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Kate Keller, Yong-Feng Yang, Ying Ying Sun, Ted Acott; ADAMTS4 activity and TIMP3 levels are modulated in trabecular meshwork cells with decreased hyaluronan synthesis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3546.
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© ARVO (1962-2015); The Authors (2016-present)
Accumulating evidence supports a role for hyaluronan (HA) in regulating matrix metalloproteinase (MMP) expression, cellular localization and activity. Here, we analyzed ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs-4) mRNA expression, proteins levels and enzyme activity in trabecular meshwork (TM) cells with depleted HA. The amount of TIMP3, the tissue inhibitor of ADAMTS4, was also assessed.
Two methods were used to reduce HA concentration: 1 mM 4-methylumbelliferone (4MU) or previously characterized short hairpin RNAi silencing (shRNA) lentivirus targeted to each individual HA synthase (HAS) gene (shHAS1, shHAS2 and shHAS3). Vehicle control and shControl, which does not target any known gene, were also included. Primary porcine TM cells were subjected to 4MU treatment for 48 or 72 hours. For infection, lentivirus was incubated for 3 days and then cells were changed to serum-free media for a further 48 or 72 hours. ADAMTS4 mRNA levels were determined by quantitative RT-PCR and protein levels in RIPA cell lysates were analyzed by Western immunoblotting. A fluorometric ADAMTS4 activity assay was used to quantitate enzyme activity. Cells were fixed and immunostained for confocal microscopy.
ADAMTS4 mRNA levels decreased with all treatments, but protein levels were not significantly altered. ADAMTS4 activity was significantly decreased with all treatments at 48 and 72 hours. TIMP3 protein levels were reduced with shHAS infection, but increased with 4MU treatment. TIMP3 was immunolocalized in the cytoplasm and at PILS (podosome or invadopodia-like structures), areas of focal ECM degradation. TIMP3 also colocalized with clusters of ADAMTS4-stained vesicles. There seemed to be a higher degree of colocalization of TIMP3 and ADAMTS4 in 4MU-treated cells.
ADAMTS4 activity appears to be dependent on HA concentration. Since HA concentration decreases in aged eyes and is reduced even further in POAG patients, the ability of TM cells to focally remodel ECM via ADAMTS4 may be compromised, which in turn could adversely affect aqueous outflow.
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