Transcriptional regulation of CXC-ELR chemokines KC and MIP-2 in mouse pancreatic acini

Abstract

Neutrophils and their chemoattractants, the CXC-ELR chemokines keratinocyte cytokine (KC) and macrophage inflammatory protein-2 (MIP-2), play a critical role in pancreatitis. While acute pancreatitis is initiated in acinar cells, it is unclear if these are a source of CXC-ELR chemokines. KC and MIP-2 have NF-κB, activator protein-1 (AP-1) sites in their promoter regions. However, previous studies have shown increased basal and reduced caerulein-induced AP-1 activation in harvested pancreatic tissue in vitro, which limits interpreting the caerulein-induced response. Moreover, recent studies suggest that NF-κB silencing in acinar cells alone may not be sufficient to reduce inflammation in acute pancreatitis. Thus the aim of this study was to determine whether acinar cells are a source of KC and MIP-2 and to understand their transcriptional regulation. Primary overnight-cultured murine pancreatic acini were used after confirming their ability to replicate physiological and pathological acinar cell responses. Upstream signaling resulting in KC, MIP-2 upregulation was studied along with activation of the transcription factors NF-κB and AP-1. Cultured acini replicated critical responses to physiological and pathological caerulein concentrations. KC and MIP-2 mRNA levels increased in response to supramaximal but not to physiological caerulein doses. This upregulation was calcium and protein kinase C (PKC), but not cAMP, dependent. NF-κB inhibition completely prevented upregulation of KC but not MIP-2. Complete suppression of MIP-2 upregulation required dual inhibition of NF-κB and AP-1. Acinar cells are a likely source of KC and MIP-2 upregulation during pancreatitis. This upregulation is dependent on calcium and PKC. MIP-2 upregulation requires both NF-κB and AP-1 in these cells. Thus dual inhibition of NF-κB and AP-1 may be a more successful strategy to reduce inflammation in pancreatitis than targeting NF-κB alone.

Fig. 1.

Acinar cells after overnight culture display dose-dependent physiological and pathological responses to caerulein. Amylase secretion (A) measured over 30 min in response to logarithmic doses of caerulein (shown on the x-axis), and expressed as a percentage of total amylase, peaks at 100 pM caerulein. Cultured acinar cells treated with 100 nM caerulein leak lactate dehydrogenase (LDH) (B) in the medium compared with control acini [basal (BAS)]. LDH leakage in the medium was measured in aliquots taken every hour over 4 h, expressed as a percentage of the total LDH content, and plotted against time (x-axis). C: comparison of 100 nM caerulein (CER)-mediated NF-kB activation with basal levels as measured by EMSA on nuclear protein extracts of cultured acinar cells. D–I: confocal immunofluorescence images of cultured acinar cells under basal conditions (D–F) and 45 min after stimulation with 100 nM caerulein (G–I), showing lack of nuclear enrichment (D and E, arrows) of p65 under basal conditions (F). Caerulein causes p65 (H) to enrich in the nucleus (arrows in G and H) with a decrease in cytoplasmic staining (dashed oval).

Fig. 2.

Supraphysiological but not physiological caerulein increases keratinocyte cytokine (KC) and macrophage inflammatory protein-2 (MIP-2) mRNA levels. A: representative images, and corresponding quantification (B), of changes in levels of MIP-2 (upper band, top) and KC (upper band, bottom) compared with 18S (lower bands) in response to 100 nM caerulein over 4.5 h in cultured acini. There is no change in basal levels (A and B). C and D: supraphysiological (0.1 μM), but not physiological, doses of caerulein (0.1 nM) or the high-affinity receptor agonist JMV (1 mM) cause an increase in KC. *P value <0.01 over basal. Representative images are below the graphs showing the results from 3 experiments.

Fig. 3.

Caerulein-induced KC and MIP-2 mRNA increase is dependent on transcription, calcium, and protein kinase C (PKC): H-89 (30 μM) (CER + H-89), 1 μM GF-109203X (CER + GFX), 20 μM BAPTA-AM (CER + BAPTA), or 5 μM actinomycin D (CER + Act.D) were added to the cultured acinar suspension 90 min before stimulation with 0.1 μM caerulein. These were then stimulated for 90 min, and the mRNA levels of KC (A) or MIP-2 (B) were measured by semiquantitative RT-PCR, with 18S as an internal standard. All but H-89 completely prevented the increase in chemokine mRNAs. C and D: cultured acini were stimulated with 0.1 μM caerulein, 1 μM 12-O-tetradecanoylphorbol-3-acetate (TPA), or 1 μM ionomycin (IONO) for 90 min, and the mRNA levels of KC (C) or MIP-2 (D) were measured by semiquantitative RT-PCR, with 18S as an internal standard. Representative images are below the graphs showing the results from 3 experiments. *P < 0.01 compared with basal.

Fig. 4.

MG-132 and 15-deoxy-Δ^12,14-prostaglandin J₂ (PGJ₂) prevent caerulein-induced NF-κB activation, inhibitory κB (IκB) degradation in the cultured acini: The cultured acini were preincubated with or without 20 μM MG-132 (CER + MG-132), or 20 μM PGJ₂ (CER + PGJ₂) for 90 min and then stimulated with 0.1 μM caerulein for 45 min. Electrophoretic mobility shift assay (EMSA) for NF-κB was run on the nuclear protein. A: bands from a representative gel; A′: densitometry; B: IκB-α degradation induced by 100 nM caerulein on Western blotting using chymotrypsin (Chymo) as a loading control, and its prevention by MG-132 and PGJ₂. Quantization of this can be seen in B′. *P < 0.05 compared with other values.

Fig. 5.

PGJ₂ inhibits activator protein-1 (AP-1) activity but does not affect stimulation secretion coupling in acini. A: cultured acini were either left unstimulated (BASAL) or were stimulated with 0.1 μM caerulein after a 90-min pretreatment with 20 μM MG-132 (CER + MG-132), 10 μM lactacystin (CER + Lacta), 25 μM SP-600125, or 20 μM PGJ₂ (CER + PGJ₂) for a total duration of 24 h. Luciferase activity (arbitrary units/mg protein) was measured in each sample, and results were plotted as a percentage of caerulein. The luciferase activity in freshly ground pancreatic tissue was also measured (PANC. TISSUE). †Significant (P < 0.05) increase compared with 100 nM caerulein. *Significant (P < 0.05) decrease compared with caerulein. Cultured acini were washed and suspended in HEPES buffer to measure amylase release (B). Some of these were then preincubated with 20 μM PGJ₂ for 15 min (filled bars), and the amylase release was measured over 30 min from acini that were either unstimulated (BAS) or in response to 0.1 nM and 0.1 μM caerulein. Open bars: amylase from acini untreated with PGJ₂. Secretion was expressed as a percentage of total amylase content of the acini.

Fig. 6.

KC upregulation is NF-κB dependent, but that of MIP-2 requires both NF-κB and AP-1. MG-132 (20 μM) (CER + MG-132), 10 μM lactacystin (CER + Lacta), 25 μM SP-600125, or 20 μM PGJ₂ (CER + PGJ₂) were added to the acinar culture before stimulating these with 0.1 μM caerulein for 90 min. mRNA levels of KC (A) or MIP-2 (B) were measured by real-time PCR. The graphs show the means ± SE from 3 separate experiments. *P < 0.01 compared with CER.