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

Abstract 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.

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

10.21203/rs.3.rs-759635/v1 ◽

2021 ◽

Author(s):

Nu-Ri IM ◽

Byoungjae Kim ◽

Kwang-Yoon Jung ◽

Seung-Kuk Baek

Keyword(s):

Epithelial Cells ◽

Matrix Metalloproteinase ◽

Laryngopharyngeal Reflux ◽

Mucosal Damage ◽

Acid Exposure ◽

Mitogen Activated Protein Kinase ◽

Signaling Mechanism ◽

Transepithelial Permeability ◽

Matrix Metalloproteinase 7 ◽

E Cadherin

Abstract 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.

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

International Journal of Molecular Sciences ◽

10.3390/ijms20215276 ◽

2019 ◽

Vol 20 (21) ◽

pp. 5276 ◽

Cited By ~ 2

Author(s):

Nu-Ri Im ◽

Doh Young Lee ◽

Byoungjae Kim ◽

Jian Kim ◽

Kwang-Yoon Jung ◽

...

Keyword(s):

Matrix Metalloproteinases ◽

Culture Media ◽

Laryngopharyngeal Reflux ◽

Acid Exposure ◽

Immunocytochemical Staining ◽

Mmp Inhibitor ◽

Transepithelial Permeability ◽

Laryngopharyngeal Reflux Disease ◽

E Cadherin

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.

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Matrilysin (Matrix Metalloproteinase-7) Mediates E-Cadherin Ectodomain Shedding in Injured Lung Epithelium

American Journal Of Pathology ◽

10.1016/s0002-9440(10)64318-0 ◽

2003 ◽

Vol 162 (6) ◽

pp. 1831-1843 ◽

Cited By ~ 208

Author(s):

John K. McGuire ◽

Qinglang Li ◽

William C. Parks

Keyword(s):

Matrix Metalloproteinase ◽

Ectodomain Shedding ◽

Lung Epithelium ◽

Injured Lung ◽

Matrix Metalloproteinase 7 ◽

E Cadherin

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Usefulness of Matrix Metalloproteinase-7 in Saliva as a Diagnostic Biomarker for Laryngopharyngeal Reflux Disease.

10.21203/rs.3.rs-490220/v1 ◽

2021 ◽

Author(s):

Nu-Ri Im ◽

Byoungjae Kim ◽

Kwang-Yoon Jung ◽

Seung-Kuk Baek

Keyword(s):

Matrix Metalloproteinase ◽

Sensitivity And Specificity ◽

Reflux Disease ◽

Laryngopharyngeal Reflux ◽

Diagnostic Methods ◽

Reflux Symptom Index ◽

Enzyme Activity Assay ◽

Symptom Index ◽

Laryngopharyngeal Reflux Disease ◽

Matrix Metalloproteinase 7

Abstract Introduction Several diagnostic methods are currently being used to diagnose LPRD (laryngopharyngeal reflux disease), but have the disadvantage of being invasive, subjective, or expensive. Objectives Our purpose in this study was to investigate the correlation between pepsin and MMP-7 (Matrix Metalloproteinase-7) in pharyngeal secretions of subjects according to RSI (Reflux Symptom Index) score to find out the diagnostic value of MMP-7. Method We recruited 173 subjects aged between 19 and 85 years who completed the RSI scale. All samples were taken after waking up, and the amount of the pepsin and MMP-7 in saliva were measured by means of an enzyme activity assay. Results There was a significant increase of pepsin and MMP-7 activity in the study group with an RSI score of 13 or higher. The sensitivity and specificity of MMP-7 for predicting the possibility of an RSI of 13 or more was higher than that of pepsin. When MMP-7 and pepsin were combined, this sensitivity and specificity increased. Conclusion An enzyme assay of MMP-7 in saliva may be a noninvasive and easy technique for diagnosing LPRD.

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