ABSTRACT
AIM: To scrutinize the role of the Wnt/β-catenin signaling pathway in the epithelial-mesenchymal transition(EMT)of lens epithelial cells under hypoxic conditions, and to further analyze the effect of Dickkopf-1(DKK-1)expression on EMT of lens epithelial cells.METHODS: Human lens epithelial cells(HLEB3 cells)were propagated in vitro and then separated into two groups: one exposed to standard oxygen levels, added DMEM culture solution containing 10% FBS(normoxic group)and another subjected to low oxygen levels(hypoxic group). The hypoxic condition was emulated by applying a concentration of 100 μmol/L cobalt chloride(CoCl2)for 6, 12, 24, and 48h. The utilization of immunofluorescence staining enabled the detection of Wnt3a and DKK-1 expressions, along with the expression and localization of β-catenin protein in these groups. The expression of DKK-1 mRNA was discerned by quantitative real-time polymerase chain reaction(qRT-PCR).RESULTS: Immunofluorescence assays indicated an escalating trend in the Wnt3a and DKK-1 protein expression, which corresponded with the increasing duration of hypoxia. Likewise, an intensified nuclear accumulation of β-catenin protein was observed to be directly proportional to the length of hypoxia treatment. The qRT-PCR demonstrated that the difference in DKK-1 mRNA expression between the normoxic group and the group exposed to hypoxia for 6h was not statistically significant(P>0.05), whereas the DKK-1 mRNA expression of the 12, 24, and 48h hypoxia groups were significantly increased(P<0.001).CONCLUSION: Hypoxia can activate Wnt/β-catenin pathway in lens epithelial cells and induce the expression of DKK-1, thus regulating the Wnt/β-catenin pathway and affecting the EMT process.
ABSTRACT
BackgroundObstruction of aqueous humor out flow pathway or abnormality of the extracellular matrix( ECM ) of trabecular meshwork cells causes high intraocular pressure. The balance of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases(TIMPs) is critical for the metabolism of ECM. Interleukin1α(IL-1α) can influence outflow of aqueous humor by regulating MMPs level. Objective This study was to investigate the effect of interleukin-1α on the expression of MMP-2,MMP-3 and TIMP-I in cultured swine trabecular meshwork cells.Methods Swine sclera with trabecular meshwork tissue was isolated from 20 swine eyes and cultured with explant cultured method. Cultured cells were passaged and third generation cells were identified by fibronectin ( FN ) and laminin ( LN ) staining. After 24 hours of serum starvation, trabecular meshwork cells treated with IL-1α at the concentration of 10 mg/L were regarded as the IL group,and serum-free culture medium used to treat trabecular meshwork cells was regarded as the control group. The expression of MMP-2, MMP-3 and TIMP-1 proteins in trabecular meshwork cells were detected by immunohistochemistry,and the expression of MMP-2 mRNA, MMP-3 mRNA and TIMP-1 mRNA were detected by RT-PCR. The examination results were compared between the two groups. ResultsThe third generation of cells were positive for FN and LM. Compared with the control group, the expression levels of MMP-3 and TIMP-1 proteins(A value) in trabecular meshwork cells were significantly higher in the IL group than the control group(t=-7. 694,t =-5. 199,P＜0. 05) ,but no obvious difference was found in the expression of MMP-2 between the two groups( t=-2. 365, P＞0.05 ). The higher expression levels in MMP-3 mRNA and TIMP-1 mRNA (A value) in trabecular meshwork cells were seen in comparison with the control group (t =-3. 025,t=-1. 921 ,P＜0. 05). However,similar results were found in the expression of MMP-2 mRNA between the two groups(t =- 1. 173, P＞0.05 ). ConclusionsThe overexpression of MMP-3 and TIMP-1 proteins and their mRNA leads to the imbalance of MMP-3/TIMP-1 and promotes the decomposition of ECM in the trabecular meshwork, and therefore increases aqueous outflow.