Abstract
Purpose :
The secreted Ly-6/uPAR-related protein-1 (SLURP1) is an immunomodulatory and anti-angiogenic protein highly expressed by the corneal epithelium (CE). We have previously shown that SLURP1 suppresses TGF-b-induced Smad2/3 phosphorylation. Here we compared the wild type (WT) and Slurp1-null (Slurp1X-/-) corneal transcriptomes and studied the effect of SLURP1 on TGF-b-induced epithelial-mesenchymal transition (EMT).
Methods :
We compared the 6-month-old WT and Slurp1X-/- mouse corneal transcriptomes by RNA-Seq, validated the RNA-Seq results by QPCR and analyzed the data using CLC-Genomics workbench and Ingenuity Pathway Analysis (IPA). Control human corneal limbal epithelial (HCLE) cells and those overexpressing SLURP1 (HCLE-SLURP1) were treated with vehicle or 5ng/ml TGF-b for 5 days and the expression of EMT markers E-cadherin, N-Cadherin, Vimentin, Slug, Zeb1 and Zeb2 was quantified by immunoblots and immunofluorescent staining. The effect of TGF-b on epithelial permeability was studied using xCELLigence Real-Time Cell Analysis (RTCA).
Results :
RNA-Seq identified 334 up- and 195 down-regulated genes (>1.5-fold with p <0.05) in Slurp1X-/- compared with the WT corneas. IPA predicted upstream regulators Spdef, Smad7, and Sox2 to be inhibited and TGF-b1, -b2, -b3, NFkB, Fgf2, Tnf-a, IkBKB, IL-17A and Twist1 to be activated. Immunofluorescent staining revealed increased phosphorylation of NFkB and IkK(a/b) in the Slurp1X-/- corneas. TGF-b treatment of HCLE-SLURP1 and control HCLE cells resulted in relatively lower increase in N-cadherin (2.9- and 4.8-fold, respectively) and vimentin (1.9- and 2.4-fold, respectively) expression, with no significant change in the expression of E-Cadherin (0.76- and 0.71-fold, respectively), Zeb1 (0.94- and 1.1-fold, respectively), and Zeb2 (1.0- and 0.96-fold, respectively). TGF-b-treated HCLE-SLURP1 cells displayed significantly increased normalized cell index values compared with the control HCLE cells.
Conclusions :
Considering that: (i) key regulators of angiogenic inflammation- TGF-b1, NFkB and IkBKB- are activated in Slurp1X-/- corneas; (ii) TGF-b-treated HCLE-SLURP1 cells display relatively subdued upregulation of EMT marker proteins; and (iii) the permeability barrier function is compromised in the TGF-b-treated control HCLE but not HCLE-SLURP1 cells, we conclude that SLURP1 protects the CE cells from TGF-b-induced EMT.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.