Profiling CCK-mediated pancreatic growth: the dynamic genetic program and the role of STATs as potential regulators

Abstract

Feeding mice with protease inhibitor (PI) leads to increased endogenous cholecystokinin (CCK) release and results in pancreatic growth. This adaptive response requires calcineurin (CN)-NFAT and AKT-mTOR pathways, but the genes involved, the dynamics of their expression, and other regulatory pathways remain unknown. Here, we examined the early (1-8 h) transcriptional program that underlies pancreatic growth. We found 314 upregulated and 219 downregulated genes with diverse temporal and functional profiles. Several new identifications include the following: stress response genes Gdf15 and Txnip, metabolic mediators Pitpnc1 and Hmges2, as well as components of growth factor response Fgf21, Atf3, and Egr1. The genes fell into seven self-organizing clusters, each with a distinct pattern of expression; a representative gene within each of the upregulated clusters (Egr1, Gadd45b, Rgs2, and Serpinb1a) was validated by qRT-PCR. Genes up at any point throughout the time course and CN-dependent genes were subjected to further bioinformatics-based networking and promoter analysis, yielding STATs as potential transcriptional regulators. As shown by PCR, qPCR, and Western blots, the active phospho-form of STAT3 and the Jak-STAT feedback inhibitor Socs2 were both increased throughout early pancreatic growth. Moreover, immunohistochemistry showed a CCK-dependent and acinar cell-specific increase in nuclear localization of p-STAT3, with >75% nuclear occupancy in PI-fed mice vs. <0.1% in controls. Thus, the study identified novel genes likely to be important for CCK-driven pancreatic growth, characterized and biologically validated the dynamic pattern of their expression and investigated STAT-Socs signaling as a new player in this trophic response.

Fig. 1.

A, B: molecular concept mapping (MCM): graphical representation of statistically derived association networks between genes of interest and color-coded molecular concepts (see color legend). Size of a node reflects number of genes in the set and the thickness of the line relates to odds ratio of the association. A: associations with gene sets up at 1,2,4 and 8 h of protease inhibitor (PI) feeding (green nodes). B: associations with gene sets down at 4 and 8 h of PI feeding (green nodes). Not enough genes were down at 1 or 2 h to conduct an analysis. C, D: a compiled Ingenuity cell and molecular function categorization analysis for 1 h, 2 h, 4 h and 8 h of PI feeding. C: top 5 functional categories altered at 1–4 h. D: top 5 functional categories altered at 4–8 h.

Fig. 2.

Left: clustering analysis of all genes (n = 533, corresponding to 608 probes) significantly altered at least once in the 1–8 h course of pancreatic growth (up or down at any point). Gene cluster into 7 groups with temporally distinct patterns (A–G). A: peak at 2–8 h; B: up at 4 h and/or 8 h; C: peak at 1–2 h; D: up at 4–8 h; E: decreased at 2–8 h; F: down at 4 h; G: slow decrease with max trough at 8 h. Green arrows point to 38 calcineurin (CN)-dependent (FK506-inhibited) genes. Right: graphical representation of 12 random genes within a cluster (black lines) and averaged time course of all genes within a cluster (red line) shown as fold induction (log 10 scale) at 1–8 h vs. fasted controls.

Fig. 3.

RT-qPCR assays for a representative gene from each upregulated cluster: Egr1 (A), Gadd45b (B), Rgs2 (C), and Serpinb1a (D). Rcan1 (E) and cyclophilin (CycloA) (F) were shown to be upregulated and unchanged, respectively; n = 4–7, **P < 0.01.

Fig. 4.

Two sets of genes analyzed using bioinformatics-based tools: 1) 314 genes up at any point of the early 1–8 h course of pancreatic growth and 2) 38 CN-dependent genes, corresponding to green arrows in Fig. 2. A: MCM association network; B: regionminer (Genomatix) transcription factor motif sites found vs expected with enrichment and Z-score. C: analogous analysis for transcription factor modules, with top 5 enriched and de-enriched modules by Z-score.

Fig. 5.

STAT3 is activated early in the course of cholecystokinin (CCK)-mediated growth of the exocrine compartment of the pancreas. A: immunohistochemistry for P-Tyr705 STAT3 and 4′6-diamidino-2-phenylindole (DAPI) nuclear stain in fasted mice or mice fasted and then refed PI-containing chow for 1, 2, or 3 h. Representative ×40 field (top) and quantitation of percent nuclear localization of P-STAT3 (bottom), with 3 mice per group (n = 3, **P < 0.01). B: representative Western blot (top) with summary of densitometry data below of p-Tyr705 STAT3 and total STAT3; 4 independent experiments (n = 4, **P < 0.01). C: immunohistochemistry for p-Tyr 705 STAT3 in exocrine pancreas of fasted (inset) and 2 h PI-fed mice (top left, A); immunohistochemistry for a ductal marker CK19 with arrows used for emphasis of duct-specific labeling (top right, B); overlay of p-Tyr705 STAT3 (green), ductal marker CK19 (red), and nuclear label DAPI (blue), showing the activation and nuclear localization of p-STAT3 occurs specifically in acinar cells, but not in islets or ducts (bottom left, C); corresponding Nomarski image (bottom right, D).

Fig. 6.

Activation of STAT3 requires an increase in endogenous CCK induced by PI feeding. A: immunohistochemistry for P-Tyr705 STAT3 and staining for DAPI in exocrine pancreas for the following conditions: fasting, ad libitum feeding, and fasting with refeeding of either control chow or chow containing PI. Representative ×40 field (top) and quantitation of percent nuclear localization of P-STAT3 (bottom), with 4 mice per group (n = 4, **P < 0.01). B: representative Western blot (top) and summary data of P-Tyr705 STAT3, total STAT3, and CycloA (control) for same conditions as listed in A; 4 mice per group (n = 4, **P < 0.01). C: representative Western blot (top) and summary data for wild-type (WT) and CCK-deficient (CCK−/−) mice that were fasted or fasted and then refed PI-containing chow; 3 mice per group (n = 3, **P < 0.01).

Fig. 7.

Socs2, a feedback regulator of Jak-STAT signaling, is activated in the early course of CCK-mediated pancreatic growth. A: RT-PCR for Socs2 expression during 1–8 h course of PI feeding; static gene CycloA served as control. B: quantitative RT-PCR for Socs2 expression during early course of PI feeding; values normalized to fasting controls (n = 4–7, P < 0.01). C: representative Western blot for protein expression of Socs2 during early course of PI feeding; CycloA as control.