Matrix metalloproteinase-7 induces E-cadherin cleavage in acid-exposed primary human pharyngeal epithelial cells via the ROS/ERK/c-Jun pathway

Laryngopharyngeal reflux disease (LPRD) is caused by pharyngeal mucosal damage due to the reflux of gastric contents, including acid, pepsin, and bile juice. Our previous study has demonstrated that LPRD is associated with the cleavage of E-cadherin, which is facilitated by the acid-activated matrix metalloproteinase-7 (MMP-7); however, the mechanism by which the acid activates MMP-7 remains unclear. The purpose of this study was to investigate the mechanism by which MMP-7 is activated in the pharyngeal epithelial cells that are exposed to acid. The levels of reactive oxygen species (ROS) were measured in the epithelial cells exposed to acid. To investigate the signaling mechanism of ROS in the expression of MMP-7, the mechanism of action of the mitogen-activated protein kinase was examined. The expression of various signaling factors was determined, according to the presence or absence of each inhibitor in the acid-exposed pharyngeal epithelial cells. To identify changes in the cleavage of E-cadherin, the integrity of the mucosal membrane was assessed using a transepithelial permeability test. We found that acid exposure increased the levels of ROS, phosphorylated-extracellular signal-regulated kinase (p-ERK) 1/2, and phosphorylated-c-Jun (p–c-Jun) in pharyngeal epithelial cells. The ROS inhibitor reduced the expression of p-ERK and MMP-7, while the ERK inhibitor reduced the expression of p–c-Jun and MMP-7. Moreover, the c-Jun inhibitor reduced the expression of MMP-7 and blocked the degradation of E-cadherin. In addition, decrease in the levels of immunostained E-cadherin and increase in transepithelial permeability after acid exposure were collectively alleviated by the inhibitors of ROS, ERK, and c-Jun. The degradation of E-cadherin that occurs after human mucosal cells are exposed to acid appears to be caused by an increase in the expression of MMP-7 via the ROS/ERK/c-Jun pathway, which is thought to be an important mechanism associated with the development of LPRD. Key messages • ROS is triggered when reflux occurs. • ROS regulates the transcription factor c-Jun via the ERK pathway. • The increase in MMP-7 that induces LPRD is induced via the ROS/ERK/c-Jun pathway. • This study revealed for the first time the expression mechanism of MMP-7, which is one of the causes of LPRD.

Matrix Metalloproteinase-7 Induces E-Cadherin Cleavage in Acid-Exposed Primary Human Pharyngeal Epithelial Cells Via The ROS/ERK/c-Jun Pathway

Introduction Laryngopharyngeal reflux disease (LPRD) is caused by pharyngeal mucosal damage due to the reflux of gastric contents, including acid, pepsin, and bile juice. Our previous study has demonstrated that LPRD is associated with the cleavage of E-cadherin, which is facilitated by the acid-activated matrix metalloproteinase-7 (MMP-7); however, the mechanism by which the acid activates MMP-7 remains unclear. The purpose of this study was to investigate the mechanism by which MMP-7 is activated in the pharyngeal epithelial cells that are exposed to acid. Methods The levels of reactive oxygen species (ROS) were measured in the epithelial cells exposed to acid. To investigate the signaling mechanism of ROS in the expression of MMP-7, the mechanism of action of the mitogen-activated protein kinase was examined. The expression of various signaling factors were determined, according to the presence or absence of each inhibitor in the acid-exposed pharyngeal epithelial cells. To identify changes in the cleavage of E-cadherin, the integrity of the mucosal membrane was assessed using a transepithelial permeability test.ResultsWe found that acid exposure increased the levels of ROS, phosphorylated-extracellular signal-regulated kinase (p-ERK) 1/2, and phosphorylated-c-Jun (p-c-Jun) in pharyngeal epithelial cells. The ROS inhibitor reduced the expression of p-ERK and MMP-7, while the ERK inhibitor reduced the expression of p-c-Jun and MMP-7. Moreover, the c-Jun inhibitor reduced the expression of MMP-7 and blocked the degradation of E-cadherin. In addition, decrease in the levels of immunostained E-cadherin and increase in transepithelial permeability after acid exposure were collectively alleviated by the inhibitors of ROS, ERK, and c-Jun. Conclusions The degradation of E-cadherin that occurs after human mucosal cells are exposed to acid appears to be caused by an increase in the expression of MMP-7 via the ROS/ERK/c-Jun pathway, which is thought to be an important mechanism associated with the development of LPRD.

