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TXNIP links innate host defense mechanisms to oxidative stress and inflammation in retinal Muller glia under chronic hyperglycemia: implications for diabetic retinopathy.

Devi TS, Lee I, Hüttemann M, Kumar A, Nantwi KD, Singh LP - Exp Diabetes Res (2012)

Bottom Line: We also found in vivo that streptozocin-induced diabetic rats have higher retinal TXNIP and innate immune response gene expression than normal rats.Thus, our results show that HG sustains TXNIP up-regulation in Muller glia and evokes a program of cellular defense/survival mechanisms that ultimately lead to oxidative stress, ER stress/inflammation, autophagy and apoptosis.TXNIP is a potential target to ameliorate blinding ocular complications of diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Wayne State University, Detroit, MI 48201, USA.

ABSTRACT
Thioredoxin Interacting Protein (TXNIP) mediates retinal inflammation, gliosis, and apoptosis in experimental diabetes. Here, we investigate the temporal response of Muller glia to high glucose (HG) and TXNIP expression using a rat Muller cell line (rMC1) in culture. We examined if HG-induced TXNIP expression evokes host defense mechanisms in rMC1 in response to metabolic abnormalities. HG causes sustained up-regulation of TXNIP (2 h to 5 days), ROS generation, ATP depletion, ER stress, and inflammation. Various cellular defense mechanisms are activated by HG: (i) NLRP3 inflammasome, (ii) ER stress response (sXBP1), (iii) hypoxic-like HIF-1α induction, (iv) autophagy/mitophagy, and (v) apoptosis. We also found in vivo that streptozocin-induced diabetic rats have higher retinal TXNIP and innate immune response gene expression than normal rats. Knock down of TXNIP by intravitreal siRNA reduces inflammation (IL-1β) and gliosis (GFAP) in the diabetic retina. TXNIP ablation in vitro prevents ROS generation, restores ATP level and autophagic LC3B induction in rMC1. Thus, our results show that HG sustains TXNIP up-regulation in Muller glia and evokes a program of cellular defense/survival mechanisms that ultimately lead to oxidative stress, ER stress/inflammation, autophagy and apoptosis. TXNIP is a potential target to ameliorate blinding ocular complications of diabetic retinopathy.

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Chronic hyperglycemia persistently upregulates TXNIP expression in rMC1 and induces IL-1β and NLRP3 inflammasome activation. rMC1 cells were cultured under HG for (a and b) 0–24 h or (c and d) 0–5 days and TXNIP proteins were detected by Western blotting. For this, cell extracts were prepared in RIPA buffer and 30 μg protein was analyzed on 12% SDS-PAGE and Western blot for cytosolic TXNIP, Pro-IL-1β, NLRP3, and pro-caspase-1 and the nuclear level of phosphorylated p65 at serine residue 276 (S276) of NF-κB. ECL detected the immunoreactive bands. Actin and tubulin were used as controls for protein loading. A representative blot for each protein is shown here from n = 3-4.
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fig3: Chronic hyperglycemia persistently upregulates TXNIP expression in rMC1 and induces IL-1β and NLRP3 inflammasome activation. rMC1 cells were cultured under HG for (a and b) 0–24 h or (c and d) 0–5 days and TXNIP proteins were detected by Western blotting. For this, cell extracts were prepared in RIPA buffer and 30 μg protein was analyzed on 12% SDS-PAGE and Western blot for cytosolic TXNIP, Pro-IL-1β, NLRP3, and pro-caspase-1 and the nuclear level of phosphorylated p65 at serine residue 276 (S276) of NF-κB. ECL detected the immunoreactive bands. Actin and tubulin were used as controls for protein loading. A representative blot for each protein is shown here from n = 3-4.

Mentions: We further examine whether the protein levels of TXNIP and pro-IL-1β protein are induced by HG and if the levels of NLRP3 and caspase-1 are important for processing pro-IL-1β to its mature form in a time-dependent manner. TXNIP expression is low in rMC1 under LG while HG upregulates TXNIP (~4-folds) significantly from 2 h to 5 days (Figures 3(a) and 3(c)). Nuclear level of phosphorylated p65 subunit of NF-κB increases at 2 and 4 h but reduces at 24 h (Figure 3(b)); however, it elevates again at day 2 through day 5 (Figure 3(d)). Pro-IL-1β is also increased at 2 and 4 h of HG exposure and reduces at 24 h, and again induces at 3 days of HG (Figures 3(c) and 3(d)), which correlates with NLRP3 expression. Procaspase 1 level is lower at 2 and 4 h and upregulated at 24 h (Figure 3(c)). This is in agreement with the increased level of active caspase-1 at 24 h shown in Figure 2(d). Pro-caspase-1 level rises again at 5 days of HG exposure (Figure 3(d)). The results demonstrate that TXNIP is a glucose-sensitive gene and its expression remains upregulated when hyperglycemia persists. On the other hand, the innate immune response to chronic hyperglycemia by IL-1β, NLRP3 and caspase-1 inflammasome is cyclical.


