Suppression of IkappaBalpha increases the expression of matrix metalloproteinase-2 in human ciliary muscle cells.
OBJECTIVE
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 (IkappaBalpha) 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 (IkappaBalpha) was transfected into HCM cells. The mRNA and protein levels of IkappaBalpha, nuclear factor-kappa B (NF-kappaB)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 IkappaBalpha siRNA transfection by real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot. The activation of NF-kappaBp65 was determined through the translocation of NF-kappaBp65 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 IkappaBalpha siRNA led to gradual downregulation of IkappaBalpha and TIMP-2 both at the mRNA and protein level after 24 h, 48 h and 72 h. The IkappaBalpha 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 IkappaBalpha 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 IkappaBalpha siRNA transfection. Although no obvious changes were seen in either mRNA or protein levels of total NF-kappaBp65 (p>0.05), the protein level of NF-kappaBp65 increased dramatically in the nucleus as revealed by western blot and fluorescence staining 24 h, 48 h, and 72 h after IkappaBalpha 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 IkappaBalpha siRNA in HCM cells through the activation of the NF-kappaB signaling pathway. Our results suggested that IkappaBalpha may therefore be a potential target for controlling the uveoscleral outflow pathway in glaucoma.
Lan
Yu-Qing
YQ
Department of Ophthalmology, The Second Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.
Zhang
Chi
C
Xiao
Jian-Hui
JH
Zhuo
Ye-Hong
YH
Guo
Hui
H
Peng
Wei
W
Ge
Jian
J
D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology*; D005260:Female; D017353:Gene Deletion; D005786:Gene Expression Regulation; D055785:Gene Knockdown Techniques; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism*; D008297:Male; D053511:Matrix Metalloproteinase 14 / Q000378:metabolism; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D032342:Muscle Cells / Q000166:cytology / Q000201:enzymology*; D000072000:NF-KappaB Inhibitor alpha; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000378:metabolism; D051996:Transcription Factor RelA / Q000378:metabolism; D014162:Transfection
D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D051996:Transcription Factor RelA; D019716:Tissue Inhibitor of Metalloproteinase-2; D000072000:NF-KappaB Inhibitor alpha; C522361:MMP2 protein, human; D020778:Matrix Metalloproteinase 2; C507086:MMP14 protein, human; D053511:Matrix Metalloproteinase 14