EGF blocks intestinal shedding via ERK.

Epidermal growth factor suppresses intestinal epithelial cell shedding both in vitro and in vivo

via a MAPK dependent pathway.

Jennifer C. Miguel1*, Adrienne A. Maxwell1*, Jonathan J. Hsieh1, Denise Al Alam2, Lukas C. Harnisch4, D. Brent Polk1,3, Ching-Ling Lien2,3, Alastair J. M. Watson4, and Mark R. Frey1,3.1Department of Pediatrics, University of Southern California Keck School of Medicine and The Saban Research Institute at Children’s Hospital Los Angeles, Los Angeles, CA2Department of Surgery, University of Southern California Keck School of Medicine and The Saban Research Institute at Children’s Hospital Los Angeles, Los Angeles, CA3Department of Biochemistry and Molecular Biology, University of Southern California Keck School of Medicine, Los Angeles, CA4Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich, United Kingdom

*Denotes equal contribution

Running title: EGF blocks intestinal cell shedding via MAPK.

Keywords: Intestinal epithelium, Inflammatory bowel disease, Epidermal growth factor receptor (EGFR), MAP kinases (MAPKs), Epithelial cell, Cell shedding

To whom correspondence should be addressed: Mark R. Frey, The Saban Research Institute at Children’s Hospital Los Angeles, 4650 Sunset Blvd., MS#137, Los Angeles, CA, USA, 90027; Tel.: (323) 361-7204; email: mfrey@chla.usc.edu; and Alastair J. M. Watson, Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK; Tel.: +(0)1603 597266; email: alastair.watson@uea.ac.uk

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Abstract

Cell shedding from the intestinal villus is a key element of tissue turnover, essential to maintain

health and homeostasis. However, the signals regulating this process are not well understood. We

asked whether shedding is controlled by epidermal growth factor receptor (EGFR), an important

driver of intestinal growth and differentiation. In 3D ileal enteroid culture and cell culture

models (MDCK, IEC-6, IPEC-J2 cells), extrusion events were suppressed by EGF, as

determined by direct counting of released cells or rhodamine-phalloidin labeling of condensed

actin rings. Blockade of MEK/ERK, but not other downstream pathways such as PI3K or PKC,

reversed EGF inhibition of shedding. These effects were not due to a change in cell viability.

Furthermore, EGF-driven MAPK signaling inhibited both caspase-independent and -dependent

shedding pathways. Similar results were found in vivo, in a novel zebrafish model for intestinal

epithelial shedding. Together, the data show that EGF suppresses cell shedding in the intestinal

epithelium through a selective, MAPK dependent pathway affecting multiple extrusion

mechanisms. EGFR signaling may be a therapeutic target for disorders featuring excessive cell

turnover, such as inflammatory bowel diseases.

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EGF blocks intestinal shedding via ERK.

Introduction

The intestinal epithelium, a monolayer of polarized cells separating the organism from

luminal gut contents, is the most rapidly renewing tissue in adult mammals . Routine turnover of

this tissue without loss of the barrier requires coordination between stem cell proliferation and

shedding/extrusion of mature cells from the upper villus or colonic surface mucosa. Accelerated

shedding, which can lead to infection and exacerbated immune responses , is associated with

inflammatory bowel diseases [IBD; ] and endotoxemia . This “pathological” shedding can be

induced in experimental models by cytokines or lipopolysaccharide . However, much less is

known about regulation of constitutive physiological shedding, and especially about signals that

repress it. Understanding the mechanisms controlling normal, physiological cell extrusion could

identify targets for correcting pathological shedding in diseases such as IBD.

Physiological cell shedding occurs through at least two mechanisms. In situ apoptosis of a

damaged cell can trigger extrusion . Alternatively, acute crowding reportedly promotes live cell

shedding through a sphingosine-1-phosphate and Rho-kinase dependent mechanism , with

apoptosis occurring after loss of attachment rather than as a cause. In either case, the process

involves Rho-driven myosin ring formation and contraction by neighboring cells and remodeling

of tight junctions . These mechanisms are conserved in several epithelial cell types , and

presumably across most vertebrates.

Endogenous regulators of constitutive shedding, including factors that restrain it, are not well

understood. In this study, we tested the possibility that epidermal growth factor (EGF) receptor

(R) is involved in this process. EGFR is a receptor tyrosine kinase that controls intestinal cell

growth, repair, and migration . The overlap in the mechanical forces and cytoskeletal alterations

in cell migration and cell extrusion suggest a possible role for EGFR in shedding; furthermore, as

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EGF blocks intestinal shedding via ERK.

EGF is an epithelial cell mitogen , EGFR activation might be expected to induce crowding and

thus increase shedding. We used coordinated in vivo (3D enteroid miniguts and cultured IEC-6,

MDCK, and IPEC-J2 cells) and in vivo (adult zebrafish gut) models to study the role of EGFR in

constitutive, non-pathological shedding. Our results show that, somewhat surprisingly, EGFR

suppresses cell extrusion via a MAPK-dependent mechanism.

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EGF blocks intestinal shedding via ERK.

