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A novel role for FGF and extracellular signal-regulated kinase in gap junction-mediated intercellular communication in the lens.

Le AC, Musil LS - J. Cell Biol. (2001)

Bottom Line: Insulin and insulin-like growth factor 1, as potent as FGF in inducing lens cell differentiation, had no effect on gap junctions.These findings support a model in which regional differences in FGF signaling through the ERK pathway lead to the asymmetry in gap junctional coupling required for proper lens function.Our results also identify upregulation of intercellular communication as a new function for sustained ERK activation and change the current paradigm that ERKs only negatively regulate gap junction channel activity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Division, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA.

ABSTRACT
Gap junction-mediated intercellular coupling is higher in the equatorial region of the lens than at either pole, a property believed to be essential for lens transparency. We show that fibroblast growth factor (FGF) upregulates gap junctional intercellular dye transfer in primary cultures of embryonic chick lens cells without detectably increasing either gap junction protein (connexin) synthesis or assembly. Insulin and insulin-like growth factor 1, as potent as FGF in inducing lens cell differentiation, had no effect on gap junctions. FGF induced sustained activation of extracellular signal-regulated kinase (ERK) in lens cells, an event necessary and sufficient to increase gap junctional coupling. We also identify vitreous humor as an in vivo source of an FGF-like intercellular communication-promoting activity and show that FGF-induced ERK activation in the intact lens is higher in the equatorial region than in polar and core fibers. These findings support a model in which regional differences in FGF signaling through the ERK pathway lead to the asymmetry in gap junctional coupling required for proper lens function. Our results also identify upregulation of intercellular communication as a new function for sustained ERK activation and change the current paradigm that ERKs only negatively regulate gap junction channel activity.

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FGF-mediated upregulation of gap junctional intercellular communication in chick lens cells requires sustained activation of ERK. (A) 3-d-old DCDMLs cultured in M199/BOTS were incubated for 15 min at 37°C in the absence (lane 1; control) or presence of 15 ng/ml FGF-2. In lanes 3–5, 15 μM UO126 was then added to inhibit further activation of ERK. At the indicated times, the cells were solubilized in SDS and whole cell lysates were assessed for phosphoERK immunoreactivity. pERK immunoreactivity is lower in lane 5 than in the lane 1 control because UO126 reduces the level of basal ERK activity in DCDML cultures after long-term (>4 h) treatment (Le and Musil, 2001). (B) 3 d after plating, DCDML cells were incubated for an additional 24 h in either the absence of FGF (24 h −FGF), in the presence of 15 ng/ml FGF-2 (24 h + FGF), or for 4, 8, or 12 h in the presence of 15 ng/ml FGF-2 before addition of 15 uM UO126 and a further 20, 16, or 12 h incubation (respectively) in FGF plus UO126. The cells were then assayed for gap junction–mediated intercellular communication as described in the legend to Fig. 1 A. Only Lucifer yellow immunofluorescence is presented; rhodamine-dextran was confined to a single row of cells immediately bordering the wound. The values given to the right of the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment; n, number of independent experiments. The asterisks denote values significantly different from control (P < 0.05) as assessed by the two-tailed paired Student's t test; P for 12 h plus FGF compared with untreated control was 0.01. Note that at least 12 h of ERK activation was required for FGF to stimulate intercellular transfer of Lucifer yellow. Bar, 50 μm.
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fig7: FGF-mediated upregulation of gap junctional intercellular communication in chick lens cells requires sustained activation of ERK. (A) 3-d-old DCDMLs cultured in M199/BOTS were incubated for 15 min at 37°C in the absence (lane 1; control) or presence of 15 ng/ml FGF-2. In lanes 3–5, 15 μM UO126 was then added to inhibit further activation of ERK. At the indicated times, the cells were solubilized in SDS and whole cell lysates were assessed for phosphoERK immunoreactivity. pERK immunoreactivity is lower in lane 5 than in the lane 1 control because UO126 reduces the level of basal ERK activity in DCDML cultures after long-term (>4 h) treatment (Le and Musil, 2001). (B) 3 d after plating, DCDML cells were incubated for an additional 24 h in either the absence of FGF (24 h −FGF), in the presence of 15 ng/ml FGF-2 (24 h + FGF), or for 4, 8, or 12 h in the presence of 15 ng/ml FGF-2 before addition of 15 uM UO126 and a further 20, 16, or 12 h incubation (respectively) in FGF plus UO126. The cells were then assayed for gap junction–mediated intercellular communication as described in the legend to Fig. 1 A. Only Lucifer yellow immunofluorescence is presented; rhodamine-dextran was confined to a single row of cells immediately bordering the wound. The values given to the right of the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment; n, number of independent experiments. The asterisks denote values significantly different from control (P < 0.05) as assessed by the two-tailed paired Student's t test; P for 12 h plus FGF compared with untreated control was 0.01. Note that at least 12 h of ERK activation was required for FGF to stimulate intercellular transfer of Lucifer yellow. Bar, 50 μm.

