September 2017
Volume 58, Issue 11
Open Access
Biochemistry and Molecular Biology  |   September 2017
Nuclear Respiratory Factor-1 (NRF-1) Regulates Transcription of the CXC Receptor 4 (CXCR4) in the Rat Retina
Author Affiliations & Notes
  • Pei Chen
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Xiaoxiao Cai
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Ying Yang
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Zhao Chen
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Jin Qiu
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Na Yu
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Mingjun Tang
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Qiyun Wang
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Jian Ge
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Keming Yu
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Jing Zhuang
    State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
  • Correspondence: Jing Zhuang, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 S. Xianlie Road, Guangzhou, China 510060; zhuangj@mail.sysu.edu.cn
  • Keming Yu, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 S. Xianlie Road, Guangzhou, China 510060; yukeming@mail.sysu.edu.cn
Investigative Ophthalmology & Visual Science September 2017, Vol.58, 4662-4669. doi:10.1167/iovs.17-22115
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      Pei Chen, Xiaoxiao Cai, Ying Yang, Zhao Chen, Jin Qiu, Na Yu, Mingjun Tang, Qiyun Wang, Jian Ge, Keming Yu, Jing Zhuang; Nuclear Respiratory Factor-1 (NRF-1) Regulates Transcription of the CXC Receptor 4 (CXCR4) in the Rat Retina. Invest. Ophthalmol. Vis. Sci. 2017;58(11):4662-4669. doi: 10.1167/iovs.17-22115.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The CXC receptor 4 (CXCR4) is required for various physiologic and pathologic processes in the eye, including stem cell trafficking, neuronal development, immune responses, and ocular neovascularization. Here, we used the rat retina models to determine the mechanisms driving CXCR4 transcription.

Methods: The expression pattern of CXCR4 and nuclear respiratory factor-1 (NRF-1) were profiled in the rat retina during the course of development. Chromatin immunoprecipitation (CHiP) assay determined the transcriptional mechanism of CXCR4 in rat retina. A rat model of oxygen-induced retinopathy (OIR) that mimics retinal ischemia-reperfusion injury was established. Under either normoxic or hypoxic conditions, CXCR4 and NRF-1 expression in rat retinas was tracked by RT-PCR and Western analysis. Immunofluorescence staining localized CXCR4 and NRF-1.

Results: Both CXCR4 and NRF-1 were highly expressed in the neonatal rat retina, down-regulated in parallel, and silenced in fully developed retinas (1 month of age). ChIP assays revealed that NRF-1 was required for CXCR4 promoter activity in rat retinas. In the OIR rat model, retinal hypoxia induced up-regulation of CXCR4 and NRF-1 concurrently.

Conclusions: Our findings suggest that NRF-1 regulates the expression of CXCR4 in normal retinal development and in pathologic processes of retinal hypoxia and neovascularization.

