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Arumugam Ramachandran Muralidharan, Rupalatha Maddala, Vasanth Rao; The Role of Growth Differentiation Factor-15 in Regulation of Contractile Properties of Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):No Pagination Specified. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
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).
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.
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.
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.
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