Limits...
TNFα inhibits IGFBP-3 through activation of p38α and casein kinase 2 in human retinal endothelial cells.

Zhang Q, Soderland D, Steinle JJ - PLoS ONE (2014)

Bottom Line: We found that TNFα significantly reduced IGFBP-3 levels and vice-versa, IGFBP-3 can lower TNFα and TNFα receptor expression.We found that TNFα significantly increased phosphorylation of P38α and CK2.Furthermore, specific inhibitors of P38α or CK2 blocked TNFα inhibition of IGFBP-3 expression, demonstrating that TNFα reduces IGFBP-3 through activation of P38α and CK2.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.

ABSTRACT
We recently reported a reciprocal relationship between tumor necrosis factor alpha (TNFα) and insulin-like receptor growth factor binding protein 3 (IGFBP-3) in whole retina of normal and IGFBP-3 knockout mice. A similar relationship was also observed in cultured retinal endothelial cells (REC). We found that TNFα significantly reduced IGFBP-3 levels and vice-versa, IGFBP-3 can lower TNFα and TNFα receptor expression. Since IGFBP-3 is protective to the diabetic retina and TNFα is causative in the development of diabetic retinopathy, we wanted to better understand the cellular mechanisms by which TNFα can reduce IGFBP-3 levels. For these studies, primary human retinal endothelial cells (REC) were used since these cells undergo TNFα-mediated apoptosis under conditions of high glucose conditions and contribute to diabetic retinopathy. We first cultured REC in normal or high glucose, treated with exogenous TNFα, then measured changes in potential signaling pathways, with a focus on P38 mitogen-activated protein kinase alpha (P38α) and casein kinase 2 (CK2) as these pathways have been linked to both TNFα and IGFBP-3. We found that TNFα significantly increased phosphorylation of P38α and CK2. Furthermore, specific inhibitors of P38α or CK2 blocked TNFα inhibition of IGFBP-3 expression, demonstrating that TNFα reduces IGFBP-3 through activation of P38α and CK2. Since TNFα and IGFBP-3 are key mediators of retinal damage and protection respectively in diabetic retinopathy, increased understanding of the relationship between these two proteins will offer new therapeutic options for treatment.

Show MeSH

Related in: MedlinePlus

TNFα phosphorylates IGFBP-3 on Serine 111/113.IGFBP-3 ELISA results for REC treated with normal glucose (NG), high glucose (HG), high glucose + TNFα (HG+TNFα), high glucose + TNFα + IGFBP-3 mutant (mutation on Serine 111/113, (HG+TNFα+BP3Ser-Ala)), and high glucose + TNFα + IGFBP-3 siRNA + IGFBP-3 mutant (HG+TNFα+BP3si+BP3Ser-Ala). *P<0.05 vs. NG, #P<0.05 vs. HG, ∧P<0.05 vs. HG+TNFα $P<0.05 vs. HG+TNFα+BP3Ser-Ala. All data are mean ± SEM. Data is from 3–4 independent experiments for each treatment.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4114743&req=5

pone-0103578-g004: TNFα phosphorylates IGFBP-3 on Serine 111/113.IGFBP-3 ELISA results for REC treated with normal glucose (NG), high glucose (HG), high glucose + TNFα (HG+TNFα), high glucose + TNFα + IGFBP-3 mutant (mutation on Serine 111/113, (HG+TNFα+BP3Ser-Ala)), and high glucose + TNFα + IGFBP-3 siRNA + IGFBP-3 mutant (HG+TNFα+BP3si+BP3Ser-Ala). *P<0.05 vs. NG, #P<0.05 vs. HG, ∧P<0.05 vs. HG+TNFα $P<0.05 vs. HG+TNFα+BP3Ser-Ala. All data are mean ± SEM. Data is from 3–4 independent experiments for each treatment.