Retinal ischemia is a pathophysiologic condition characteristic of several vision-threatening diseases, such as diabetic retinopathy and retinopathy caused by premature birth,1,2 where ischemia disrupts the balance between proangiogenic and antiangiogenic factors and produces inflammation and oxidative stress, thereby triggering pathologic retinal angiogenesis.1 To halt overgrowth of pathologic retinal vasculature, anti-VEGF therapy is currently considered the most promising treatment,3 but it remains undetermined whether anti-VEGF therapy will show long-term efficacy in persistent avascular retinal and recurrent intravitreal neovascularization. To develop optimal therapies, it will likely be useful to identify other genes that govern and initiate pathologic retinal angiogenesis. 
One candidate for preventing abnormal retinal vascularization is CXCR4. Interactions between CXCR4 (a seven-transmembrane-spanning-domain, G-protein-coupled receptor) and the chemokine, stromal-derived factor 1 (SDF-1) drive various fundamental physiologic processes, including immune responses, cell migration, and neuron development.4 In the central nervous system, CXCR4 is not only essential for axon guidance and to maintain neural precursors during development,5 it also regulates neurotransmission and neurologic interaction in mature neurocytes.6 Mice deficient in CXCR4 exhibit neural abnormalities and brain dysfunction, as well as vascular defects.7,8 In the retina (which is part of the central nervous system), CXCR4 is required for survival of embryonic ganglion cells9 and functions as a morphogen in modulating retinal vasculogenesis10; throughout retinal development, CXCR4 regulates stem cell trafficking.11 Recent observations also determined that, in the eyes of human embryos, CXCR4 is more highly expressed in the avascular inner retina than in the vascularized retina, and its expression declines with canalization of new vessels.12 Together, these data suggest CXCR4 involves in a variety of developmental processes, including regulating retinal vascular development. 
Accumulating evidence also implicates the CXCR4/SDF-1 axis in the pathogenic infiltration of inflammatory cells and tumor genesis and metastasis. The CXCR4 antagonist, AMD3100, has long been prescribed as clinical treatment for tumors.13 Likewise, several studies in eye tissues correlated abnormal activation of CXCR4 with ophthalmic diseases, such as primary open-angle glaucoma, pathologic angiogenesis, and ocular inflammation.14,15 Specifically, in neovascular diseases that cause retinal ischemia, both inflammation and angiogenesis initiate and then drive the pathologic process. Previous studies have implicated that CXCR4 signaling is stimulated by VEGF and augments its angiogenic effects of VEGF in the retina via the YY1 and Akt signaling pathways.16,17 
The CXCR4/SDF-1 axis recruits endothelial progenitor cells to ischemic sites and induces the phosphorylation and disruption of vascular endothelial-cadherin, thereby regulating angiogenesis and increasing vascular permeability.18,19 Previous studies, in alkali-induced corneal injury or laser-induced animal models of choroidal neovascularization, CXCR4 antagonists had a therapeutic effect on ocular neovascularization.20,21 Accordingly, we demonstrated that pharmacologic blockade of CXCR4 modulated extracellular matrix (ECM) accumulation, suppressing cytoskeleton remodeling, inhibiting angiogenesis, and ultimately protecting retinal neurocytes.14,15,21 Thus, identifying the transcriptional mechanism of CXCR4 might offer a new strategy for antiangiogenesis therapy. 
Under ischemic conditions, oxidative stress and reactive oxygen species (ROS) likely contribute to loss of retinal neurons.22 NRF-1, a mitochondrial-specific transcription factor, is implicated in the pathogenesis of ischemic reperfusion injury23; in the rat cerebrum, under ischemic conditions in vivo, NRF-1 expression is drastically elevated.24 Nevertheless, whether NRF-1 is involved in the pathogenesis of retinal ischemia remains unclear. 
Here, we profiled the transcriptional regulation and developmental expression pattern of CXCR4. In an oxygen-induced retinopathy (OIR) rat model, which mimics retinal ischemia-reperfusion injury,25 our data showed CXCR4 and NRF-1 were up-regulated in response to ischemia reperfusion damage in the retina. Thus, CXCR4 and NRF-1 might be new candidate genes for targeted therapy in ocular neovascularization diseases. 
Materials and Methods
Ethics Statement
Sprague-Dawley (SD) rats were treated in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Zhongshan Ophthalmic Center (Permit SYXK [YUE] 2010-0058). Rats were housed in an air-conditioned room with proper ambient temperature and relative humidity in the Ophthalmic Animal Laboratory of Zhongshan Ophthalmic Center. Rats were killed by an intraperitoneal injection of chloral hydrate (P3761, 60 mg/kg; Sigma-Aldrich Corp., St. Louis, MO, USA) before eyes were harvested and retinas dissected for experimentation. 
RT-PCR Assays
Total RNA from rat retinas was isolated using Trizol reagent (Sigma-Aldrich Corp.). RT-PCR was performed using a PrimeScript RT-PCR Kit (TaKaRa, Tokyo, Japan) in accordance with the manufacturer's instructions. The following primer pairs were used: CXCR4, 5′-CCTCTGAGGCGTTTGGTGCTC-3′ (sense) and 5′-TAGATGGTGGGCAGGAAGATC-3′ (antisense); for NRF-1, 5′-TAGCCCATCTCGTACCATCAC-3′ (sense) and 5′-TTTGTTCCACCTCTCCATCAG-3′ (antisense); for β-actin, 5′-TCACCCACACTGTGCCCAT-3′ and 5′-TCTTTAATGTCACGCACGATT-3′. PCR products were electrophoresed on agarose gels and visualized using ethidium bromide (EB) staining. β-actin mRNA was tracked, as an internal control. 
Western Blotting
Retina tissues were lysed with radioimmunoprecipitation (RIPA) buffer supplemented with a protease inhibitor cocktail. Total protein was extracted and loaded onto a SDS-PAGE gel for separation and then transferred to a nitrocellulose (PVDF) membrane. Blots were incubated with polyclonal rabbit antibodies recognizing CXCR4 (1:1000; Abcam, Cambridge, MA, USA), NRF-1 (1:1000; Abcam) or GAPDH (1:10,000; Proteintech, Rosemont, IL, USA), overnight at 4°C. Immunoreactive species were visualized with horseradish peroxidase–conjugated, secondary anti-rabbit (CST, Danvers, MA, USA) and an enhanced chemiluminescence (ECL) chemiluminescence system. 
