To investigate whether forskolin, a protein kinase A agonist, regulates toll-like receptor 4 actions on retinal endothelial cell permeability in vitro. We also evaluated whether PKA could regulate TLR4 signaling independent of exchange protein activated by cAMP in REC in culture. REC were grown in normal (5 mM) or high (25 mM) glucose. Cells were treated with forskolin to increase PKA levels, siRNA against TLR4, siRNA against myeloid differentiation primary response 88, siRNA against translocating chain associated membrane protein 1, siRNA against epac1, or scrambled siRNA, or a combination of these treatments. Western blotting was done for zonula occludens 1 and occludin protein levels, as well as TLR4 signaling cascade proteins. Permeability measurements were done for REC in culture following inhibition of TLR4 or its signaling cascades. Forskolin restored high glucose-associated decreases in ZO-1 and occludin, which was associated with improved in vitro permeability levels. Both forskolin and TLR4 inhibition reduced high glucose-induced increases in REC permeability, but the actions were not cooperative. Forskolin regulated both MyD88-dependent and -independent signaling pathways, independent of Epac1. Finally, blockade of MyD88 or TRAM1 reduced permeability in REC grown in high glucose. A PKA agonist regulated TLR4 signaling independent of Epac1. PKA agonism or TLR4 inhibition is effective at reducing high glucose-induced permeability in REC in vitro. These studies offer new avenues for therapeutic development.
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This work was funded by R01EY030284 (JJS), P30EY04068 (LDH), and an Unrestricted Grant to the Department of Ophthalmology from Research to Prevent Blindness (Kresge Eye Institute). The funders did not influence the design or execution of these studies.
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Supplemental Figure 1. MyD88-dependent and -independent signaling both regulate REC permeability. Western blot showing successful knockdown by siRNA of MyD88 (A) and TRAM (B). Data are from retinal endothelial cells (REC) grown in normal glucose (NG), high glucose (HG) alone or high glucose+ MyD88 siRNA, high glucose+TRAM siRNA, or high glucose+ scrambled siRNA. *P<0.05 vs. NG, #P<0.05 vs. HG. N=5-10. (JPG 90 kb)
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Liu, L., Jiang, Y. & Steinle, J.J. Forskolin regulates retinal endothelial cell permeability through TLR4 actions in vitro. Mol Cell Biochem 476, 4487–4492 (2021). https://doi.org/10.1007/s11010-021-04252-9