Results and Discussion

EGF suppresses cell shedding in vitroTo model intestinal turnover, we first generated ileal

epithelial enteroids from mice expressing the Lifeact-EGFP cytoskeleton-labeling construct .

Shedding events in these enteroids (Fig. 1A) show the characteristic early saccular and funnel

morphologies described in vivo , and can be viewed in real time (Supplementary Video

Shed1.mov; box encloses an event). Shed cells per unit distance of epithelial perimeter can be

counted over time. Cultures treated with EGF showed a 40% decrease in shedding per unit

distance versus control (Fig. 1A).

Similar results were observed in cell culture. MDCK cells on Transwells were treated with

EGF (10 ng/mL) or the EGFR inhibitor AG1478 (150 nM) for 4 h, and stained with rhodamine-

phalloidin. EGF reduced the number of shedding events (0.55 vs. 3.0 per field in control;

p<0.01), identified as a cell surrounded by a condensed actin ring/funnel and neighboring cells

assembled in a characteristic “rosette” pattern [ and see Fig. 1B]. By counting nuclei displaced

above the plane of the monolayer in orthogonal projection (Figure 1C), we also observed that

EGF reduced and AG1478 induced shedding (4.5 or 19.8 vs. 12.5 in control displaced

nuclei/field; p<0.01). Interestingly, phosphorylated (activated) EGFR in unstimulated MDCK

monolayers was only found in cells distant to a shedding event (Fig. 1D), consistent with the

notion that EGFR activation restrains shedding.

To develop a convenient model for mechanistic studies, we live-labeled cells with DAPI ,

washed away debris, and returned cultures to growth medium for up to 4 h. Over time, extruded

cells were collected and counted by fluorescence microscopy (example images, Figure 1E).

Results from this method were consistent with enteroids and Transwell cultures; EGF reduced

shedding, while AG1478 caused a consistent but nonsignificant trend towards more cell

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EGF blocks intestinal shedding via ERK.

extrusion (Figure 1F). Suppression appears to be an early event in the shedding process, as an

EGF pulse followed by wash-out and chase did not provoke a synchronized wave of shedding

(Figure 1G). Thus, EGF is likely blocking the onset of the process rather than arresting it

midway. Consistent with decreased extrusion and trophic effects on the epithelium in vivo , 48 h

EGF exposure (Figure 1H) resulted in increased cell density, though it should be noted that the

effects of suppressed shedding and increased proliferation cannot be separated over this longer

period. EGF also reduced shedding of IEC-6 rat intestinal and IPEC-J2 pig jejunal epithelial cells

(Figure 1I, J). Overall, these results show that EGFR-mediated suppression of cell extrusion is

conserved in cell culture models.

MEK/ERK signaling is required for EGFR suppression of sheddingTo examine the

molecular mechanisms of this effect, we used inhibitors to signaling intermediates which might

impact caspase or Rho-kinase activity. Labeled cells were treated with EGF +/- inhibitors to

MEK1/2 (5 M U0126), PKC (1 M BisI), or PI3K (5 M LY294002), and shed cells were

collected and counted. MEK/ERK inhibition reversed EGF’s suppression of shedding in both

MDCK and IEC-6 cells (Figure 2A-C). In contrast, neither PKC nor PI3K inhibition had any

effect. Consistent with a role for MAPK signaling, constitutively shed cells showed low basal

ERK activation versus attached cells (Figure 2D, E). Furthermore, treatment with neuregulin-1

(NRG1) or fibroblast growth factor 10 (FGF10), both of which stimulate ERK , suppressed

shedding (Figure 2F, G). Together these data suggest a model in which MAPK signaling tone,

which can be stimulated by multiple growth factors, regulates shedding. While off-target effects

of U0126 are a possibility, identical results were obtained with a second inhibitor (PD98059, not

shown), and the apparent increase in shedding beyond baseline in the presence of inhibitor is

likely due to loss of the basal MAPK activity in untreated cells.

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EGFR regulates both caspase- and Rho-dependent sheddingPhysiological cell shedding

from epithelial monolayers includes both apoptotic cells (a caspase-dependent mechanism) and

Rho-kinase driven extrusion of live cells . Trypan Blue staining of shed MDCK cells showed no

difference in cell viability with EGF or U0126 (Figure 3A; control 85.3 +/- 3.6% viable; EGF

89.2 +/- 1.0%; U0126 83.4 +/- 1.3%; U0126+EGF 79.5 +/-9.2%; ANOVA p=0.31), suggesting

that the effects of EGF and MAPK are not simply due to blocking cell death. To further explore

which pathways are impacted, we performed shedding experiments using caspase (Z-VAD-

FMK, 1 M) or Rho-kinase (Y27632, 20 M) inhibitors in the presence or absence of EGF

and/or U0126. None of the inhibitors affected the bulk density of cultures in the short term (Fig

3B, C), showing that shedding changes are not simply due to altered cell numbers. Both caspase

and Rho-kinase inhibitors reduced MDCK shedding (Figure 3D, E). However, EGF did not

augment this reduction. Together these results suggest that EGFR is blocking both the apoptotic

caspase and the Rho-Kinase-dependent myosin contraction shedding pathways. Interestingly,

MEK inhibition stimulated shedding even at baseline or in the presence of caspase plus Rho-

kinase inhibitors (Figure 3E), suggesting that EGF->MEK->ERK signaling blocks shedding

downstream of both of these pathways. Similarly, the EGFR inhibitor AG1478 reversed

suppression of baseline shedding by Z-VAD-FMK + Y27632 (Figure 3F). As MEK/ERK signals

are known to contribute to tight junction maintenance in the intestine , it may be that an initial

step in the mechanical shedding process requires loss of MAPK activity in the target cell to

dissolve cell-cell junctions.

