Tacrolimus Modulates TGF-β Signaling-Related Genes and MicroRNAs in Human Retinal Pigment Epithelial Cells Activated by Lipopolysaccharide

Abstract

The retinal pigment epithelium (RPE) plays a crucial role in maintaining retinal homeostasis, and dysregulation of the transforming growth factor-beta (TGF-β) signaling pathways contributes to retinal fibrosis and inflammatory diseases, including proliferative vitreoretinopathy (PVR). Tacrolimus (FK506), an immunosuppressant, has shown potential antifibrotic properties, but its effects on TGF-β-related genes and microRNAs (miRNAs) in RPE cells remain unclear. Human RPE (H-RPE) cells were treated with lipopolysaccharide (LPS) to induce inflammation and subsequently exposed to tacrolimus. Gene and miRNA expression profiling related to TGF-β signaling pathways were conducted using microarrays, followed by Quantitative Reverse-Transcription Polymerase Chain Reaction (RT-qPCR) validation. Protein levels were assessed via enzyme-linked immunosorbent assay (ELISA), and interactions were analyzed using STRING database network analysis. Tacrolimus modulated key components of the TGF-β pathway, upregulating TGF-β2, TGF-β3, SMAD2, and SMAD4 while downregulating TGF-βR1 and SMAD7. JAK/STAT and MAPK pathways were also affected, indicating broad regulatory effects. miRNA profiling identified hsa-miR-200a-3p, hsa-miR-589-3p, hsa-miR-21, and hsa-miR-27a-5p as key regulators. STRING analysis confirmed strong functional interactions within the TGF-β network. In conclusion, tacrolimus modulates both canonical (upregulation of SMAD2/4 and downregulation of SMAD7) and non-canonical (JAK/STAT and MAPK) TGF-β signaling pathways in LPS-stimulated RPE cells. These changes collectively suggest a dual anti-inflammatory and anti-fibrotic effect. The increased TGF-β2 and decreased SMAD7 levels, alongside altered miRNA expression (e.g., downregulation of miR-200a-3p), indicate that tacrolimus may inhibit key profibrotic mechanisms underlying PVR. These findings support the potential therapeutic repurposing of tacrolimus in PVR and warrant further in vivo validation.

Keywords: lipopolysaccharide (LPS); proliferative vitreoretinopathy; retinal pigment epithelium; tacrolimus; transforming growth factor-beta (TGF-β) signaling pathway.

Figure 1

Results of cytotoxicity assay (A) Cell viability after exposure to increasing concentrations of LPS (1, 2, 10 µg/mL) at 6, 12, and 24 h; (B) Cell viability after exposure to tacrolimus (0.1, 1, 10, 100 ng/mL) at 6, 12, and 24 h; (C) Cell viability following sequential treatment: LPS (1 µg/mL for 6 h) followed by tacrolimus (0.1–100 ng/mL) for an additional 6, 12, or 24 h. Data are presented as mean ± SD; n = three independent experiments. Viability was assessed as a percentage of control (untreated) cells. * p < 0.05 vs. control at the same time point (one-way ANOVA with Scheffé’s post hoc test); LPS, lipopolysaccharide.

Figure 2

Venn diagram illustrating time-dependent differential expression of TGF-β-related genes in H-RPE cells treated with LPS and tacrolimus. C, control culture; H_6, H_12, H_24, culture exposed to tacrolimus for 6, 12, 24 h.

Figure 3

Expression of 20 mRNA in the H-RPE treated with LPS or tacrolimus (RT-qPCR results) presented as fold change in expression when compared to a control culture. Data are presented as mean ± SD; TGF-β2, Transforming Growth Factor Beta 2; TGF-β3, Transforming Growth Factor Beta 3; TGF-βR1, Transforming Growth Factor Beta Receptor 1; TGF-βR3, Transforming Growth Factor Beta Receptor 3; SMAD2, Mothers Against Decapentaplegic Homolog 2; SMAD4, Mothers Against Decapentaplegic Homolog 4; MAPK3, Mitogen-Activated Protein Kinase 3 (ERK1); JAK1, Janus Kinase 1; JAK2, Janus Kinase 2; JAK3, Janus Kinase 3; STAT3, Signal Transducer and Activator of Transcription 3; EGFR, Epidermal Growth Factor Receptor; SMAD7, Mothers Against Decapentaplegic Homolog 7; IFNG, Interferon Gamma; IL-6, Interleukin 6; CASP3, Caspase 3; IL-2, Interleukin 2; CASP8, Caspase 8; KDR, Kinase Insert Domain Receptor (Vascular Endothelial Growth Factor Receptor 2, VEGFR-2); PSAP, Prosaposin.

Figure 4

Expression of 20 mRNA in the H-RPE treated with LPS and tacrolimus (RT-qPCR results) presented as fold change in expression when compared to a control culture. Data are presented as mean ± SD; TGF-β2, Transforming Growth Factor Beta 2; TGF-β3, Transforming Growth Factor Beta 3; TGF-βR1, Transforming Growth Factor Beta Receptor 1; TGF-βR3, Transforming Growth Factor Beta Receptor 3; SMAD2, Mothers Against Decapentaplegic Homolog 2; SMAD4, Mothers Against Decapentaplegic Homolog 4; MAPK3, Mitogen-Activated Protein Kinase 3 (ERK1); JAK1, Janus Kinase 1; JAK2, Janus Kinase 2; JAK3, Janus Kinase 3; STAT3, Signal Transducer and Activator of Transcription 3; EGFR, Epidermal Growth Factor Receptor; SMAD7, Mothers Against Decapentaplegic Homolog 7; IFNG, Interferon Gamma; IL-6, Interleukin 6; CASP3, Caspase 3; IL-2, Interleukin 2; CASP8, Caspase 8; KDR, Kinase Insert Domain Receptor (Vascular Endothelial Growth Factor Receptor 2, VEGFR-2); PSAP, Prosaposin.

Figure 5

Protein interaction network for TGF- β system differentiation-related genes generated using the STRING database. TGF-β2, Transforming Growth Factor Beta 2; TGF-β3, Transforming Growth Factor Beta 3; TGF-βR1, Transforming Growth Factor Beta Receptor 1; TGF-βR3, Transforming Growth Factor Beta Receptor 3; SMAD2, Mothers Against Decapentaplegic Homolog 2; SMAD4, Mothers Against Decapentaplegic Homolog 4; MAPK3, Mitogen-Activated Protein Kinase 3 (ERK1); JAK1, Janus Kinase 1; JAK2, Janus Kinase 2; JAK3, Janus Kinase 3; STAT3, Signal Transducer and Activator of Transcription 3; EGFR, Epidermal Growth Factor Receptor; SMAD7, Mothers Against Decapentaplegic Homolog 7; IFNG, Interferon Gamma; IL-6, Interleukin 6; CASP3, Caspase 3; IL-2, Interleukin 2; CASP8, Caspase 8; KDR, Kinase Insert Domain Receptor (Vascular Endothelial Growth Factor Receptor 2, VEGFR-2); PSAP, Prosaposin.