Immunofluorescence Assays
After phosphate-buffered formalin perfusion, rat eyes were collected and fixed in 4% paraformaldehyde (PFA) for 24 hours and then embedded in optimal cutting temperature compound (OCT) compound. Tissues were sectioned (5 μm) and permeabilized by 0.5% Triton X-100 (Sigma-Aldrich Corp.) for 10 minutes and immersed for 30 minutes in 10% normal goat serum (Boster, Wuhan, China). Afterward, the slides were incubated with primary antibodies against CXCR4 (1:300; Abcam), NRF-1 (1:300; Abcam), or glial fibrillary acidic protein (GFAP) (1:100; Boster) overnight at 4°C. The next day, secondary antibodies (1:500; CST) were added, at room temperature. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were captured by a confocal microscope (LSM 510; Carl Zeiss Meditec, Oberkochen, Germany). 
ChIP Assays
ChIP was performed using a ChIP assay kit in accordance with the manufacturer's instructions.26 Briefly, retinas from P1 or 1 month (1M) rats were cross-linked by adding formaldehyde to a final concentration of 1%, for 10 minutes at room temperature. The reaction was terminated with glycine (125 mM final concentration), and retinal cells were harvested and incubated with SDS lysis buffer, supplemented with protease inhibitors, for 10 minutes on ice. Afterward, the lysate was sonicated to shear DNA and then centrifuged at 13,000 rpm for 10 minutes at 4°C. Supernatants were diluted with buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, and protease inhibitors) and precleared with protein G-agarose; 10% of the supernatant was reserved as an “input” control. Supernatants were incubated with 2 μg rabbit NRF-1 antibody (Abcam) or rabbit normal IgG (Sigma-Aldrich Corp.) overnight at 4°C with rotation and then incubated with protein G-agarose for 3 hours. Immune complexes were washed and eluted with elution buffer on a rotating platform (1% SDS, 0.1 M NaHCO3, and 200 mM NaCl). Cross-linking was reversed by warming at 65°C overnight. RNA was degraded with RNase A for 30 minutes, and protein was degraded with proteinase K for 2 hours. DNA was purified by phenol/chloroform extraction and ethanol precipitation and amplified by PCR using primers spanning the activated transcription factor (ATF) site of the CXCR4 promoter (forward, 5′-GCTCTCCGACTTCTGTTTGTG-3′; reverse, 5′-AGAGGGTCACTGCTACCTGCT-3′). 
OIR Animal Model
Ischemic retinopathy was induced in neonatal SD rats, as described previously.27 Seven-day-old (P7) rats, weighting 20 to 25 g, were randomly divided into a control group and an OIR group and housed with nursing mothers. The OIR rats were moved into an airtight incubator, in which the oxygen fraction was 75%, for 5 days. At postnatal day 12 (P12), OIR rats were removed from the high-oxygen incubators and euthanized at P15 or P18. The control group was not exposed to the high-oxygen environment, and their retinas were collected at the same time point as the OIR rats. When OIR rats are removed from the high-oxygen environment, their retinas show revascularization of the avascular central retinal area, a symptom associated with pathologic neovascularization at the border of the avascular and vascular zones. 
Quantification of Retinal Neovascularization in Flat-Mounted Retinas
Images of intravitreal injected, fluorescein-labeled dextran (n = 5 for each group) were used to quantify retinal neovascularization. At P18, rats were killed and perfused with a solution of 50 mg/mL fluorescein-labeled dextran in sodium chloride. Eyes were then enucleated and fixed in 4% formaldehyde for 4 hours at room temperature. The anterior segment was then cut, and the retina was carefully removed, cut radially, and flat-mounted in glycerin. Retinal whole mounts were examined by fluorescence microscopy (BH2-RFC; Olympus, Hamburg, Germany). Total images of flat-mounted retina were produced from 9 to 12 pieces of images acquired using a fluorescence microscope (BX50; Olympus, Tokyo, Japan). 
Statistical Analysis
All in vivo experiments were performed at least in triplicate. Data depicted in the figures are presented as mean ± SD. Statistical comparisons between two groups were evaluated by using the Student's t-test, and statistical comparisons between different time points were evaluated using 1-way ANOVA with the least significant difference (LSD) multiple comparison test. P < 0.05 was considered statistically significant. 
Results
Developmental Expression Profile of CXCR4 in the Rat Retina
CXCR4 expression and localization profiles in the rat retina changed after birth (Figs. 1A, 1B): in the neonatal rat retina, expression of CXCR4 mRNA was strong but developmentally down-regulated over time (for mRNA, P1: 2.74 ± 0.43, P3: 1.8 ± 0.57, P5: 1.45 ± 0.39, 1M: 0.59 ± 0.26, n = 3; 1-way ANOVA with LSD multiple comparison test, P < 0.05). Western analysis of CXCR4 reflected the age-related decline of the mRNA (P1: 0.78 ± 0.21, P3: 0.69 ± 0.17, P5: 0.54 ± 0.12, 1M: 0.11 ± 0.07, n = 3; 1-way ANOVA with LSD multiple comparison test, P < 0.05; Figs. 1C, 1D). Immunofluorescence at postnatal day 1 (P1; Fig. 1E) detected CXCR4 in the membrane of retinal cells and distributed throughout entire retinal layer. As development progressed, CXCR4 expression was markedly down-regulated, and by P5, staining was faint and restricted to the ganglion cell layer (GCL) and inner nuclear layer (INL). By 1M after birth, the immunofluorescence signal was silent. Our immunofluorescence assay was done in the eye tissue harvested after phosphate-buffered formalin perfusion, thus eliminating interference by blood cells in retinal vessels. An analysis of total protein by Western blotting would not have excluded this kind of contamination. Indeed, in 1-month-old rats, discrepancies in the Western results were likely caused by blood cells contaminating the retinal tissue. 
Figure 1
 
