imitation Switch family and plays an essential role in neural tissues and gonads. In our previous studies, we have demonstrated that the basal transcription of human SNF2L gene is regulated by two cis-elements, cAMP response element (CRE)- and Sp1-binding sites. Recent studies suggested that cyclic adenosine monophosphate (cAMP) stimulation significantly up-regulated SNF2L expression in ovarian granulose cells. These data suggested that protein kinase-mediated signal pathways might also regulate SNF2L expression in neural cells. We therefore investigated the effects of agents that activate protein kinases A on SNF2L gene expression in neural cells.
MATERIALS AND METHODS
To increase intracellular cAMP levels, all neural cells were treated with forskolin and dbcAMP, two cAMP response activators. We exmined the effects of cAMP on the promoter activity of human SNF2L gene by luciferase reporter gene assays, and further examined the effects of cAMP on endogenous SNF2L mRNA levels by qPCR.
RESULTS
Transient expression of a luciferase fusion gene under the control of the SNF2L promoter was significantly increased by treatment of rat primary neurons with forskolin or dbcAMP, but not PC12, C6 and SH-SY5Y cells. Consistently, treatment with forskolin or dbcAMP could enhance endogenous SNF2L mRNA levels also only in rat primary neurons.
CONCLUSION
These results suggest that the CRE consensus sequence in the SNF2L proximal promoter most likely confers constitutive activation and regulation by cAMP in neural cells.

Keyword

SNF2L; CRE; cAMP stimulation; neural cells

MeSH Terms

Animals
Bucladesine/pharmacology
Cell Line
Colforsin/pharmacology
Cyclic AMP/*metabolism
DNA-Binding Proteins/chemistry/*genetics/metabolism
*Gene Expression Regulation
Humans
Luciferases/analysis
Neurons/*metabolism
PC12 Cells
Promoter Regions, Genetic
RNA, Messenger/metabolism
Rats
Rats, Wistar
Recombinant Fusion Proteins/analysis
*Response Elements
Transcription Factors/chemistry/*genetics/metabolism
DNA-Binding Proteins
RNA, Messenger
Recombinant Fusion Proteins
Transcription Factors
Colforsin
Bucladesine
Cyclic AMP
Luciferases

Figure

• 

  Fig. 1 Effects of forskolin or dbcAMP on the promoter activity of human SNF2L gene in PC12, C6, SH-SY5Y and rat primary neurons. We investigated the activity of the CRE-Wild and CRE-Mut promoters in response to cAMP activator forskolin or dbcAMP. (A) The addition of forskolin or dbcAMP to transiently transfected cells resulted in no activation of the CRE-Wild and CRE-Mut constructs in PC12 cells, and the same effects were deteted in C6 and SH-SY5Y cells. (B) Treatment with forskolin or dbcAMP increased luciferase activity nearly 4.0-fold in the CRE-Wild construct, In contrast, the response to cAMP activator was diminished in the CRE-Mut construct. *Values of statistical significance (Student t-test; p<0.05).

• 

  Fig. 2 Effect of cAMP on endogenous SNF2L gene expression in PC12, C6, SH-SY5Y and rat primary neurons. The SNF2L mRNA levels in neuron cells were detected by qPCR, with or without cAMP activator forskolin or dbcAMP treatment. Treatment with forskolin or dbcAMP did not enhance endogenous SNF2L mRNA levels in PC12, C6 and SH-SY5Y cells, but the SNF2L mRNA levels with cAMP induced showed more than 4.0-fold higher than the levels unstimulated. *Values of statistical significance (Student t-test; p<0.05).

• 

  Fig. 3 Effects of the cAMP activator forskolin or dbcAMP. Western blot analyses of nuclear extracts from neuron cells treated with or without cAMP activator for 24 h. Whether in SH-SY5Y cells or rat primary neurons, levels of phosphorylated CREB significantly increased with forskolin or dbcAMP treatment, but this effects were not detected in total CREB levels.

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