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Cross-talk between TGF-beta1 and IL-6 in human trabecular meshwork cells.

Liton PB, Li G, Luna C, Gonzalez P, Epstein DL - Mol. Vis. (2009)

Bottom Line: Incubation of HTM cells with TGF-beta1 (5 ng/ml) resulted in a significant increase in the protein and mRNA levels of IL-6, which was significantly diminished in the presence of the inhibitors for p38 MAPK or JNK.In addition, the presence of inhibitors for the p38 MAPK, ERK, and TGF-beta1 pathways significantly decreased the increased expression of IL-6 by cyclic mechanical stress.We also found that p38 MAPK might play a contributing role in the maintenance of such a loop.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Duke University, Durham, NC 27710, USA. paloma.liton@duke.edu

ABSTRACT

Purpose: To investigate the relationship between transforming growth factor beta-1 (TGF-beta1) and interleukin-6 (IL-6) in human trabecular meshwork (HTM) cells as well as to identify the signaling pathway/s involved in the increased IL-6 expression that occurs in response to mechanical stress and TGF-beta1.

Methods: All experiments were performed in confluent monolayers of HTM cells at passage 3. Secreted IL-6 was quantified by ELISA. Levels of IL-6 mRNA were evaluated by polymerase chain reaction (PCR) analysis. Activation of the IL-6 and TGF-beta1 promoters was monitored by measuring secreted alkaline phosphatase protein (SEAP) released into the culture medium by HTM cells infected with an adenovirus expressing the SEAP reporter gene that was controlled by either the IL-6 promoter (AdIL6-SEAP) or the TGF-beta1 promoter (AdTGFbeta1-SEAP). Cyclic mechanical stress (5% elongation, one cycle per second) was applied using the Flexcell System. Reagents used in this study included human TGF-beta1, human IL-6, and the inhibitors for the p38 mitogen-activated protein kinase (MAPK; SB202190), c-jun NH2-terminal kinase (JNK; SP600125), extracellular-regulating kinase (ERK; PD98059), and TGF type I activin receptor (SB431542).

Results: Incubation of HTM cells with TGF-beta1 (5 ng/ml) resulted in a significant increase in the protein and mRNA levels of IL-6, which was significantly diminished in the presence of the inhibitors for p38 MAPK or JNK. No transcriptional activation of the exogenous IL-6 promoter was observed following TGF-beta1 treatment. In addition, the presence of inhibitors for the p38 MAPK, ERK, and TGF-beta1 pathways significantly decreased the increased expression of IL-6 by cyclic mechanical stress. Furthermore, exposure of HTM cells to IL-6 (100 ng/ml) demonstrated the transcriptional activation of TGF-beta1 promoter, which was severely impaired by blocking the p38 MAPK pathway.

Conclusions: Our results indicate that TGF-beta1 participates in the regulation of basal expression and the stretch-induced expression of IL-6 and suggest the possible existence in cultured HTM cells of an autocrine loop between IL-6 and TGF-beta1. We also found that p38 MAPK might play a contributing role in the maintenance of such a loop.

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Effect of MAPK on the TGF-β1 induced expression of IL-6. Three independent primary cultures of HTM cells were serum-starved for 16 h, pre-treated for 2 h with the MAPK inhibitors, and then incubated in serum-free media with TGF-β1 (5 ng/ml) in the presence of the MAPK inhibitors (10 μM). A: Secreted IL-6 quantified by ELISA at 16 h post-treatment is shown. B: Differential IL-6 mRNA expression compared to control-DMSO, calculated at 6 h post-treatment. The asterisk indicates that p is greater than 0.001, and a double asterisk denotes that p is less than 0.001. Statistical significance between groups was assessed by the paired Student's t-test (n=3).
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f3: Effect of MAPK on the TGF-β1 induced expression of IL-6. Three independent primary cultures of HTM cells were serum-starved for 16 h, pre-treated for 2 h with the MAPK inhibitors, and then incubated in serum-free media with TGF-β1 (5 ng/ml) in the presence of the MAPK inhibitors (10 μM). A: Secreted IL-6 quantified by ELISA at 16 h post-treatment is shown. B: Differential IL-6 mRNA expression compared to control-DMSO, calculated at 6 h post-treatment. The asterisk indicates that p is greater than 0.001, and a double asterisk denotes that p is less than 0.001. Statistical significance between groups was assessed by the paired Student's t-test (n=3).

