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Inhibition of NUCKS Facilitates Corneal Recovery Following Alkali Burn

Poon, Ming-Wai, Jiang, Dan, Qin, Peng, Zhang, Yuelin, Qiu, Beiying, Chanda, Sumit, Tergaonkar, Vinay, Li, Qing, Wong, Ian Y., Yu, Zhendong, Tse, Hung-Fat, Wong, David S. H., Lian, Qizhou·2017·Scientific Reports
DOI 10.1038/srep41224PMC PMC5247723
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Abstract

Corneal wound healing involves a complex cascade of cytokine-controlled cellular events, including inflammatory and angiogenesis responses that are regulated by transcriptional chromatin remodeling. Nuclear Ubiquitous Casein and cyclin-dependent Kinase Substrate (NUCKS) is a key chromatin modifier and transcriptional regulator of metabolic signaling. In this study, we investigated the role of NUCKS in corneal wound healing by comparing its effects on corneal alkali burn in NUCKS knockout (NKO) and NUCKS wild-type (NWT) mice. Our data showed that following alkali-injury, inhibition of NUCKS (NKO) accelerated ocular resurfacing and suppressed neovascularization; the cytokine profile of alkali burned corneas in NKO mice showed suppressed expression of inflammation cytokines (IL1A & IL1B); upregulated expression of antiangiogenic factor (Pigment Epithelium-derived Factor; PEDF); and downregulated expression of angiogenic factor (Vascular Endothelial Growth Factor, VEGF); in vitro, following LPS-induced NFκB activation, NKO corneal cells showed reduced expression of IL6, IP10 and TNFα. In vitro, corneal epithelial cells showed reduced NF-κb activation on silencing of NUCKS and corresponding NFκB-mediated cytokine expression was reduced. Here, we illustrate that inhibition of NUCKS played a role in cytokine modulation and facilitated corneal recovery. This reveals a potential new effective strategy for ocular burn treatment.

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Figure 1 Inhibition of NUCKS Promotes Corneal Resurfacing in Alkali Injured Corneas in Vivo. (A) Bright field images (upper rows of NWT and NKO columns) and fluorescein images (lower rows of NWT and NKO columns) were captured by slit lamp biomicroscopy. Representative bright field images are shown for NWT (i, iii, vii, xi and xv) and NWT mice and (ii, v, ix, xiii, xvii) at specific time points (pre-injury, days 0, 3, 7 and 14). NKO mice exhibited less severe oedema than NWT mice at the end point, day 14. (NWT*oedema, N = 4; NKO no oedema, N = 4; Scale bars: 2 mm). Representative fluorescein images are shown for NWT (iv, viii, xii and xvi) and NKO mice (vi, x, xiv and xviii). Corneal defeat recovery in NKO mice was significantly faster than that for NWT mice at the end point, post injury day 14. (B) Percentage of retained corneal defeat was calculated as follows: the area of defeat retained at a specific time point divided by the area of defeat measured on post injury day 0 (initial point following injury). On day 3, NWT mice showed 96.56 ± 3.89% and NKO mice 27.75 ± 6.61% defeat, indicating that NKO mice achieved better initial healing. As early as day 7, NKO mice showed no defeats 0 ± 0% compared with NWT mice who showed 84.03 ± 9.67% defeat. At the end point on day 14, NWT mice retained 50.18 ± 7.05% defeat and NKO mice exhibited no defeats 0 ± 0%. (*P < 0.01; NWT N = 4; NKO N = 4; Scale bars: 2 mm).
