# Phosphorylation of the MNK1 substrate eIF4E is not required for response to acute pancreatitis

Amandine Alard, Manon Strehaïano, David Müller, Charline Lasfargues, Stéphanie Cassant-Sourdy, Christine Jean, Corinne Bousquet, Yvan Martineau, Stéphane Pyronnet

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Amandine Alard, Manon Strehaïano, David Müller, Charline Lasfargues, Stéphanie Cassant-Sourdy, et al.. Phosphorylation of the MNK1 substrate eIF4E is not required for response to acute pancreatitis. Pancreatology, 2021, 21 (4), pp.677-681. 10.1016/j.pan.2021.02.013. hal-04823535

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# Phosphorylation of the MNK1 substrate eIF4E is not required for response to acute pancreatitis

Amandine Alard<fnref n="1" />, Manon Strehaïano<fnref n="1" />, David Müller<fnref n="1" />, Charline Lasfargues, Stéphanie Cassant-Sourdy, Christine Jean, Corinne Bousquet, Yvan Martineau, Stéphane Pyronnet*

INSERM UMR-1037, Université de Toulouse, Centre de Recherches en Cancérologie de Toulouse (CRCT), Equipe Labellisée Ligue Contre le Cancer, Laboratoire d'Excellence Toulouse Cancer (TOUCAN), Toulouse, France

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

**Article history:**

Received 14 September 2020

Received in revised form

21 January 2021

Accepted 10 February 2021

Available online 19 February 2021

**Keywords:**

Acute pancreatitis

eIF4E

MNK1

## ABSTRACT

**Background:** The MNK1 protein kinase is directly activated by the MAPK pathway and is specifically expressed in pancreatic acinar cells. Both the MNK1 kinase and the MAPK pathway are required for response to pancreatitis, suggesting that their pharmacological targeting would be of therapeutic interest. Because the mRNA cap-binding protein and translation initiation factor eIF4E is the major known MNK1 substrate, one could anticipate that the protective function of MNK1 in pancreatitis is mediated by eIF4E phosphorylation.

**Methods:** Acute pancreatitis was induced by the intraperitoneal administration of cerulein in wild-type mice and in transgenic mice carrying two non-phosphorylatable Eif4e alleles. The expression and phosphorylation of proteins of the MNK1-eIF4E pathway was visualized by western blotting. The severity of pancreatitis was monitored by the measure of serum amylase levels and by histopathology and immunohistochemistry using apoptosis and immune infiltrate markers.

**Results:** Despite a strong induction in MNK1 kinase activity in both wild-type and transgenic mice, precluding eIF4E phosphorylation has no impact on the severity of acute pancreatitis. Serum amylase is equally induced in both mouse genotypes and neither acinar cell apoptosis nor immune infiltrate is exacerbated.

**Conclusion:** eIF4E phosphorylation is not required for response to pancreatitis indicating that the acinar-cell-specific MNK1 kinase acts in acute pancreatitis *via* another substrate.

© 2021 The Authors. Published by Elsevier B.V. on behalf of IAP and EPC. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

The MNK1 protein kinase is directly phosphorylated by ERK1/2 or p38 MAPKs [1] and has been shown to be specifically expressed in pancreatic acinar cells where it is the unique kinase phosphorylating the mRNA translation initiation factor eIF4E [2,3]. Both the MAPK pathway [4] and MNK1 [2] are required for a proper response to acute pancreatitis. The biological function and regulation of eIF4E are well characterized. Through its interaction with eIF4G, another translation initiation factor serving as a docking site for its kinase MNK1 [5], eIF4E recruits the ribosome to capped mRNAs thus stimulating mRNA translation into proteins [6]. In contrast, the

eIF4E-binding proteins 1 and 2 (4E-BP1/2) compete with the eIF4G/MNK1 complex for binding to eIF4E thus inhibiting eIF4E phosphorylation and mRNA translation [7]. The phosphorylation of eIF4E is an excellent read-out of MNK1 activity. It was used as such earlier to validate that eIF4E is exclusively phosphorylated by MNK1 in the pancreas where eIF4E phosphorylation is totally abrogated in mice knocked-out for the two Mnk1 alleles (named Mnk1-KO) [2]. We expected that mice genetically engineered to express only a non-phosphorylatable mutant of eIF4E protein would recapitulate the pancreatitis aggravating phenotype of Mnk1-KO mice.