Mentions: To determine whether the observed correlation between FGF-mediated sustained ERK activation and upregulation of intercellular coupling reflected a cause-and-effect relationship, the duration of ERK activity was varied using the MEK inhibitor UO126 (Fig. 7) . 15 ng/ml FGF-2 was added to lens cells and, after 4, 8, or 12 h, UO126 was included and the incubation continued for a total of 24 h in FGF. PhosphoERK blots verified that addition of UO126 reduced the level of activated ERK to near that of untreated controls within 90 min (Fig. 7 A, lane 3), and that this inhibition persisted for over 8 h (lane 5). Scrape-load dye transfer analysis revealed that ERK had to be active for at least 12 h in order for FGF to increase gap junctional coupling (Fig. 7 B). Given that expression of CA-MEK also induces long-term activation of ERKs and increased junctional coupling (Fig. 5), these findings establish a new role for sustained ERK activation in the regulation of intercellular communication.


A novel role for FGF and extracellular signal-regulated kinase in gap junction-mediated intercellular communication in the lens.

Le AC, Musil LS - J. Cell Biol. (2001)

FGF-mediated upregulation of gap junctional intercellular communication in chick lens cells requires sustained activation of ERK. (A) 3-d-old DCDMLs cultured in M199/BOTS were incubated for 15 min at 37°C in the absence (lane 1; control) or presence of 15 ng/ml FGF-2. In lanes 3–5, 15 μM UO126 was then added to inhibit further activation of ERK. At the indicated times, the cells were solubilized in SDS and whole cell lysates were assessed for phosphoERK immunoreactivity. pERK immunoreactivity is lower in lane 5 than in the lane 1 control because UO126 reduces the level of basal ERK activity in DCDML cultures after long-term (>4 h) treatment (Le and Musil, 2001). (B) 3 d after plating, DCDML cells were incubated for an additional 24 h in either the absence of FGF (24 h −FGF), in the presence of 15 ng/ml FGF-2 (24 h + FGF), or for 4, 8, or 12 h in the presence of 15 ng/ml FGF-2 before addition of 15 uM UO126 and a further 20, 16, or 12 h incubation (respectively) in FGF plus UO126. The cells were then assayed for gap junction–mediated intercellular communication as described in the legend to Fig. 1 A. Only Lucifer yellow immunofluorescence is presented; rhodamine-dextran was confined to a single row of cells immediately bordering the wound. The values given to the right of the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment; n, number of independent experiments. The asterisks denote values significantly different from control (P < 0.05) as assessed by the two-tailed paired Student's t test; P for 12 h plus FGF compared with untreated control was 0.01. Note that at least 12 h of ERK activation was required for FGF to stimulate intercellular transfer of Lucifer yellow. Bar, 50 μm.
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fig7: FGF-mediated upregulation of gap junctional intercellular communication in chick lens cells requires sustained activation of ERK. (A) 3-d-old DCDMLs cultured in M199/BOTS were incubated for 15 min at 37°C in the absence (lane 1; control) or presence of 15 ng/ml FGF-2. In lanes 3–5, 15 μM UO126 was then added to inhibit further activation of ERK. At the indicated times, the cells were solubilized in SDS and whole cell lysates were assessed for phosphoERK immunoreactivity. pERK immunoreactivity is lower in lane 5 than in the lane 1 control because UO126 reduces the level of basal ERK activity in DCDML cultures after long-term (>4 h) treatment (Le and Musil, 2001). (B) 3 d after plating, DCDML cells were incubated for an additional 24 h in either the absence of FGF (24 h −FGF), in the presence of 15 ng/ml FGF-2 (24 h + FGF), or for 4, 8, or 12 h in the presence of 15 ng/ml FGF-2 before addition of 15 uM UO126 