HB-EGF Signaling Is Required for Glucose-Induced Pancreatic β-Cell Proliferation in Rats
Authors: Gabriel N Brill
Lee B Honig
Liora S Katz
Vincent Poitout
Grace Fergusson
Melanie Ethier
Donald K Scott
Mallikarjuna R Metukuri
Hasna Maachi
Julien Ghislain
Journal: Diabetes (2020)
Discipline: CLINICAL MEDICINE
Keywords: β-cell compensation; metabolic stress; epidermal growth factor receptor (EGFR); heparin-binding EGF-like growth factor (HB-EGF); glucose
Affiliations: Montreal Diabetes Research Center, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montreal, Quebec, Canada., Department of Pharmacology and Physiology, University of Montreal, Montreal, Quebec, Canada., Diabetes, Obesity, and Metabolism Institute, Icahn School of Medicine at Mount Sinai, New York, NY., Division of Endocrinology, University of Pittsburgh School of Medicine, Pittsburgh, PA., Montreal Diabetes Research Center, Centre de Recherche du Centre Hospitalier de l'Université de Montréal, Montreal, Quebec, Canada ., Department of Medicine, University of Montreal, Montreal, Quebec, Canada.

Abstract:  The molecular mechanisms of β-cell compensation to metabolic stress are poorly understood. We previously observed that nutrient-induced β-cell proliferation in rats is dependent on epidermal growth factor receptor (EGFR) signaling. The aim of this study was to determine the role of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) in the β-cell proliferative response to glucose, a β-cell mitogen and key regulator of β-cell mass in response to increased insulin demand. We show that exposure of isolated rat and human islets to HB-EGF stimulates β-cell proliferation. In rat islets, inhibition of EGFR or HB-EGF blocks the proliferative response not only to HB-EGF but also to glucose. Furthermore, knockdown of HB-EGF in rat islets blocks β-cell proliferation in response to glucose ex vivo and in vivo in transplanted glucose-infused rats. Mechanistically, we demonstrate that HB-EGF mRNA levels are increased in β-cells in response to glucose in a carbohydrate-response element-binding protein (ChREBP)-dependent manner. In addition, chromatin immunoprecipitation studies identified ChREBP binding sites in proximity to the HB-EGF gene. Finally, inhibition of Src family kinases, known to be involved in HB-EGF processing, abrogated glucose-induced β-cell proliferation. Our findings identify a novel glucose/HB-EGF/EGFR axis implicated in β-cell compensation to increased metabolic demand. 

