Yonsei Med J.  2018 Dec;59(10):1222-1231. 10.3349/ymj.2018.59.10.1222.

German Cockroach Extract Induces Matrix Metalloproteinase-1 Expression, Leading to Tight Junction Disruption in Human Airway Epithelial Cells

• Lee KE
• Jee HM
• Hong JY
• Kim MN
• Oh MS
• Kim YS
• Kim KW
• Kim KE
• Sohn MH

• KMID: 2426337
• DOI: http://doi.org/10.3349/ymj.2018.59.10.1222

Abstract

PURPOSE
Cockroach exposure is a pivotal cause of asthma. Tight junctions are intercellular structures required for maintenance of the barrier function of the airway epithelium, which is impaired in this disease. Matrix metalloproteinases (MMPs) digest extracellular matrix components and are involved in asthma pathogenesis: MMP1 is a collagenase with a direct influence on airway obstruction in asthmatics. This study aimed to investigate the mechanism by which German cockroach extract (GCE) induces MMP1 expression and whether MMP1 release alters cellular tight junctions in human airway epithelial cells (NCI-H292).
MATERIALS AND METHODS
mRNA and protein levels were determined using real-time PCR and ELISA. Tight junction proteins were detected using immunofluorescence staining. Epithelial barrier function was measured by transepithelial electrical resistance (TEER). The binding of a transcription factor to DNA molecules was determined by electrophoretic mobility shift assay, while the levels of tight junction proteins and phosphorylation were determined using Western blotting.
RESULTS
GCE was shown to increase MMP1 expression, TEER, and tight junction degradation. Both an inhibitor and small interfering RNA (siRNA) of MMP1 significantly decreased GCE-induced tight junction disruption. Furthermore, transient transfection with ETS1 and SP1 siRNA, and anti-TLR2 antibody pretreatment prevented MMP1 expression and tight junction degradation. An extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) inhibitor also blocked MMP1 release, ETS1/SP1 DNA binding, and tight junction alteration.
CONCLUSION
GCE treatment increases MMP1 expression, leading to tight junction disruption, which is transcriptionally regulated and influenced by the ERK/MAPK pathway in airway epithelial cells. These findings may contribute to developing novel therapeutic strategies for airway diseases.

Keyword

German cockroach; asthma; matrix metalloproteinase; airway epithelial cell; tight junction

MeSH Terms

Airway Obstruction
Asthma
Blattellidae*
Blotting, Western
Cockroaches
Collagenases
DNA
Electric Impedance
Electrophoretic Mobility Shift Assay
Enzyme-Linked Immunosorbent Assay
Epithelial Cells*
Epithelium
Extracellular Matrix
Fluorescent Antibody Technique
Humans*
Matrix Metalloproteinase 1*
Matrix Metalloproteinases
Phosphorylation
Phosphotransferases
Protein Kinases
Real-Time Polymerase Chain Reaction
RNA, Messenger
RNA, Small Interfering
Tight Junction Proteins
Tight Junctions*
Transcription Factors
Transfection
Collagenases
DNA
Matrix Metalloproteinase 1
Matrix Metalloproteinases
Phosphotransferases
Protein Kinases
RNA, Messenger
RNA, Small Interfering
Tight Junction Proteins
Transcription Factors

Figure

• 

  Fig. 1 Expression of matrix metalloproteinases (MMPs) and TIMP in human airway epithelial cells (NCI-H292) treated with German cockroach extract (GCE) for up to 48 h. (A) MMP1, 2, 7, 9, and 12 mRNA expressions at 6 h after GCE treatment. Time-dependent mRNA expression of (B) MMP1, (C) MMP9, and (D) TIMP1 was analyzed using real-time PCR. (E) MMP1 and (F) MMP9 protein expression were analyzed in the cell culture supernatants. Data are presented as the mean±SEM obtained in three independent experiments. *p<0.05, †p<0.01, ‡p<0.001, compared with control (ctrl, media only) or cells at 0 h of media only. NS, not significant.

