A Lactobacillus rhamnosus GG-derived Soluble Protein, p40, Stimulates Ligand Release from Intestinal Epithelial Cells to Transactivate Epidermal Growth Factor Receptor*

Background: p40 is a Lactobacillus rhamnosus GG-derived protein. Results: p40 stimulates ADAM17 activation and HB-EGF release, which is required for EGF receptor transactivation, prevention of apoptosis, and preservation of barrier function in intestinal epithelial cells. Conclusion: p40 transactivates the EGF receptor through ADAM17-mediated HB-EGF release in intestinal epithelial cells. Significance: These results define a mechanism of p40 in modulating intestinal epithelial cell homeostasis. p40, a Lactobacillus rhamnosus GG (LGG)-derived soluble protein, ameliorates intestinal injury and colitis, reduces apoptosis, and preserves barrier function by transactivation of the EGF receptor (EGFR) in intestinal epithelial cells. The aim of this study is to determine the mechanisms by which p40 transactivates the EGFR in intestinal epithelial cells. Here we show that p40-conditioned medium activates EGFR in young adult mouse colon epithelial cells and human colonic epithelial cell line, T84 cells. p40 up-regulates a disintegrin and metalloproteinase domain-containing protein 17 (ADAM17) catalytic activity, and broad spectrum metalloproteinase inhibitors block EGFR transactivation by p40 in these two cell lines. In ADAM17-deficient mouse colonic epithelial (ADAM17−/− MCE) cells, p40 transactivation of EGFR is blocked, but can be rescued by re-expression with WT ADAM17. Furthermore, p40 stimulates release of heparin binding (HB)-EGF, but not transforming growth factor (TGF)α or amphiregulin, in young adult mouse colon cells and ADAM17−/− MCE cells overexpressing WT ADAM17. Knockdown of HB-EGF expression by siRNA suppresses p40 effects on transactivating EGFR and Akt, preventing apoptosis, and preserving tight junction function. The effects of p40 on HB-EGF release and ADAM17 activation in vivo are examined after administration of p40-containing pectin/zein hydrogel beads to mice. p40 stimulates ADAM17 activity and EGFR activation in colonic epithelial cells and increases HB-EGF levels in blood from WT mice, but not from mice with intestinal epithelial cell-specific ADAM17 deletion. Thus, these data define a mechanism of a probiotic-derived soluble protein in modulating intestinal epithelial cell homeostasis through ADAM17-mediated HB-EGF release, leading to transactivation of EGFR.

[1]  Elaheh Vahabnezhad,et al.  Lactobacillus bacteremia associated with probiotic use in a pediatric patient with ulcerative colitis. , 2013, Journal of clinical gastroenterology.

[2]  Christina K. Chan,et al.  Endothelial deletion of ADAM17 in mice results in defective remodeling of the semilunar valves and cardiac dysfunction in adults , 2013, Mechanisms of Development.

[3]  M. Washington,et al.  Epidermal growth factor receptor inhibits colitis-associated cancer in mice. , 2012, The Journal of clinical investigation.

[4]  C. Elson,et al.  Host-microbiota interactions in inflammatory bowel disease , 2012, Gut microbes.

[5]  S. Lebeer,et al.  Analysis of the Peptidoglycan Hydrolase Complement of Lactobacillus casei and Characterization of the Major γ-D-Glutamyl-L-Lysyl-Endopeptidase , 2012, PloS one.

[6]  W. D. de Vos,et al.  Genetic and Biochemical Characterization of the Cell Wall Hydrolase Activity of the Major Secreted Protein of Lactobacillus rhamnosus GG , 2012, PloS one.

[7]  M. Freeman,et al.  Tumor Necrosis Factor Signaling Requires iRhom2 to Promote Trafficking and Activation of TACE , 2012, Science.

[8]  C. Blobel,et al.  iRhom2 Regulation of TACE Controls TNF-Mediated Protection Against Listeria and Responses to LPS , 2012, Science.

