Abstract
Purpose :
Growth differentiation factor-15 (GDF-15), a member of the transforming growth factor (TGF)-β superfamily of proteins, is implicated in several physiologic and pathological processes. Our recent studies using mass spectrometry detected the consistent presence of GDF-15 in the extracellular matrix (ECM) fraction derived from primary cultures of human trabecular meshwork (HTM) cells, suggesting a possible role for this protein in TM biology. Here we investigated the potential role of GDF-15 in HTM cell contractile activity and fibronectin production in the context of homeostasis of aqueous humor (AH) outflow and intraocular pressure (IOP).
Methods :
For secondary confirmation of GDF-15 expression by TM cells, RT-PCR analysis was performed using RNA derived from HTM cells. The time-dependent effects of recombinant human GDF-15 (rhGDF-15) on actin cytoskeletal organization, focal adhesions (vinculin), phosphorylation of myosin light chain (MLC), paxillin, myosin phosphatase target subunit 1 (MYPT1) and SMAD2/3, and levels of total SMAD and fibronectin in serum-starved human TM cells were determined by immunofluorescence and immunoblot analyses using either the respective phosphospecific or other antibodies.
Results :
In addition to mass spectrometry-based detection of GDF-15 in the ECM fraction of HTM cells, GDF-15 expression in HTM cells was confirmed by RT-PCR analysis. Recombinant GDF-15 (20 ng/ml) induced formation of actin stress fibers and focal adhesions in serum starved HTM cells within 3 hrs, based on immunofluorescence analysis. Moreover, rhGDF-15 treatment led to significant increase in protein levels of phosphorylated MLC, paxillin, MYPT1 and SMAD2/3, and αSMA in serum starved human TM cells at both 3 and 24 hrs post treatment compared to serum starved control TM cells. The GDF-15 treated HTM cells showed increased immunostaining for fibronectin and αSMA under serum starved conditions compared to untreated controls.
Conclusions :
To the best of our knowledge, this is the first study to detect the presence of GDF-15 in TM cells and to investigate its effects on TM cell contractile properties and fibronectin production. These results reveal that GDF-15 influences the contractile properties of TM cells, and elicits focal adhesion formation and expression of αSMA and fibronectin, indicating a potential role for this protein in homeostasis of AH outflow and IOP.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.