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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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Vitreous humor contains a gap junctional intercellular communication-promoting activity with properties of an FGF. 50 ng/ml FGF-2 in M199/BOTS or E10 chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH) was incubated with heparin-conjugated Affigel beads in the presence of 0.1 M NaCl or 0.6 M NaCl as described in Materials and methods. The beads were pelleted, and after removal of the supernatant (heparin 0.1 M or 0.6 M NaCl unbound), FGF-like activity was eluted with 2.5 M NaCl (heparin 2.5 M NaCl eluate). The fractions were then brought to 0.15 M NaCl by repeated rounds of concentration and dilution with M199 medium. DCDMLs were cultured for 2 d in M199/BOTS with no additions (control), in M199/BOTS with unfractionated FGF-2 (50 ng/ml FGF-2 input), in unfractionated 30% VH (30% VH input), or with the indicated heparin-Affigel fraction. Gap junctional intercellular communication was assessed as described in the legend to Fig. 1 A; only Lucifer yellow immunofluorescence is presented. In all cases, rhodamine-dextran was confined to a single row of cells immediately bordering the wound. Representative results from three independent experiments; similar results were obtained with vitreous body conditioned medium (not shown). Note that the heparin beads quantitatively bound the communication-promoting activity of even the highest concentration of FGF tolerated by lens cells (50 ng/ml). The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment. Only the values obtained for the 30% VH input and the heparin 2.5 M NaCl eluate were significantly higher than control (P < 0.03). Bar, 50 μm.
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fig9: Vitreous humor contains a gap junctional intercellular communication-promoting activity with properties of an FGF. 50 ng/ml FGF-2 in M199/BOTS or E10 chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH) was incubated with heparin-conjugated Affigel beads in the presence of 0.1 M NaCl or 0.6 M NaCl as described in Materials and methods. The beads were pelleted, and after removal of the supernatant (heparin 0.1 M or 0.6 M NaCl unbound), FGF-like activity was eluted with 2.5 M NaCl (heparin 2.5 M NaCl eluate). The fractions were then brought to 0.15 M NaCl by repeated rounds of concentration and dilution with M199 medium. DCDMLs were cultured for 2 d in M199/BOTS with no additions (control), in M199/BOTS with unfractionated FGF-2 (50 ng/ml FGF-2 input), in unfractionated 30% VH (30% VH input), or with the indicated heparin-Affigel fraction. Gap junctional intercellular communication was assessed as described in the legend to Fig. 1 A; only Lucifer yellow immunofluorescence is presented. In all cases, rhodamine-dextran was confined to a single row of cells immediately bordering the wound. Representative results from three independent experiments; similar results were obtained with vitreous body conditioned medium (not shown). Note that the heparin beads quantitatively bound the communication-promoting activity of even the highest concentration of FGF tolerated by lens cells (50 ng/ml). The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment. Only the values obtained for the 30% VH input and the heparin 2.5 M NaCl eluate were significantly higher than control (P < 0.03). Bar, 50 μm.

Mentions: A defining characteristic of all members of the FGF family is their high affinity for heparin, which distinguishes them from other growth factors known or suspected to be present in the eye (Ornitz, 2000). Control experiments verified that the gap junctional communication-promoting activity of 50 ng/ml recombinant FGF was quantitatively bound to heparin-Affigel beads in 0.1 M NaCl and was eluted from the beads only when the salt concentration was raised to 2.5 M (Fig. 9 , top row). The ability of 30% VH (or, not shown, vitreous body conditioned medium) to enhance intercellular transfer of Lucifer yellow was abolished if the sample was incubated with immobilized heparin in 0.1 M NaCl, and only an insignificant amount of activity remained if the adsorption was carried out in 0.6 M salt (Fig. 9, bottom row). Incubation of the vitreous-treated heparin beads with 2.5 M NaCl released an activity that increased intercellular dye transfer an average of 2.1-fold and (not shown) induced sustained activation of ERK. This elution behavior is typical of an FGF (Seed et al., 1988) and has been used to purify FGFs from bovine (Schulz et al., 1993) and chick (Mascarelli et al., 1987) vitreous humor. The δ-crystallin–promoting activity of vitreous also binds to heparin with high affinity (Le and Musil, 2001), strongly supporting (although not unequivocally proving) a role for one or more of the ∼23 members of the FGF family in both fiber differentiation and upregulation 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)

