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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 IL-6 on the TGF-β1 promoter activity. Three independent primary cultures of HTM cells were infected with AdTGFβ1-SEAP (m.o.i=20 pfu/cell) and serum-starved for 16 h at day 1 post-infection. A: Cells were incubated in serum-free media with IL-6 (100 ng/ml). SEAP activity was assayed in the culture media at the indicated times. B: Cells were pre-treated for 2 h with the MAPK inhibitors and then incubated in serum-free media with IL-6 (100 ng/ml) in the presence of the inhibitors (10 μM). SEAP activity in the culture media was assayed at 6 h. The asterisk indicates that p is less than 0.05. Statistical significance between groups was assessed by the paired Student's t-test (n=3).
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f5: Effect of IL-6 on the TGF-β1 promoter activity. Three independent primary cultures of HTM cells were infected with AdTGFβ1-SEAP (m.o.i=20 pfu/cell) and serum-starved for 16 h at day 1 post-infection. A: Cells were incubated in serum-free media with IL-6 (100 ng/ml). SEAP activity was assayed in the culture media at the indicated times. B: Cells were pre-treated for 2 h with the MAPK inhibitors and then incubated in serum-free media with IL-6 (100 ng/ml) in the presence of the inhibitors (10 μM). SEAP activity in the culture media was assayed at 6 h. The asterisk indicates that p is less than 0.05. Statistical significance between groups was assessed by the paired Student's t-test (n=3).

Mentions: Several works have reported a cross talk between IL-6 and TGF-β1 signaling pathways in different cell lines [17-20]. We wondered whether IL-6 might upregulate TGF-β1 expression in HTM cells and thus initiate an autocrine loop similar to the one described in activated pancreatic stellate cells [21]. For this purpose, we treated three different HTM primary cultures previously infected with the recombinant adenovirus, AdTGFβ1-SEAP, with IL-6 (100 ng/ml). Such IL-6 concentration, which significantly increased the permeability of SC endothelial cells as well as outflow facility in perfused porcine anterior segments [9], has been used for similar studies in other cell types [17]. As shown in Figure 5A, exogenous IL-6 quickly induced the transcriptional activation of TGF-β1 promoter at 3 h (112.79±33.4 relative fluorescence units [RFU] in IL-6-treated cultures versus 52.34±12.79 RFU in control cultures, p=0.042) and 6 h post treatment (393.44±132.45 RFU  in IL-6-treated cultures versus 7.96±26.07 RFU in control cultures, p=0.034) compared to control levels. To identify the intracellular pathways participating in such activation, we repeated the experiment in the presence of MAPK inhibitors. Whereas PD98059 and SP600125 did not exert any major effect, SB202190 significantly decreased the percentage of induction in SEAP activity compared to the control (394.75±74.2 RFU in vehicle-treated cultures versus 168.43±53.8 RFU in SB202190-treated cultures, p=0.012), thus suggesting a role of p38 MAPK in the upregulation of TGF-β1 by IL-6 (Figure 5B).


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 IL-6 on the TGF-β1 promoter activity. Three independent primary cultures of HTM cells were infected with AdTGFβ1-SEAP (m.o.i=20 pfu/cell) and serum-starved for 16 h at day 1 post-infection. A: Cells were incubated in serum-free media with IL-6 (100 ng/ml). SEAP activity was assayed in the culture media at the indicated times. B: Cells were pre-treated for 2 h with the MAPK inhibitors and then incubated in serum-free media with IL-6 (100 ng/ml) in the presence of the inhibitors (10 μM). SEAP activity in the culture media was assayed at 6 h. The asterisk indicates that p is less than 0.05. Statistical significance between groups was assessed by the paired Student's t-test (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Effect of IL-6 on the TGF-β1 promoter activity. Three independent primary cultures of HTM cells were infected with AdTGFβ1-SEAP (m.o.i=20 pfu/cell) and serum-starved for 16 h at day 1 post-infection. A: Cells were incubated in serum-free media with IL-6 (100 ng/ml). SEAP activity was assayed in the culture media at the indicated times. B: Cells were pre-treated for 2 h with the MAPK inhibitors and then incubated in serum-free media with IL-6 (100 ng/ml) in the presence of the inhibitors (10 μM). SEAP activity in the culture media was assayed at 6 h. The asterisk indicates that p is less than 0.05. Statistical significance between groups was assessed by the paired Student's t-test (n=3).
Mentions: Several works have reported a cross talk between IL-6 and TGF-β1 signaling pathways in different cell lines [17-20]. We wondered whether IL-6 might upregulate TGF-β1 expression in HTM cells and thus initiate an autocrine loop similar to the one described in activated pancreatic stellate cells [21]. For this purpose, we treated three different HTM primary cultures previously infected with the recombinant adenovirus, AdTGFβ1-SEAP, with IL-6 (100 ng/ml). Such IL-6 concentration, which significantly increased the permeability of SC endothelial cells as well as outflow facility in perfused porcine anterior segments [9], has been used for similar studies in other cell types [17]. As shown in Figure 5A, exogenous IL-6 quickly induced the transcriptional activation of TGF-β1 promoter at 3 h (112.79±33.4 relative fluorescence units [RFU] in IL-6-treated cultures versus 52.34±12.79 RFU in control cultures, p=0.042) and 6 h post treatment (393.44±132.45 RFU  in IL-6-treated cultures versus 7.96±26.07 RFU in control cultures, p=0.034) compared to control levels. To identify the intracellular pathways participating in such activation, we repeated the experiment in the presence of MAPK inhibitors. Whereas PD98059 and SP600125 did not exert any major effect, SB202190 significantly decreased the percentage of induction in SEAP activity compared to the control (394.75±74.2 RFU in vehicle-treated cultures versus 168.43±53.8 RFU in SB202190-treated cultures, p=0.012), thus suggesting a role of p38 MAPK in the upregulation of TGF-β1 by IL-6 (Figure 5B).

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