The ERK1/2 Inhibitor U0126 Attenuates Diabetes-Induced Upregulation of MMP-9 and Biomarkers of Inflammation in the Retina

This study was conducted to determine the expression of matrix metalloproteinase-9 (MMP-9) and tissue inhibitor of metalloproteinase-1 (TIMP-1) in a time-dependent manner and the effect of extracellular-signal-regulated kinases-1/2 (ERK1/2) inhibition on the expressions of MMP-9, TIMP-1, and inflammatory biomarkers in the retinas of diabetic rats. The expression of MMP-9 was quantified by zymography, and the mRNA level of MMP-9 and TIMP-1 was quantified by RT-PCR. The expression of inducible nitric oxide synthase (iNOS), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) was examined by Western blot analysis. MMP-9 expression was significantly higher in diabetic rat retinas compared to controls at all time points.TIMP-1 expression was nonsignificantly upregulated at 1week of diabetes and was significantly downregulated at 4 and 12 weeks of diabetes. Intravitreal administration of the ERK1/2 inhibitor U0126 prior to induction of diabetes decreased ERK1/2 activation, attenuated diabetes-induced upregulation of MMP-9, iNOS, IL-6, and TNF-α and upregulated TIMP-1 expression. In MMP-9 knockout mice, diabetes had no effect on retinal iNOS expression and its level remained unchanged. These data provide evidence that ERK1/2 signaling pathway is involved in MMP-9, iNOS, IL-6, and TNF-α induction in diabetic retinas and suggest that ERK1/2 can be a novel therapeutic target in diabetic retinopathy.

Matrix metalloproteinases (MMPs) are a large family of proteinases that remodel extracellular matrix components and play an important role in the regulation of numerous physiological processes including vascular remodeling and angiogenesis. Altered MMPs activities have been implicated in many diseases, including diabetes. Diabetes is believed to stimulate the secretion of several MMPs which participate in both macrovascular and microvascular diseases such as coronary artery disease, peripheral arterial disease, stroke, nephropathy, neuropathy, and retinopathy [18][19][20]. MMP-9, the largest and most complex member of the MMP family, regulates a variety of cellular functions, including proliferation, differentiation, and angiogenesis. Endogenous tissue inhibitors of MMPs (TIMPs) regulate their activation, and TIMP-1 shows greater preference for MMP-9 than any other MMP [21]. The published literature and our previously published data demonstrated elevated levels of MMP-9 in the epiretinal membranes and vitreous fluid from patients with proliferative diabetic retinopathy (PDR) and increased MMP-9 expression in the retinas of diabetic rodents [13,15,16,[22][23][24]. In addition, our previous data also suggested a pro-apoptotic role of MMP-9 in the pathogenesis of DR [13,15]. Recently, several studies documented that MMP-9 expression is regulated by extracellular-signal-regulated kinases-(ERK-) dependent pathways [24][25][26][27] and in the retina RAF protooncogene serine/threonine-protein kinase-(Raf-) mitogen-activated protein kinase kinase (MEK)-ERK cascade is activated by diabetes [13,24]. Therefore, inhibitors of Raf-MEK-ERK pathway represent a unique opportunity to prevent MMP-9 induction in the retina induced by diabetes. Previously, it was shown that 1 week after diabetes was induced, the retinal ERK 1/2 , vascular endothelial growth factor (VEGF), ICAM-1, leukostasis, and retinal vascular permeability are significantly upregulated [28][29][30]. Based on these observations, this study was designed to investigate the potential therapeutic role of ERK 1/2 -specific inhibitor U0126 on the retinas at one week of diabetes in rats. We, therefore, investigated the time course change in the expression of MMP-9 and TIMP-1 in the retinas of diabetic rats and examined the effect of intravitreal administration of U0126 on the expression of MMP-9, TIMP-1, iNOS, IL-6, and TNFin the diabetic retinas.  [28]. After 24 hours, diabetes was induced with STZ as mentioned above. The animals were sacrificed one week after diabetes was induced, and the retinas were carefully dissected, snap frozen in liquid nitrogen, and stored at −80 ∘ C to be analyzed by PCR and western blotting.