Figure 1 Inhibition of NUCKS Promotes Corneal Resurfacing in Alkali Injured Corneas in Vivo. (A) Bright field images (upper rows of NWT and NKO columns) and fluorescein images (lower rows of NWT and NKO columns) were captured by slit lamp biomicroscopy. Representative bright field images are shown for NWT (i, iii, vii, xi and xv) and NWT mice and (ii, v, ix, xiii, xvii) at specific time points (pre-injury, days 0, 3, 7 and 14). NKO mice exhibited less severe oedema than NWT mice at the end point, day 14. (NWT*oedema, N = 4; NKO no oedema, N = 4; Scale bars: 2 mm). Representative fluorescein images are shown for NWT (iv, viii, xii and xvi) and NKO mice (vi, x, xiv and xviii). Corneal defeat recovery in NKO mice was significantly faster than that for NWT mice at the end point, post injury day 14. (B) Percentage of retained corneal defeat was calculated as follows: the area of defeat retained at a specific time point divided by the area of defeat measured on post injury day 0 (initial point following injury). On day 3, NWT mice showed 96.56 ± 3.89% and NKO mice 27.75 ± 6.61% defeat, indicating that NKO mice achieved better initial healing. As early as day 7, NKO mice showed no defeats 0 ± 0% compared with NWT mice who showed 84.03 ± 9.67% defeat. At the end point on day 14, NWT mice retained 50.18 ± 7.05% defeat and NKO mice exhibited no defeats 0 ± 0%. (*P < 0.01; NWT N = 4; NKO N = 4; Scale bars: 2 mm).CC BY · PubMed Central
Figure 2 Corneal Epithelial Cells Cultures Isolated from (A) NWT and (B) NKO mice were Positively Stained with Corneal Epithelial Specific Marker, Cytokeratin K3 on Immunofluorescence Staining. (A) Staining for NWT culture. (i) cytokeratin K3 (K3), (ii) DAPI, (iii) combination of K3 with DAPI. (iv) cytokeratin K19 (K19), (v) DAPI, (vi) combination of K19 with DAPI. NWT cell cultures were positively stained with K3 (i, ii, iii), and negatively stained with K19 (iv, v, vi) (NWT, N = 3; NKO, N = 3. (B) Staining for NKO culture. (i) K3, (ii) DAPI, (iii) combined K3 and DAPI. (iv) K19, (v) DAPI, (vi) combined K19 with DAPI. NKO mice cell cultures were positively stained with K3 (i, ii, iii) and negatively stained with K19 (iv, v, vi) (NWT, N = 3; NKO, N = 3). Our data confirmed that the isolated cultures from NWT and NKO mice were (~100% K3-positive) pure corneal epithelial cells.
Figure 2 Corneal Epithelial Cells Cultures Isolated from (A) NWT and (B) NKO mice were Positively Stained with Corneal Epithelial Specific Marker, Cytokeratin K3 on Immunofluorescence Staining. (A) Staining for NWT culture. (i) cytokeratin K3 (K3), (ii) DAPI, (iii) combination of K3 with DAPI. (iv) cytokeratin K19 (K19), (v) DAPI, (vi) combination of K19 with DAPI. NWT cell cultures were positively stained with K3 (i, ii, iii), and negatively stained with K19 (iv, v, vi) (NWT, N = 3; NKO, N = 3. (B) Staining for NKO culture. (i) K3, (ii) DAPI, (iii) combined K3 and DAPI. (iv) K19, (v) DAPI, (vi) combined K19 with DAPI. NKO mice cell cultures were positively stained with K3 (i, ii, iii) and negatively stained with K19 (iv, v, vi) (NWT, N = 3; NKO, N = 3). Our data confirmed that the isolated cultures from NWT and NKO mice were (~100% K3-positive) pure corneal epithelial cells.CC BY · PubMed Central
Figure 3 Inhibition of NUCKS Facilities Healing in the Classic Scratched Wound in Vitro. Scratched cultures of cells from NWT and NKO mice were monitored for 24 hours and the process was recorded at specific time points (0 hr, 8 hrs, 12 hrs and 24 hrs) under microscopy. (A) Representative images were captured and are presented for: (i, iii, v, vii) NWT group and (ii, iv, vi, viii) NKO group at particular time points: 0, 8, 12, and 24 hours. NKO group showed faster wound healing than the NWT group and recovery at 24 hours (NWT, N = 3; NKO, N = 3). Scale bars: 200 μm. (B) Graphic representation of the data. NWT group showed 58.82 ± 2.20% retention, while NKO group showed 0.03 ± 0.50% retention after 24 hours. (*P < 0.01; NWT, N = 3; NKO, N = 3).
Figure 3 Inhibition of NUCKS Facilities Healing in the Classic Scratched Wound in Vitro. Scratched cultures of cells from NWT and NKO mice were monitored for 24 hours and the process was recorded at specific time points (0 hr, 8 hrs, 12 hrs and 24 hrs) under microscopy. (A) Representative images were captured and are presented for: (i, iii, v, vii) NWT group and (ii, iv, vi, viii) NKO group at particular time points: 0, 8, 12, and 24 hours. NKO group showed faster wound healing than the NWT group and recovery at 24 hours (NWT, N = 3; NKO, N = 3). Scale bars: 200 μm. (B) Graphic representation of the data. NWT group showed 58.82 ± 2.20% retention, while NKO group showed 0.03 ± 0.50% retention after 24 hours. (*P < 0.01; NWT, N = 3; NKO, N = 3).CC BY · PubMed Central
Figure 4 Inhibition of NUCKS Resulted in Reduced Expression of the Key Inflammatory Cytokines, (A) IL1A and (B) IL1B. Corneas were harvested from NWT and NKO mice at particular time points. The level of expression of IL1A (3.33-fold reduction; **P < 0.001) and IL1B (2.34- fold reduction; **P < 0.001) was reduced in the NKO group on day 7. Level of IL1A and IL1B returned to baseline on by day 14 (NWT N = 12; NKO N = 12).