• 

  Fig. 2 GCE induces tight junction and its proteins change. (A) Occludin and ZO-1 localization on GCE-stimulated cells was detected by immunofluorescence staining with anti-occludin or ZO-1 and observed under confocal microscopy. Scale bar represents 10 µm. (B) Transepithelial electrical resistance was measured after 24 hours of GCE treatment. (C) Occludin and (D) ZO-1 mRNA expression by time-dependent GCE treatment. *p<05 and †p<0.01, compared with control (ctrl, cells in media only). (E) Occludin and (F) ZO-1 protein expression by dose-dependent GCE treatment probed with anti-occludin or ZO-1. Control (Ctrl) is protein from cells in media only probed with anti-occludin or ZO-1. Data are presented as the mean±SEM obtained in three independent experiments. Representative images are presented. GCE, German cockroach extract; ZO, zonula occludens; NS, not significant.

• 

  Fig. 3 GCE-induced MMP1 expression influences tight junction disruption. (A) Representative images show occludin and ZO-1 localization in cells pretreated with MMP inhibitor GM6001 and treated with GCE. Scale bar represents 10 µm. (B) Occludin and (C) ZO-1 protein was detected in cells transiently transfected with MMP1 siRNA and treated with GCE by anti-occludin or ZO-1. Representative images of three independent experiments are presented. (D) MMP1 protein expression abundance was measured in the supernatant of cells transiently transfected with MMP1 siRNA and treated with GCE by ELISA. *p<05, compared with the GCE only-treated cells. GCE, German cockroach extract; MMP, matrix metalloproteinase; ZO, zonula occludens; siRNA, small interfering RNA.

• 

  Fig. 4 GCE-induced MMP1 transcriptional regulation influences tight junction disruption. (A) ETS1 and (B) SP1 mRNA was expressed in GCE-treated cells. (C and D) Nuclear extracts were collected to assess the translocation of ETS1 and SP1. (E and F) MMP1 protein was measured in transiently transfected cells with ETS1, ETS2, or SP1 siRNA and treated with GCE. (G and H) Cell lysates were collected to assess the expression of occludin and ZO-1 protein. Data are represented as the mean±SEM obtained in three independent experiments. *p<05, †p<0.01, compared with cells in media only or treated with GCE. Images are representative of three individual experiments. GCE, German cockroach extract; MMP, matrix metalloproteinase; ZO, zonula occludens; siRNA, small interfering RNA; NS, not significant.

• 

  Fig. 5 ERK/MAPK pathway modulates MMP1 transcriptional regulation and tight junction disruption. (A) Phosphorylated and total ERK1/2 was detected in H292 cells pretreated with PD98059 for 1 h and treated with GCE for 24 h. (B) MMP1 protein was measured in the supernatant. Data are presented as the mean±SEM obtained in three independent experiments. *p<05, compared with the GCE only-stimulated cells. (C) ETS1 and (D) SP1 translocation were identified by the biotin-labeled probe of ETS1 or SP1. (E and F) Occludin and ZO-1 protein were immunoblotted with anti-occludin and ZO-1 in cells pretreated with PD98059 and treated with GCE. Images are representative of the results obtained in three independent experiments. MMP, matrix metalloproteinase; GCE, German cockroach extract; ZO, zonula occludens; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase.

• 

  Fig. 6 TLR2 regulates MMP1 expression and might affect tight junction disruption. (A) MMP1 was measured in cells pretreated with anti-TLR2 antibody and treated GCE. Data are presented as the mean±SEM obtained in three independent experiments. †p<0.01, compared with the GCE only-treated cells. (B and C) Occludin and ZO-1 protein was detected in cells pretreated with anti-TLR2 and treated GCE by anti-occludin and ZO-1 antibody. Images are representative of the results obtained in three independent experiments. TLR2, Toll-like receptor 2; MMP, matrix metalloproteinase; GCE, German cockroach extract; ZO, zonula occludens.

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