Modulation of corneal and stromal matrix metalloproteinase by the mannose-induced Acanthamoeba cytolytic protein.
The involvement of the mannose-induced Acanthamoeba cytopathic protein (MIP-133) in tissue injury and activation of metalloproteinase of corneal and stromal cells was examined in vitro. Activation of MMP-1, MMP-2, MMP-3, and MMP-9 induced by MIP-133 on human corneal epithelial and stromal cell cultures was examined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA. MMP-1, MMP-2, MMP-3, and MMP-9 mRNA were expressed in both cultured human corneal epithelial and stromal cells. When the epithelial cells were exposed to MIP-133 protein, the mRNA expression for MMP-1 and MMP-9 was unchanged. However, the transcript for MMP-2 and MMP-3 was decreased by 2-fold. By contrast, the expression of MMP-2 and MMP-3 was significantly upregulated (2- to 4-fold) in the corneal stromal cells 1, 4, and 8h after MIP-133 stimulation. At the protein level, there was no significant difference in the level of MMPs between the corneal epithelial cells before and after stimulation with MIP-133. By contrast, the levels of MMP-2 and MMP-3 were significantly higher in the corneal stromal cells stimulated with MIP-133. The supernatants from corneal stromal cells stimulated with MIP-133 were incubated with PMSF and MIP-133 antibody and the level of MMP-2 was measured by ELISA. Activation of MMP-2 by MIP-133 was inhibited in the supernatants pretreated with the serine protease inhibitor, PMSF, and anti-MIP-133. Supernatants pretreated with the cysteine protease inhibitor E6 or control antibody produced the same amount of MMP-2 as the untreated supernatants. To verify possible homology between MMPs and Acanthamoeba castellanii proteases, the mRNA from A. castellanii was prepared and analyzed for the expression of MMP genes by PT-PCR. The results showed that A. castellanii did not express mRNA for MMP-1, MMP-2, MMP-3, or MMP-9. Thus, A. castellanii mRNA does not cross-react with human MMPs. Furthermore, ELISA was used to determine the cross-reactivity of MMP antibodies with the MIP-133 protein. Monoclonal antibodies against MMPs did not cross-react with either the MIP-133 protein or BSA (negative control antigen). The results indicate that the MIP-133 protein modulates MMP-2 and -3 expression differently in human corneal epithelial and stromal cells.
Alizadeh
Hassan
H
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9057, USA. hassan.alizadeh@utsouthwestern.edu
Li
Haochuan
H
Neelam
Sudha
S
Niederkorn
Jerry Y
JY
D000048:Acanthamoeba / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D003319:Corneal Stroma / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D003429:Cross Reactions; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism*; D015800:Protozoan Proteins / Q000378:metabolism / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods
D015800:Protozoan Proteins; D012333:RNA, Messenger; C534367:mannose-induced cytopathic protein 133, Acanthamoeba; D020782:Matrix Metalloproteinases