Suppression of IκBα increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells

PURPOSE: An increase of matrix metalloproteinase-2 (MMP-2) has been found to improve outflow through the uveoscleral pathway. This experiment was designed to test whether reduction of inhibitor of nuclear factor kappa B alpha (IκBα) levels could enhance MMP-2 expression in human ciliary muscle (HCM) cells in vitro. METHODS: The small interfering RNA (siRNA) targeting inhibitor of nuclear factor kappa B (IκBα) was transfected into HCM cells. The mRNA and protein levels of IκBα, nuclear factor-kappa B (NF-κB)p65, MMP-2, tissue inhibitor of metalloproteinase-2 (TIMP-2), and membrane-type 1 matrix metalloproteinase (MT1-MMP) in HCM cells were examined 24 h, 48 h, and 72 h after IκBα siRNA transfection by real-time reverse transcription polymerase chain reaction (RT–PCR) and western blot. The activation of NF-κBp65 was determined through the translocation of NF-κBp65 by fluorescence microscope. Gelatin zymography was used to detect the secretion and activity of MMP-2. RESULTS: Real-time RT–PCR and western blot showed that transfection of IκBα siRNA led to gradual downregulation of IκBα and TIMP-2 both at the mRNA and protein level after 24 h, 48 h and 72 h. The IκBα and TIMP-2 mRNA levels decreased 92.7%±1.6% and 70.3%±13.1%, respectively, and the protein levels were reduced 87.3%±2.0% and 62.9%±0.8% (p<0.01), respectively, when compared to the control 72 h after siRNA transfection. Conversely, the MMP-2 and MT1-MMP mRNA and protein levels increased in the time-dependent manner after IκBα siRNA transfection. The MMP-2 and MT1-MMP mRNA levels increased 178%±4.6% and 165%±8.2%, respectively, while protein levels were raised to 162%±3.7% and 157.6%±5.7% (p<0.01), respectively, when compared to the control 72 h after IκBα siRNA transfection. Although no obvious changes were seen in either mRNA or protein levels of total NF-κBp65 (p>0.05), the protein level of NF-κBp65 increased dramatically in the nucleus as revealed by western blot and fluorescence staining 24 h, 48 h, and 72 h after IκBα siRNA transfection. Moreover, gelatin zymography indicated that the secretion and activity of MMP-2 in treated cells were higher than those in the control cells. The maximum increases of pro-MMP-2 and active-MMP-2 were 172%±15% and 151%±14% (p<0.01), respectively, when compared to the control at the experiment’s conclusion 72 h after siRNA transfection. CONCLUSIONS: Expression and activity of MMP-2 was enhanced by the IκBα siRNA in HCM cells through the activation of the NF-κB signaling pathway. Our results suggested that IκBα may therefore be a potential target for controlling the uveoscleral outflow pathway in glaucoma.