GLP-1 and Autocrine TGF-β/BMP SMAD Synergy in Step-Wise Initiation and Maturation during β-Cell Differentiation of AR42J Cells
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Type 1 diabetes mellitus in Australia and around the globe is reaching epidemic proportions. Our newfound capacity to transplant islets with good engraftment using a relatively innocuous immunosuppression regimen shifts the pressure for research onto a need for a vast supply of islets to cure or ameliorate the disease worldwide. The logical source for these islets is engineered -cells from a stem or progenitor cell population. In order to engineer such cells, the proper environmental or extracellular signals must be developed to channel the precursors towards the pancreatic -cell phenotype. Clearly, an empirical or alchemy approach of testing different growth factor cocktails or recipes to induce progenitor cells to form pancreatic -cells could be successful and likely to yield a long-lasting and ideal solution. With recent advances in the knowledge of two key areas, allogeneic islet transplantation and -cell replacement therapy, there is a growing opportunity to better understand the forces that control differentiation of early embryonic stem cells to become insulin-secreting -cells for the treatment of Type 1 diabetes. Increasing success with such microsurgical/cell engineering approaches will mandate an increased source of insulin-producing cells. Furthermore, while pancreatic stem cells may be a good source for the transplantations, this requires understanding how to induce these stem cells to become a useful population of pancreatic -cells. The signaling of Transforming Growth Factor- (TGF-) isoforms, together with their intracellular mediators, the Smad proteins, and GLP-1/Exendin-4 provide a proinsulin inductive capacity in pancreatic acinar AR42J cells. However, no relationship had previously been established between TGF- signaling and GLP-1 signaling. In this group of investigations, evidence of a novel interrelationship between these two signaling pathways has been provided. Most importantly, interdependence between them was shown to be necessary for -cell formation. Moreover, it has been shown that stimulation by Exendin-4, a potent long-acting agonist of GLP-1, leads to a specific autocrinemediated intracellular transcription factor activation of insulin-positive differentiation by AR42J cells. Like TGF- isoforms, Bone Morphogenetic Proteins (BMPs), members of the TGF- superfamily of proteins, have also been shown to regulate a wide range of biological and cellular responses during early embryogenesis. The intracellular mechanisms existing between these two signaling pathways have been dissected. It was found that BMP signaling represents a novel downstream target of Exendin-4 (GLP-1) signaling and serves as an upstream regulator of TGF- isoform signaling to differentiate the AR42J cells into insulin-expressing -cells. Preliminary results indicated that the induction of insulin-positive AR42J cell differentiation occurring in response to Exendin-4 was mediated through TGF- isoforms and BMP ligands, as well as their downstream activation of Smad proteins. Further inhibitory studies using either pan-neutralizing antibodies or morpholino antisense oligonucleotides, and a synergistic augmentation of insulin differentiation with the addition of exogenous ligands, both strongly point toward a critical role for TGF- and BMP signaling in driving insulin-positive differentiation of AR42J cells. In short, an interaction existing between the TGF- isoform signaling pathways and the GLP-1/Exendin-4 pathway leading to insulin-positive differentiation in AR42J cells has been defined. This novel synergistic relationship is necessary for endocrine - cell differentiation and maturation. In addition, manipulation of this system led to greatly enhanced insulin gene expression, and hence has great significance for cellular engineering therapy. The exact mechanism by which this interplay occurs was also elaborated, in terms of both receptors and Smad intracellular signaling molecules that mediate the response. Similar signaling pathways operating during signaling by other TGF- superfamily members have been explored that also induce the insulin-positive differentiation of AR42J cells. In particular, BMP signaling plays an analogous and importantly related role. However, competition between these two signaling pathways at multiple levels significantly impacts on the selection of differentiation pathways to mature pancreatic -cells. Thus, the novel research findings presented in this thesis have important implications for the engineering of pancreatic progenitor cells to become functional pancreatic -cells for the treatment of insulin-dependent Type 1 diabetes mellitus.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
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Appendix 1 which consists of reprints of published papers has not been published here.
Type 1 diabetes mellitus
Transforming growth factor-β isoforms