Vitreous humor contains a gap junctional intercellular communication-promoting activity with properties of an FGF. 50 ng/ml FGF-2 in M199/BOTS or E10 chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH) was incubated with heparin-conjugated Affigel beads in the presence of 0.1 M NaCl or 0.6 M NaCl as described in Materials and methods. The beads were pelleted, and after removal of the supernatant (heparin 0.1 M or 0.6 M NaCl unbound), FGF-like activity was eluted with 2.5 M NaCl (heparin 2.5 M NaCl eluate). The fractions were then brought to 0.15 M NaCl by repeated rounds of concentration and dilution with M199 medium. DCDMLs were cultured for 2 d in M199/BOTS with no additions (control), in M199/BOTS with unfractionated FGF-2 (50 ng/ml FGF-2 input), in unfractionated 30% VH (30% VH input), or with the indicated heparin-Affigel fraction. Gap junctional intercellular communication was assessed as described in the legend to Fig. 1 A; only Lucifer yellow immunofluorescence is presented. In all cases, rhodamine-dextran was confined to a single row of cells immediately bordering the wound. Representative results from three independent experiments; similar results were obtained with vitreous body conditioned medium (not shown). Note that the heparin beads quantitatively bound the communication-promoting activity of even the highest concentration of FGF tolerated by lens cells (50 ng/ml). The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment. Only the values obtained for the 30% VH input and the heparin 2.5 M NaCl eluate were significantly higher than control (P < 0.03). Bar, 50 μm.
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Related In: Results  -  Collection

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fig9: Vitreous humor contains a gap junctional intercellular communication-promoting activity with properties of an FGF. 50 ng/ml FGF-2 in M199/BOTS or E10 chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH) was incubated with heparin-conjugated Affigel beads in the presence of 0.1 M NaCl or 0.6 M NaCl as described in Materials and methods. The beads were pelleted, and after removal of the supernatant (heparin 0.1 M or 0.6 M NaCl unbound), FGF-like activity was eluted with 2.5 M NaCl (heparin 2.5 M NaCl eluate). The fractions were then brought to 0.15 M NaCl by repeated rounds of concentration and dilution with M199 medium. DCDMLs were cultured for 2 d in M199/BOTS with no additions (control), in M199/BOTS with unfractionated FGF-2 (50 ng/ml FGF-2 input), in unfractionated 30% VH (30% VH input), or with the indicated heparin-Affigel fraction. Gap junctional intercellular communication was assessed as described in the legend to Fig. 1 A; only Lucifer yellow immunofluorescence is presented. In all cases, rhodamine-dextran was confined to a single row of cells immediately bordering the wound. Representative results from three independent experiments; similar results were obtained with vitreous body conditioned medium (not shown). Note that the heparin beads quantitatively bound the communication-promoting activity of even the highest concentration of FGF tolerated by lens cells (50 ng/ml). The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment. Only the values obtained for the 30% VH input and the heparin 2.5 M NaCl eluate were significantly higher than control (P < 0.03). Bar, 50 μm.
Mentions: A defining characteristic of all members of the FGF family is their high affinity for heparin, which distinguishes them from other growth factors known or suspected to be present in the eye (Ornitz, 2000). Control experiments verified that the gap junctional communication-promoting activity of 50 ng/ml recombinant FGF was quantitatively bound to heparin-Affigel beads in 0.1 M NaCl and was eluted from the beads only when the salt concentration was raised to 2.5 M (Fig. 9 , top row). The ability of 30% VH (or, not shown, vitreous body conditioned medium) to enhance intercellular transfer of Lucifer yellow was abolished if the sample was incubated with immobilized heparin in 0.1 M NaCl, and only an insignificant amount of activity remained if the adsorption was carried out in 0.6 M salt (Fig. 9, bottom row). Incubation of the vitreous-treated heparin beads with 2.5 M NaCl released an activity that increased intercellular dye transfer an average of 2.1-fold and (not shown) induced sustained activation of ERK. This elution behavior is typical of an FGF (Seed et al., 1988) and has been used to purify FGFs from bovine (Schulz et al., 1993) and chick (Mascarelli et al., 1987) vitreous humor. The δ-crystallin–promoting activity of vitreous also binds to heparin with high affinity (Le and Musil, 2001), strongly supporting (although not unequivocally proving) a role for one or more of the ∼23 members of the FGF family in both fiber differentiation and upregulation 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