TXNIP links innate host defense mechanisms to oxidative stress and inflammation in retinal Muller glia under chronic hyperglycemia: implications for diabetic retinopathy.

Devi TS, Lee I, Hüttemann M, Kumar A, Nantwi KD, Singh LP - Exp Diabetes Res (2012)

Chronic hyperglycemia persistently upregulates TXNIP expression in rMC1 and induces IL-1β and NLRP3 inflammasome activation. rMC1 cells were cultured under HG for (a and b) 0–24 h or (c and d) 0–5 days and TXNIP proteins were detected by Western blotting. For this, cell extracts were prepared in RIPA buffer and 30 μg protein was analyzed on 12% SDS-PAGE and Western blot for cytosolic TXNIP, Pro-IL-1β, NLRP3, and pro-caspase-1 and the nuclear level of phosphorylated p65 at serine residue 276 (S276) of NF-κB. ECL detected the immunoreactive bands. Actin and tubulin were used as controls for protein loading. A representative blot for each protein is shown here from n = 3-4.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3313582&req=5

fig3: Chronic hyperglycemia persistently upregulates TXNIP expression in rMC1 and induces IL-1β and NLRP3 inflammasome activation. rMC1 cells were cultured under HG for (a and b) 0–24 h or (c and d) 0–5 days and TXNIP proteins were detected by Western blotting. For this, cell extracts were prepared in RIPA buffer and 30 μg protein was analyzed on 12% SDS-PAGE and Western blot for cytosolic TXNIP, Pro-IL-1β, NLRP3, and pro-caspase-1 and the nuclear level of phosphorylated p65 at serine residue 276 (S276) of NF-κB. ECL detected the immunoreactive bands. Actin and tubulin were used as controls for protein loading. A representative blot for each protein is shown here from n = 3-4.
Mentions: We further examine whether the protein levels of TXNIP and pro-IL-1β protein are induced by HG and if the levels of NLRP3 and caspase-1 are important for processing pro-IL-1β to its mature form in a time-dependent manner. TXNIP expression is low in rMC1 under LG while HG upregulates TXNIP (~4-folds) significantly from 2 h to 5 days (Figures 3(a) and 3(c)). Nuclear level of phosphorylated p65 subunit of NF-κB increases at 2 and 4 h but reduces at 24 h (Figure 3(b)); however, it elevates again at day 2 through day 5 (Figure 3(d)). Pro-IL-1β is also increased at 2 and 4 h of HG exposure and reduces at 24 h, and again induces at 3 days of HG (Figures 3(c) and 3(d)), which correlates with NLRP3 expression. Procaspase 1 level is lower at 2 and 4 h and upregulated at 24 h (Figure 3(c)). This is in agreement with the increased level of active caspase-1 at 24 h shown in Figure 2(d). Pro-caspase-1 level rises again at 5 days of HG exposure (Figure 3(d)). The results demonstrate that TXNIP is a glucose-sensitive gene and its expression remains upregulated when hyperglycemia persists. On the other hand, the innate immune response to chronic hyperglycemia by IL-1β, NLRP3 and caspase-1 inflammasome is cyclical.

Bottom Line: We also found in vivo that streptozocin-induced diabetic rats have higher retinal TXNIP and innate immune response gene expression than normal rats.Thus, our results show that HG sustains TXNIP up-regulation in Muller glia and evokes a program of cellular defense/survival mechanisms that ultimately lead to oxidative stress, ER stress/inflammation, autophagy and apoptosis.TXNIP is a potential target to ameliorate blinding ocular complications of diabetic retinopathy.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Wayne State University, Detroit, MI 48201, USA.

ABSTRACT
Thioredoxin Interacting Protein (TXNIP) mediates retinal inflammation, gliosis, and apoptosis in experimental diabetes. Here, we investigate the temporal response of Muller glia to high glucose (HG) and TXNIP expression using a rat Muller cell line (rMC1) in culture. We examined if HG-induced TXNIP expression evokes host defense mechanisms in rMC1 in response to metabolic abnormalities. HG causes sustained up-regulation of TXNIP (2 h to 5 days), ROS generation, ATP depletion, ER stress, and inflammation. Various cellular defense mechanisms are activated by HG: (i) NLRP3 inflammasome, (ii) ER stress response (sXBP1), (iii) hypoxic-like HIF-1α induction, (iv) autophagy/mitophagy, and (v) apoptosis. We also found in vivo that streptozocin-induced diabetic rats have higher retinal TXNIP and innate immune response gene expression than normal rats. Knock down of TXNIP by intravitreal siRNA reduces inflammation (IL-1β) and gliosis (GFAP) in the diabetic retina. TXNIP ablation in vitro prevents ROS generation, restores ATP level and autophagic LC3B induction in rMC1. Thus, our results show that HG sustains TXNIP up-regulation in Muller glia and evokes a program of cellular defense/survival mechanisms that ultimately lead to oxidative stress, ER stress/inflammation, autophagy and apoptosis. TXNIP is a potential target to ameliorate blinding ocular complications of diabetic retinopathy.

Show MeSH
Related in: MedlinePlus