ITF2357 regulates NF-κB signaling pathway to protect barrier integrity in retinal pigment epithelial cells

Abstract

The robust integrity of the retinal pigment epithelium (RPE), which contributes to the outer brain retina barrier (oBRB), is compromised in several retinal degenerative and vascular disorders, including diabetic macular edema (DME). This study evaluates the role of a new generation of histone deacetylase inhibitor (HDACi), ITF2357, in regulating outer blood-retinal barrier function and investigates the underlying mechanism of action in inhibiting TNFα-induced damage to RPE integrity. Using the immortalized RPE cell line (ARPE-19), ITF2357 was found to be non-toxic between 50 nM and 5 μM concentrations. When applied as a pre-treatment in conjunction with an inflammatory cytokine, TNFα, the HDACi was safe and effective in preventing epithelial permeability by fortifying tight junction (ZO-1, -2, -3, occludin, claudin-1, -2, -3, -5, -19) and adherens junction (E-cadherin, Nectin-1) protein expression post-TNFα stress. Mechanistically, ITF2357 depicted a late action at 24 h via attenuating IKK, IκBα, and p65 phosphorylation and ameliorated the expression of IL-1β, IL-6, and MCP-1. Also, ITF2357 delayed IκBα synthesis and turnover. The use of Bay 11-7082 and MG132 further uncovered a possible role for ITF2357 in non-canonical NF-κB activation. Overall, this study revealed the protection effects of ITF2357 by regulating the turnover of tight and adherens junction proteins and modulating NF-κB signaling pathway in the presence of an inflammatory stressor, making it a potential therapeutic application for retinal vascular diseases such as DME with compromised outer blood-retinal barrier.

Keywords: HDAC inhibitor; ITF2357; NF-κB; diabetic macular edema; inflammation; outer blood-retinal barrier; retinal pigment epithelium.

Figure 1.. ITF2357 is safe and effective in micromolar concentrations in ARPE-19 cells.

Real time monitoring of cell adhesion and proliferation with xCELLigence system showed that (A) ITF2357 did not cause any change in cell adhesion (0–5h), spreading (5–10h), or proliferation (10–24h) at different concentrations between 0.05µM and 5µM. (B) Relative cell survival at 12h and 24h remained constant for all drug concentrations. (C) Cell viability was unaffected by cell seeding density (0.5–5.0×10⁴ cells/96well), as seen by PrestoBlue assay and (D) MTT assay after 24h treatment. (E) No apoptotic cells were seen in TUNEL assay with either treatment, which was corroborated by no significant change in Caspase-3 (F) expression and (G) activity. (H) ITF2357 resulted in hyperacetylation of lysine residue in nuclear proteins and histone H3 even in the presence of TNFα, indicating the HDACi to be effective in ARPE-19 cells. Scale bar, 10μm. All experiments were repeated twice with n=3.

Figure 2.. ITF2357 increased TER and reduced permeability in ARPE-19 cells.

(A) ARPE-19 cells were seeded on polycarbonate filters and maintained in 1% RPE media for 2 weeks. Real-time TEER (Ωcm²) was measured on the cellZscope system. After 24h for baseline measurement, ITF2357 (2.5μM) and TNFα (10ng/ml) were added together to the apical chamber, and TER monitored over 4 days. ITF2357 drastically elevated the TER reduced by TNFα. (B) Fluorescein permeability across the ARPE-19 monolayer was also increased after TNFα treatment but attenuated with ITF2357. (C) SEM show clear demarcation of tight junctions (red arrow) around APRE-19 cells, which was lost in TNFα and restored with ITF2357. (D) Similarly, TEM showed tight junctions between RPE cells were enhanced in ITF2357 treated groups. All experiments were repeated twice with n=3.

Figure 3.. ITF2357 restored tight junction protein expression.

Representative immunostaining images of (A) ZO-1, (B) ZO-2, (C) ZO-3, (D) Occludin, (E) Claudin-1, and (F) Claudin-5 showed disrupted junctional proteins after 24h TNFα stimulus, which was restored with ITF2357. n=3. Scale bar, 10μm. (G-R) Real-time PCR showed similar downregulation of tight junction gene expression with TNFα, and reversal to that of the control or more after ITF2357 pre-treatment. (Q) JAM-B was the lone exception to the trend, increasingly with TNFα and decreasing with ITF2357. (S, T) Protein expression of ZO-2, ZO-3 and Occludin were upregulated with ITF2357 alone, while JAM-B was expressed only in the presence of TNFα. ***, p<0.001 compared to control; ^⌘⌘, p<0.01, ^⌘⌘⌘, p<0.001 against TNFα. All experiments were repeated twice with n=3.

Figure 4.. ITF2357 modulates adherens junction proteins.