Developmental expression profile of CXCR4 in the rat retina. (A) CXCR4 mRNA level was developmentally down-regulated in the rat retina. (B) Relative mRNA expression of CXCR4 in rat retina was quantified by densitometry, and the data are presented graphically. (C) Age-related expression pattern of CXCR4 was observed in rat retina, as evidenced by Western blot. (D) Relative expression of CXCR4 in rat retina was quantified by densitometry, and the data are presented graphically. (E) Immunofluorescence staining revealed that CXCR4 was expressed in the cell membrane of retinal cells and constitutively distributed in the whole layer of the neonatal rat retina, but gradually down-regulated with age and weakly expressed in the fully developed rat retina. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
Figure 1
 
Developmental expression profile of CXCR4 in the rat retina. (A) CXCR4 mRNA level was developmentally down-regulated in the rat retina. (B) Relative mRNA expression of CXCR4 in rat retina was quantified by densitometry, and the data are presented graphically. (C) Age-related expression pattern of CXCR4 was observed in rat retina, as evidenced by Western blot. (D) Relative expression of CXCR4 in rat retina was quantified by densitometry, and the data are presented graphically. (E) Immunofluorescence staining revealed that CXCR4 was expressed in the cell membrane of retinal cells and constitutively distributed in the whole layer of the neonatal rat retina, but gradually down-regulated with age and weakly expressed in the fully developed rat retina. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
NRF-1 Expression Correlates With That of CXCR4 During Retinal Development
The mechanisms regulating CXCR4 transcription differ among tissues.28 A previous study in human rhabdomyosarcomas cells found CXCR4 promoter activity was regulated by NRF-1,29 and in silico scanning detected the expected transcription factor binding motifs. To investigate whether NRF-1 regulated CXCR4 expression in retina, we assayed the expression profile and subcellular localization of NRF-1 in developing rat retina. As shown in Figures 2A and 2B, NRF-1 mRNA expression was developmentally down-regulated in the rat retina, over time (relative mRNA level at P1: 2.45 ± 0.51, P3: 1.66 ± 0.37, P5: 0.98 ± 0.32, 1M: 0.69 ± 0.18, n = 3; 1-way ANOVA with LSD multiple comparison test, P < 0.05), which is consistent with down-regulation of CXCR4. Western blot assays showed protein and mRNA levels decreased in parallel (relative protein level at P1: 1.73 ± 0.46, P3: 1.45 ± 0.3, P5: 0.96 ± 0.13, 1M: 0.25 ± 0.09; n = 3; 1-way ANOVA with LSD multiple comparison test, P < 0.05; Figs. 2C, 2D). Moreover, whereas at P1 the entire retinal layer stained intensely with NRF-1 (in the cell nuclei), as the animal developed, retinal staining progressively diminished, as evidenced by immunofluorescence (Fig. 2E). This pattern of NRF-1 expression parallels the expression of CXCR4 in rat retinal development, suggesting that CXCR4 and NRF-1 might together drive physiologic regulation of rat retinal development. 
Figure 2
 
Developmental expression profile of NRF-1 in the rat retina. (A) mRNA expression of NRF-1 was developmentally down-regulated in the rat retina with age, as evidenced by RT-PCR. (B) Relative expression level of NRF-1 mRNA presented as a histogram. (C) Age-related expression pattern of NRF-1 was observed in rat retina, as evidenced by Western blot. (D) Relative expression of NRF-1 in rat retina was quantified by densitometry, and the data presented graphically. (E) Intense staining of NRF-1 in the cell nucleus labeled the entire layer of a P1 rat retina was gradually down-regulated with age and weakly expressed in the fully developed rat retina. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
Figure 2
 
Developmental expression profile of NRF-1 in the rat retina. (A) mRNA expression of NRF-1 was developmentally down-regulated in the rat retina with age, as evidenced by RT-PCR. (B) Relative expression level of NRF-1 mRNA presented as a histogram. (C) Age-related expression pattern of NRF-1 was observed in rat retina, as evidenced by Western blot. (D) Relative expression of NRF-1 in rat retina was quantified by densitometry, and the data presented graphically. (E) Intense staining of NRF-1 in the cell nucleus labeled the entire layer of a P1 rat retina was gradually down-regulated with age and weakly expressed in the fully developed rat retina. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
NRF-1 Binds the ATF Site of the CXCR4 Promoter
Double immunofluorescence staining of P1 retinal ganglion cells that labeled with microtubule-associated protein-2 (MAP-2, green) detected a strong CXCR4 signal (red) at the cell membrane and a strong NRF-1 signal (red) in the nucleus (Fig. 3A). Moreover, at P1, the cellular distribution of CXCR4 and NRF-1 was uniform within each retinal layer, with the signal in the GCL and INL being more prominent than that detected at the outer nuclear layer (ONL). To test whether NRF-1 directly controls transcription of CXCR4, we analyzed CXCR4 promoters by ChIP assays. As shown in Figure 3B, ChIP of rat retinal neurocytes suggested NRF-1 was indeed bound to the CXCR4 promoter at P1, but by 1M, binding was only weakly detected. These data were consistent with previous reports29 and implicated NRF-1 in the transcriptional activation of CXCR4 in developing retinal neurocytes. 
Figure 3
 