Figure 4 Inhibition of NUCKS Resulted in Reduced Expression of the Key Inflammatory Cytokines, (A) IL1A and (B) IL1B. Corneas were harvested from NWT and NKO mice at particular time points. The level of expression of IL1A (3.33-fold reduction; **P < 0.001) and IL1B (2.34- fold reduction; **P < 0.001) was reduced in the NKO group on day 7. Level of IL1A and IL1B returned to baseline on by day 14 (NWT N = 12; NKO N = 12).CC BY · PubMed Central
Figure 5 Inhibition of NUCKS Resulted in a Reduced Angiogenic Response in Vivo. (A) Representative (dorsal and lateral) corneal neovascularization photographs at specific time points (post injury day 0, 7, and 14) of (i, ii, v, vi, ix and x) NWT and (iii, iv, vii, viii, xi and xii) NKO groups are shown. NKO group showed less severe angiogenic response compared with NWT group on day 7 and day 14 (neovascularization marked by arrows; Scale bars: 2 mm). (B) Graphic representation of the mean total area of neovascularization. ImageJ was used for the analysis. Compared with NWT, NKO mice exhibited a notable reduction in angiogenic response on day 7 (NWT and NKO; 1.017 ± 0.124 mm2 and 0.466 ± 0.125 mm2; *P < 0.01) and day 14 (NWT and NKO; 0.868 ± 0.066 mm2 and 0.341 ± 0.043 mm2; *P < 0.01) (NWT, N = 4; NKO, N = 4).
Figure 5 Inhibition of NUCKS Resulted in a Reduced Angiogenic Response in Vivo. (A) Representative (dorsal and lateral) corneal neovascularization photographs at specific time points (post injury day 0, 7, and 14) of (i, ii, v, vi, ix and x) NWT and (iii, iv, vii, viii, xi and xii) NKO groups are shown. NKO group showed less severe angiogenic response compared with NWT group on day 7 and day 14 (neovascularization marked by arrows; Scale bars: 2 mm). (B) Graphic representation of the mean total area of neovascularization. ImageJ was used for the analysis. Compared with NWT, NKO mice exhibited a notable reduction in angiogenic response on day 7 (NWT and NKO; 1.017 ± 0.124 mm2 and 0.466 ± 0.125 mm2; *P < 0.01) and day 14 (NWT and NKO; 0.868 ± 0.066 mm2 and 0.341 ± 0.043 mm2; *P < 0.01) (NWT, N = 4; NKO, N = 4).CC BY · PubMed Central
Figure 6 Inhibition of NUCKS Resulted in Reduced Expression of Antiangiogenic and Angiogenic Cytokines, (A) PEDF and (B) VEGF. Following injury corneas were harvested from NWT and NKO mice at particular time points. Compared with NWT mice, the relative expression level of PEDF (~2.63-fold, **P < 0.001) was upregulated in NKO mice on day 7 and returned to normal on day 14. Furthermore, the relative expression level of VEGF was downregulated (~1.86-fold, **P < 0.001) in NKO mice on day 14 (NWT, N = 12; NKO, N = 12).
