Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2008 Jan;19(1):198-206.
doi: 10.1091/mbc.e07-05-0430. Epub 2007 Oct 31.

Cholecystokinin activates pancreatic calcineurin-NFAT signaling in vitro and in vivo

Grzegorz T Gurda  1 , LiLi Guo, Sae-Hong Lee, Jeffery D Molkentin, John A Williams
Affiliations

Cholecystokinin activates pancreatic calcineurin-NFAT signaling in vitro and in vivo

Grzegorz T Gurda et al. Mol Biol Cell. 2008 Jan.

Abstract

Elevated endogenous cholecystokinin (CCK) release induced by protease inhibitors leads to pancreatic growth. This response has been shown to be mediated by the phosphatase calcineurin, but its downstream effectors are unknown. Here we examined activation of calcineurin-regulated nuclear factor of activated T-cells (NFATs) in isolated acinar cells, as well as in an in vivo model of pancreatic growth. Western blotting of endogenous NFATs and confocal imaging of NFATc1-GFP in pancreatic acini showed that CCK dose-dependently stimulated NFAT translocation from the cytoplasm to the nucleus within 0.5-1 h. This shift in localization correlated with CCK-induced activation of NFAT-driven luciferase reporter and was similar to that induced by a calcium ionophore and constitutively active calcineurin. The effect of CCK was dependent on calcineurin, as these changes were blocked by immunosuppressants FK506 and CsA and by overexpression of the endogenous protein inhibitor CAIN. Parallel NFAT activation took place in vivo. Pancreatic growth was accompanied by an increase in nuclear NFATs and subsequent elevation in expression of NFAT-luciferase in the pancreas, but not in organs unresponsive to CCK. The changes also required calcineurin, as they were blocked by FK506. We conclude that CCK activates NFATs in a calcineurin-dependent manner, both in vitro and in vivo.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Expression pattern of calcineurin-regulated NFAT genes (NFATc1-c4) in exocrine pancreas. (A) Semiquantitative RT-PCR using primer pairs specific for NFATc1-c4 genes, as described in Materials and Methods. Lane 1, RT control; lane 2, kidney; lane 3, liver; lane 4, pancreas, lane 5, pancreatic acini, and lane 6, acinar cell-derived AR42J cell line. Cyclophillin A was included as a control. (B) Western blot analysis of NFAT expression at the protein level. Lanes 1 and 2, cytoplasmic [C] and nuclear [N] extracts of pancreatic acini (30 μg/lane); Lanes 3 and 4, C and N extracts of whole pancreas (30 μg/lane); lanes 5 and 6, C and N extracts of Jurkat T-cell line (positive control, 15 μg/lane), separated slightly for clarity. Cyclophilin A (CypA) was used as a loading control.
Figure 2.
Figure 2.
Stimulation by CCK leads to an increase in nuclear occupancy of endogenous NFATs. (A) Isolated acinar cells were left untreated (CTL) or treated with 1 μM of A23187, 10 μM of carbachol and 30 or 100 pM of CCK for 1 h. Cytoplasmic [C] and nuclear [N] extracts were prepared, loaded at 30 μg/lane and analyzed by Western blotting, using antibodies for NFATc1-c3. Representative blots, along with nuclear (lamin A/C) loading control are shown. Blots of CCK-treated extracts (lanes 7–10) were separated for clarity. (B) Quantitation of nuclear occupancy based on densitometry measurements of Western blots such as the one shown in A and adjusted for total protein as described in Materials and Methods. Values are the mean and SE for 3–6 experiments. *p ≤ 0.05 versus control.
Figure 3.
Figure 3.
CCK promotes nuclear translocation of NFATc1-GFP. Isolated mouse acini were incubated overnight with 107 PFU/ml either NFATc1-GFP or βgal/GFP adenovirus, loaded with DRAQ5 nuclear-labeling dye and imaged on a Cell-Tak–coated glass coverslip using an Olympus FluoView 500 confocal microscope. (A) Time course of a single isolated mouse acinus before (top) and after (bottom) 30-min treatment with 100 pM CCK. (B) Sample images of acini treated for 30 min with agents as listed. In experiments using FK506 and CaCN, acini were pretreated for 30 min and overnight, respectively. (C) Randomly chosen medium-sized (5–20 cell) DRAQ5-loaded acini expressing GFP (approx. 95% of all acinar cells) were used to quantitate % of nuclei showing NFATc1 signal, using colocalization of DRAQ5 and GFP. Data are a mean ± SE of 3–5 independent acinar isolations with averaged counts from at least 10 acini per preparation. *p < 0.01 compared with vehicle-treated control.
