Abstract
BackgroundOur prior characterization of RasGrf1 deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf1 to the generation of those “pancreatic” phenotypes.ResultsHere, we compared the transcriptional profile of highly purified pancreatic islets from RasGrf1 KO mice to that of WT control animals using commercial oligonucleotide microarrays. RasGrf1 elimination resulted in differential gene expression of numerous components of MAPK- and Calcium-signaling pathways, suggesting a relevant contribution of this GEF to modulation of cellular signaling in the cell lineages integrating the pancreatic islets. Whereas the overall transcriptional profile of pancreatic islets was highly specific in comparison to other organs of the same KO mice, a significant specific repression of Pttg1 was a common transcriptional alteration shared with other tissues of neuroectodermal origin. This observation, together with the remarkable pancreatic phenotypic similarities between RasGrf1 KO and Pttg1 KO mice suggested the possibility of proximal functional regulatory links between RasGrf1 and Pttg1 in pancreatic cell lineages expressing these proteins.Analysis of the mPttg1 promoter region identified specific recognition sites for numerous transcription factors which were also found to be differentially expressed in RasGrf1 KO pancreatic islets and are known to be relevant for Ras-ERK signaling as well as beta cell function. Reporter luciferase assays in BT3 insulinoma cells demonstrated the ability of RasGrf1 to modulate mPttg1 promoter activity through ERK-mediated signals. Analysis of the phenotypic interplay between RasGrf1 and Pttg1 in double knockout RasGrf1/Pttg1 mice showed that combined elimination of the two loci resulted in dramatically reduced values of islet and beta cell count and glucose homeostasis function which neared those measured in single Pttg1 KO mice and were significantly lower than those observed in individual RasGrf1 KO mice.ConclusionsThe specific transcriptional profile and signaling behavior of RasgGrf1 KO pancreatic islets, together with the dominance of Pttg1 over RasGrf1 with regards to the generation of these phenotypes in mouse pancreas, suggest that RasGrf1 is an important upstream component of signal transduction pathways regulating Pttg1 expression and controlling beta cell development and physiological responses.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1019) contains supplementary material, which is available to authorized users.
Highlights
Our prior characterization of RasGrf1 deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf1 to the generation of those “pancreatic” phenotypes
Room temperature (RT)-PCR analysis of purified RNA from wild type and RasGrf1 KO pancreatic islets showed that RasGrf1 mRNA is strongly expressed in wild type islets and, as expected, its expression was totally lost in the KO islets (Figure 1A)
A similar RasGrf1 expression pattern was observed at the protein level, as Western blot analysis of islet protein immunoprecipitates allowed detection of the full size 140 kDa RasGrf1 protein band in wild type islets, which was completely absent in the RasGrf1 KO samples
Summary
Our prior characterization of RasGrf deficient mice uncovered significant defects in pancreatic islet count and size as well as beta cell development and signaling function, raising question about the mechanisms linking RasGrf to the generation of those “pancreatic” phenotypes. The RasGrf proteins act by coupling different upstream stimuli to activation of various intracellular pathways through the specific contribution of some of its structural domains, including the CDC25-H domain capable of activating Ras family members, the Ca+2/Calmodulin interacting IQ domain [5,10], two PH domains, or a DH (Dbl homology) domain capable of activating Rho/Rac family GTPases [10]. Due to this complex domain structure, RasGrf is able to couple Ras activation to different signal transduction processes, such as those mediated by Ca2+/calmodulin [1,11], heterotrimeric G proteins [12], cAMP activity [13], CDC42 [14], or even non-receptor tyrosine kinases [15]. RasGrf elimination is associated to defective eye phenotypes including impaired retinal photoreception [18] and altered lens growth [19]
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