EGFR suppresses constitutive intestinal epithelial cell shedding in vivo in zebrafish via

MEK/ERKTo test our findings in vivo, we established the adult zebrafish intestine as a

shedding model. On rhodamine-phalloidin stained sections of adult zebrafish midgut, all stages

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EGF blocks intestinal shedding via ERK.

of the shedding process—including actin “funnels” characteristic of cytoskeletal rearrangements

in epithelial cells preparing to undergo extrusion—are clearly visible (Figure 4A). To test

EGFR’s role in constitutive shedding, we injected adult fish i.p. with 20 L vehicle (0.01%

DMSO), EGF (1 g/ml; final 60 g/kg), or AG1478 (200 nM; final 388 g/kg). After 4 h,

intestines were collected and stained with rhodamine-phalloidin to detect F-actin-rich shedding

funnels. EGF suppressed and AG1478 induced shedding compared to control (1.7 or 4.3 vs. 2.5

shedding events/mm tissue perimeter; p<0.05, Figure 4B). Comparing the location of shedding

events, we found that a greater proportion of events in AG1478 treated fish were in the lower

half of the villus folds or in the inter-fold region (61.4 +/- 10.2% in AG1478 versus 45.1 +/-

12.1% in control) suggesting a mislocalization with EGFR blockade. As MAPK was essential for

EGF-mediated suppression of cell shedding in vitro (Figure 2), we tested it in our in vivo

zebrafish model. Fish were injected with vehicle, EGF, U0126 (10 M; final 228 g/kg), or both

for 4 h. Intestinal sections were stained for shedding funnels. Similar to our in vitro results,

MEK/ERK inhibition abrogated EGF reduction in detectable shedding events on intestinal villi

of the fish (Figure 4C).

Together these data indicate that EGFR suppresses constitutive intestinal epithelial cell

shedding, and position EGFR ligands as a suite of soluble factors potentially used by the tissue to

regulate its own turnover. The relevant physiological ligands for controlling shedding have not

yet been defined, but as discussed above any ligand that stimulates MAPK is potentially

important. Endogenous EGF from salivary and Brunner’s glands is present in the intestinal

lumen , but its availability to EGFR on the basolateral membranes of enterocytes may be

limited, and thus for example TGF- released from basolateral surfaces may be more relevant

under homeostatic conditions. Ligands are also produced by subepithelial myofibroblasts and

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Paneth cells , possibly creating growth factor gradients along the crypt-villus axis. This could

explain why extrusion is normally restricted to the upper villi. Consistent with this notion,

weaning pigs exhibit increased intestinal epithelial turnover coincident with a loss of EGFR

expression on the villi . Studies in Drosophila gut indicate that intestinal stem cells and

enterocytes communicate to coordinate generation of new cells with loss of old ones , and EGF

plays an important role in maintaining the intestinal stem cell compartment . Thus, EGFR ligand

gradients within the epithelium could provide a mechanism for coordination between the stem

cell compartment and the shedding zone on the upper villus.

In summary, we have shown that EGF participates in regulation of epithelial homeostasis

through suppression of constitutive cell extrusion. This occurs through a MEK/ERK signaling

mechanism. Ongoing work is focused on understanding the fundamental cellular mechanisms

targeted by this signaling pathway, and on understanding whether EGFR also regulates

pathologic cell shedding under inflammatory conditions. Several investigators have reported

reduced EGFR ligand expression in IBD . Furthermore, tumor necrosis factor, a key cytokine

involved in Crohn’s Disease which promotes shedding and the formation of epithelial gaps in the

mouse small intestine , can also inhibit EGFR activation in intestinal epithelial cells in vitro and

in vivo . Thus, suppressed activation of MAPK by EGFR could contribute to the dysregulated

shedding seen in inflammatory conditions including IBD.

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Materials and Methods

Zebrafish Animal use was approved and monitored by the Children’s Hospital Los Angeles

IACUC. Adult zebrafish were maintained under standard conditions with a 14 h dark/10h light

cycle at 28.5C, anaesthetized with tricaine (Sigma-Aldrich), injected i.p. with vehicle (DMSO),

EGF, AG1478, or U0126, and sacrificed after 4 h. Intestines were collected and cryosectioned (4

m). Midgut was stained with rhodamine-phalloidin (Molecular Probes) and mounted in

Vectashield with DAPI (Vector Laboratories, Inc.). Condensed actin funnels surrounding cells in

early stages of shedding were identified, imaging at least 50 villi/fish. Shedding events per mm

epithelial perimeter were traced using ImageJ (National Institutes of Health).