Mice.
Wild-type (WT) and MMP-9 knockout (MMP-9 KO) mice [31] (a generous gift from Professor Ghislain Opdenakker, Rega Institute for Medical Research, university of Leuven, Belgium) were backcrossed 13 times into C57BL6 background to maximally reduce confounding small but additive genetic influences. To illustrate the latter, only in the 13th backcross generation, the brown fur coat of the knockout mice became black. The WT and KO mice were made diabetic by intraperitoneal injection of streptozotocin for five consecutive days. Mice with blood glucose above 200 mg/dL, 3 days after the last injection of streptozotocin, were considered as diabetic. Age-matched normal WT mice served as controls.
To investigate long-term effects on the retina, 3 months after induction of diabetes, mice were sacrificed by pentobarbital overdose, and the retinas were removed from one eye, snap frozen in liquid nitrogen, and stored at −80 ∘ C for RNA isolation. All experiments were performed in accordance to the Association of Research in Vision and Ophthalmology (ARVO) and King Saud University's Animal Care and Use Committee Guidelines on handling and treatment of animals for basic science research.

Real-Time Reverse Transcription Polymerase Chain Reaction (RT-PCR).
Total RNA was extracted from retina using TRI reagent (Ambion, TX, USA), according to manufacturer's protocol. cDNA were synthesized from 1 g RNA, using an high capacity cDNA reverse transcription kit (Applied Biosystem, CA, USA) following manufacturer's instruction. Real-time RT-PCR was performed using a SYBR green PCR master mix. The PCR primers for rats were MMP-9 forward 5 -GCAACGGAGACGGCAAACC-3 reverse   The standard PCR conditions included 2 minutes at 50 ∘ C and 10 min at 95 ∘ C followed by 40 cycles of extension at 95 ∘ C for 15 seconds and one minute at 60 ∘ C. Threshold lines were automatically adjusted to intersect amplification lines in the linear portion of the amplification curves, and cycles to threshold (Ct) were recorded automatically. Data were normalized with -actin mRNA level (housekeeping gene), and the fold change in gene expression relative to normal was calculated using the ddCt method as previously described [19].

Statistical Analysis.
Each measurement was made in duplicate, and the assays were repeated three or more times. Data are expressed as mean±SD. The Mann-Whitney U test was used to compare means from two independent groups. A value less than ≤0.05 indicated statistical significance. SPSS version 12.0 was used for the statistical analyses. (d) Figure 2: Effect of ERK 1/2 inhibitor (U0126) on diabetes induced retinal ERK 1/2 activation and MMP-9 and TIMP-1 expressions. Protein expressions of ERK 1/2 activation (phosphorylation) were quantified by western blotting using phosphospecific antibody and were adjusted to the protein levels of unphosphorylated antibody in each sample. The expression of (b) MMP-9 gelatinase activity was quantified by using zymography technique. Gene expressions of (c) MMP-9 and (d) TIMP-1 were quantified by RT-PCR using the specific primers and were adjusted to the mRNA levels of -actin in each sample. Each measurement was performed at least three times. Results are expressed as mean ± s.d. of at least six rats in each group. * < 0.05 compared with normal rats and # < 0.05 compared to diabetic rats. N, D, and D + U0126 = normal, 1-week diabetic and U0126 pretreated diabetic rat.

Time-Dependent Changes in MMP-9 Expression in Dia-
betic Retinas. The MMP-9 gelatinase levels in the retinas of diabetic rats were increased by about 40%, 55%, 85%, and 50% at 1, 4, 8, and 12 weeks, respectively, after the onset of diabetes compared to nondiabetic rats (Figure 1(a)). The relative mRNA levels of MMP-9 were detected by real-time RT-PCR analysis at 1, 4, 8, and 12 weeks after the onset of diabetes. The MMP-9 mRNA levels of the control groups at all time points remained at very constant levels (1 ± 0.3-fold). However, a significant increase ( < 0.05) in MMP-9 mRNA levels by about 0.5-to 2-fold was detected in the retinas from 1, 4, 8, and 12 week diabetic rats compared with nondiabetic rats (Figure 1(b)). Figure 1 about 70% and 46% ( < 0.01) in the retinas from 4-and 12week diabetic rats compared to the control group, but there was no significant difference between the control and diabetic animals at week 8. Interestingly, there was a nonsignificant increase in TIMP-1 mRNA in the retinas from 1-week diabetic rats compared to the nondiabetic rats.

