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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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Related in: MedlinePlus

FGF, but not insulin, increases gap junctional intercellular communication in chick lens DCDMLs. 1-d-old DCDML cultures prepared from E10 chick lenses were incubated for an additional 48 h in either the absence (control) or presence of the indicated growth factor. Gap junction–mediated intercellular communication was then assessed using the scrape-loading/dye transfer assay as described in Materials and methods. Cells were scrape-loaded with rhodamine-dextran mixed with either Lucifer yellow (A) or biocytin (B). After either 8 (A) or 2 min (B), the cells were fixed and the dyes visualized by fluorescence microscopy. The Mr = 10 kD rhodamine-dextran (A and B; RD) remained confined to the cells at the wound edge into which dye had been directly introduced during the scrape-loading process. In contrast, Lucifer yellow (A; LY) and biocytin (B; biocytin) were transferred to adjacent cells via open gap junctional channels. Each panel depicts a portion of the right half of the scrape/load wound. Superposition of the staining pattern of the two dyes is shown to the immediate right of the rhodamine-dextran images (LY/RD and biocytin/RD) Graphs at far right: averaged fluorescence plot profiles of the corresponding Lucifer yellow (A) or biocytin (B) images. In each case, the solid line represents the average of three fluorescence profiles generated as described in Material and methods, and the dotted lines indicate standard deviation (blue and magenta, respectively). The distance of gap junction–mediated dye transfer into the monolayer (d) is indicated; d for FGF-treated monolayers was essentially the same if the concentration of Lucifer yellow was reduced from 1 to 0.04% (data not shown). Bars, 100 μm.
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fig1: FGF, but not insulin, increases gap junctional intercellular communication in chick lens DCDMLs. 1-d-old DCDML cultures prepared from E10 chick lenses were incubated for an additional 48 h in either the absence (control) or presence of the indicated growth factor. Gap junction–mediated intercellular communication was then assessed using the scrape-loading/dye transfer assay as described in Materials and methods. Cells were scrape-loaded with rhodamine-dextran mixed with either Lucifer yellow (A) or biocytin (B). After either 8 (A) or 2 min (B), the cells were fixed and the dyes visualized by fluorescence microscopy. The Mr = 10 kD rhodamine-dextran (A and B; RD) remained confined to the cells at the wound edge into which dye had been directly introduced during the scrape-loading process. In contrast, Lucifer yellow (A; LY) and biocytin (B; biocytin) were transferred to adjacent cells via open gap junctional channels. Each panel depicts a portion of the right half of the scrape/load wound. Superposition of the staining pattern of the two dyes is shown to the immediate right of the rhodamine-dextran images (LY/RD and biocytin/RD) Graphs at far right: averaged fluorescence plot profiles of the corresponding Lucifer yellow (A) or biocytin (B) images. In each case, the solid line represents the average of three fluorescence profiles generated as described in Material and methods, and the dotted lines indicate standard deviation (blue and magenta, respectively). The distance of gap junction–mediated dye transfer into the monolayer (d) is indicated; d for FGF-treated monolayers was essentially the same if the concentration of Lucifer yellow was reduced from 1 to 0.04% (data not shown). Bars, 100 μm.

