Johanson-blizzard syndrome: pathophysiology of ubiquitin ligase deficiency in pancreatic acinar cells

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<h3>Introduction</h3> Johanson-Blizzard syndrome (JBS) is an autosomal recessive multisystem disorder including exocrine pancreatic inflammation and insufficiency. JBS is associated with mutations in the UBR1 gene, which encodes an E3 ubiquitin ligase of the N-end rule pathway. One of its substrates is RGS4, a GTPase activating protein that down-regulates specific G proteins. The UBR1- and RGS4-dependent mechanisms of pancreatic damage in JBS are unknown and were studied here. <h3>Methods</h3> From wild type (wt) and <i>ubr-1</i> knock-out mice (ubr1ko) pancreatic acini were freshly prepared by collagenase digestion. Intracellular trypsin activation was measured fluorometrically with (CBZ-Ile-Pro-Arg)₂-R110 (10µM). G-protein-controlled Ca²⁺ signalling was monitored with FURA2-AM (2µM) by ratio imaging. <h3>Results</h3> In isolated pancreatic acini of ubr1ko mice we found a significant increase in intracellular trypsin activation upon physiological stimulation (caerulein, 0.1nM) associated with a significant disruption of cell-cell contacts. Calcium analysis after physiological stimulation revealed an increase of pathological Ca²⁺ signalling events, i.e., significant decrease of spike number and significant increase of spike duration. Pre-incubation of ubr1ko acini with a specific RGS4 inhibitor (CCG-4986, 10µM) normalised Ca²⁺ pattern and prevented premature trypsin activation and acinar disintegration. CCG-4986 did not affect trypsin activity itself. <h3>Conclusion</h3> In acinar cells of ubr1ko mice, pathophysiological signalling pathways were followed by acinar destruction, which could be abolished by RGS4 inhibition, suggesting a vital function of ubr1 and RGS4 in the defense against pathological damage– and JBS as an inflammatory disorder due to an inadequate UBR1 defence.