Enteroid culturesIleal epithelial crypts from LifeAct-EGFP mice were isolated by Ca2+

chelation and shaking, then grown in Matrigel plus R-spondin (500 ng/ml), noggin (100 ng/ml),

and EGF (20 ng/ml) as previously described . Growth factors were removed for 24 h before

experiments. Confocal Z-stacks of control and EGF-treated cultures were recorded every 5

minutes for 24 h.

Growth Factors and Inhibitors Recombinant EGF was from Peprotech. Recombinant

NRG1, FGF10, R-spondin, and noggin were from R&D Systems. Inhibitors were from:

AG1478, Cayman Chemical; U0126, LY294002, and BisI, Calbiochem; Z-VAD-FMK, G

Biosciences; Y27632, Enzo Life Sciences.

Cell culture Rat intestinal epithelial cells (IEC-6) were purchased from ATCC (#CRL-

1592) and maintained in Dulbecco’s Modified Eagle Medium (DMEM; Mediatech Inc.) with

10% FBS, 100 U/ml penicillin and streptomycin, and insulin-transferrin-selenium (BD

Biosciences) at 37C in 5% CO2 atmosphere. MDCK canine kidney epithelial cells were

maintained in DMEM with 10% FBS and 100 U/ml penicillin and streptomycin. IPEC-J2 cells ,

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a kind gift of Dr. Anthony Blikslager (North Carolina State University), were cultured in

DMEM/F12 with 5% FBS, 100 U/ml penicillin and streptomycin, and insulin-transferrin-

selenium. Density of some cultures was confirmed by resazurin reduction assay (Promega) or

Delaunay analysis of mean internuclear distance.

Confocal analysis of sheddingMDCK cells seeded on 0.4-μm-pore polycarbonate tissue

culture inserts (Transwells; Corning Biosciences) were treated, fixed, stained with DAPI and

rhodamine-phalloidin, and mounted on glass slides. Confocal Z-stacks of 15-20 consecutive 0.4

μm slices were acquired using a Zeiss LSM700 microscope. Four regions per sample were

imaged. Shedding cells were counted in two ways: visualization of condensed F-actin “rosettes”

surrounding a shedding cell in en face view , or of nuclei leaving the plane of the monolayer in

orthogonal projection.

Immunofluorescence analysis of EGFR activation Cultures fixed in 4% formaldehyde were

immunostained using anti-E-cadherin (1:100; RR1; Developmental Studies Hybridoma Bank,

University of Iowa) and PY-845-EGFR (1:100; Cell Signaling Technology); secondary

antibodies were anti-mouse Alexa Fluor 488 and anti-rabbit Alexa Fluor 555 (Invitrogen).

Direct counting of shed cellsCells were seeded in 24-well cell culture plates (250,000

cells/well) and grown for 24 h. The next day, cultures were labeled 15 min with DAPI (0.1

g/ml; Sigma-Aldrich), washed with PBS, and supplemented with fresh media. Media were

collected at defined times. Shed cells were collected by centrifugation and counted by wide-field

fluorescence. A minimum of 4 images were captured/analyzed from each well.

Statistical analysisAll data are representative of at least 3 individual zebrafish per

treatment or 3 independent cell culture experiments. Statistical analyses were performed using

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Prism software (GraphPad, Inc.). Significance of differences from controls was assessed by

ANOVA with appropriate post-test analysis. Error bars represent the standard error of means.

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Supplementary Movie

(Shed1.mov) Live imaging movie of an enteroid showing shedding.

Acknowledgments

We thank Edward J. Kronfli III, Arthela Osorio, Sean McCann, and Joshua Billings for technical

assistance. We are also grateful for assistance from the Dixon Cellular Imaging Core at The

Saban Research Institute.

Competing Interests

None.

Author Contributions

Conceived and designed the experiments: JCM DBP CLL AJMW MRF. Performed the

experiments and analyzed the data: JCM AAM JJH DAA LCH MRF. Wrote the manuscript:

JCM AAM CLL AJMW MRF.

Funding

The grants supporting this work were: (to MRF) NIH K01DK077956, R03DK090295, and

R01DK095004, a Senior Research Award from the Crohn’s and Colitis Foundation of America,

and a Research Scholar Grant from the American Cancer Society; (to AJMW) Wellcome Trust

grant WT0087768MA and BBSRC grant BB/J004529/1; (to CLL) NIH R01HL096121; (to DBP)

NIH R01DK056008 and R01DK54993.

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REFERENCES

1. E. Sancho, E. Batlle, H. Clevers, Signaling pathways in intestinal development and cancer. Annu Rev Cell Dev Biol 20, 695-723 (2004).