NRF-1 binds to ATF site of the CXCR4 promoter. (A) Distribution of CXCR4 (cell membrane, red) and NRF-1 (cell nuclear, red) in the P1 rat retina is identical: uniformly distributed in all retinal layers and more prominent in the GCL and INL than the ONL. The retinal ganglion cells were labeled with MAP-2 (green), and the cell nuclei were stained by DAPI (blue). (B) ChIP assays demonstrate that the DNA fragments were amplified from the precipitates of P1 rat retinal neurocytes, whereas in 1M rat retina, DNA was only weakly detected, indicating that NRF-1 bound the promoter region of CXCR4.
Figure 3
 
NRF-1 binds to ATF site of the CXCR4 promoter. (A) Distribution of CXCR4 (cell membrane, red) and NRF-1 (cell nuclear, red) in the P1 rat retina is identical: uniformly distributed in all retinal layers and more prominent in the GCL and INL than the ONL. The retinal ganglion cells were labeled with MAP-2 (green), and the cell nuclei were stained by DAPI (blue). (B) ChIP assays demonstrate that the DNA fragments were amplified from the precipitates of P1 rat retinal neurocytes, whereas in 1M rat retina, DNA was only weakly detected, indicating that NRF-1 bound the promoter region of CXCR4.
CXCR4 and NRF-1 Are Up-Regulated in the Rat Retina During OIR
Previous reports showed CXCR4 was up-regulated in the ischemic rat retina30; therefore, we used a rat model of oxygen-induction ischemia to test whether CXCR4 up-regulates NRF-1 during ocular neovascularization. Here, P12 rats exposed to oxygen showed retinal vessel loss within 8 hours after being returned to normal air. Afterward, retinal revascularization was accompanied by pathologic neo-vascularization. Fluorescein angiography with FITC-dextran revealed typical pathologic changes of ischemic retinopathy in the P18 OIR rat, including obvious blood vessel tortuosity, neovascularization, and a distinct avascular area between the dilated retinal vessels (Fig. 4A). 
Figure 4
 
Both CXCR4 and NRF-1 were abnormally activated in developed retina of OlR rats. (A) Typical pathologic changes of ischemic retinopathy were observed in the retina of the P18 OIR rat, as evidenced by fluorescein angiography with FITC-dextran. (B) Both CXCR4 and NRF-1 were up-regulated in the retina of OIR rats, as evidenced by RT-PCR and Western blot. (C) Fold change of both CXCR4 and NRF-1 expression, presented as a histogram. (D) In avascular ischemic retina of OIR rats, CXCR4 and NRF-1 were up-regulated in parallel and with prolonged hypoxia. (E) Fold change of both CXCR4 and NRF-1 expression, presented as a histogram. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
Figure 4
 
Both CXCR4 and NRF-1 were abnormally activated in developed retina of OlR rats. (A) Typical pathologic changes of ischemic retinopathy were observed in the retina of the P18 OIR rat, as evidenced by fluorescein angiography with FITC-dextran. (B) Both CXCR4 and NRF-1 were up-regulated in the retina of OIR rats, as evidenced by RT-PCR and Western blot. (C) Fold change of both CXCR4 and NRF-1 expression, presented as a histogram. (D) In avascular ischemic retina of OIR rats, CXCR4 and NRF-1 were up-regulated in parallel and with prolonged hypoxia. (E) Fold change of both CXCR4 and NRF-1 expression, presented as a histogram. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
In the retina of control rats, basal mRNA and protein expression of CXCR4 and NRF-1 was low; however, with OIR, the avascular ischemic retina expressed much higher levels of CXCR4 mRNA and protein (Figs. 4B, 4C, the relative change of mRNA was 3.49-fold; for protein it was 4.12-fold; Test/Control (T/C), n = 3; 1-way ANOVA with Dunnett's test, P < 0.05). The same was found for NRF-1 expression (Figs. 4B, 4C, for mRNA, the change was 1.92-fold; for protein, 2.91-fold; T/C, n = 3; 1-way ANOVA with Dunnett's test, P < 0.05). Moreover, in the avascular ischemic retina of OIR rats, CXCR4 and NRF-1 expression was up-regulated in parallel and increased with prolonged hypoxia (Figs. 4D, 4E, for CXCR4: Con:1, P15:1.67-fold, P17:3.18-fold; for NRF-1: Con:1, P15:1.51-fold, P17:3.98-fold; T/C, n = 3; 1-way ANOVA with LSD multiple comparison test, P < 0.05). This evidence implicates both CXCR4 and NRF-1 in the pathologic processes of ischemic damage. 
Using immunohistochemistry, we tracked intermittent GFAP staining above the ganglion cell layer and along the border of the inner retina, which is a pattern characteristic of astrocytes. Consistent with previous reports demonstrating pathological activation of glial cell signaling in response to retinal injury,30 we detected increased GFAP immunofluorescence staining in the inner retina of OIR rats undergoing ischemic reperfusion injury (Fig. 5A). As mentioned above, both CXCR4 and NRF-1 are only weakly expressed in the nonischemic P18 control rat retina. In OIR rats, however, CXCR4 was strongly expressed throughout the inner retinal layer (GCL and INL; Fig. 5B). In addition, NRF-1 stained intensely in the nuclear of retinal cell in OIR rats, consistent with the localization pattern of CXCR4 (Fig. 5C). Importantly, the most prominent increase in both CXCR4 and NRF-1 immunolabeling in retina damaged by hypoxia was in the ganglion cell layer and inner nuclear cell layer. This evidence further implies that NRF-1 might transcriptionally regulate the CXCR4 promoter during ischemic damage. 
Figure 5
 
