advertisement
Abstract #24637 Published in IGR 11-4
Suppression of I(kappa)B(alpha) increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells
Lan Y-Q; Zhang C; Xiao J-H; Zhuo Y-H; Guo H; Peng W; Ge JMolecular Vision 2009; 15: 1977-1987
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 (I(kappa)B(alpha)) 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 (I(kappa)B(alpha)) was transfected into HCM cells. The mRNA and protein levels of I(kappa)B(alpha), nuclear factor-kappa B (NF-(kappa)B)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 I(kappa)B(alpha) siRNA transfection by real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot. The activation of NF-(kappa)Bp65 was determined through the translocation of NF-(kappa)Bp65 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 I(kappa)B(alpha) siRNA led to gradual downregulation of I(kappa)B(alpha) and TIMP-2 both at the mRNA and protein level after 24 h, 48 h and 72 h. The I(kappa)B(alpha) and TIMP-2 mRNA levels decreased 92.7%(plus or minus)1.6% and 70.3%(plus or minus)13.1%, respectively, and the protein levels were reduced 87.3%(plus or minus)2.0% and 62.9% (plus or minus)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 I(kappa)B(alpha) siRNA transfection. The MMP-2 and MT1-MMP mRNA levels increased 178%(plus or minus)4.6% and 165%(plus or minus)8.2%, respectively, while protein levels were raised to 162%(plus or minus)3.7% and 157.6%(plus or minus)5.7% (p<0.01), respectively, when compared to the control 72 h after I(kappa)B(alpha) siRNA transfection. Although no obvious changes were seen in either mRNA or protein levels of total NF-(kappa)Bp65 (p>0.05), the protein level of NF-(kappa)Bp65 increased dramatically in the nucleus as revealed by western blot and fluorescence staining 24 h, 48 h, and 72 h after I(kappa)B(alpha) 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%(plus or minus)15% and 151%(plus or minus)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 I(kappa)B(alpha) siRNA in HCM cells through the activation of the NF-(kappa)B signaling pathway. Our results suggested that I(kappa)B(alpha) may therefore be a potential target for controlling the uveoscleral outflow pathway in glaucoma.
J. Ge. Department of Glaucoma, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Xianlienan Road 54, Guangzhou, Guangdong, China. gejian@mail.sysu.edu.cn
Classification:
2.9 Ciliary body (Part of: 2 Anatomical structures in glaucoma)
3.6 Cellular biology (Part of: 3 Laboratory methods)
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