Mentions: After demonstrating that TNFα increased phosphorylation of P38α and CK2, and ultimately caused a decrease in IGFBP-3 levels, our next step was to determine if specific phosphorylation sites on IGFBP-3 were targeted by CK2. We wanted to ascertain whether TNFα regulated IGFBP-3 levels through phosphorylation of Serine 111 and Serine 113 on IGFBP-3, as these are key sites for CK2 phosphorylation [17]. Unfortunately, no phospho-specific antibodies exist for these two sites. To answer our question without antibodies, we first performed site directed mutagenesis for Serine 111 and Serine 113 on IGFBP-3 NB plasmid to create IGFBP-3 NBSer-Ala. We then wanted to verify that IGFBP-3 NBSer-Ala plasmid was able to increase total IGFBP-3 levels even in the presence of TNFα (Fig. 4), which include a control to verify successful reduction of IGFBP-3 with IGFBP-3 siRNA. As expected, levels of endogenous IGFBP-3 were decreased in response to high glucose and were further decreased when TNFα was added. In contrast, cells transfected with IGFBP-3 NBSer-Ala plasmid and treated with HG and TNFα had significantly higher levels of total IGFBP-3, which would be expected to include any remaining endogenous IGFBP-3 as well as mutated IGFBP-3 NBSer-Ala. In order to distinguish between endogenous IGFBP-3 and mutated IGFBP-3 NBSer-Ala, we pretreated cells with IGFBP-3 siRNA to block transcription of endogenous IGFBP-3. Under these conditions, levels of IGFBP-3 (presumably only IGFBP-3 NBSer-Ala) remained significantly elevated even in the presence of TNFα, suggesting that the absence of active serine sites reduced the inhibitory effects of TNFα. Some may expect that transfection with the IGFBP-3 mutant would have reduced IGFBP-3 levels compared to wildtype IGFBP-3. However, we found that transfection with either the mutant or wildtype plasmid increased total IGFBP-3 levels. Others have reported this for other proteins [18]. The difference between mutant and wildtype IGFBP-3 levels would like be noted in the phospho-IGFBP-3 levels rather than total IGFBP-3 (measured here). Unfortunately, antibodies are not available to investigate this. Therefore, we then compared the HG+TNFa+BP3M with the HG+TNFa+BP3M+BP3siRNA, which should limit endogenous IGFBP-3 (wildtype IGFBP-3). When you compare these 2 bars, there is a significant difference. Therefore, we feel that these 2 data points suggest that TNFa does regulate IGFBP-3 through Serine111/113. Based on these findings, we conclude that TNFα acts on Serine 111 and Serine 113 phosphorylation sites to decrease IGFBP-3. A more direct test of this hypothesis will require production of site-specific antibodies.


TNFα inhibits IGFBP-3 through activation of p38α and casein kinase 2 in human retinal endothelial cells.

Zhang Q, Soderland D, Steinle JJ - PLoS ONE (2014)

TNFα phosphorylates IGFBP-3 on Serine 111/113.IGFBP-3 ELISA results for REC treated with normal glucose (NG), high glucose (HG), high glucose + TNFα (HG+TNFα), high glucose + TNFα + IGFBP-3 mutant (mutation on Serine 111/113, (HG+TNFα+BP3Ser-Ala)), and high glucose + TNFα + IGFBP-3 siRNA + IGFBP-3 mutant (HG+TNFα+BP3si+BP3Ser-Ala). *P<0.05 vs. NG, #P<0.05 vs. HG, ∧P<0.05 vs. HG+TNFα $P<0.05 vs. HG+TNFα+BP3Ser-Ala. All data are mean ± SEM. Data is from 3–4 independent experiments for each treatment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4114743&req=5