Localization of CXCR4 and NRF-1 in the OIR rat retina. (A) The OIR rat retina showed stronger staining of GFAP (red) than that of control rat retina and demonstrated pathologic activation of glial cell signaling. (B) Both CXCR4 and NRF-1 are weakly expressed in the nonischemic, P18 control rat retina. However, increased immunolabeling of CXCR4 (red) was detected throughout the inner layer of retina in OIR rats. (C) Intensive NRF-1 staining (green) was observed in the nuclear of retinal cell of OIR rats and was consistent with the localization pattern of CXCR4 (compared with that of control rats). The nuclei were stained with DAPI (blue).
Figure 5
 
Localization of CXCR4 and NRF-1 in the OIR rat retina. (A) The OIR rat retina showed stronger staining of GFAP (red) than that of control rat retina and demonstrated pathologic activation of glial cell signaling. (B) Both CXCR4 and NRF-1 are weakly expressed in the nonischemic, P18 control rat retina. However, increased immunolabeling of CXCR4 (red) was detected throughout the inner layer of retina in OIR rats. (C) Intensive NRF-1 staining (green) was observed in the nuclear of retinal cell of OIR rats and was consistent with the localization pattern of CXCR4 (compared with that of control rats). The nuclei were stained with DAPI (blue).
Discussion
A persistent state of retinal hypoxia will trigger recurrent ocular neovascularization, despite therapeutic attempts to control growth of new vessels with anti-VEGF therapy or laser and cryosurgery. Because the chemokine factor, CXCR4, is known to contribute to pathologic ocular angiogenesis30 and is up-regulated by inflammatory cytokines and angiogenic factors,17,31 this receptor and its up- and downstream signaling molecules might offer new therapeutic targets for controlling neovascularization.13,14,20,31,32 Here, our in silico scanning analysis identified NRF-1 as a transcriptional regulator of CXCR4. ChIP assays confirmed NRF-1 binding to the ATF site of the CXCR4 promoter, correlating NRF-1 with the transcription mechanisms of CXCR4. Moreover, our data showed CXCR4 and NRF-1 were expressed in parallel in the rat retina, under physiologic and pathologic conditions: both decreased with age during development, and both were up-regulated in response to ischemic stimulation. These data strongly suggest NRF-1 transcriptionally regulates CXCR4 during retinal development and the pathogenesis of ischemia reperfusion injury. 
CXCR4 is highly conserved across species33 and plays a key role in regulating several fundamental physiologic processes, such as hematopoiesis, leukocyte trafficking, neuron development, and neurogenesis.4 Here we demonstrated that, although CXCR4 was distributed throughout the newborn rat retinal layer, it is developmentally down-regulated with age. Consistently, Hasegawa et al. implicated that CXCR4 expression declines with the development and canalization of retinal vasculature in embryonic human eyes.10,12 Likewise, a decrease and eventual loss of CXCR4 expression by endothelial cells was observed by a previous study, indicating its apparent importance in vascular development.34 Extensive evidence implicated CXCR4/SDF-1 axis as required for not only maintenance of neuron progenitor cells but also cell migration in central neural system.6 Specifically, McLeod et al. reported that CXCR4 is essential for retina development as well as survival of retinal ganglion cells.10 Moreover, our results have shown that NRF-1 expression is also developmentally regulated in rat retina. Accordingly, in zebra fish retina, NRF-1 is highly expressed in the period before neuronal differentiation and decreases sharply shortly thereafter.35 NRF-1 deletion causes early embryonic lethality, severe oxidative stress, and progressive neuronal dysfunction.36,37 Taken together, these observations suggest CXCR4 and NRF-1 play a crucial role in retinal development, both in neuron differentiation and vasculature formation. 
In embryonic human eyes, CXCR4 is primarily distributed in the inner retinal layer.10,12 In the present study in rats, CXCR4 staining was more intense in the inner layer versus the outer layer, but the whole layer of the neonatal rat retina expressed CXCR4. This discrepancy might be due to different developmental progressions among different species. 
By using an OIR rat model, we confirmed that, in the ischemic rat retina, CXCR4 and NRF-1 were abnormally activated in parallel and in a time-dependent manner. Our data further showed immunostaining of both CXCR4 and NRF-1 were most prominent in the GCL and INL of the retina, suggesting the inner retina might be more vulnerable to the ischemic conditions. These observations are consistent with previous studies of CXCR4 in OIR.30 In vivo and in vitro evidence supports that NRF-1 drives some pathogenic outcomes triggered by ischemia.23 Collectively, these studies and our data in vivo support that NRF-1 might transcriptionally regulate CXCR4 during retinal development and also during the pathogenesis of ischemic reperfusion injury. A NRF-1 gene knockout/mutant model is warranted to further confirm the regulative relation between the NRF-1 and CXCR4 and their possible biological function during retinal development. 
