Effect of zymosan on the expression and function of the gap-junction protein connexin 43 in human corneal fibroblasts

Abstract

AIM: To study the effect of zymosan, a ligand found on the surface of fungi, on gap junctional intercellular communication (GJIC) in cultured human corneal fibroblasts (HCFs). METHODS: Zymosan was added to the medium of cultured HCFs with or without the administration of mitogen-activated protein kinase (MAPK) inhibitors or the inhibitor kappa B kinase 2 (IKK2) inhibitor IV. The protein and mRNA levels of connexin 43 (Cx43) in HCFs were measured by Western blot, immunofluorescence, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analyses. The GJIC activity was tested using a dye-coupling assay. RESULTS: The reduction of Cx43 protein and mRNA levels as well as a significant decrease in GJIC activity were observed in cultured HCFs when zymosan was added into the culture medium. Compared with controls (no zymosan), the protein level of Cx43 was reduced by 45% and 54% in the presence of zymosan at 200 and 600 μg/mL, respectively (P<0.05); and it was reduced by 45%, 48%, and 75% in the presence of zymosan (600 μg/mL) for 24, 36, and 48h, respectively (P<0.05). The mRNA expression of Cx43 was reduced by 98% in the presence of zymosan (P<0.05). The effects of zymosan on Cx43 expression and GJIC activity were attenuated by the administration of PD98059 [an extracellular signal-regulated kinase (ERK) signaling inhibitor] (P<0.05), c-Jun NH2-terminal kinase (JNK) inhibitor II (P<0.05), and IKK2 inhibitor IV (P<0.05). CONCLUSION: Zymosan inhibits the activity of GJIC in cultured HCFs. This effect is likely regulated via the nuclear factor-κB (NF-κB), MAPK/ERK, and JNK signaling pathways. The inhibitory effects of zymosan on Cx43 expression and GJIC activity in HCFs may induce damage of corneal stroma during corneal fungal infection.

Keywords

• corneal fibroblast
• zymosan
• gap junction
• connexin 43
• mapks