Representative images of ARPE-19 cells labelled with (A) E-cadherin, (B) β-catenin and (C) Nectin-1 showed loss of membrane localization after TNFα and prevention with ITF2357 pre-treatment. Scale bar, 10μm. (G-L) Real-time PCR showed similar trends, with significant restoration of (I) E-cadherin and (K) Nectin-1 with ITF2357. In contrast, (H) β-catenin and (J) N-cadherin were increased after TNFα stimulus but reduced back to that of control with ITF2357. (M) ITF2357 alone resulted in E-cadherin protein expression, with slight increase also seen in Nectin-1. β-catenin protein expression was elevated with TNFα, similar to tra nscript (H), but was seen predominantly in cytoplasm of cells (B). *, p<0.05, **, p<0.01, ***, p<0.001 vs control; ^⌘⌘, p<0.01, ^⌘⌘⌘, p<0.001 vs TNFα. All experiments were repeated twice with n=3.

Figure 5.. TNFα-induced NF-κB nuclear translocation is inhibited with ITF2357.

(A-D) ARPE-19 cells pre-treated with 2.5μM ITF2357 (2h) and 10ng/ml TNFα for 24h were stained with p65. Control and ITF2357 alone treated cells show cytosolic localization of p65 subunit, which was markedly increased in the nucleus after TNFα stimulus. ITF2357 resulted in p65 sequestration in the cytoplasm. Scale bar, 10μm. (E) Nuclear and cytosolic fractions isolated from treated ARPE-19 confirmed p65 enrichment in the nuclear fraction after TNFα treatment, which is reduced by ITF2357. Cytosolic content of p65 was slightly increased in the presence of ITF2357, suggesting production of p65 which were retained in the cytoplasm. (F) Nuclear extract subjected to NF-κB activity assay further validated increased activity after TNFα stimulus and significant attenuation with ITF2357 pre-treatment. ***, p<0.001 vs control; ^⌘⌘⌘, p<0.001 vs TNFα. All experiments were repeated twice with n=3.

Figure 6.. ITF2357 reduced NF-κB target inflammatory genes and IκBα resynthesis.

Polarized ARPE-19 cell lysate were collected 30min and 24h after treatment for protein and transcript analysis. (A) Transcript levels of p65, IKKα, IKKβ and IKKγ were unaffected, but IκBα expression was significantly upregulated after stimulus at both timepoints. (B) Phosphorylated (p)IKKα/β, p-p65 and IKKγ was elevated at 30min, and persisted at 24h after TNFα stimulus, indicating acute and persistent activation of NF-κB signaling. Attenuation by ITF2357 was minimal at 30min, but successful by 24h. Meanwhile, TNFα resulted in almost total phosphorylation of IκBα at 30min, and resynthesis by 24h. However, ITF2357 treated cells showed repressed IκBα levels both alone and with TNFα. (C) NF-κB target genes were dramatically upregulated with TNFα, albeit at different time points. Pretreatment with ITF2357 significantly inhibited IL-6, IL-1β and MCP-1 expression. p<0.001 vs control; ^⌘⌘⌘, p<0.001 vs TNFα.

Figure 7.. ITF2357 inhibits TNFα-induced NF-κB activation via IKK complex.

NF-κB inhibitor (BAY 11–7082, 10μM) was given as a pretreatment with ITF2357 to polarized ARPE-19 for 2h prior to TNFα stimulus. Cells were collected at 30min and 24h timepoints to quantify effects at transient and persistent phases. At 30min and 24h, BAY 11–7082 and TNFα treatment alone (lane 7) showed reduction in IKKα/β and p65 basal levels, along with complete inhibition of their phosphorylation. ITF2357 treatment (lane 8) increased both basal and phosphorylated levels of IKK and p65, and targeted IκBα for degradation in the transient phase. After 24h, BAY 11–7082 together with ITF2357 increased basal IKKα/β levels and relieved suppression of IκBα resynthesis delay (lane 8).

Figure 8.. ITF2357 transcriptionally modulates IκBα turnover in TNFα-treated ARPE-19 cells.

Polarized ARPE-19 was treated with proteasome inhibitor, MG-132 (20μM), with or without 2.5μM ITF2357 for 2h prior to TNFα. Cells were lysed at 30min and 24h post treatment. MG-132 resulted in accumulation of pIκBα at 30min, leading to p-p65 activation even in cells not treated with TNFα (lanes 5, 6). At the 24h time point, excessive pIKKα/β depleted total IκBα (lanes 7, 8), and ITF2357 reduced IκBα levels regardless of TNFα (lanes 6, 8).

Figure 9.. Schematic of ITF2357 effects in ARPE-19 cells.

TNFα initiated downstream phosphorylation of IKKα/β, IκBα and p65 were attenuated by ITF2357, resulting in cytosolic retention of p65/p50 complex in and reduced NF-κB-mediated transcription of cytokines IL-1β, IL-6, MCP-1 and TNFα. In addition, IκBα resynthesis was delayed, leading to delayed action of ITF2357 in the persistent phase of NF-κB activation. Moreover, pre-treatment with ITF2357 elevated expression and plasma membrane localization of tight and adherens junctions, improving TEER and ameliorating paracellular transport, leading to enhanced epithelial barrier properties.