A Pdx-1-Regulated Soluble Factor Activates Rat and Human Islet Cell Proliferation

Abstract

The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in ␤-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a ␤-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both ␣ and ␤ cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor ␤ (TGF-␤) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and ␤-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and ␤ cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation.T ype 1 and type 2 diabetes ultimately arise from loss of functional islet ␤-cell mass (1-3). Islet transplantation and pharmaceutical activation of ␤-cell regeneration have been considered strategies for treatment of these diseases, but both approaches are hindered by the lack of druggable pathways that reliably induce human ␤-cell replication (4). Furthermore, factors that induce ␤-cell growth must do so without altering key ␤-cell functions, such as glucose-stimulated insulin secretion (GSIS), or causing cellular or DNA damage (5). For example, overexpression of cyclin D or cyclin-dependent kinase 6 (CDK6) induces human ␤-cell proliferation with retention of function (6) but leads to DNA damage, as measured by staining for ␥-H2A member histone family member X (␥-H2AX) (5).Our laboratory has demonstrated that Pdx-1 and Nkx6.1, two homeobox domain transcription factors well known for key roles in islet ␤-cell development, activate proliferation when overexpressed in rat or human islets while maintaining and enhancing GSIS, respectively (7-10). Importantly, the increase in proliferation induced by either factor has little to no impact on ␥-H2AX expression, suggesting that these factors engage "safe" pathways of islet cell replication. In adult islets, Pdx-1 expression is restricted to ␤ and ␦ cells, and Nkx6.1 expression is restricted to ␤ cells. Importantly, expression of these transcription factors is low or absent in nonislet cells, except in the central nervous system, suggesting that deeper knowledge of the molecular pathways that they activate might be an entrée to...

The Transcription Factor Nfatc2 Regulates β-Cell Proliferation and Genes Associated with Type 2 Diabetes in Mouse and Human Islets

“…Thus, using the non-cell type specific promoter to study the ca-NFATs provided a tool to overexpress the construct in all islet cells. Finally, it is also possible that cell-type specific expression of ca-NFAT would nonetheless affect non-transduced cells through the production and secretion of soluble factors, similar to what has recently been reported for β-cell specific expression of Pdx-1 in rat and human islets [42]. Future studies that would utilize targeted expression of Nfatc1 and Nfatc2 are required to fully assess their cell-type specific roles to gene regulation and islet function.…”

Insulin Signaling Regulates the FoxM1/PLK1/CENP-A Pathway to Promote Adaptive Pancreatic β Cell Proliferation

“…A decreased β-cell mass is a consistent feature in T2DM (Meier et al, 2008; Rahier et al, 2008) and provides a strong rationale to explore the cellular mechanism(s) that regulate β-cell mass especially in the context of an adaptive response to insulin resistance. In addition to acute glucose stimulation, which promotes β-cell proliferation and survival by modulating proteins in the growth factor (insulin/insulin-like growth factor-1) signaling pathway (Assmann et al, 2009; Demozay et al, 2011; Shirakawa et al, 2013), recent studies have implicated several other proteins to enhance human β-cell replication some of which act by modulating proteins in the insulin/IGF-1 signaling pathway (Dhawan et al, 2016; Dirice et al, 2016; El Ouaamari et al, 2016; Hayes et al, 2016; Kondegowda et al, 2015; Shirakawa and Kulkarni, 2016; Wang et al, 2015). In pancreatic islets obtained from T2DM donors, the mRNA expression of several proteins in the insulin/IGF-1 signaling cascade are reduced compared with islets from non-diabetes controls (Folli et al, 2011; Gunton et al, 2005; Muller et al, 2006).…”

Creation of versatile cloning platforms for transgene expression and dCas9-based epigenome editing

“…Within this framework, we created a toolbox of >100 pENTR MultiSite Gateway Pro building blocks and 35 custom Gateway destination vectors (Figure 3, Supplemental Tables S1 and S3), enabling the creation of >108,000 unique vectors for a gene of interest. To drive gene expression, we incorporated various ubiquitous (CMV, EF1a) and cell-type specific (RIP (8), TBG (23), cTnT (41)) promoters, as well as our recently-published self-contained Tet-On inducible expression system (8), where the Tet-on activator is driven by CMV, EF1a, or RIP (Figure 3D, E, K, S). For transgene detection, an assortment of epitope tags (HA, FLAG, OLLAS, V5, myc, 6x-His) are provided for fusion to either the N-terminus or C-terminus of the recombinant protein of interest (Figure 3F, N).…”

“…Our group was the first to demonstrate that cultured pancreatic islets could be efficiently transduced with recombinant serotype 5 adenoviruses (Ad5) (2), and since that time, Ad5 vectors have been used to study the impact of manipulation of specific genes on pancreatic islet cell function (2–6), replication (7–10), and survival (5,11). Whereas Ad5 vectors have proven to be an important tool to gain insights into an otherwise difficult model system, virus construction, especially for cell-type specific applications, is still laborious and time-consuming (8). Furthermore, the difficulty in engineering new Ad5 vectors hampers rapid adoption of new technologies and approaches, such as the recent advances in dCas9-mediated epigenetic engineering.…”