Mentions: We next examined the intracellular signaling pathway through which TGF-β1 stimulates IL-6 expression and secretion by HTM cells. For this, three different cultures of HTM cells that were serum-deprived for 16 h were pre-treated for 2 h with either p38 inhibitor (SB 202190, 10 μM), MEK1 inhibitor (PD98059, 10 μM), or JNK inhibitor (SP600125, 10 μM) and then exposed to TGF-β1 (5 ng/ml) in the presence of the inhibitors. As shown in Figure 3, pre-treatment of HTM with the p38 MAPK inhibitor led to a significant decrease in the TGF-β1 induced expression of IL-6 at both the protein (513.43±72.53 pg/ml secreted IL-6 in SB202190-treated cultures versus 996.41±166.19 pg/ml in vehicle-treated cultures, p=0.009) and mRNA levels (1.31±0.37 fold IL-6 mRNA expression in SB202190-treated cultures versus 3.03±0.67 fold in vehicle-treated cultures, p=0.017), quantified at 16 h and 6 h, respectively. Our results also showed a significant reduction in the levels of secreted IL-6 induced by TGF-β1 with the JNK inhibitor (474.41±55.68 pg/ml secreted IL-6 in SP600125-treated cultures versus 996.41±166.19 pg/ml in vehicle-treated cultures, p=0.007; Figure 3A). Although it did not reach statistical significance, IL-6 mRNA was found to be downregulated with SP600125 (2.1±0.5 IL-6 mRNA fold expression in SP600125-treated cultures versus 3.03±0.67 fold in vehicle-treated cultures, p=0.126). These data indicate a role of p38 MAPK and JNK pathways in IL-6 induction by exogenous TGF-β1.


Cross-talk between TGF-beta1 and IL-6 in human trabecular meshwork cells.

Liton PB, Li G, Luna C, Gonzalez P, Epstein DL - Mol. Vis. (2009)

Effect of MAPK on the TGF-β1 induced expression of IL-6. Three independent primary cultures of HTM cells were serum-starved for 16 h, pre-treated for 2 h with the MAPK inhibitors, and then incubated in serum-free media with TGF-β1 (5 ng/ml) in the presence of the MAPK inhibitors (10 μM). A: Secreted IL-6 quantified by ELISA at 16 h post-treatment is shown. B: Differential IL-6 mRNA expression compared to control-DMSO, calculated at 6 h post-treatment. The asterisk indicates that p is greater than 0.001, and a double asterisk denotes that p is less than 0.001. Statistical significance between groups was assessed by the paired Student's t-test (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2637787&req=5

f3: Effect of MAPK on the TGF-β1 induced expression of IL-6. Three independent primary cultures of HTM cells were serum-starved for 16 h, pre-treated for 2 h with the MAPK inhibitors, and then incubated in serum-free media with TGF-β1 (5 ng/ml) in the presence of the MAPK inhibitors (10 μM). A: Secreted IL-6 quantified by ELISA at 16 h post-treatment is shown. B: Differential IL-6 mRNA expression compared to control-DMSO, calculated at 6 h post-treatment. The asterisk indicates that p is greater than 0.001, and a double asterisk denotes that p is less than 0.001. Statistical significance between groups was assessed by the paired Student's t-test (n=3).
Mentions: We next examined the intracellular signaling pathway through which TGF-β1 stimulates IL-6 expression and secretion by HTM cells. For this, three different cultures of HTM cells that were serum-deprived for 16 h were pre-treated for 2 h with either p38 inhibitor (SB 202190, 10 μM), MEK1 inhibitor (PD98059, 10 μM), or JNK inhibitor (SP600125, 10 μM) and then exposed to TGF-β1 (5 ng/ml) in the presence of the inhibitors. As shown in Figure 3, pre-treatment of HTM with the p38 MAPK inhibitor led to a significant decrease in the TGF-β1 induced expression of IL-6 at both the protein (513.43±72.53 pg/ml secreted IL-6 in SB202190-treated cultures versus 996.41±166.19 pg/ml in vehicle-treated cultures, p=0.009) and mRNA levels (1.31±0.37 fold IL-6 mRNA expression in SB202190-treated cultures versus 3.03±0.67 fold in vehicle-treated cultures, p=0.017), quantified at 16 h and 6 h, respectively. Our results also showed a significant reduction in the levels of secreted IL-6 induced by TGF-β1 with the JNK inhibitor (474.41±55.68 pg/ml secreted IL-6 in SP600125-treated cultures versus 996.41±166.19 pg/ml in vehicle-treated cultures, p=0.007; Figure 3A). Although it did not reach statistical significance, IL-6 mRNA was found to be downregulated with SP600125 (2.1±0.5 IL-6 mRNA fold expression in SP600125-treated cultures versus 3.03±0.67 fold in vehicle-treated cultures, p=0.126). These data indicate a role of p38 MAPK and JNK pathways in IL-6 induction by exogenous TGF-β1.

