Functional significance of SPINK1 promoter variants in chronic pancreatitis

Abstract

Chronic pancreatitis is a progressive inflammatory disorder of the pancreas, which often develops as a result of genetic predisposition. Some of the most frequently identified risk factors affect the serine protease inhibitor Kazal type 1 (SPINK1) gene, which encodes a trypsin inhibitor responsible for protecting the pancreas from premature trypsinogen activation. Recent genetic and functional studies indicated that promoter variants in the SPINK1 gene might contribute to disease risk in carriers. Here, we investigated the functional effects of 17 SPINK1 promoter variants using luciferase reporter gene expression assay in four different cell lines, including three pancreatic acinar cell lines (rat AR42J with or without dexamethasone-induced differentiation and mouse 266-6) and human embryonic kidney 293T cells. We found that most variants caused relatively small changes in promoter activity. Surprisingly, however, we observed significant variations in the effects of the promoter variants in the different cell lines. Only four variants exhibited consistently reduced promoter activity in all acinar cell lines, confirming previous reports that variants c.-108G>T, c.-142T>C, and c.-147A>G are risk factors for chronic pancreatitis and identifying c.-52G>T as a novel risk variant. In contrast, variant c.-215G>A, which is linked with the disease-associated splice-site mutation c.194 + 2T>C, caused increased promoter activity, which may mitigate the overall effect of the pathogenic haplotype. Our study lends further support to the notion that sequence evaluation of the SPINK1 promoter region in patients with chronic pancreatitis is justified as part of the etiological investigation.

Keywords: acinar cell; chronic pancreatitis; luciferase reporter; promoter mutation; trypsin inhibitor.

Fig. 1.

The serine protease inhibitor Kazal type 1 (SPINK1) promoter sequence used in this study as cloned into the pGL3-Basic plasmid. The sequence corresponds to the region from c.–541 to c.35 of the human SPINK1 gene. The positions of the promoter variants are underlined and emboldened. The original SPINK1 translation initiation codon is highlighted in green. Note that this codon is not in frame with the downstream luciferase start codon. The major transcriptional start site at c.–60 is shown in red. Binding sites for transcription factors hepatocyte nuclear factor 1 and pancreas-specific transcription factor 1 are indicated in blue and gray, respectively.

Fig. 2.

Luciferase reporter gene expression by the SPINK1 promoter. The indicated cell lines were transfected with the pGL3-SPINK1 plasmid or the promoterless pGL3-Basic plasmid, and luciferase expression was determined as described in materials and methods. The ratios of the luciferase activities (SPINK1 over control) are indicated as fold change with pGL3-Basic values arbitrarily set at unity. Means ± SD (n = 10–45 transfections) are shown. Note that measured firefly luciferase activities varied among cell lines; typical values with pGL3-Basic transfections were in the 10,000–50,000 relative light units range for AR42J, AR42J + dexamethasone (Dexa), and 266-6 cells, whereas about 2–7 million relative light units were detected with HEK 293T cells.

Fig. 3.

Effect of SPINK1 promoter variants on luciferase expression. The indicated cell lines were transfected with SPINK1 promoter constructs, and luciferase expression was determined as described in materials and methods. Relative luciferase activities were expressed as percentages of the wild-type value. Means ± SD (n = 3–6 transfections) are shown. Data for 4 variants in the dexamethasone-differentiated AR42J cells were taken from Hegyi et al. (14). *P < 0.05, using Student's t-test. Dexa, dexamethasone.