Physiological growth of the pancreas takes place in response to high protein diet, hyperphagia, pregnancy and lactation20
. The molecular mechanisms that govern this adaptive response, however, are poorly understood. Here, we examined the expression profile of CCK-mediated pancreatic growth. We focused on CN-NFAT signaling axis, identifying several novel growth-related, NFAT-regulated genes. We also demonstrate that one of these genes, Rcan1, functions as a CN/NFAT-dependent feedback inhibitor that controls growth plasticity of adult pancreas.
The results from our expression profile both validate and extend past work. Sets of genes significantly altered in our expression profile significantly overlap with molecular concepts linked to pathways previously shown to be activated CCK, including MAPK-AP114
. Aside from CN-NFAT-Rcan1, our work also points to JAK-STAT and its inhibitors Socs2 and Socs3 as yet another signal regulating this response (Suppl. Table I
and unpublished data). Several of our 38 CN-dependent genes have been tied to regulation of growth, differentiation and metabolism. FGF21 has been shown to be important for hepatic regeneration and β-cell survival23
, HB-EGF in β-cell transdifferentiation and pancreatic cancer24
, whereas Socs3 for proliferation and angiogenisis in the liver25
. We posit that these and other membrane-bound paracrine/autocrine mediators alter local microenvironment, drive hyperplasia or differentiation and thereby direct pancreatic growth. Analogous mechanisms have already been shown in the liver following a partial resection26
and the pancreas following pancreatitis27
. Broadly, our results argue that CCK-mediated pancreatic growth has much in common with these regenerative responses.
NFATs are best known as transcriptional mediators of inflammation. Their role as regulators of growth or tissue homeostasis in general, however, is not as clear. Our work is one of the most detailed analyses of genome-wide expression that underlies an adaptive response linked to CN-NFAT signals. We predict a sizable new set of NFAT-regulated genes related to pancreatic growth and show that NFATc1 binds to promoters of selected high quality identifications. The time frame of increased NFATc1 binding by ChIP corresponds to CCK-stimulated nuclear shuttling of NFATc1-GFP in acini8
and the kinetics of NFAT translocation in other cell types. Lastly, NFATs have intrinsically low affinity for DNA and therefore function within complexes or modules. Known partners of NFATs include GATA-4 in the heart, C/EBP in lung, MEF2 in muscle and AP-1/Foxp3 in immune cells28
. Complex-dependent effects dictate timing, affinity and outcome of NFAT binding in a tissue or stimulus-dependent manner. Here, we identify several enriched transcriptional modules among CN-dependent genes, including the well-known synergistic NFAT/AP-1 complex29
. As a site of convergence, transcriptional complexes may explain why inhibition of a single pathway can be sufficient to disrupt an entire physiological response.
Rcan1 expression increased throughout the early course of pancreatic growth and endogenous Rcan1 strongly inhibited CCK-induced CN-NFAT signaling. This led us to formulate the central paradigm of this study: CCK-mediated activation of CN leads to nuclear translocation and activation of NFATs, NFAT-dependent induction of Rcan1 and finally RCAN1-mediated inhibition of CN. The exact means whereby CN-NFAT-RCAN1 axis constrains growth, however, remains unknown. One rationale is that RCAN1 blocks proliferation; an alternative may be that RCAN1 limits angiogenesis. NFATc1 has been shown to regulate vascular endothelial growth factor (VEGF) expression30
and mice that overexpress Rcan1 showed decreased tumor formation due attenuated angiogenesis31
. Rcan1 is also expressed at a higher level in Down Syndrome (Rcan1 is also known as Down Syndrome Critical Region 1 or DSCR1) and may decrease susceptibility to solid tumors in these individuals. CCK also increased expression and NFATc1 binding to the promoter of Leukemia Inhibitory Protein (Lif). Lif has recently been shown to regulate microvessel density and VEGF expression in retinopathy32
. Direct or indirect role of Rcan1 in regulation of angiogenesis is certainly a promising area for future research.
Finally, our work here shows that transgenic overexpression of Rcan1 is sufficient to completely block pancreatic growth. The pre-emptive expression of Rcan1 clamps the CN-NFAT-Rcan1 signal in a permanent “off” position, independent of CCK. This tissue-specific genetic approach circumvents side effects of pharmacological agents like immune suppression, nephro and neurotoxicity, whereas inducible overexpression preempts possible developmental defects or compensatory changes. Rcan1 also inhibited many, but not all of the genes blocked by CN inhibitor FK506. Interestingly, Rcan1 appeared to significantly inhibit only CN-dependent genes which we also computationally predicted to be NFAT-regulated. NFAT-specific skewing of the inhibitory effect of Rcan1 on CN has been suggested elsewhere, but primarily based on biochemical evidence33
Recent advances in manipulation of cell fate and plasticity of pancreatic tissue brought renewed promise of treatments for pancreatitis, pancreatic cancer and diabetes. Our work and the expression profiles of pancreatic development34
and pancreatic cancer36
are grounds for further integrative research. Relevantly, NFATc1 has been shown to be inappropriately activated in pancreatic cancer37
. The role of Rcan1 has yet to be examined, but may well prove to be important based on significant downregulation of its message in at least two microarrays of human pancreatic cancer samples38–39
In summary, we examined the expression profile of an early point of CCK-mediated pancreatic growth. We computationally and experimentally examined several growth related genes, focusing on endogenous CN regulator Rcan1. Rcan1 showed a strong, sustained expression throughout the growth response and significantly inhibited CCK-simulated CN-NFAT signaling. Most notably, inducible, exocrine-specific expression of Rcan1 in vivo completely blocked acinar cell hyperplasia and the accompanying CCK-driven pancreatic growth. Our work identifies an important new feedback mechanism that delimits adaptive pancreatic growth and provides firm evidence that CN-NFAT axis is necessary for this hormonally-mediated, adaptive response.