Of note, according to our results, NRF-1 was completely silenced in 1-month-old rat retinas (Figs. 2B, 2C), but CXCR4 was still detectable by Western blot (Fig. 1C). This discrepancy might be due to hemocytes in the retina vessel, where CXCR4 is highly expressed.34 
Signaling via CXCR4 and NRF-1 likely plays a role in inflammation and neovascularization in the ischemic retina, which up-regulates GFAP (Fig. 5A), a marker of glial remolding. Similarly, previous studies in rat optic nerves crush injury reported that activated glial cells might trigger up-regulation of CXCR4 and SDF-1.30,38 Lima et al. also identified glial cells as the predominant endogenous retinal cells involved in activation of the CXCR4/SDF-1 axis.30 Moreover, both CXCR4 and NRF-1 are multifunctional genes that are involved in various physiologic and pathologic processes and expressed in different cell types.4,37 The up-regulation of CXCR4 and NRF-1 in the OIR rat retina may possible be expressed in astrocytes, Muller glia, or the neuro retina (ganglion cell). 
In conclusion, the present study indicated that CXCR4 and NRF-1 were independently associated with physiologic retinal development and the pathologic processes of retinal hypoxia and neovascularization. Moreover, our study implicates NRF-1 as a transcriptional activator of CXCR4 in the retina. Going forward with this new insight, further investigation might improve strategies for anti-CXCR4 therapy, particularly in ocular diseases. Clearly, further comprehensive research is warranted to explore the strategy of anti-CXCR4 treatment and determine the benefit of CXCR4 antagonist therapy in ocular diseases. 
Acknowledgments
Supported by the National Natural Science Foundation, China Grants 81370987 and 81470626. 
Disclosure: P. Chen, None; X. Cai, None; Y. Yang, None; Z. Chen, None; J. Qiu, None; N. Yu, None; M. Tang, None; Q. Wang, None; J. Ge, None; K. Yu, None; J. Zhuang, None 
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Figure 1
 
Developmental expression profile of CXCR4 in the rat retina. (A) CXCR4 mRNA level was developmentally down-regulated in the rat retina. (B) Relative mRNA expression of CXCR4 in rat retina was quantified by densitometry, and the data are presented graphically. (C) Age-related expression pattern of CXCR4 was observed in rat retina, as evidenced by Western blot. (D) Relative expression of CXCR4 in rat retina was quantified by densitometry, and the data are presented graphically. (E) Immunofluorescence staining revealed that CXCR4 was expressed in the cell membrane of retinal cells and constitutively distributed in the whole layer of the neonatal rat retina, but gradually down-regulated with age and weakly expressed in the fully developed rat retina. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
Figure 1
 
Developmental expression profile of CXCR4 in the rat retina. (A) CXCR4 mRNA level was developmentally down-regulated in the rat retina. (B) Relative mRNA expression of CXCR4 in rat retina was quantified by densitometry, and the data are presented graphically. (C) Age-related expression pattern of CXCR4 was observed in rat retina, as evidenced by Western blot. (D) Relative expression of CXCR4 in rat retina was quantified by densitometry, and the data are presented graphically. (E) Immunofluorescence staining revealed that CXCR4 was expressed in the cell membrane of retinal cells and constitutively distributed in the whole layer of the neonatal rat retina, but gradually down-regulated with age and weakly expressed in the fully developed rat retina. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
Figure 2
 
Developmental expression profile of NRF-1 in the rat retina. (A) mRNA expression of NRF-1 was developmentally down-regulated in the rat retina with age, as evidenced by RT-PCR. (B) Relative expression level of NRF-1 mRNA presented as a histogram. (C) Age-related expression pattern of NRF-1 was observed in rat retina, as evidenced by Western blot. (D) Relative expression of NRF-1 in rat retina was quantified by densitometry, and the data presented graphically. (E) Intense staining of NRF-1 in the cell nucleus labeled the entire layer of a P1 rat retina was gradually down-regulated with age and weakly expressed in the fully developed rat retina. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
Figure 2
 
