Modulation of transcription factor expression induces adult human pancreatic exocrine-endocrine transdifferentiation and protects rat beta-cells against cytokine-induced apoptosis

Scriptie/Masterproef: Doctoral Thesis

Samenvatting

Given its recent clinical success, interest in treating insulin-dependent diabetes by transplantation of human insulin-producing beta-cells has been renewed. However, the severe limitation of the available amount of donor islet tissue only allows treatment of a very small fraction of patients. Because mature differentiated beta-cells cannot be expanded significantly in vitro, the need for innovative methods to establish an abundant supply of transplantable, insulin-producing tissue represents a highly relevant international research topic. A potential solution is to generate novel insulin-producing cells via differentiation of embryonic or adult stem cells or via transdifferentiation of mature cells.
Although so far no conclusive evidence regarding the location of pancreatic cells with endocrine (trans)differentiation potential has been obtained, nor of the underlying mechanisms controlling their (trans)differentiation, indications for an endocrine (trans)differentiation potential in pancreatic duct cells urged us to examine their potential as a source for new beta-cells. Given the general idea that cell-specific transcription factors regulating the mature, differentiated phenotype of a particular cell are often also required for its embryonic development, we performed a direct comparison of the transcription factor expression profile of isolated adult human endocrine cells versus duct cells. The absence of an endocrine-specific transcription factor from the duct cell population would suggest its importance as an essential molecular endocrine developmental switch and, therefore, its usefulness as a target for experimental manipulation. In view of the fact that; (i) Foxa2, Pdx1/Ipf1 and Hnf6 are present in islet and duct cells, (ii) neurogenin-3 (ngn3) functions transiently immediately downstream from these factors during embryonic development of the mouse endocrine pancreas, and (iii) ngn3 is expressed at very low levels in duct and islet cells, we focused on the basic Helix-Loop-Helix (bHLH)-factor ngn3. We hypothesized that its low expression level in duct cells might constitute a physiological block to initiate an endocrine developmental program. Therefore, we designed ngn3 gain-of-function experiments, based on an adenoviral transduction system.
Our work demonstrates the capacity for induction of an endocrine transdifferentiation program in isolated adult human duct cells. Adenoviral-mediated expression of ngn3 in adult duct cells recapitulates the embryonic pancreatic endocrine developmental program, culminating in the induction of insulin expression in a significant fraction of transduced duct cells. During this process, a hierarchical induction of the transcription factors Pax4, NeuroD1, Nkx6.1 and Pax6 - all known to be acting downstream from ngn3 during embryonic islet formation - is observed. Furthermore, a duct-to-beta-cell phenotypic switch is evident by the induction of endocrine and beta-cell specific markers (i.e. synaptophysin, chromogranin A, prohormone convertase 1/3, glucokinase and insulin). However, from a theoretical therapeutic standpoint, both the extent and yield of the endocrine transdifferentation process towards fully mature, glucose-sensitive insulin-producing cells needs to be greatly improved.
To our knowledge, this is the first study demonstrating endocrine transdifferentiation of adult human duct cells induced by a single gene transfer. Furthermore, we show unambiguously that ngn3 is the switch for induction of a transcription factor-dependent program, involved in endocrine pancreas differentiation. Causative sequential activation of the factors downstream from ngn3 had never been demonstrated so far. Therefore, we provide a simple in vitro model to study early endocrine pancreas development and endocrine transdifferentiation, supplementing the proposed hierarchy model of transcription factors, as established by transgenic and knockout mouse analysis. Similar studies of MyoD and related bHLH proteins in cell culture have led to considerable progress in understanding the regulation of myogenesis. Understanding the hierarchy of the molecular events involved in islet formation is a prerequisite for the generation of transplantable insulin-producing tissue.
Ultimately, from a clinical transplantation standpoint, new sources of insulin-producing cells will need to resist the host's immune responses. Although improved immunosuppression protocols are being developed, the risks associated with life-long generalized, non-specific immunosuppression do not appear justified by the potential benefits of islet transplantation. Novel immunoprotective strategies will need to be developed for beta-cell transplantation therapy in insulin-dependent diabetes.
The hallmark of the autoimmune process operating in type I diabetes is a chronic inflammatory mononuclear cell infiltration of the islets. In the course of insulitis, activated macrophages and T-cells secrete soluble mediators, including IL-1-beta, IFN-gamma and TNF-alpha. In response to these cytokines, Nuclear Factor-kappaB (NF-kappaB) modifies beta-cell gene-expression. Several of the responsive beta-cell genes have an essential role in the outcome of the inflammation process, be it positive (beta-cell protection) or negative (beta-cell death). However, the major outcome of cytokine-induced NF-kappaB activation in beta-cells remainded elusive. In order to elucidate its role, we blocked the activity of NF-kappaB in cytokine-exposed primary rat beta-cells by transduction with a mutant, non-degradable form of its inhibitor I-kappaB, thereby locking NF-kappaB in an inactive cytosolic protein complex. Our experiments demonstrate that blocking the activity of NF-kappaB in cytokine-exposed beta-cells prevents gene expression of several NF-kappaB targets, including iNOS, Fas and MnSOD. Furthermore, beta-cell survival during cytokine-exposure is significantly improved, mainly through inhibition of the apoptotic pathway. Our results thus indicate that NF-kappaB activation upon cytokine-exposure has primarily a pro-apoptotic function in beta-cells. Together with the forthcoming identification of additional NF-kappaB-regulated genes by microarray analysis, novel therapeutic targets for the prevention of recurrent autoimmune-mediated cell destruction after transplantation of insulin-producing cells may be disclosed.
Datum prijsjun 2003
Originele taalEnglish
BegeleiderHenry Heimberg (Promotor), Luc Bouwens (Co-promotor), Luc Leyns (Jury), Zhidong Ling (Jury), Ivan Van Riet (Jury), Elisabeth Peters (Jury), Pedro Herrera (Jury), Chantal Mathieu (Jury) & Bernard Peers (Jury)

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