[9]  H. Szajewska,et al.  Meta‐analysis: the effects of Lactobacillus rhamnosus GG supplementation for the prevention of healthcare‐associated diarrhoea in children , 2011, Alimentary pharmacology & therapeutics.

[10]  Wei-Li Di,et al.  Inflammatory skin and bowel disease linked to ADAM17 deletion. , 2011, The New England journal of medicine.

[11]  D. Polk,et al.  Probiotics and immune health , 2011, Current opinion in gastroenterology.

[12]  M. Washington,et al.  Colon-specific delivery of a probiotic-derived soluble protein ameliorates intestinal inflammation in mice through an EGFR-dependent mechanism. , 2011, The Journal of clinical investigation.

[13]  V. Monedero,et al.  Functional Analysis of the p40 and p75 Proteins from Lactobacillus casei BL23 , 2010, Journal of Molecular Microbiology and Biotechnology.

[14]  C. Blobel,et al.  MyD88 signaling in nonhematopoietic cells protects mice against induced colitis by regulating specific EGF receptor ligands , 2010, Proceedings of the National Academy of Sciences.

[15]  P. Schirmacher,et al.  Critical role of the disintegrin metalloprotease ADAM17 for intestinal inflammation and regeneration in mice , 2010, The Journal of experimental medicine.

[16]  D. Polk,et al.  Probiotics: progress toward novel therapies for intestinal diseases , 2010, Current opinion in gastroenterology.

[17]  R. Derynck,et al.  Direct activation of TACE-mediated ectodomain shedding by p38 MAP kinase regulates EGF receptor-dependent cell proliferation. , 2010, Molecular cell.

[18]  S. Basu,et al.  Efficacy of High-dose Lactobacillus rhamnosus GG in Controlling Acute Watery Diarrhea in Indian Children: A Randomized Controlled Trial , 2009, Journal of clinical gastroenterology.

[19]  C. Blobel,et al.  The role of protease activity in ErbB biology. , 2009, Experimental cell research.

[20]  T. Venkatesan.  Probiotic Prophylaxis in Predicted Severe Acute Pancreatitis: A Randomised, Double-Blind, Placebo-Controlled Trial. , 2008, Nutrition in clinical practice : official publication of the American Society for Parenteral and Enteral Nutrition.

[21]  Charles P B Vanderpool,et al.  Mechanisms of probiotic action: Implications for therapeutic applications in inflammatory bowel diseases , 2008, Inflammatory bowel diseases.

[22]  W. Tong,et al.  Transactivation of EGF receptor and ErbB2 protects intestinal epithelial cells from TNF-induced apoptosis , 2008, Proceedings of the National Academy of Sciences.

[23]  R. Rao,et al.  Probiotics ameliorate the hydrogen peroxide-induced epithelial barrier disruption by a PKC- and MAP kinase-dependent mechanism. , 2008, American journal of physiology. Gastrointestinal and liver physiology.

[24]  R. Whitehead,et al.  Epidermal growth factor stimulates Rac activation through Src and phosphatidylinositol 3-kinase to promote colonic epithelial cell migration. , 2008, American journal of physiology. Gastrointestinal and liver physiology.

[25]  M. Washington,et al.  Soluble proteins produced by probiotic bacteria regulate intestinal epithelial cell survival and growth. , 2007, Gastroenterology.

[26]  P. Dempsey,et al.  ADAMs as mediators of EGF receptor transactivation by G protein-coupled receptors. , 2006, American journal of physiology. Cell physiology.

[27]  M. Pop,et al.  Metagenomic Analysis of the Human Distal Gut Microbiome , 2006, Science.

[28]  A. Gasbarrini,et al.  Efficacy of Lactobacillus GG in maintaining remission of ulcerative colitis , 2006, Alimentary pharmacology & therapeutics.