and a further 20, 16, or 12 h incubation (respectively) in FGF plus UO126. The cells were then assayed for gap junction–mediated intercellular communication as described in the legend to Fig. 1 A. Only Lucifer yellow immunofluorescence is presented; rhodamine-dextran was confined to a single row of cells immediately bordering the wound. The values given to the right of the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment; n, number of independent experiments. The asterisks denote values significantly different from control (P < 0.05) as assessed by the two-tailed paired Student's t test; P for 12 h plus FGF compared with untreated control was 0.01. Note that at least 12 h of ERK activation was required for FGF to stimulate intercellular transfer of Lucifer yellow. Bar, 50 μm.
Mentions: To determine whether the observed correlation between FGF-mediated sustained ERK activation and upregulation of intercellular coupling reflected a cause-and-effect relationship, the duration of ERK activity was varied using the MEK inhibitor UO126 (Fig. 7) . 15 ng/ml FGF-2 was added to lens cells and, after 4, 8, or 12 h, UO126 was included and the incubation continued for a total of 24 h in FGF. PhosphoERK blots verified that addition of UO126 reduced the level of activated ERK to near that of untreated controls within 90 min (Fig. 7 A, lane 3), and that this inhibition persisted for over 8 h (lane 5). Scrape-load dye transfer analysis revealed that ERK had to be active for at least 12 h in order for FGF to increase gap junctional coupling (Fig. 7 B). Given that expression of CA-MEK also induces long-term activation of ERKs and increased junctional coupling (Fig. 5), these findings establish a new role for sustained ERK activation in the regulation of intercellular communication.

Bottom Line: Insulin and insulin-like growth factor 1, as potent as FGF in inducing lens cell differentiation, had no effect on gap junctions.These findings support a model in which regional differences in FGF signaling through the ERK pathway lead to the asymmetry in gap junctional coupling required for proper lens function.Our results also identify upregulation of intercellular communication as a new function for sustained ERK activation and change the current paradigm that ERKs only negatively regulate gap junction channel activity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Medicine Division, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201, USA.

ABSTRACT
Gap junction-mediated intercellular coupling is higher in the equatorial region of the lens than at either pole, a property believed to be essential for lens transparency. We show that fibroblast growth factor (FGF) upregulates gap junctional intercellular dye transfer in primary cultures of embryonic chick lens cells without detectably increasing either gap junction protein (connexin) synthesis or assembly. Insulin and insulin-like growth factor 1, as potent as FGF in inducing lens cell differentiation, had no effect on gap junctions. FGF induced sustained activation of extracellular signal-regulated kinase (ERK) in lens cells, an event necessary and sufficient to increase gap junctional coupling. We also identify vitreous humor as an in vivo source of an FGF-like intercellular communication-promoting activity and show that FGF-induced ERK activation in the intact lens is higher in the equatorial region than in polar and core fibers. These findings support a model in which regional differences in FGF signaling through the ERK pathway lead to the asymmetry in gap junctional coupling required for proper lens function. Our results also identify upregulation of intercellular communication as a new function for sustained ERK activation and change the current paradigm that ERKs only negatively regulate gap junction channel activity.

Show MeSH
Related in: MedlinePlus