Developmental expression profile of NRF-1 in the rat retina. (A) mRNA expression of NRF-1 was developmentally down-regulated in the rat retina with age, as evidenced by RT-PCR. (B) Relative expression level of NRF-1 mRNA presented as a histogram. (C) Age-related expression pattern of NRF-1 was observed in rat retina, as evidenced by Western blot. (D) Relative expression of NRF-1 in rat retina was quantified by densitometry, and the data presented graphically. (E) Intense staining of NRF-1 in the cell nucleus labeled the entire layer of a P1 rat retina was gradually down-regulated with age and weakly expressed in the fully developed rat retina. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
Figure 3
 
NRF-1 binds to ATF site of the CXCR4 promoter. (A) Distribution of CXCR4 (cell membrane, red) and NRF-1 (cell nuclear, red) in the P1 rat retina is identical: uniformly distributed in all retinal layers and more prominent in the GCL and INL than the ONL. The retinal ganglion cells were labeled with MAP-2 (green), and the cell nuclei were stained by DAPI (blue). (B) ChIP assays demonstrate that the DNA fragments were amplified from the precipitates of P1 rat retinal neurocytes, whereas in 1M rat retina, DNA was only weakly detected, indicating that NRF-1 bound the promoter region of CXCR4.
Figure 3
 
NRF-1 binds to ATF site of the CXCR4 promoter. (A) Distribution of CXCR4 (cell membrane, red) and NRF-1 (cell nuclear, red) in the P1 rat retina is identical: uniformly distributed in all retinal layers and more prominent in the GCL and INL than the ONL. The retinal ganglion cells were labeled with MAP-2 (green), and the cell nuclei were stained by DAPI (blue). (B) ChIP assays demonstrate that the DNA fragments were amplified from the precipitates of P1 rat retinal neurocytes, whereas in 1M rat retina, DNA was only weakly detected, indicating that NRF-1 bound the promoter region of CXCR4.
Figure 4
 
Both CXCR4 and NRF-1 were abnormally activated in developed retina of OlR rats. (A) Typical pathologic changes of ischemic retinopathy were observed in the retina of the P18 OIR rat, as evidenced by fluorescein angiography with FITC-dextran. (B) Both CXCR4 and NRF-1 were up-regulated in the retina of OIR rats, as evidenced by RT-PCR and Western blot. (C) Fold change of both CXCR4 and NRF-1 expression, presented as a histogram. (D) In avascular ischemic retina of OIR rats, CXCR4 and NRF-1 were up-regulated in parallel and with prolonged hypoxia. (E) Fold change of both CXCR4 and NRF-1 expression, presented as a histogram. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
Figure 4
 
Both CXCR4 and NRF-1 were abnormally activated in developed retina of OlR rats. (A) Typical pathologic changes of ischemic retinopathy were observed in the retina of the P18 OIR rat, as evidenced by fluorescein angiography with FITC-dextran. (B) Both CXCR4 and NRF-1 were up-regulated in the retina of OIR rats, as evidenced by RT-PCR and Western blot. (C) Fold change of both CXCR4 and NRF-1 expression, presented as a histogram. (D) In avascular ischemic retina of OIR rats, CXCR4 and NRF-1 were up-regulated in parallel and with prolonged hypoxia. (E) Fold change of both CXCR4 and NRF-1 expression, presented as a histogram. Error bars represent SD from the mean. The asterisks indicate statistically significant differences between the control and experimental rats.
Figure 5
 
Localization of CXCR4 and NRF-1 in the OIR rat retina. (A) The OIR rat retina showed stronger staining of GFAP (red) than that of control rat retina and demonstrated pathologic activation of glial cell signaling. (B) Both CXCR4 and NRF-1 are weakly expressed in the nonischemic, P18 control rat retina. However, increased immunolabeling of CXCR4 (red) was detected throughout the inner layer of retina in OIR rats. (C) Intensive NRF-1 staining (green) was observed in the nuclear of retinal cell of OIR rats and was consistent with the localization pattern of CXCR4 (compared with that of control rats). The nuclei were stained with DAPI (blue).
Figure 5
 
Localization of CXCR4 and NRF-1 in the OIR rat retina. (A) The OIR rat retina showed stronger staining of GFAP (red) than that of control rat retina and demonstrated pathologic activation of glial cell signaling. (B) Both CXCR4 and NRF-1 are weakly expressed in the nonischemic, P18 control rat retina. However, increased immunolabeling of CXCR4 (red) was detected throughout the inner layer of retina in OIR rats. (C) Intensive NRF-1 staining (green) was observed in the nuclear of retinal cell of OIR rats and was consistent with the localization pattern of CXCR4 (compared with that of control rats). The nuclei were stained with DAPI (blue).
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