Mentions: Although cells isolated from the lenses of several vertebrate species will take on fiber-like characteristics when maintained ex vivo, epithelial-to-fiber differentiation is most completely recapitulated in primary cultures of embryonic chick lens epithelial cells (Menko et al., 1984, 1987; TenBroek et al., 1994). This system also most faithfully reiterates fiber-type gap junction formation (FitzGerald and Goodenough, 1986; Menko et al., 1987; Jiang et al., 1993). DCDML cultures prepared from E10 chick lenses as described by Menko et al. (1984) (termed DCDMLs) are enriched in cells originating from the equatorial region of the lens, the area that undergoes both fiber differentiation and upregulation of gap junctional intercellular coupling in vivo. We have shown previously that DCDML cultures continue to divide and differentiate in a defined, growth factor–free medium (M199/BOTS), albeit to a more limited extent than in the presence of serum. They also remain coupled by gap junctions and express all three known chick lens connexins (Cx43, Cx45.6, and Cx56; Le and Musil, 1998). Addition of either purified recombinant FGF, insulin, or IGF-1 to serum-free DCDML cultures stimulates both cell proliferation and the expression of fiber differentiation markers (Le and Musil, 2001). We tested whether any of these growth factors affected gap junctional intercellular communication using the scrape-loading/dye transfer assay (el-Fouly et al., 1987). In the experiments depicted in Fig. 1 A, the membrane impermeant, low-molecular weight fluorescent dye Lucifer yellow was introduced into DCDML cultures by scraping the monolayer with a 27-gauge needle. As evaluated by the spread of Lucifer yellow from cells at the scrape border to adjoining unwounded cells, cultures maintained in the absence of added growth factor were moderately well coupled by gap junctions. Intercellular transfer of Lucifer yellow was increased an average of 2.72-fold (± 0.48; Fig. 2 A) when cells were cultured for 2 d in 15 ng/ml FGF-2. In contrast, neither 1 μg/ml insulin nor (not shown) 15 ng/ml of the IGF-1 analogue R3IGF-1, although comparable to 15 ng/ml FGF-2 in their ability to upregulate expression of fiber differentiation markers (Le and Musil, 2001), had any detectable effect on Lucifer yellow transfer. Qualitatively similar results were obtained with biocytin, a gap junction–permeable compound with physical properties distinct from those of Lucifer yellow that is a more sensitive tracer of gap junctional communication in lens cells (Le and Musil, 1998; Fig. 1 B). Cultures treated with FGF were indistinguishable from those exposed to insulin/IGF with regard to cell number, protein content, and incorporation of [35S]methionine into proteins synthesized during a 4 h pulse (data not shown). The increase in intercellular dye transfer in FGF-treated cells was prevented by the gap junction blocker 18β-GA (Davidson et al., 1986; Le and Musil, 1998), indicating a bona fide increase in gap junction–mediated intercellular communication (Fig. 2 A).


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, but not insulin, increases gap junctional intercellular communication in chick lens DCDMLs. 1-d-old DCDML cultures prepared from E10 chick lenses were incubated for an additional 48 h in either the absence (control) or presence of the indicated growth factor. Gap junction–mediated intercellular communication was then assessed using the scrape-loading/dye transfer assay as described in Materials and methods. Cells were scrape-loaded with rhodamine-dextran mixed with either Lucifer yellow (A) or biocytin (B). After either 8 (A) or 2 min (B), the cells were fixed and the dyes visualized by fluorescence microscopy. The Mr = 10 kD rhodamine-dextran (A and B; RD) remained confined to the cells at the wound edge into which dye had been directly introduced during the scrape-loading process. In contrast, Lucifer yellow (A; LY) and biocytin (B; biocytin) were transferred to adjacent cells via open gap junctional channels. Each panel depicts a portion of the right half of the scrape/load wound. Superposition of the staining pattern of the two dyes is shown to the immediate right of the rhodamine-dextran images (LY/RD and biocytin/RD) Graphs at far right: averaged fluorescence plot profiles of the corresponding Lucifer yellow (A) or biocytin (B) images. In each case, the solid line represents the average of three fluorescence profiles generated as described in Material and methods, and the dotted lines indicate standard deviation (blue and magenta, respectively). The distance of gap junction–mediated dye transfer into the monolayer (d) is indicated; d for FGF-treated monolayers was essentially the same if the concentration of Lucifer yellow was reduced from 1 to 0.04% (data not shown). Bars, 100 μm.
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Related In: Results  -  Collection