2. L. A. Knodler, B. A. Vallance, J. Celli, S. Winfree, B. Hansen, M. Montero, O. Steele-Mortimer, Dissemination of invasive Salmonella via bacterial-induced extrusion of mucosal epithelia. Proceedings of the National Academy of Sciences of the United States of America 107, 17733-17738 (2010).

3. M. Hausmann, How bacteria-induced apoptosis of intestinal epithelial cells contributes to mucosal inflammation. Int J Inflam 2010, 574568 (2010).

4. J. J. Liu, K. Wong, A. L. Thiesen, S. J. Mah, L. A. Dieleman, B. Claggett, J. R. Saltzman, R. N. Fedorak, Increased epithelial gaps in the small intestines of patients with inflammatory bowel disease: density matters. Gastrointest Endosc 73, 1174-1180 (2011).

5. R. Kiesslich, C. A. Duckworth, D. Moussata, A. Gloeckner, L. G. Lim, M. Goetz, D. M. Pritchard, P. R. Galle, M. F. Neurath, A. J. Watson, Local barrier dysfunction identified by confocal laser endomicroscopy predicts relapse in inflammatory bowel disease. Gut 61, 1146-1153 (2012).

6. S. F. Assimakopoulos, A. C. Tsamandas, G. I. Tsiaoussis, E. Karatza, C. Triantos, C. E. Vagianos, I. Spiliopoulou, V. Kaltezioti, A. Charonis, V. N. Nikolopoulou, C. D. Scopa, K. C. Thomopoulos, Altered intestinal tight junctions' expression in patients with liver cirrhosis: a pathogenetic mechanism of intestinal hyperpermeability. Eur J Clin Invest 42, 439-446 (2012).

7. A. M. Marchiando, L. Shen, W. V. Graham, K. L. Edelblum, C. A. Duckworth, Y. Guan, M. H. Montrose, J. R. Turner, A. J. Watson, The epithelial barrier is maintained by in vivo tight junction expansion during pathologic intestinal epithelial shedding. Gastroenterology 140, 1208-1218 e1201-1202 (2011).

8. C. W. Lai, T. L. Sun, W. Lo, Z. H. Tang, S. Wu, Y. J. Chang, C. C. Wu, S. C. Yu, C. Y. Dong, L. W. Chen, Shedding-induced gap formation contributes to gut barrier dysfunction in endotoxemia. J Trauma Acute Care Surg 74, 203-213 (2013).

9. M. Pentecost, G. Otto, J. A. Theriot, M. R. Amieva, Listeria monocytogenes invades the epithelial junctions at sites of cell extrusion. PLoS Pathog 2, e3 (2006).

10. T. W. Marshall, I. E. Lloyd, J. M. Delalande, I. Nathke, J. Rosenblatt, The tumor suppressor adenomatous polyposis coli controls the direction in which a cell extrudes from an epithelium. Molecular biology of the cell 22, 3962-3970 (2011).

11. T. F. Bullen, S. Forrest, F. Campbell, A. R. Dodson, M. J. Hershman, D. M. Pritchard, J. R. Turner, M. H. Montrose, A. J. Watson, Characterization of epithelial cell shedding from human small intestine. Laboratory investigation; a journal of technical methods and pathology 86, 1052-1063 (2006).

12. D. Andrade, J. Rosenblatt, Apoptotic regulation of epithelial cellular extrusion. Apoptosis 16, 491-501 (2011).

13. G. T. Eisenhoffer, P. D. Loftus, M. Yoshigi, H. Otsuna, C. B. Chien, P. A. Morcos, J. Rosenblatt, Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. Nature 484, 546-549 (2012).

14. Y. Guan, A. J. Watson, A. M. Marchiando, E. Bradford, L. Shen, J. R. Turner, M. H. Montrose, Redistribution of the tight junction protein ZO-1 during physiological

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1

1

2

3456789

101112131415161718192021222324252627282930313233343536373839404142434445

2

EGF blocks intestinal shedding via ERK.

shedding of mouse intestinal epithelial cells. Am J Physiol Cell Physiol 300, C1404-1414 (2011).

15. J. L. Madara, Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: physiological rearrangement of tight junctions. J Membr Biol 116, 177-184 (1990).

16. J. Rosenblatt, M. C. Raff, L. P. Cramer, An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr Biol 11, 1847-1857 (2001).

17. D. B. Polk, Epidermal growth factor receptor-stimulated intestinal epithelial cell migration requires phospholipase C activity. Gastroenterology 114, 493-502 (1998).

18. M. R. Frey, R. S. Dise, K. L. Edelblum, D. B. Polk, p38 kinase regulates epidermal growth factor receptor downregulation and cellular migration. The EMBO journal 25, 5683-5692 (2006).

19. G. Sheng, K. Q. Bernabe, J. Guo, B. W. Warner, Epidermal growth factor receptor-mediated proliferation of enterocytes requires p21waf1/cip1 expression. Gastroenterology 131, 153-164 (2006).

20. T. Sato, D. E. Stange, M. Ferrante, R. G. Vries, J. H. Van Es, S. Van den Brink, W. J. Van Houdt, A. Pronk, J. Van Gorp, P. D. Siersema, H. Clevers, Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium. Gastroenterology 141, 1762-1772 (2011).

