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Mariah A. Schumacher, Lezlie Jones, Mahesh Shivanna, Clark Springs, Udaya B. Kompella, SP Srinivas; Paclitaxel Modulates TGF-β1 Signaling In The Corneal Endothelium. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6697.
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TGF-ß family cytokines, which are found in the cornea and aqueous humor, modulate cell proliferation and fibrotic response of the corneal endothelium during wound healing. In this study, we have examined whether Smad signaling downstream of TGF-ß1 can be influenced by paclitaxel (Taxol) in the corneal endothelium.
Cultured bovine corneal endothelial cells (BCEC) were used for all the experiments. The effect of TGF-ß on BCEC was characterized by electric cell-substrate impedance sensing (ECIS). The impedance, which is sensitive to changes in cellular morphology and breakdown of the apical junctional complex, was measured using ECIS1600R (Applied Biophysics, Inc.,Troy, NY) with cells grown on gold electrodes to an AC current of 1 µA at 4 KHz and reported as normalized transendothelial electrical resistance (TER). Cytochalasin D (1 µg/mL), an actin severing agent was used as a positive control to assess the ECIS measurements.
Cells were serum starved for an hour and then exposed to TGF-ß1 (20 ng/mL; >24 hrs; n = 6). TER, which was measured in realtime, showed a sustained decrease (17.7 + 3.4%) when compared to untreated cells. The effect of cytochalasin D (1µg/mL), which disrupts the peri-junctional actomyosin ring, on the decrease in TER was much more pronounced (> 30%) and maximum response took < 3 h. The effect of the cytokine was opposed significantly by co-treatment with paclitaxel (5 µM) at 24 hrs (TGF-ß1: 22% vs. Paclitaxel +TGF-ß1:6.22%; n = 5). Effect of elevated cAMP, which is known to oppose TGF-ß1 signaling, was tested by pre-exposure to forskolin (10 µM) + Rolipram (50 µM). This pre-treatment also reduced the effect of the cytokine (TGF-ß1: 21% vs Forskolin + Rolipram +TGF-ß1: 10%; n = 5). The cytokine also induced a noticeable disassembly of the microtubules at 3 h which was opposed by forskolin and rolipram as well as paclitaxel.
Our findings suggest that the effect of TGF-ß1 can be opposed by stabilizing microtubules and by elevated cAMP in the corneal endothelium. In addition to epithelial to mesenchymal transformation associated with TGF-ß1, the cytokine also breaks down the barrier integrity of corneal endothelium as implied by the significant decrease in TER.
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