pone-0103578-g004: TNFα phosphorylates IGFBP-3 on Serine 111/113.IGFBP-3 ELISA results for REC treated with normal glucose (NG), high glucose (HG), high glucose + TNFα (HG+TNFα), high glucose + TNFα + IGFBP-3 mutant (mutation on Serine 111/113, (HG+TNFα+BP3Ser-Ala)), and high glucose + TNFα + IGFBP-3 siRNA + IGFBP-3 mutant (HG+TNFα+BP3si+BP3Ser-Ala). *P<0.05 vs. NG, #P<0.05 vs. HG, ∧P<0.05 vs. HG+TNFα $P<0.05 vs. HG+TNFα+BP3Ser-Ala. All data are mean ± SEM. Data is from 3–4 independent experiments for each treatment.
Mentions: After demonstrating that TNFα increased phosphorylation of P38α and CK2, and ultimately caused a decrease in IGFBP-3 levels, our next step was to determine if specific phosphorylation sites on IGFBP-3 were targeted by CK2. We wanted to ascertain whether TNFα regulated IGFBP-3 levels through phosphorylation of Serine 111 and Serine 113 on IGFBP-3, as these are key sites for CK2 phosphorylation [17]. Unfortunately, no phospho-specific antibodies exist for these two sites. To answer our question without antibodies, we first performed site directed mutagenesis for Serine 111 and Serine 113 on IGFBP-3 NB plasmid to create IGFBP-3 NBSer-Ala. We then wanted to verify that IGFBP-3 NBSer-Ala plasmid was able to increase total IGFBP-3 levels even in the presence of TNFα (Fig. 4), which include a control to verify successful reduction of IGFBP-3 with IGFBP-3 siRNA. As expected, levels of endogenous IGFBP-3 were decreased in response to high glucose and were further decreased when TNFα was added. In contrast, cells transfected with IGFBP-3 NBSer-Ala plasmid and treated with HG and TNFα had significantly higher levels of total IGFBP-3, which would be expected to include any remaining endogenous IGFBP-3 as well as mutated IGFBP-3 NBSer-Ala. In order to distinguish between endogenous IGFBP-3 and mutated IGFBP-3 NBSer-Ala, we pretreated cells with IGFBP-3 siRNA to block transcription of endogenous IGFBP-3. Under these conditions, levels of IGFBP-3 (presumably only IGFBP-3 NBSer-Ala) remained significantly elevated even in the presence of TNFα, suggesting that the absence of active serine sites reduced the inhibitory effects of TNFα. Some may expect that transfection with the IGFBP-3 mutant would have reduced IGFBP-3 levels compared to wildtype IGFBP-3. However, we found that transfection with either the mutant or wildtype plasmid increased total IGFBP-3 levels. Others have reported this for other proteins [18]. The difference between mutant and wildtype IGFBP-3 levels would like be noted in the phospho-IGFBP-3 levels rather than total IGFBP-3 (measured here). Unfortunately, antibodies are not available to investigate this. Therefore, we then compared the HG+TNFa+BP3M with the HG+TNFa+BP3M+BP3siRNA, which should limit endogenous IGFBP-3 (wildtype IGFBP-3). When you compare these 2 bars, there is a significant difference. Therefore, we feel that these 2 data points suggest that TNFa does regulate IGFBP-3 through Serine111/113. Based on these findings, we conclude that TNFα acts on Serine 111 and Serine 113 phosphorylation sites to decrease IGFBP-3. A more direct test of this hypothesis will require production of site-specific antibodies.

Bottom Line: We found that TNFα significantly reduced IGFBP-3 levels and vice-versa, IGFBP-3 can lower TNFα and TNFα receptor expression.We found that TNFα significantly increased phosphorylation of P38α and CK2.Furthermore, specific inhibitors of P38α or CK2 blocked TNFα inhibition of IGFBP-3 expression, demonstrating that TNFα reduces IGFBP-3 through activation of P38α and CK2.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.

ABSTRACT
We recently reported a reciprocal relationship between tumor necrosis factor alpha (TNFα) and insulin-like receptor growth factor binding protein 3 (IGFBP-3) in whole retina of normal and IGFBP-3 knockout mice. A similar relationship was also observed in cultured retinal endothelial cells (REC). We found that TNFα significantly reduced IGFBP-3 levels and vice-versa, IGFBP-3 can lower TNFα and TNFα receptor expression. Since IGFBP-3 is protective to the diabetic retina and TNFα is causative in the development of diabetic retinopathy, we wanted to better understand the cellular mechanisms by which TNFα can reduce IGFBP-3 levels. For these studies, primary human retinal endothelial cells (REC) were used since these cells undergo TNFα-mediated apoptosis under conditions of high glucose conditions and contribute to diabetic retinopathy. We first cultured REC in normal or high glucose, treated with exogenous TNFα, then measured changes in potential signaling pathways, with a focus on P38 mitogen-activated protein kinase alpha (P38α) and casein kinase 2 (CK2) as these pathways have been linked to both TNFα and IGFBP-3. We found that TNFα significantly increased phosphorylation of P38α and CK2. Furthermore, specific inhibitors of P38α or CK2 blocked TNFα inhibition of IGFBP-3 expression, demonstrating that TNFα reduces IGFBP-3 through activation of P38α and CK2. Since TNFα and IGFBP-3 are key mediators of retinal damage and protection respectively in diabetic retinopathy, increased understanding of the relationship between these two proteins will offer new therapeutic options for treatment.

Show MeSH
Related in: MedlinePlus