21. J. Riedl, K. C. Flynn, A. Raducanu, F. Gartner, G. Beck, M. Bosl, F. Bradke, S. Massberg, A. Aszodi, M. Sixt, R. Wedlich-Soldner, Lifeact mice for studying F-actin dynamics. Nat Methods 7, 168-169 (2010).

22. B. Daniel, M. A. DeCoster, Quantification of sPLA2-induced early and late apoptosis changes in neuronal cell cultures using combined TUNEL and DAPI staining. Brain Res Brain Res Protoc 13, 144-150 (2004).

23. J. Berlanga-Acosta, R. J. Playford, N. Mandir, R. A. Goodlad, Gastrointestinal cell proliferation and crypt fission are separate but complementary means of increasing tissue mass following infusion of epidermal growth factor in rats. Gut 48, 803-807 (2001).

24. M. R. Frey, V. C. Hilliard, M. T. Mullane, D. B. Polk, ErbB4 promotes cyclooxygenase-2 expression and cell survival in colon epithelial cells. Laboratory investigation; a journal of technical methods and pathology 90, 1415-1424 (2010).

25. A. Yamada, M. Futagi, E. Fukumoto, K. Saito, K. Yoshizaki, M. Ishikawa, M. Arakaki, R. Hino, Y. Sugawara, M. Ishikawa, M. Naruse, K. Miyazaki, T. Nakamura, S. Fukumoto, Connexin 43 Is Necessary for Salivary Gland Branching Morphogenesis and FGF10-induced ERK1/2 Phosphorylation. The Journal of biological chemistry 291, 904-912 (2016).

26. T. Kinugasa, T. Sakaguchi, X. Gu, H. C. Reinecker, Claudins regulate the intestinal barrier in response to immune mediators. Gastroenterology 118, 1001-1011 (2000).

27. K. Goishi, P. Lee, A. J. Davidson, E. Nishi, L. I. Zon, M. Klagsbrun, Inhibition of zebrafish epidermal growth factor receptor activity results in cardiovascular defects. Mech Dev 120, 811-822 (2003).

28. M. Schweiger, M. Steffl, W. M. Amselgruber, Differential expression of EGF receptor in the pig duodenum during the transition phase from maternal milk to solid food. J Gastroenterol 38, 636-642 (2003).

15

1

123456789

101112131415161718192021222324252627282930313233343536373839404142434445

2

EGF blocks intestinal shedding via ERK.

29. J. F. Thompson, M. van den Berg, P. C. Stokkers, Developmental regulation of epidermal growth factor receptor kinase in rat intestine. Gastroenterology 107, 1278-1287 (1994).

30. L. A. Scheving, R. A. Shiurba, T. D. Nguyen, G. M. Gray, Epidermal growth factor receptor of the intestinal enterocyte. Localization to laterobasal but not brush border membrane. The Journal of biological chemistry 264, 1735-1741 (1989).

31. R. J. Playford, N. A. Wright, Why is epidermal growth factor present in the gut lumen? Gut 38, 303-305 (1996).

32. R. J. Playford, A. M. Hanby, S. Gschmeissner, L. P. Peiffer, N. A. Wright, T. McGarrity, The epidermal growth factor receptor (EGF-R) is present on the basolateral, but not the apical, surface of enterocytes in the human gastrointestinal tract. Gut 39, 262-266 (1996).

33. P. J. Dempsey, K. S. Meise, R. J. Coffey, Basolateral sorting of transforming growth factor-alpha precursor in polarized epithelial cells: characterization of cytoplasmic domain determinants. Experimental cell research 285, 159-174 (2003).

34. J. Shao, H. Sheng, Amphiregulin promotes intestinal epithelial regeneration: roles of intestinal subepithelial myofibroblasts. Endocrinology 151, 3728-3737 (2010).

35. T. Sato, J. H. van Es, H. J. Snippert, D. E. Stange, R. G. Vries, M. van den Born, N. Barker, N. F. Shroyer, M. van de Wetering, H. Clevers, Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469, 415-418 (2011).

36. S. Bulow, P. Skov Olsen, S. S. Poulsen, P. Kirkegaard, Is epidermal growth factor involved in development of duodenal polyps in familial polyposis coli? Am J Gastroenterol 83, 404-406 (1988).

37. S. S. Poulsen, E. Nexo, P. S. Olsen, J. Hess, P. Kirkegaard, Immunohistochemical localization of epidermal growth factor in rat and man. Histochemistry 85, 389-394 (1986).

38. T. Skrzypek, J. L. Valverde Piedra, H. Skrzypek, J. Wolinski, W. Kazimierczak, S. Szymanczyk, M. Pawlowska, R. Zabielski, Light and scanning electron microscopy evaluation of the postnatal small intestinal mucosa development in pigs. J Physiol Pharmacol 56 Suppl 3, 71-87 (2005).

39. L. E. O'Brien, S. S. Soliman, X. Li, D. Bilder, Altered modes of stem cell division drive adaptive intestinal growth. Cell 147, 603-614 (2011).