[29]  G. Ruthel,et al.  Pectin/Zein Beads for Potential Colon-Specific Drug Delivery: Synthesis and in Vitro Evaluation , 2006, Drug delivery.

[30]  A. Fornace,et al.  Gene Mutations Are Associated with Decreased Sensitivity of Human Lymphoma Cells to DNA Damaging Agents , 2006 .

[31]  D. Snydman,et al.  Lactobacillus GG: bacteriology and clinical applications. , 2005, Gastroenterology clinics of North America.

[32]  S. Soond,et al.  ERK-mediated phosphorylation of Thr735 in TNFα-converting enzyme and its potential role in TACE protein trafficking , 2005, Journal of Cell Science.

[33]  J. Cnota,et al.  Lactobacillus Sepsis Associated With Probiotic Therapy , 2005, Pediatrics.

[34]  C. Blobel,et al.  ADAMs: key components in EGFR signalling and development , 2005, Nature Reviews Molecular Cell Biology.

[35]  Daniel Metzger,et al.  Tissue‐specific and inducible Cre‐mediated recombination in the gut epithelium , 2004, Genesis.

[36]  C. Blobel,et al.  Stimulated Shedding of Vascular Cell Adhesion Molecule 1 (VCAM-1) Is Mediated by Tumor Necrosis Factor-α-converting Enzyme (ADAM 17)* , 2003, Journal of Biological Chemistry.

[37]  Joseph Kost,et al.  Pectin-based systems for colon-specific drug delivery via oral route. , 2003, Biomaterials.

[38]  R. Playford,et al.  Epidermal growth factor enemas with oral mesalamine for mild-to-moderate left-sided ulcerative colitis or proctitis. , 2003, The New England journal of medicine.

[39]  J. Arribas,et al.  Shedding of plasma membrane proteins. , 2003, Current topics in developmental biology.

[40]  D. Polk,et al.  Probiotic Bacterium Prevents Cytokine-induced Apoptosis in Intestinal Epithelial Cells* , 2002, The Journal of Biological Chemistry.

[41]  T. Kirkegaard,et al.  Tumour necrosis factor α converting enzyme (TACE) activity in the colonic mucosa of patients with inflammatory bowel disease , 2002 .

[42]  E. Dı́az-Rodrı́guez,et al.  Extracellular signal-regulated kinase phosphorylates tumor necrosis factor alpha-converting enzyme at threonine 735: a potential role in regulated shedding. , 2002, Molecular biology of the cell.

[43]  O. Hurtado,et al.  Implication of TNF-alpha convertase (TACE/ADAM17) in inducible nitric oxide synthase expression and inflammation in an experimental model of colitis. , 2001, Cytokine.

[44]  Y. Yarden.  The EGFR family and its ligands in human cancer. signalling mechanisms and therapeutic opportunities. , 2001, European journal of cancer.

[45]  Y. Yarden,et al.  Untangling the ErbB signalling network , 2001, Nature Reviews Molecular Cell Biology.

[46]  C. Blobel,et al.  Intracellular maturation and localization of the tumour necrosis factor alpha convertase (TACE). , 2000, The Biochemical journal.

[47]  S. Rockman,et al.  Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans. , 1999, Gastroenterology.

[48]  Sir Isaac Knight Newton,et al.  The Discovery of Lactobacillus GG , 1996 .

[49]  K. Salminen.  Editorial: The First Functional Probiotic Food , 1996 .

[50]  K. Kohn,et al.  p53 gene mutations are associated with decreased sensitivity of human lymphoma cells to DNA damaging agents. , 1994, Cancer research.

[51]  M. Noble,et al.  Establishment of conditionally immortalized epithelial cell lines from both colon and small intestine of adult H-2Kb-tsA58 transgenic mice. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[52]  C. Blobel,et al.  Intracellular maturation and localization of the tumour necrosis factor α convertase ( TACE ) , 2022 .