<footnote>* Corresponding author. INSERM UMR-1037, CRCT, 2 avenue Hubert Curien, 31037 Toulouse, France.</footnote>

<footnote>E-mail address: stephane.pyronnet@inserm.fr (S. Pyronnet).</footnote>

<footnote n="1">¹ These authors contributed equally.</footnote>
# Methods

## C57Bl/6J mouse strains and experimental pancreatitis

Mice were maintained in a specified-pathogen-free barrier facility under a 12-h light cycle and euthanized according to ethical regulations. 4E-WT and 4E-KI mice carrying a homozygous substitution of Ser209 into Ala209 in the eIF4E polypeptide were described earlier [8]. To allow a direct comparison with the data obtained next to the use of Mnk1-KO [2] and Eif4ebp1/2-DKO [9] mice, two protocols of acute pancreatitis were performed: one "short" protocol as in Ref. [2] and one "long" protocol as in Ref. [9]. Experiments were performed with 2- to 5-month-old ("short" protocol) and with 10- to 14-month-old ("long" protocol) mice.

The "short" protocol of acute pancreatitis consisted in one series of 6 hourly intraperitoneal injections with either 0.9% sodium chloride (saline) or 50 µg/kg cerulein. Blood was sampled either before injections (0h), during the acute phase of pancreatitis (2h, 4h, 8h and 24h) or after recovery (48h). Mice were sacrificed during the acute phase (24h) and after recovery (48h).

The "long" protocol of consisted two series separated by a one-day break of 6 hourly intraperitoneal injections with either 0.9% sodium chloride (saline) or 75 µg/kg cerulein. Blood was sampled either before injections (0h), during the acute phases of pancreatitis (8h and 24h) or after recovery (96h). Mice were sacrificed during the acute phase (72h) and after recovery (96h).

All experiments using animals were done in accordance with the principles and guidelines established by INSERM Anexplo UMS006 and were approved by the institutional and regional animal care and use committees.

## Serum amylase level

Serum amylase activity was measured in triplicates for each time point using the Phadebas Amylase Test (Magle) according to the manufacturer's instructions.

## Antibodies

Antibodies used for western blotting (WB): anti-eIF4E (#2067), anti-MNK1 (#2195), anti-phospho-MNK1 (#2111S) and anti-4E-BP1 (#9452) and were from Cell Signaling Technologies; anti-phospho-eIF4E (#76256) and anti-PTF1A (#62818) were from Abcam; anti-actin (A2228) and anti-amylase (A8273) were from Sigma.

Antibodies used for immunohistochemistry (IHC): anti-eIF4E and anti-phospho-eIF4E were as above; and anti-cleaved-Caspase3 (#9665) was from Cell Signaling Technologies; anti-CD45 (#10558) was from Abcam.

## SDS-PAGE and WB

Pancreas were collected, frozen in liquid nitrogen and crushed using cold mortar and pestle. Organ powders were then lysed in 150 mM NaCl, 50 mM TrisHCl pH7.5, 1 mM EDTA pH 8.0, 1% Triton x100, 2 mM NaF, 2 mM Na3VO4, 0.5 mM DTT supplemented with protease inhibitors (Roche) and subjected to sonication. Protein concentration was estimated using the Bradford method (Protein Assay kit, Biorad). Equal amounts of proteins were subjected to SDS-PAGE and transferred onto nitrocellulose membranes (Bio-TraceNT; Pall Corp). Membranes were then saturated in 5% non-fat dry milk TBS-T solution (Tris buffer saline: 150 mM NaCl, 10 mM Tris-HCl pH 7.6, 0.1% Tween20) before incubation overnight with primary antibodies diluted in 5% BSA (Bovine Serum Albumin) TBS-T. After several washes and incubation with HRP-coupled secondary antibodies diluted in TBS-T, membranes were revealed using

the SuperSignal West Pico Chemiluminescent Substrate (Pierce) and visualized thanks to a PXi Imager (Syngene).

## Pancreas staining and IHC

Seven micrometers formalin-fixed, paraffin-embedded serial pancreatic sections were first deparaffined and rehydrated. Antigen retrieval and immunostaining were conducted using standard methods Sections were incubated with primary antibodies diluted in Antibody diluent (Dako) for one night at 4 °C. Endogenous peroxidase were blocked using peroxidase blocking solution (PBS, 10% methanol, 0.3% H₂O₂) and signal amplification was performed by incubating samples with peroxidase-conjugated secondary antibodies using either Signal stain boost (Cell Signaling Technologies) for detection of rabbit antibodies or ImmPRESS anti-mouse Ig polymer detection reagent (Vector) for detection of mouse antibodies. Bound peroxidase was visualized using AEC + Substrate-Chromogen or DAB + reagent (Dako). Sections were washed, counterstained with Mayer's hematoxylin solution (Sigma-Aldrich) and mounted using Glycer gel Mounting Medium (Dako). One representative slide per mouse was imaged using either an Eclipse E400 microscope (Nikon Corporation) or a Hamamatsu Nano-zoomer 2-slide scanner (Hamamatsu Photonics). Image processing was done with Explora Nova Morpho Expert software (Explora Nova). Quantification of the cleaved-caspase3 and CD45 positive cells was done on five random pictures per pancreatic section using the ImageJ software (NIH).