Pollen Proteases Play Multiple Roles in Allergic Disorders

Allergic diseases are a major health concern worldwide. Pollens are important triggers for allergic rhinitis, conjunctivitis and asthma. Proteases released upon pollen grain hydration appear to play a major role in the typical immunological and inflammatory responses that occur in patients with allergic disorders. In this study, we aimed to identify specific proteolytic activity in a set of pollens with diverse allergenic potential. Diffusates from Chenopodium album, Plantago lanceolata and Eucalyptus globulus were added to a confluent monolayer of Calu-3 cells grown in an air-liquid interface system. We identified serine proteases and metalloproteinases in all pollen diffusates investigated. Proteases found in these pollen diffusates were shown to compromise the integrity of the lung epithelial barrier by disrupting transmembrane adhesion proteins E-cadherin, claudin-1 and Occludin, as well as, the cytosolic complex zonula occludens-1 (ZO-1) resulting in a time-dependent increase in transepithelial permeability. Tight junction disruption and increased transepithelial permeability facilitates allergen exposure to epithelial sub-layers contributing to the sensitization to a wide range of allergens. These pollen extracts also induced an increase in the release of interleukin 6 (IL-6) and interleukin 8 (IL-8) cytokines measured by flow cytometry possibly as a result of the activation of protease-activated receptors 2 (PAR-2).

Role of Matrix Metalloproteinases 7 in the Pathogenesis of Laryngopharyngeal Reflux: Decreased E-cadherin in Acid exposed Primary Human Pharyngeal Epithelial Cells

Cleavage of E-cadherin and the resultant weakness in the cell-cell links in the laryngeal epithelium lining is induced by exposure to acidic contents of the refluxate. Herein, we aimed to evaluate the role of matrix metalloproteinases (MMPs) in inducing E-cadherin level changes following acid exposure to the human pharyngeal mucosal cells. E-cadherin levels were inversely correlated with the duration of acid exposure. Treatment with actinonin, a broad MMP inhibitor, inhibited this change. Immunocytochemical staining and transepithelial permeability test revealed that the cell surface staining of E-cadherin decreased and transepithelial permeability increased after acid exposure, which was significantly inhibited by the MMP inhibitor. Among the various MMPs analyzed, the mRNA for MMP-7 in the cellular component was upregulated, and the secretion and enzymatic activity of MMP-7 in the culture media increased with the acid treatment. Consequently, MMP-7 plays a significant role in the degradation of E-cadherin after exposure to a relatively weak acidic condition that would be similar to the physiologic condition that occurs in Laryngopharyngeal reflux disease patients.

Effect of surface hydrophobicity of therapeutic protein loaded in polyelectrolyte nanoparticles on transepithelial permeability

Oral delivery of protein drugs is greatly limited by low hydrophobicity, an important determinant for intestinal epithelial permeation and bioavailability. Herein, surface properties of recombinant erythropoietin were investigated using the fluorescent dye bis-ANS to monitor relative hydrophobicity for correlation with permeabilities with Caco-2 cells. At various pHs, bis-ANS fluorescence intensity indicated different surface hydrophobicities of erythropoietin molecules. Erythropoietin incorporated in chitosan or chitosan-trimethylchitosan (CS-TMC) nanoparticles prepared by polyelectrolyte complexation and ionotropic gelation with tripolyphosphate also showed different surface hydrophobicities. Chitosan nanoparticles with erythropoietin provided the most hydrophobic surface, followed by free erythropoietin (in water) and that loaded into CS-TMC nanoparticles. Chitosan nanoparticles were more effective than CS-TMC nanoparticles for permeation of erythropoietin across Caco-2 cell monolayers; the lowest permeability was shown by erythropoietin itself. Thus, hydrophilic protein molecules complexed with polyelectrolytes can provide more hydrophobic surfaces that enhance transepithelial permeability. This bis-ANS method also provides valuable information for the design of polyelectrolyte nanoparticules for oral delivery of protein drugs.