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NIH Award from the National Institute of Dental & Craniofacial Research

Targeting Tight Junctions to Treat Mucositis

  • Principal Investigator: Frederick Toback, MD, PhD, Professor, Section of Nephrology, Department of Medicine
  • Start Date: May 1, 2009
  • Total Award Amount: $195,000 (first year) $234,000 (second year)

Public Health Relevance

Radiation therapy and chemotherapy in cancer patients often damages the mucosal lining of the gastrointestinal (GI) tract - especially the mouth and throat, a complication called mucositis. We have identified and characterized a fragment of a protein made by GI cells that targets and strengthens the connections between cells that line the mouth and GI tract. This project is aimed at learning how this protein fragment exerts its biological effects because when it is given to mice or hamsters with experimental oral mucositis, it protects against and speeds recovery from injury.

Project Description

We have identified a novel peptide that acts as an effective therapeutic agent in mouse and hamster models of radiation-induced oral mucositis by targeting tight junctions (TJs) that connect adjacent epithelial cells, thereby protecting the mucosal barrier. This peptide is derived from a central domain of an 18 kD Antrum Mucosal Protein (AMP-18) that is expressed in human gastric epithelial cells. The synthetic 21-mer peptide stimulates growth of human keratinocytes (HaCaT line) used to model the oral mucosa, and also has protective, and motogenic properties in cell culture.

Recombinant human AMP-18 binds to the plasma membrane of keratinocytes in normal human oral mucosal tissue suggesting that its effects are receptor mediated. Both AMP peptide and the recombinant protein protect barrier function and structure in human epithelial cell cultures injured by an oxidant, indomethacin, or low-calcium stress. AMP-18 appears to exert its protective effects by limiting the loss of TJ proteins after injury, enhancing assembly of these proteins into new TJs, and also stabilizing perijunctional actin. In cell culture, AMP-18 activates Akt and protein kinase C-zeta (PKC6) which could mediate the observed translocation of cytosolic proteins such as Par6 and Cdc42 into junctional domains thereby facilitating protein assembly for new or damaged junctions after TJ disruption. In murine mucosa in vivo and human epithelial cell cultures, AMP peptide increased accumulation of TJ proteins (occludin, ZO-1), and stimulated the phosphorylation of p38 MAP kinase and heat shock protein 25 which could stabilize actin filaments.

To determine if AMP peptide could protect the oral mucosa in vivo, radiation- induced injuries of the mouse tongue and hamster buccal mucosa were studied in models of oral mucositis. AMP peptide treatment protected the surface epithelium and connective tissue of the mouse tongue, and delayed the appearance and reduced the extent of ulcer formation in the buccal mucosa of hamsters.

The goal of this project is to find out how AMP-18 exerts its novel effects, as it is the only agent of which we are aware that increases accumulation and facilitates assembly of specific TJ proteins, and protects against their loss following injury. To determine how AMP-18 targets epithelial cell TJs in the oral and gastrointestinal (GI) mucosa we have two specific aims: (1) identify and characterize the cell surface AMP-18 receptor in oral mucosal cells, and (2) identify down stream targets of AMP-18 receptor activation required for assembly of TJ proteins. A pull down (affinity) strategy has been used to identify a putative 80 kD AMP-18 binding protein, and biopanning of a human phage-display cDNA library has identified a candidate sequence that could represent a ligand-binding domain within the AMP-18 receptor. We will determine if one or both of these candidate receptors can accept AMP-18 as a ligand.

Achieving our aims will provide a rationale for developing a strategy using AMP peptide to protect and repair TJs in the oral mucosa of cancer patients who develop mucositis following radiation and chemotherapy.

This award is funded under the American Recovery and Reinvestment Act of 2009, NIH Award number: 1R21DE018811-01A2

Frederick Toback

Frederick Toback, MD, PhD,
Professor, Section of Nephrology, Department of Medicine