## Statistical analysis

Quantitative data are expressed as the mean ± SD. Statistical analyses were performed by unpaired Student's t-test two-tailed using the GraphPad program. *P* values < 0.05 were considered statistically significant.

# Results

## Abrogating eIF4E phosphorylation does not alter the exocrine pancreas phenotype

Before looking at the potential role of eIF4E phosphorylation in experimental pancreatitis, we first evaluated the exocrine pancreas phenotype of homozygous mice engineered to express only a non phosphorylatable mutant of eIF4E protein (Eif4e<sup>5209A/5209A</sup> or 4E-KI mice). These mice were generated earlier and exhibit no altered global phenotype [8]. To date however, no data are available concerning a potential more specific phenotype in the pancreatic exocrine function. A WB analysis first confirmed that eIF4E phosphorylation is undetectable in the pancreas of 4E-KI mice as compared to that of 4E-WT mice (Fig. 1A). Similarly, despite that IHC-staining of pancreatic serial sections showed no signal for phosphorylated eIF4E in 4E-KI mice, the 4E-KI pancreatic architecture revealed no abnormality as compared to that of 4E-WT mice (Fig. 1B). Together with the fact that amylase secretion by isolated acini is not significantly affected (not shown), these data are consistent with a modest (if any) role of eIF4E phosphorylation in pancreatic exocrine function (as is the case for Mnk1-KO mice [2]).

## MNK1 activation and induction of eIF4E phosphorylation upon acute pancreatitis

Knocking-out either the eIF4E protein kinase MNK1 (Mnk1-KO) [2] or its two inhibitory proteins 4E-BP1 and 4E-BP2 (Eif4ebp1/2-DKO) [9] engenders similar phenotypes under cerulein-induced pancreatitis in mice. Each knock-out aggravates the severity of
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Abrogating eIF4E phosphorylation does not alter the exocrine pancreas phenotype. (A) WB of 4E-WT (mice # 1 and 2) and 4E-KI (mice # 2 and 3) whole pancreas extracts showing no phosphorylation of eIF4E in the 4E-KI genotype as compared to 4E-WT. (B) Immunohistochemistry (IHC) of pancreatic serial sections showing no eIF4E phosphorylation and no morphologic abnormality in the pancreas of 4E-KI mice as compared to 4E-WT mice. Scale bar: 20 µm.</figcaption></figure>

To test whether lacking eIF4E phosphorylation also exerts an exacerbating effect, the two distinct protocols of cerulein-induced pancreatitis set earlier for *Mnk1*-KO ("short" protocol [2]) and *Eif4ebp1/2*-DKO ("long" protocol [9]) mice were here reproduced but using 4E-WT and 4E-KI mice (as described in Fig. 2A). The data confirmed that, at the protein level, MNK1 activity and eIF4E phosphorylation (first visualized by phospho-specific antibodies in the "short" protocol) increase massively but transiently upon pancreatitis in wild-type (WT) mice, while, as expected, no eIF4E phosphorylation is detected in 4E-KI mice despite a strong induction in MNK1 activity (Fig. 2B). In addition, 4E-BP1 is similarly hypophosphorylated in 4E-WT and 4E-KI mice (Supplementary Fig. 1), a feature which, through eIF4E sequestration, contributes to the inhibition of protein synthesis observed in the acute phase of pancreatitis [9,10]. Very similar results were observed when using the "long" protocol (not shown).

*Induction of eIF4E phosphorylation does not contribute to the severity of acute pancreatitis*

Given the massive induction of MNK1 activity upon acute

pancreatitis, one could anticipate that precluding phosphorylation of its substrate eIF4E would recapitulate the aggravating phenotype observed in *Mnk1*-KO mice. To test for this hypothesis, different markers of pancreatitis severity were monitored in 4E-KI vs 4E-WT mice. Surprisingly, genetic abrogation of eIF4E phosphorylation has no impact (or even a non-significant decrease) on plasma amylase levels measured in both "short" (Fig. 3A, left) and "long" (Fig. 3A, right) protocols of cerulein-induced pancreatitis. Consistently, while both *Mnk1*-KO [2] and *Eif4ebp1/2*-DKO [9] mice showed earlier a more altered phenotype than WT mice, 4E-KI mice do not. Indeed, 4E-WT and 4E-KI mice exhibit equal alterations in pancreatic tissue architecture such as disordered acinar organization, presence of metaplastic lesions and interstitial expansion (Fig. 3B, top left). Also, apoptosis (visualized by cleaved caspase-3-specific antibodies) (Fig. 3B, middle left) and immune infiltrate (visualized by CD45-specific antibodies) (Fig. 3B, bottom left) in 4E-WT and 4E-KI mice of both "short" and "long" protocols reveals similar inductions after quantification (Fig. 3B, right). These data indicate that, as opposed to MNK1 activation or 4E-BP1/2 hypophosphorylation, phosphorylation of eIF4E is not involved in the severity of pancreatitis.