Figure 4.
Figure 4.
CCK activates NFATs in isolated acinar cells (in vitro). Isolated acinar cells were incubated for 6.5 hours with 5 × 106 PFU/ml NFAT-luciferase (NFAT-luc) adenovirus, treated, lysed, and assayed for luciferase activity. For adenoviruses (CaCN, FLAG-CAIN, or βgal/GFP), treatments were carried out overnight with 107 PFU/ml; for all other agents, cells were treated for 5.5 h. (A) Dose-response activation of NFAT-luc reporter with 10 pM–1 nM of CCK. (B) Dose-response inhibition of CCK-induced NFAT-luc activation by specific calcineurin inhibitors FK506 (100 pM–10 nM) and CsA (1 nM–100 nM). (C) Inhibition of CCK- and CaCN-induced NFAT-luc activation by overexpression of truncated form of endogenous calcineurin inhibitor CAIN (Flag-CAIN). (D) Dose-response activation of NFAT-luciferase with A23187, bombesin, and carbachol. Relative luciferase activity (RLU) was normalized to protein concentration in the lysates. Data represent a mean ± SE of 3–8 independent acinar isolations with averaged counts of four wells per preparation. *p < 0.05 or **p < 0.01 compared with vehicle-treated controls and #p < 0.05 or ##p < 0.01 when compared with agonist (100 pM CCK)-treated controls.
Figure 5.
Figure 5.
Elevation in endogenous CCK secretion promotes increase in nuclear NFATs in vivo. (A) Mice adapted to powdered diet were fasted overnight and fed with a chow containing 0.1% camostat for 0–1.5 h. Animals treated with FK506 were injected with 3 mg/kg twice a day for 2 d before fasting. Nuclear protein was extracted and analyzed by Western blotting. Blots shown are representative of at least three independent experiments for NFATc1-c3, along with nuclear (lamin A/C) loading control. (B) Short-term treatment with camostat-containing diet does not alter total NFAT protein expression in the pancreas. Whole cell extracts were loaded with 60 μg/lane and analyzed by Western blotting using antibodies for NFATc1-c3. Representative blots, along with loading control (Lamin A/C), are shown.
Figure 6.
Figure 6.
Elevation in endogenous CCK leads to NFAT activation in vivo. (A) FVBN male NFAT-luciferase mice and their nontransgenic littermates were fed with either a control chow or a chow containing 0.1% camostat for 7 d. The mice were then killed, and their livers and pancreases were quickly excised and homogenized. Animals treated with FK506 were injected with 3 mg/kg twice a day for 2 d before and throughout the time of treatment with camostat. (A) Feeding camostat-containing diet increases NFAT-luciferase reporter expression in the pancreases of transgenic NFAT-luc mice but not in wild-type littermate controls; in the liver there is no difference in reporter expression due to camostat treatment in either group. (B) Injection of FK506 completely blocked the camostat-induced increase in NFAT-luciferase reporter expression in the pancreases of NFAT-luc transgenic mice. Liver expression was not affected. n = 3–8 animals per group. **p < 0.01 compared with the control chow-fed group; ##p < 0.01 compared with the saline-injected, camostat-fed group.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Asagiri M., et al. Autoamplification of NFATc1 expression determines its essential role in bone homeostasis. J. Exp. Med. 2005;202:1261–1269. - PMC - PubMed
    1. Bentrem D. J., Joehl R. J. Pancreas: healing response in critical illness. Crit. Care Med. 2003;31:S582–589. - PubMed
    1. Berube F. L., Benrezzak O., Vanier M., Morisset J. Effects of cerulein and epidermal growth factor on pancreatic growth in the reserpinized rat model. J. Pediatr. Gastroenterol. Nutr. 1993;17:39–48. - PubMed
    1. Brackmann P., Rosemeyer D. Oral foy in the treatment of chronic pancreatitis. Ric. Clin. Lab. 1984;14:435–437. - PubMed
    1. Brannon P. M. Adaptation of the exocrine pancreas to diet. Annu. Rev. Nutr. 1990;10:85–105. - PubMed

Publication types

MeSH terms

LinkOut - more resources