Figure 6 Inhibition of NUCKS Resulted in Reduced Expression of Antiangiogenic and Angiogenic Cytokines, (A) PEDF and (B) VEGF. Following injury corneas were harvested from NWT and NKO mice at particular time points. Compared with NWT mice, the relative expression level of PEDF (~2.63-fold, **P < 0.001) was upregulated in NKO mice on day 7 and returned to normal on day 14. Furthermore, the relative expression level of VEGF was downregulated (~1.86-fold, **P < 0.001) in NKO mice on day 14 (NWT, N = 12; NKO, N = 12).CC BY · PubMed Central
Figure 7 NUCKS regulates NFκB Activation in in Vitro Studies. Cells were transfected with luciferase reporter and the corresponding luciferase activity represented intracellular NFκB/ISRE activity. Next, the NUCKS expressing construct (NUCKS) or empty vector (Control) was transfected to the cells, followed by (A) TNFα treatment or no treatment (UT); NFκB activation was measured (B) lipopolysaccharide (LPS) or no treatment (UT); (i) NFκB activation was measured and (ii) interferon stimulated response element (ISRE) activation was measured. (C) Poly-inosinic-cytidylic (polyIC) treatment or no treatment (UT); (i) NFκB activation was measured and (ii) interferon stimulated response element (ISRE) activation was measured. (D) On NUCKS silencing and LPS-induced-NFκB activation, the level of expression of (i) TNFα, (ii) IL8 and (iii) MCP1 was tested. (E) On NUCKS silencing and LPS-induced-NFκB activation, protein expression level of the phosphorylated form of IKK, P65 and IκBα was analyzed.
Figure 7 NUCKS regulates NFκB Activation in in Vitro Studies. Cells were transfected with luciferase reporter and the corresponding luciferase activity represented intracellular NFκB/ISRE activity. Next, the NUCKS expressing construct (NUCKS) or empty vector (Control) was transfected to the cells, followed by (A) TNFα treatment or no treatment (UT); NFκB activation was measured (B) lipopolysaccharide (LPS) or no treatment (UT); (i) NFκB activation was measured and (ii) interferon stimulated response element (ISRE) activation was measured. (C) Poly-inosinic-cytidylic (polyIC) treatment or no treatment (UT); (i) NFκB activation was measured and (ii) interferon stimulated response element (ISRE) activation was measured. (D) On NUCKS silencing and LPS-induced-NFκB activation, the level of expression of (i) TNFα, (ii) IL8 and (iii) MCP1 was tested. (E) On NUCKS silencing and LPS-induced-NFκB activation, protein expression level of the phosphorylated form of IKK, P65 and IκBα was analyzed.CC BY · PubMed Central
Figure 8 NKO Corneal Epithelial Cells upon LPS-induced-NFκB-Activation Showed Reduced Expression of Phosphorylated IκB (P IκB), IL6, IP10 and TNFα Compared to NWT Group. (A) (i) Corneal epithelial cells isolated from NWT and NKO mice were treated with LPS in vitro; expression of PIκB was measured (ii) Graphic representation for PIκB data in (i) is shown. (B) (i) Corneal epithelial cells were treated with LPS or LPS + NFκB inhibitor, SC 154. Expression of PIκB and TNFα was tested. (ii and iii) Graphic representation of the relative expression of (ii) PIκB and (iii) TNFα, respectively in (i) is shown. (C) On treatment with LPS, expression of various inflammatory cytokines (IL6, IL10, INFγ, IP10, TNFα and IL12) was compared and analyzed by flow cytometry analysis. Results for (i) NWT group and (ii) NKO group are shown.
Figure 8 NKO Corneal Epithelial Cells upon LPS-induced-NFκB-Activation Showed Reduced Expression of Phosphorylated IκB (P IκB), IL6, IP10 and TNFα Compared to NWT Group. (A) (i) Corneal epithelial cells isolated from NWT and NKO mice were treated with LPS in vitro; expression of PIκB was measured (ii) Graphic representation for PIκB data in (i) is shown. (B) (i) Corneal epithelial cells were treated with LPS or LPS + NFκB inhibitor, SC 154. Expression of PIκB and TNFα was tested. (ii and iii) Graphic representation of the relative expression of (ii) PIκB and (iii) TNFα, respectively in (i) is shown. (C) On treatment with LPS, expression of various inflammatory cytokines (IL6, IL10, INFγ, IP10, TNFα and IL12) was compared and analyzed by flow cytometry analysis. Results for (i) NWT group and (ii) NKO group are shown.CC BY · PubMed Central

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Metadata

Authors
Poon, M.-W., Jiang, D., Qin, P., Zhang, Y., Qiu, B., Chanda, S., Tergaonkar, V., Li, Q., Wong, I. Y., Yu, Z., Tse, H.-F., Wong, D. S. H., & Lian, Q.
Venue
Scientific Reports
Publisher
Springer Science and Business Media LLC
Published
2017-01-20
Volume
7
Issue
1
ISSN
2045-2322
DOI
10.1038/srep41224
PMC
PMC5247723
Cited by
25
References
41
Language
en
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