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fig1: FGF, but not insulin, increases gap junctional intercellular communication in chick lens DCDMLs. 1-d-old DCDML cultures prepared from E10 chick lenses were incubated for an additional 48 h in either the absence (control) or presence of the indicated growth factor. Gap junction–mediated intercellular communication was then assessed using the scrape-loading/dye transfer assay as described in Materials and methods. Cells were scrape-loaded with rhodamine-dextran mixed with either Lucifer yellow (A) or biocytin (B). After either 8 (A) or 2 min (B), the cells were fixed and the dyes visualized by fluorescence microscopy. The Mr = 10 kD rhodamine-dextran (A and B; RD) remained confined to the cells at the wound edge into which dye had been directly introduced during the scrape-loading process. In contrast, Lucifer yellow (A; LY) and biocytin (B; biocytin) were transferred to adjacent cells via open gap junctional channels. Each panel depicts a portion of the right half of the scrape/load wound. Superposition of the staining pattern of the two dyes is shown to the immediate right of the rhodamine-dextran images (LY/RD and biocytin/RD) Graphs at far right: averaged fluorescence plot profiles of the corresponding Lucifer yellow (A) or biocytin (B) images. In each case, the solid line represents the average of three fluorescence profiles generated as described in Material and methods, and the dotted lines indicate standard deviation (blue and magenta, respectively). The distance of gap junction–mediated dye transfer into the monolayer (d) is indicated; d for FGF-treated monolayers was essentially the same if the concentration of Lucifer yellow was reduced from 1 to 0.04% (data not shown). Bars, 100 μm.
Mentions: Although cells isolated from the lenses of several vertebrate species will take on fiber-like characteristics when maintained ex vivo, epithelial-to-fiber differentiation is most completely recapitulated in primary cultures of embryonic chick lens epithelial cells (Menko et al., 1984, 1987; TenBroek et al., 1994). This system also most faithfully reiterates fiber-type gap junction formation (FitzGerald and Goodenough, 1986; Menko et al., 1987; Jiang et al., 1993). DCDML cultures prepared from E10 chick lenses as described by Menko et al. (1984) (termed DCDMLs) are enriched in cells originating from the equatorial region of the lens, the area that undergoes both fiber differentiation and upregulation of gap junctional intercellular coupling in vivo. We have shown previously that DCDML cultures continue to divide and differentiate in a defined, growth factor–free medium (M199/BOTS), albeit to a more limited extent than in the presence of serum. They also remain coupled by gap junctions and express all three known chick lens connexins (Cx43, Cx45.6, and Cx56; Le and Musil, 1998). Addition of either purified recombinant FGF, insulin, or IGF-1 to serum-free DCDML cultures stimulates both cell proliferation and the expression of fiber differentiation markers (Le and Musil, 2001). We tested whether any of these growth factors affected gap junctional intercellular communication using the scrape-loading/dye transfer assay (el-Fouly et al., 1987). In the experiments depicted in Fig. 1 A, the membrane impermeant, low-molecular weight fluorescent dye Lucifer yellow was introduced into DCDML cultures by scraping the monolayer with a 27-gauge needle. As evaluated by the spread of Lucifer yellow from cells at the scrape border to adjoining unwounded cells, cultures maintained in the absence of added growth factor were moderately well coupled by gap junctions. Intercellular transfer of Lucifer yellow was increased an average of 2.72-fold (± 0.48; Fig. 2 A) when cells were cultured for 2 d in 15 ng/ml FGF-2. In contrast, neither 1 μg/ml insulin nor (not shown) 15 ng/ml of the IGF-1 analogue R3IGF-1, although comparable to 15 ng/ml FGF-2 in their ability to upregulate expression of fiber differentiation markers (Le and Musil, 2001), had any detectable effect on Lucifer yellow transfer. Qualitatively similar results were obtained with biocytin, a gap junction–permeable compound with physical properties distinct from those of Lucifer yellow that is a more sensitive tracer of gap junctional communication in lens cells (Le and Musil, 1998; Fig. 1 B). Cultures treated with FGF were indistinguishable from those exposed to insulin/IGF with regard to cell number, protein content, and incorporation of [35S]methionine into proteins synthesized during a 4 h pulse (data not shown). The increase in intercellular dye transfer in FGF-treated cells was prevented by the gap junction blocker 18β-GA (Davidson et al., 1986; Le and Musil, 1998), indicating a bona fide increase in gap junction–mediated intercellular communication (Fig. 2 A).

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