Bottom Line: Incubation of HTM cells with TGF-beta1 (5 ng/ml) resulted in a significant increase in the protein and mRNA levels of IL-6, which was significantly diminished in the presence of the inhibitors for p38 MAPK or JNK.In addition, the presence of inhibitors for the p38 MAPK, ERK, and TGF-beta1 pathways significantly decreased the increased expression of IL-6 by cyclic mechanical stress.We also found that p38 MAPK might play a contributing role in the maintenance of such a loop.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology, Duke University, Durham, NC 27710, USA. paloma.liton@duke.edu

ABSTRACT

Purpose: To investigate the relationship between transforming growth factor beta-1 (TGF-beta1) and interleukin-6 (IL-6) in human trabecular meshwork (HTM) cells as well as to identify the signaling pathway/s involved in the increased IL-6 expression that occurs in response to mechanical stress and TGF-beta1.

Methods: All experiments were performed in confluent monolayers of HTM cells at passage 3. Secreted IL-6 was quantified by ELISA. Levels of IL-6 mRNA were evaluated by polymerase chain reaction (PCR) analysis. Activation of the IL-6 and TGF-beta1 promoters was monitored by measuring secreted alkaline phosphatase protein (SEAP) released into the culture medium by HTM cells infected with an adenovirus expressing the SEAP reporter gene that was controlled by either the IL-6 promoter (AdIL6-SEAP) or the TGF-beta1 promoter (AdTGFbeta1-SEAP). Cyclic mechanical stress (5% elongation, one cycle per second) was applied using the Flexcell System. Reagents used in this study included human TGF-beta1, human IL-6, and the inhibitors for the p38 mitogen-activated protein kinase (MAPK; SB202190), c-jun NH2-terminal kinase (JNK; SP600125), extracellular-regulating kinase (ERK; PD98059), and TGF type I activin receptor (SB431542).

Results: Incubation of HTM cells with TGF-beta1 (5 ng/ml) resulted in a significant increase in the protein and mRNA levels of IL-6, which was significantly diminished in the presence of the inhibitors for p38 MAPK or JNK. No transcriptional activation of the exogenous IL-6 promoter was observed following TGF-beta1 treatment. In addition, the presence of inhibitors for the p38 MAPK, ERK, and TGF-beta1 pathways significantly decreased the increased expression of IL-6 by cyclic mechanical stress. Furthermore, exposure of HTM cells to IL-6 (100 ng/ml) demonstrated the transcriptional activation of TGF-beta1 promoter, which was severely impaired by blocking the p38 MAPK pathway.

Conclusions: Our results indicate that TGF-beta1 participates in the regulation of basal expression and the stretch-induced expression of IL-6 and suggest the possible existence in cultured HTM cells of an autocrine loop between IL-6 and TGF-beta1. We also found that p38 MAPK might play a contributing role in the maintenance of such a loop.

Show MeSH
Related in: MedlinePlus