U0126 Attenuates Diabetes-Induced ERK 1/2 Phosphorylation in Diabetic
Retinas. U0126 is a potent ERK 1/2 antagonist and decreases ERK 1/2 activity in diabetic retina [28]. We employed U0126 to investigate the anti-inflammatory function in the retinas of diabetic rats. Rats that were pretreated with U0126 followed by induction of diabetes showed significant ( < 0.05) attenuation of ERK 1/2 activation as compared to untreated diabetic rats (Figure 2(a)).

Effect of U0126 on the Expression of MMP-9 and TIMP-1.
The expression of MMP-9 was significantly attenuated in the U0126-treated diabetic rat retinas as compared to untreated diabetic rats (Figures 2(b) and 2(c)). U0126 pretreatment of the diabetic rats significantly upregulated TIMP-1 expression compared to nondiabetic rats (Figure 2(d)).

Effect of U0126 on the Expression of the Inflammatory
Biomarkers, iNOS, IL-6, and TNF-. Diabetes significantly increased the retinal expressions of iNOS by 40%, IL-6 by 60%, and TNF-by 35% as compared to nondiabetic rat retinas. The results as in Figure 3 showed that pretreatment with U0126 significantly attenuated diabetes-induced increase in the expressions of iNOS, IL-6, and TNF-as compared to untreated diabetic rats.

Effect of MMP-9 Inhibition on the mRNA Level of iNOS.
Diabetes at ∼3 months in WT mice, as expected, increased the expressions of iNOS in the retina by about twofold compared to the WT normal mice. In contrast, in MMP-9 KO mice diabetes had no effect on retinal iNOS expression; the values obtained from diabetic MMP-9 KO mice retina were significantly lower compared to those obtained from WTdiabetic mice (Figure 4).