40. N. Xu, S. Q. Wang, D. Tan, Y. Gao, G. Lin, R. Xi, EGFR, Wingless and JAK/STAT signaling cooperatively maintain Drosophila intestinal stem cells. Developmental biology 354, 31-43 (2011).

41. Q. C. Cheung, Z. Yuan, P. W. Dyce, D. Wu, K. DeLange, J. Li, Generation of epidermal growth factor-expressing Lactococcus lactis and its enhancement on intestinal development and growth of early-weaned mice. Am J Clin Nutr 89, 871-879 (2009).

42. C. Jansen, I. Ihse, Axelson, Epidermal growth factor induces increased mucosal thickness of the small intestine in mouse. Eur Surg Res 29, 447-454 (1997).

43. P. Kang, D. Toms, Y. Yin, Q. Cheung, J. Gong, K. De Lange, J. Li, Epidermal growth factor-expressing Lactococcus lactis enhances intestinal development of early-weaned pigs. J Nutr 140, 806-811 (2010).

44. R. T. Zijlstra, J. Odle, W. F. Hall, B. W. Petschow, H. B. Gelberg, R. E. Litov, Effect of orally administered epidermal growth factor on intestinal recovery of neonatal pigs infected with rotavirus. J Pediatr Gastroenterol Nutr 19, 382-390 (1994).

45. V. H. Lugo-Martinez, C. S. Petit, S. Fouquet, J. Le Beyec, J. Chambaz, M. Pincon-Raymond, P. Cardot, S. Thenet, Epidermal growth factor receptor is involved in

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2

EGF blocks intestinal shedding via ERK.

enterocyte anoikis through the dismantling of E-cadherin-mediated junctions. American journal of physiology. Gastrointestinal and liver physiology 296, G235-244 (2009).

46. J. J. Liu, K. L. Madsen, P. Boulanger, L. A. Dieleman, J. Meddings, R. N. Fedorak, Mind the gaps: confocal endomicroscopy showed increased density of small bowel epithelial gaps in inflammatory bowel disease. J Clin Gastroenterol 45, 240-245 (2011).

47. K. Hormi, G. Cadiot, S. Kermorgant, V. Dessirier, M. Le Romancer, M. J. Lewin, M. Mignon, T. Lehy, Transforming growth factor-alpha and epidermal growth factor receptor in colonic mucosa in active and inactive inflammatory bowel disease. Growth Factors 18, 79-91 (2000).

48. R. J. Alexander, A. Panja, E. Kaplan-Liss, L. Mayer, R. F. Raicht, Expression of growth factor receptor-encoded mRNA by colonic epithelial cells is altered in inflammatory bowel disease. Dig Dis Sci 40, 485-494 (1995).

49. A. M. Marchiando, L. Shen, W. V. Graham, C. R. Weber, B. T. Schwarz, J. R. Austin, 2nd, D. R. Raleigh, Y. Guan, A. J. Watson, M. H. Montrose, J. R. Turner, Caveolin-1-dependent occludin endocytosis is required for TNF-induced tight junction regulation in vivo. J Cell Biol 189, 111-126 (2010).

50. J. M. Williams, C. A. Duckworth, A. J. Watson, M. R. Frey, J. C. Miguel, M. D. Burkitt, R. Sutton, K. R. Hughes, L. J. Hall, J. H. Caamano, B. J. Campbell, D. M. Pritchard, A mouse model of pathological small intestinal epithelial cell apoptosis and shedding induced by systemic administration of lipopolysaccharide. Disease models & mechanisms 6, 1388-1399 (2013).

51. G. C. Kaiser, D. B. Polk, Tumor necrosis factor alpha regulates proliferation in a mouse intestinal cell line. Gastroenterology 112, 1231-1240 (1997).

52. S. J. McElroy, M. R. Frey, F. Yan, K. L. Edelblum, J. A. Goettel, S. John, D. B. Polk, Tumor necrosis factor inhibits ligand-stimulated EGF receptor activation through a TNF receptor 1-dependent mechanism. American journal of physiology. Gastrointestinal and liver physiology 295, G285-293 (2008).

53. Y. Feng, D. H. Teitelbaum, Epidermal growth factor/TNF-alpha transactivation modulates epithelial cell proliferation and apoptosis in a mouse model of parenteral nutrition. American journal of physiology. Gastrointestinal and liver physiology 302, G236-249 (2012).

54. L. I. Larsson, Distribution of E-cadherin and beta-catenin in relation to cell maturation and cell extrusion in rat and mouse small intestines. Histochem Cell Biol 126, 575-582 (2006).

55. E. Sahai, C. J. Marshall, RHO-GTPases and cancer. Nat Rev Cancer 2, 133-142 (2002).56. G. Slattum, K. M. McGee, J. Rosenblatt, P115 RhoGEF and microtubules decide the

direction apoptotic cells extrude from an epithelium. J Cell Biol 186, 693-702 (2009).57. R. Pinkas-Kramarski, M. Shelly, B. C. Guarino, L. M. Wang, L. Lyass, I. Alroy, M.