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** MNK1 activation and induction of eIF4E phosphorylation upon acute pancreatitis. (A) "Short" (as in Ref. [2], 2 days d1-d2) and "long" (as in Ref. [9], 4 days d1-d4) protocols for cerulein-induced acute pancreatitis. 4E-WT and 4E-KI mice (3 in each group and for each time point) were subjected to either one series (horizontal purple arrow) or two series separated by a one-day break (horizontal yellow arrow) of 6 hourly intraperitoneal injections of cerulein (vertical red arrows) and sacrificed as described in the Methods section. Blood sampling was performed at several time points during each protocols (vertical black arrows) as indicated in the Methods section. (B) WB of 4E-WT and 4E-KI whole pancreas extracts from mice of the "short" cerulein protocol showing MNK1 activation in both 4E-WT and 4E-KI mice, but eIF4E phosphorylation increase only in 4E-WT mice.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Induction of eIF4E phosphorylation does not contribute to the severity of acute pancreatitis. (A) Kinetics of serum amylase levels from mice of the "short" (left) and "long" (right) cerulein protocols showing no significant difference between 4E-WT and 4E-KI mice upon cerulein-induced pancreatitis. Values are the means ± SD and expressed as fold induction of the mean value obtained for 4E-WT mice at time 0h. ns: non-significant. (B) Representative H&E staining + IHC images of serial pancreatic sections from mice sacrificed during the acute phase (24h) of the "short" cerulein protocol (left). Image J quantifications showing no difference in either cCASP3 or CD45 staining level between 4E-WT and 4E-KI mice sacrificed during the acute phase of both "short" (24h; purple dots) and "long" (72h; yellow dots) cerulein protocols. Scale bar: 50 µm.</figcaption></figure>

Discussion

As compared to other organs, the mouse pancreas expresses
very high amounts of *Mnk1* mRNA and protein in part due to a
direct interaction of *Mnk1* promoter with the pancreatic acinar cell-
specific transcription factor PTF1A [2]. We here confirm this
observation. However, what can appear quite intriguing is that the
pancreas does not express corresponding high levels of *Eif4e* mRNA
and protein (Supplementary Fig. 2). Our data may reconcile this
apparent discrepancy since they strongly suggest that MNK1 acts in
the pancreas through a substrate other than eIF4E. Furthermore, in
part due to 4E-BP1 hypophosphorylation and consequent seque-
tration of eIF4E away from productive translation initiation com-
plexes, protein synthesis is well known to be repressed during
cerulein-induced acute pancreatitis [9,10]. However, MNK1 is
robustly activated. Given the protein synthesis stimulating role
generally attributed to eIF4E phosphorylation by MNK1 [6], these
two events appear also counterintuitive. Yet, they further argue in
favor of distinct roles for MNK1 activation and eIF4E phosphory-
lation during pancreatitis. Although phosphorylation of eIF4E is not
necessary for a proper response to pancreatitis, it is very likely that
the control of eIF4E protein availability (independently of its
phosphorylation status) is required. This assumption is supported

by the indirect, but strong, evidences showing that precluding
eIF4E sequestration through the knock-out of its two inhibitory
proteins 4E-BP1 and 4E-BP2 heavily aggravates the severity of
pancreatitis [9].

Thus, our observations reveal that although MNK1 plays a crit-
ical protective role in the response to pancreatitis [2], phosphory-
lation of its major substrate eIF4E is not required. These findings
have important consequences in a clinical context. They indicate
that targeting eIF4E phosphorylation *per se* may not be a good
option for treating pancreatitis. They however suggest the exis-
tence of (an)other critical MNK1 substrate(s) whose identification
will permit a better characterization of the MNK1 pathway in a
therapeutic perspective.

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgments

We thank Prof. Nahum Sonenberg for sharing 4E-KI mice. The
Genotype-Tissue Expression (GTEx) project was supported by the
Common Fund of the Office of the Director of the National Institutes
of Health, and by NCI, NHGRI, NHLBI, NIDA, NIMH, and NINDS. The
data used for the analyses described in this manuscript were ob-
tained from the GTEx Portal on 09/09/2020.

**Appendix A. Supplementary data**

Supplementary data to this article can be found online at
https://doi.org/10.1016/j.pan.2021.02.013.

Grant support

Support for this study came from INSERM #U1037SE17BA (SP),
Fondation de France #RAF18006BBA (SP) and Fondation Toulouse
Cancer Santé #RAK17026BBA (SP).

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