Retracted: No

PubPeer comments:
 In this article, authors claim that they have discovered a ChREBP binding site (carbohydrate response element, ChoRE) 30,000 bp upstream of HBEGF transcription start site (TSS). They are also making a huge claim about finding a glucose-regulated enhancer upstream of HBEGF TSS. They cite a paper by Schmidt et al. (Cell Reports 16, 2359u20132372, August 30, 2016) as the original work that shows ChREBP binding site (ChoRE) in HBEGF promoter. However, we did not find any results in the above mentioned paper by Schmidt et al that supports authorsu2019 claim. They also do not provide the sequence of ChoRE in the HBEGF promoter, which is necessary to compare with the consensus ChoRE. Authors did not rigorously tested if this site is actually a ChoRE. To confirm that this site is indeed a ChoRE, more tests like EMSA, reporter gene assay (with wild type and mutant ChoRE) and ChREBP ChIP assay in siChREBP treated cells are necessary and are lacking in this paper. The Fig. 6C., shows the results on 3C assay that is performed to demonstrate the presence of an enhancer element 30,000 bp upstream of HBEGF TSS that physically interacts with TSS. Firstly, there is no details how they performed this assay. Since this group never performed this 3C assay before, they should have provided detailed method. This is a 3C assay specific to HBEGF gene promoter that requires digestion with restriction enzymes specific to the HBEGF promoter and the authors did not provide that information in the paper. Additionally, since PCR is notoriously known to produce non-specific product, they should have tested the primers using BAC DNA carrying the HBEGF promoter. They also needed to sequence the 3C assay product to confirm that the ligated products are indeed the physically interacting components of HBEGF promoter. These pieces of evidence should have been included with the paper. In the absence all these data, these claims by the authors less believable. They also need to clarify how they determined the interaction frequency that increases with high glucose stimulation. , 
 Thank you for your comments on our manuscript. Here is a point-by-point response to the issues you raised: *u201cHowever, we did not find any results in the above mentioned paper by Schmidt et al that supports authorsu2019 claimu201d* The data from this paper comes from the GEO accession and is reproduced in Figure 6C. There is a dependence on ChREBP u2013 ChREBP binds to a site 30,000 bp away. Interaction with the start site is increased with glucose. *u201cThey also do not provide the sequence of ChoRE in the HBEGF promoter, which is necessary to compare with the consensus ChoRE.u201d* If its 30,000 bp away, it is not a promoter u2013 it is an enhancer. The sequence easily found in the ChIP-seq data: Proximal ChORE: CACGTGGGTCCCACATG Distal ChORE : CTCTTGAGTTTCCTGTG *u201cAuthors did not rigorously tested if this site is actually a ChoRE. To confirm that this site is indeed a ChoRE, more tests like EMSA, reporter gene assay (with wild type and mutant ChoRE) and ChREBP ChIP assay in siChREBP treated cells are necessary and are lacking in this paper.u201d* We agree that we could have done EMSAs and luciferase assays in vitro (as we have done many times in the past) but found that ChREBP did not bind to any region near the start site u2013 so it does not bind to the promoter in cellulo. We felt it was important to establish the binding in cellulo, which is more relevant to the living system, to a site at distance (definition of an enhancer) and that it ultimately interacts with the TSS u2013 which is more biologically interesting. We also agree that inhibiting the binding of ChREBP to one or both of the binding sites in 6C - and only those sites - using a CRISPR approach would be the best way to confirm our conclusion. We have developed an adenoviral approach to do exactly that, but it was not available when we published this paper. Perhaps we should have concluded that HBEGF is likely a direct target of ChREBP through these elements located at a distance based on our accumulated data. Nevertheless, we believe our data are sound and our conclusion is likely correct. *u201cSince this group never performed this 3C assay before, they should have provided detailed method. This is a 3C assay specific to HBEGF gene promoter that requires digestion with restriction enzymes specific to the HBEGF promoter and the authors did not provide that information in the paper. Additionally, since PCR is notoriously known to produce non-specific product, they should have tested the primers using BAC DNA carrying the HBEGF promoter. They also needed to sequence the 3C assay product to confirm that the ligated products are indeed the physically interacting components of HBEGF promoter. These pieces of evidence should have been included with the paper. In the absence all these data, these claims by the authors less believable. They also need to clarify how they determined the interaction frequency that increases with high glucose stimulation.u201d* While not our first 3C publication, we have published extensively with ChIP and nuclear run-ons and other molecular biological techniques detailing mechanisms of transcription factor regulation of transcription. The 3C experiment was performed exactly as described in Nature Protocols (Nat Protoc 2007;2:1722u20131733). We could have included more information about how we did the 3C, the digestion pattern of dpnII on the 30,000 bp, and on the crucial controls that we performed, including checking restriction digestion and ligation efficiencies, and the other controls included in the Nature Protocols article. , 
 According to UCSC genome browser, rat HBEGF gene located in the Chr18 chr18:29,329,831-29,340,184. We looked in the GSE81628, file GSE81628_ChREBP_peakfile.txt.gz in the GEO Access Viewer that has the ChREBP ChIPseq data in INS1E rat insulinoma cells submitted by Schmidt et al. However, we did not find any peak ID that is about 30,000bp upstream of HBEGF gene. We request the authors to provide the peak ID in the GSE81628_ChREBP_peakfile.txt.gz that they are referring to have the ChORE upstream of HBEGF. The journal u201cDiabetesu201d where this work is published requires to include these information in the article to help the readers to completely understand the results. We are pointing this because when senior authors of the article who are editorial board members and reviewers of u201cDiabetesu201d, they have greater onus when following these rules. , 
 To visualize the data as we did in the paper, please go to: https: www.ncbi.nlm.nih.gov geo download ?acc=GSE81628 Download the file labeled: Series supplementary data archive (contains BEDGRAPH files): GSE81628_RAW.tar There will be bedgraph files (compressed) representing the various treatment groups (2 h and 12 h 25 mM glucose, vs 5 mM glucose treatments) as well as ChREBP, Med1 and DNase datasets suitable for uploading into the free IGV genome browser available from the Broad institute at: https: software.broadinstitute.org software igv download It is important to use the rat Rn5 as the reference genome. ,