Korean J Physiol Pharmacol.  2009 Dec;13(6):409-416. 10.4196/kjpp.2009.13.6.409.

Altered Gene Expression in Cerulein-Stimulated Pancreatic Acinar Cells: Pathologic Mechanism of Acute Pancreatitis

• Yu JH
• Lim JW
• Kim H

• KMID: 2285374
• DOI: http://doi.org/10.4196/kjpp.2009.13.6.409

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.

Keyword

Cerulein; Pancreatitis; Acinar cells; DNA microarray

MeSH Terms

Acinar Cells
Animals
Antibodies
Blotting, Western
Caerulein
Cathepsin C
Cytoskeleton
Gene Expression
Guanylate Cyclase
Lithostathine
Myosin-Light-Chain Kinase
Oligonucleotide Array Sequence Analysis
Pancreatitis
Proteins
Rats
Real-Time Polymerase Chain Reaction
Ribosomal Proteins
Trypsin
Antibodies
Caerulein
Cathepsin C
Guanylate Cyclase
Lithostathine
Myosin-Light-Chain Kinase
Proteins
Ribosomal Proteins
Trypsin

Figure

• 

  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.

• 

  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.

• 

  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.

Reference

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