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Mark S. Filla, Donna M Peters; Dysregulation of β3 integrin signaling increases extracelluar matrix deposition by immortalized human trabecular meshwork cells.. Invest. Ophthalmol. Vis. Sci. 2014;55(13):5673.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the role of β3 integrins in regulating the incorporation of proteins such as fibronectin (FN) and laminin (LN) into the extracellular matrix (ECM) of human trabecular meshwork (HTM) cultures. Excessive ECM deposition within the TM has been suggested as a factor in the pathogenesis of primary open angle glaucoma (POAG). Understanding how the assembly of the ECM is regulated could suggest ways in which to treat glaucoma.
Three HTM cell lines, derived from immortalized TM-1 cells, were used. The cells overexpressed either a wild type (WT) β3 integrin subunit or a constitutively active (CA) β3 integrin subunit. Control cells that expressed an empty vector (EV) were also used. The incorporation of FN and/or LN into the insoluble ECM was analyzed using a differential extraction of cell cultures with deoxycholic acid (DOC) together with immunoblotting, immunofluorescence (IF) microscopy and/or On-cell western analysis techniques. Additionally, cells were treated with a FN-binding peptide (pUR4) derived from the Streptococcus pyogenes F1 adhesin protein which has been previously shown to block FN fibril formation and subsequent matrix assembly.
As determined by On-cell western analysis, FN levels from unextracted cell monolayers were comparable in all three cell lines. However, TM-1 CAβ3 cells incorporated ~ 2-fold more FN into the DOC-insoluble ECM compared to the other two cell lines. pUR4 (500 nM) reduced the incorporation of FN into the DOC-insoluble ECM by at least 50% in all three cell lines after a 24 hour exposure. Even though pUR4 is not known to directly interact with LN, IF microscopy found that treatment with pUR4 (500 nM) blocked the incorporation of LN into the ECM of all three cell lines.
The data suggest that dysregulation of β3 integrin signaling could contribute to excessive deposition of FN into the HTM ECM. Incorporation of FN and LN were both inhibited by pUR4 treatment suggesting that FN may mediate the incorporation of LN into the ECM of the TM. Finally, these data suggest that pUR4 has the potential to be developed into a treatment for POAG and possibly other glaucomas.
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