Discussion
Inflammation represents a highly coordinated set of events that allow tissues to respond to injury, and it requires the participation of various cell types expressing and reacting to diverse mediators in a sequential manner. In the development of DR, biomarkers of inflammation such as IL-6, iNOS, TNF-, and MMP-9 are elevated in the retina [9,[11][12][13]. One of the specific objectives of this current study was to determine the role of U0126, an ERK 1/2 inhibitor, in the regulation of MMP-9 and inflammatory mediators in the diabetic retinas. Salient features of the current study are as follows: (i) in the retina, diabetes upregulates MMP-9 and downregulates TIMP-1 expression in a time-dependent manner; (ii) U0126, a specific inhibitor of ERK 1/2 , attenuates early diabetes-induced upregulation of MMP-9 and enhanced TIMP-1 expression; (iii) U0126 attenuates diabetes-induced upregulation of iNOS, IL6, and TNF-in the retina; (iv) knockdown of MMP-9 prevents diabetes-induced upregulation of iNOS level in the retina.
The mitogen-activated protein kinases (MAPKs) play a critical role in the regulation of cell growth and differentiation and in the control of cellular responses to cytokines and stressors. MAPK cascades are a series of cytosolic enzymes that can transmit extracellular signals to the nucleus [32]. These cascades consist of at least three protein kinases that are activated sequentially: a MAPK kinase kinase such as Raf-1 activates a MAPK kinase such as MEK1, which in turn activates a MAPK such as ERK. The activated ERK can translocate to the nucleus [33,34], where it can phosphorylate or induce transcription factors leading to the activation of genes and the expression of proteins needed for differentiation or proliferation. Growing body of evidence supports the hypothesis that damaging effect of elevated glucose in the retina may, in part, be due to its ability to increase MAPK signaling pathway in the retina [35][36][37][38]. ERK 1/2 is the most extensively characterized member of MAPK family proteins. It plays an important role in cell growth and differentiation, but recent several reports suggested that ERK 1/2 pathway can also be related to inflammation, apoptosis, and cell injury [39,40]. A strong relation between ERK 1/2 activation and MMP-9 is observed in various pathological conditions including diabetes. Our previous work has indicated that Hras/Raf MEK/ERK 1/2 mechanisms play a crucial role in the development of diabetic retinopathy [13,38]. In the present study, we observed that MMP-9 level increases at all time points; however, TIMP-1 expression was downregulated on a time-dependent manner in the retinas of diabetic rats compared to nondiabetic rats. This observation is consistent with previous studies that demonstrated the upregulation of MMP-9 in the retina of diabetic rodents as well as in vitreous of PDR patients [15,16,23,24]. Our findings are also consistent with a previous study that demonstrated decreased TIMP-1 expression in the retina and its microvasculature at both 2 months and 12 months of diabetes [24]. These data suggest a role of TIMP-1-free MMP-9 in the development and progression of DR. Previously, it was demonstrated that the TIMP-1-free MMP-9, readily available for rapid release by neutrophils upon their influx into target tissue, is the major factor determining the high levels of its angiogenic response [41,42]. We showed here that in the retina, diabetes activates ERK 1/2 and that UO126 attenuates diabetes-induced activation of ERK 1/2 . Our results are in agreement with a recent report showing increased ERK 1/2 activity in the retina of one-week diabetic rats compared to normal rats and pretreatment of the retina with U0126 attenuating diabetesinduced ERK 1/2 activation [28]. Recent studies demonstrated that the activation of ERK 1/2 plays an essential role in induction of inflammation [40]. ERK 1/2 activation is required for cytokine signaling, and inhibition of ERK 1/2 prevents nuclear transcription factor Kappa B (NF-B) activation [43]. NF-B has been localized to the inner nuclear layer and ganglion cells of the retina [44], and NF-B-regulated inflammatory gene products are reported to be upregulated in the retinas during diabetes such as cyclooxygenase-2 [45,46], iNOS (45,47), ICAM-1 [47,48], and TNF- [49]. We demonstrated that the therapies that inhibit ERK 1/2 activation significantly attenuated MMP-9 expression and upregulated TIMP-1 in the retinas of one-week diabetic rats. Similarly, a previous report demonstrated that ERK 1/2 activation makes a significant contribution to induction of MMP-9 in the rat cortical astrocytes via NF-B activation [50]. Moreover, selective inhibition of the ERK 1/2 pathways by U0126 significantly attenuated the recombinant human erythropoietin-induced MMP-9 secretion in mouse brain endothelial cells and neural progenitor cells [51]. iNOS, IL-6, and TNF-are known to be important inflammatory mediators [52].
Strong evidence indicates that chronic low-grade inflammation is implicated in the pathogenesis of diabetic retinopathy. Diabetic retinal vascular leakage, capillary nonperfusion, and endothelial cell damage are associated with leukocyte recruitment and adhesion to the retinal vasculature, findings that correlate with the increased expression of ICAM-1 and the leukocyte integrin CD18. Inhibition of ICAM-1 activity in animals deficient in the gene encoding for ICAM-1 or by a neutralizing antibody suppresses both retinal leukostasis and vascular leakage [10,53]. The causal relationship between inflammation and angiogenesis is now widely accepted [54]. Previously, various studies have documented that diabetes enhanced the production of inflammatory mediators such as iNOS, IL6, and TNF-in the retina [9,11,45,49,55]. In agreement with these studies, we found a significant upregulation of iNOS, IL6, and TNF-in one-week diabetic rat retinas.
In addition, we also demonstrated here that inhibition of ERK 1/2 by U0126 significantly ameliorates diabetes-induced upregulation of iNOS, IL6, and TNF-in the retina. Recent several reports demonstrated the beneficial effect of ERK 1/2 inhibition on various inflammatory parameters and on the production of inflammatory cytokines [56,57]. Maddahi and Edvinsson demonstrated that U0126 significantly inhibits the iNOS, IL-6, and TNF-secretion in rat model of cerebral ischemia [58]. The expression of iNOS in the retina of diabetic mice with manipulated MMP-9 gene remains normal. Similarly, a previous report demonstrated that inhibition of MMP-9 suppresses lipopolysaccharide-induced expression of proinflammatory cytokines and iNOS in microglia [59].
In conclusion, these results indicate that ERK 1/2 pathway is an upstream signal for MMP-9 production and induction of inflammation in the diabetic retina, and targeting ERK 1/2 pathway can be a novel therapeutic strategy for the treatment of DR.

Conflict of Interests
The authors declare no conflict of interests.