Journal List > Korean J Physiol Pharmacol > v.13(6) > 1025635

Yu, Lim, and Kim: Altered Gene Expression in Cerulein-Stimulated Pancreatic Acinar Cells: Pathologic Mechanism of Acute Pancreatitis

Abstract

Acute pancreatitis is a multifactorial disease associated with the premature activation of digestive enzymes. The genes expressed in pancreatic acinar cells determine the severity of the disease. The present study determined the differentially expressed genes in pancreatic acinar cells treated with cerulein as an in vitro model of acute pancreatitis. Pancreatic acinar AR42J cells were stimulated with 10–8 M cerulein for 4 h, and genes with altered expression were identified using a cDNA microarray for 4,000 rat genes and validated by real-time PCR. These genes showed a 2.5-fold or higher increase with cerulein: lithostatin, guanylate cyclase, myosin light chain kinase 2, cathepsin C, progestin-induced protein, and pancreatic trypsin 2. Stathin 1 and ribosomal protein S13 showed a 2.5-fold or higher decreases in expression. Real-time PCR analysis showed time-dependent alterations of these genes. Using commercially available antibodies specific for guanylate cyclase, myosin light chain kinase 2, and cathepsin C, a time-dependent increase in these proteins were observed by Western blotting. Thus, disturbances in proliferation, differentiation, cytoskeleton arrangement, enzyme activity, and secretion may be underlying mechanisms of acute pancreatitis.

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Fig. 1.
A representative scatter plot of cDNA microarray analysis and modified Venn diagram according to gene function. (A) AR42J cells stimulated with cerulein (labeled with Cy5) or without cerulein (labeled with Cy3) were labeled and hybridized to the cDNA microarray. Cy5/Cy3 ratios indicate relative expression levels. (B) Venn diagram of genes shows functional overlap. Cerulein changed genes related to cell proliferation and differentiation, carcinogenesis, enzyme activity and secretion and cytoskeleton arrangement.
kjpp-13-409f1.tif
Fig. 2.
Time-dependent mRNA expression after cerulein treatment for 8 genes. Relative mRNA expression in AR42J cells treated with cerulein (10–8 M) was assessed by real-time RT-PCR. The internal standard (GAPDH) was coamplified with each gene.
kjpp-13-409f2.tif
Fig. 3.
Western blot analysis for guanylate cyclase, myosin light chain kinase 2, and cathepsin C. Cells were cultured with cerulein for 6 h, harvested, lysed, and extracted. Whole cell extracts (50 μg of protein/lane) were loaded, separated by 8∼10% SDS-polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes by electroblotting. The membranes were blocked with 5% nonfat dry milk in TBS-T. The proteins were detected with specific antibodies. After washing in TBS-T, the immunoreactive proteins were visualized using secondary antibodies conjugated to horseradish peroxidase, followed by enhanced chemiluminescence. Actin was used as a loading control.
kjpp-13-409f3.tif
Table 1.
Altered genes by cerulein
No. Gene Primer sequencesa Foldb
  Up-regulated genes   Cy5/Cy3
1 Regeneration protein, lithostatin (Pancreatic stone protein) (F) ACACCTTGTATCTGTGCTCAATGTAG 7.86
    (R) CAAACTAAAGCTGTTTGCTGTCTGGTA  
2 Guanylate cyclase 2C (F) GTGACATTGTCGGTTTCACG 6.13
    (R) CAAGGCCATCTTGGAAATGT  
3 Myosin light chain kinase 2 (F) CTGACAAGACGGACATGTGG 5.71
    (R) AAGTCTTTGGCCTCGTCTGA  
4 Cathepsin C (F) TCAGACCCCAATCCTGAGTC 3.76
    (R) AACGGAGGCAGTTTTCCTTT  
5 Progestin-induced protein (F) CTGGCAAAAACACAGAAGCA 3.35
    (R) AGCATCGGCATCTGAACTCT  
6 Pancreatic trypsin 2 (F) GGAGGATACACCTGCCAAGA 2.83
    (R) TCCTATCGAAGTTGGGATGC  
  Down-regulated genes   Cy3/Cy5
1 Stathin 1 (F) AAGGATCTTTCCCTGGAGGA 2.66
    (R) TTCTCCTCTGCCATTTTGCT  
2 Ribosomal protein S13 (F) ACCGGCTGGCTCGATACTA 2.50
    (R) GCTTGTGTACGCAACAGCAT  

a Gene sequences used as forward (F) and reverse (R) primers for real-time PCR,

b fold is the ratio of Cy5/Cy3for up-regulated genes and Cy3/Cy5 for down-regulated genes.

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