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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 correlates with sustained activation of ERK. (A and B) The indicated growth factor was added directly to the culture medium of 3-d-old DCDMLs plated and maintained in M199/BOTS. After the indicated period (15 min to 48 h) at 37°C, the cultures were solubilized in SDS and whole cell lysates were assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Control cells received no added growth factor. After quantitation of the pERK bands by densitometry, the blots were stripped and reprobed for total ERK (both activated and inactive; totERK) to verify that the level of ERK protein remained constant throughout the experiment; results are shown for 15 ng/ml FGF-2 and 1 μg/ml insulin. The data are graphed as the fold increase in pERK immunoreactivity in treated cells relative to the amount of pERK in untreated time 0 controls (first panel in each time course) within the same experiment. The data shown are representative of three independent experiments. Note that the time points analyzed in Fig. 6 A differ between the various treatments.
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fig6: FGF-mediated upregulation of gap junctional intercellular communication in chick lens cells correlates with sustained activation of ERK. (A and B) The indicated growth factor was added directly to the culture medium of 3-d-old DCDMLs plated and maintained in M199/BOTS. After the indicated period (15 min to 48 h) at 37°C, the cultures were solubilized in SDS and whole cell lysates were assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Control cells received no added growth factor. After quantitation of the pERK bands by densitometry, the blots were stripped and reprobed for total ERK (both activated and inactive; totERK) to verify that the level of ERK protein remained constant throughout the experiment; results are shown for 15 ng/ml FGF-2 and 1 μg/ml insulin. The data are graphed as the fold increase in pERK immunoreactivity in treated cells relative to the amount of pERK in untreated time 0 controls (first panel in each time course) within the same experiment. The data shown are representative of three independent experiments. Note that the time points analyzed in Fig. 6 A differ between the various treatments.

Mentions: The demonstration that ERK activation is both necessary and sufficient to upregulate intercellular coupling in DCDMLs created an apparent paradox in that insulin and IGF-1 also stimulate ERKs in chick and rodent lens cells (Le and Musil, 2001), yet have no effect on gap junctions. One of the most important determinants of the biological outcome of MAP kinase signaling is the length of time that a stimulus activates ERKs (Marshall, 1995). As assessed by quantitative antiphosphoERK immunoblotting, 1–15 ng/ml FGF-2, FGF-1 with or without its cofactor heparin, 1 μg/ml insulin, or (not shown) 15 ng/ml IGF-1 all comparably activated ERK within 15 min of addition to DCDML cultures (Fig. 6) . Only the treatments capable of upregulating junctional communication (15 ng/ml FGF-2 or FGF-1 plus heparin; Fig. 2 A), however, sustained ERK activation for more than 8 h.


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 correlates with sustained activation of ERK. (A and B) The indicated growth factor was added directly to the culture medium of 3-d-old DCDMLs plated and maintained in M199/BOTS. After the indicated period (15 min to 48 h) at 37°C, the cultures were solubilized in SDS and whole cell lysates were assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Control cells received no added growth factor. After quantitation of the pERK bands by densitometry, the blots were stripped and reprobed for total ERK (both activated and inactive; totERK) to verify that the level of ERK protein remained constant throughout the experiment; results are shown for 15 ng/ml FGF-2 and 1 μg/ml insulin. The data are graphed as the fold increase in pERK immunoreactivity in treated cells relative to the amount of pERK in untreated time 0 controls (first panel in each time course) within the same experiment. The data shown are representative of three independent experiments. Note that the time points analyzed in Fig. 6 A differ between the various treatments.
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Related In: Results  -  Collection

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fig6: FGF-mediated upregulation of gap junctional intercellular communication in chick lens cells correlates with sustained activation of ERK. (A and B) The indicated growth factor was added directly to the culture medium of 3-d-old DCDMLs plated and maintained in M199/BOTS. After the indicated period (15 min to 48 h) at 37°C, the cultures were solubilized in SDS and whole cell lysates were assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Control cells received no added growth factor. After quantitation of the pERK bands by densitometry, the blots were stripped and reprobed for total ERK (both activated and inactive; totERK) to verify that the level of ERK protein remained constant throughout the experiment; results are shown for 15 ng/ml FGF-2 and 1 μg/ml insulin. The data are graphed as the fold increase in pERK immunoreactivity in treated cells relative to the amount of pERK in untreated time 0 controls (first panel in each time course) within the same experiment. The data shown are representative of three independent experiments. Note that the time points analyzed in Fig. 6 A differ between the various treatments.
Mentions: The demonstration that ERK activation is both necessary and sufficient to upregulate intercellular coupling in DCDMLs created an apparent paradox in that insulin and IGF-1 also stimulate ERKs in chick and rodent lens cells (Le and Musil, 2001), yet have no effect on gap junctions. One of the most important determinants of the biological outcome of MAP kinase signaling is the length of time that a stimulus activates ERKs (Marshall, 1995). As assessed by quantitative antiphosphoERK immunoblotting, 1–15 ng/ml FGF-2, FGF-1 with or without its cofactor heparin, 1 μg/ml insulin, or (not shown) 15 ng/ml IGF-1 all comparably activated ERK within 15 min of addition to DCDML cultures (Fig. 6) . Only the treatments capable of upregulating junctional communication (15 ng/ml FGF-2 or FGF-1 plus heparin; Fig. 2 A), however, sustained ERK activation for more than 8 h.

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