Alimandi, A. Kuo, J. D. Moyer, S. Lavi, M. Eisenstein, B. J. Ratzkin, R. Seger, S. S. Bacus, J. H. Pierce, G. C. Andrews, Y. Yarden, ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network. Molecular and cellular biology 18, 6090-6101 (1998).

58. J. M. Rhoads, W. Chen, P. Chu, H. M. Berschneider, R. A. Argenzio, A. M. Paradiso, L-glutamine and L-asparagine stimulate Na+ -H+ exchange in porcine jejunal enterocytes. The American journal of physiology 266, G828-838 (1994).

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Figure Legends

Figure 1. EGF suppresses cell shedding in vitro. (A) Ileal enteroid cultures were generated

from Lifeact-EGFP mice. 24 h before experiments, growth factors were removed. Cultures were

then treated with vehicle (PBS) or 10 ng/ml EGF and live-imaged for 24 h. Characteristic

morphological stages of apical cell shedding and number of events per μm epithelial perimeter

per 24 h are shown. (B, C) MDCK cells were treated with vehicle (Control), EGF (10 ng/ml), or

EGFR inhibitor AG1478 (150 nM) for 4 h, fixed, and stained with rhodamine-phalloidin (red)

and DAPI (blue). See text for quantification. (B) Example image showing cell “rosettes”

(arrows) indicating shedding events. Arrowhead in top panel, nucleus from shedding cell. n=6.

(C) Orthogonal projections of confocal Z-stacks; arrows, shedding nuclei above the monolayer.

n=6. (D) MDCK cells stained with anti-E-cadherin, anti-PY-845-EGFR, and DAPI. Cell rosettes

are sites of shedding, example in magnified boxes. Arrowheads, cells positive for PY-845-

EGFR. Scale bar=50 μm. (E, F) MDCK (n=8 independent experiments) cells were labeled with

DAPI and treated with vehicle, EGF, or AG1478; shed cells were collected and counted;

representative images in D. (G) MDCK cultures were exposed to EGF for 4 h, EGF was washed

out, and then cells cultured either with or without EGF and shed cells counted at 4 h. (H)

Relative cell numbers after 48 h in control and EGF-treated cultures, determined by resazurin

reduction assay. (I) IEC-6 and (J) IPEC-J2 cells were cultured with or without EGF and shed

cells at 30 min or 4 h were counted (n=4). *, p<0.05 vs. control; **, p<0.01 vs. control.

Figure 2. MEK1/2 activity is required for EGF suppression of epithelial cell shedding in

vitro. (A,B) MDCK (n≥5) and (C) IEC-6 (n=5) cells were labeled then treated with vehicle

(Control) or EGF, +/- 5 M U0126 (MEK1/2 inhibitor), 5 M LY294002 (PI3K inhibitor), or 1

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M BisI (PKC inhibitor) for 30 min or 4 h. Shed cells were collected and counted. (D,E)

Attached and shed cells from control cultures (no EGF) collected over 4 h were subjected to

western blot for phospho-ERK. (F, G) MDCK cells (n=4) were treated with 10 ng/ml NRG1 or

2.5 ng/ml FGF10 and shed cells were counted. *, p<0.05; **, p<0.01, ***, p<0.001.

Figure 3. MEK inhibition reverses suppression of cell shedding by EGF, caspase inhibitor,

or Rho-kinase inhibitor. (A) Shed MDCK cells in vehicle (Control), EGF, and U0126 treated

cultures were collected after 4 h and viability assessed by Trypan Blue exclusion. (B) MDCK

cells were treated as indicated for 4 h and DAPI-stained to show density. (C) Delauny analysis of

mean internuclear distance on cultures. (D) MDCK (n≥5) cells were labeled with DAPI then

treated with vehicle, EGF, Z-VAD-FMK (caspase inhibitor; 1 μM), or Y27632 (Rho-kinase

inhibitor; 20 μM) for 4 h; shed cells were collected and counted. *, p<0.05 vs. control. (E)

MDCK (n=5) cells were labeled then treated with vehicle, EGF, U0126, Z-VAD-FMK, or

Y27632 for 30 min or 4 h. Shed cells were collected and counted. ***, p<0.001 vs. control; ***

brackets, p<0.001 between columns.

Figure 4. EGF inhibits cell shedding in zebrafish intestine via MAPK signaling. (A)

Zebrafish midgut tissue was fixed and stained with rhodamine-phalloidin (red, F-actin) and

DAPI (blue, nuclei). Examples of (i) early, (ii) late, and (iii) completion of shedding process

shown. (B) Fish were treated i.p. with vehicle (Control), EGF, or AG1478 for 4 h. Early-stage

shedding funnels (arrows) per mm of tissue perimeter were counted as shown in (C). Bars=50

m; ***, p<0.001 vs. control, n4 per condition. (D) Fish were treated i.p. with vehicle

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(Control), EGF, or U0126 for 4 h. Tissue was fixed, stained, and shedding funnels counted. ***,

p<0.001; n3 per condition.

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