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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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ERK activation is required for FGF to increase gap junctional intercellular communication in chick lens cells. (A) 3-d-old DCDMLs, cultured in M199/BOTS, were incubated for 15 min at 37°C with no additions (control), with 15 ng/ml FGF-2, or with 15 μM UO126 plus 15 ng/ml FGF-2 after a 30 min pretreatment with 15 μM UO126 alone. The samples were then immediately solubilized in SDS and whole cell lysates assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Molecular mass markers are indicated on the right. Note that chick lens cells express only the 42 kD (ERK2) MAP kinase species. (B) 1-d-old DCDMLs were incubated for 30 min with or without 15 μM UO126, after which they were incubated for an additional 48 h with no additions (control), 15 ng/ml FGF-2, or 15% fetal calf serum, in the absence or presence of 15 μM UO126 as indicated. 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 (see Fig. 1). 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. P for UO126 +FGF-2 compared with +FGF-2 was 0.0004; UO126 +FCS was not significantly different from +FCS (P = 0.55). UO126 did not affect the amount of total (activated plus inactive) ERK protein, nor did it influence the basal level of intercellular dye transfer (not shown). Bar, 50 μm.
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fig4: ERK activation is required for FGF to increase gap junctional intercellular communication in chick lens cells. (A) 3-d-old DCDMLs, cultured in M199/BOTS, were incubated for 15 min at 37°C with no additions (control), with 15 ng/ml FGF-2, or with 15 μM UO126 plus 15 ng/ml FGF-2 after a 30 min pretreatment with 15 μM UO126 alone. The samples were then immediately solubilized in SDS and whole cell lysates assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Molecular mass markers are indicated on the right. Note that chick lens cells express only the 42 kD (ERK2) MAP kinase species. (B) 1-d-old DCDMLs were incubated for 30 min with or without 15 μM UO126, after which they were incubated for an additional 48 h with no additions (control), 15 ng/ml FGF-2, or 15% fetal calf serum, in the absence or presence of 15 μM UO126 as indicated. 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 (see Fig. 1). 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. P for UO126 +FGF-2 compared with +FGF-2 was 0.0004; UO126 +FCS was not significantly different from +FCS (P = 0.55). UO126 did not affect the amount of total (activated plus inactive) ERK protein, nor did it influence the basal level of intercellular dye transfer (not shown). Bar, 50 μm.

Mentions: In the lens, as in many other tissues, FGF activates the ERK subclass of MAP kinases (Chow et al., 1995; Le and Musil, 2001). Activation of ERKs by growth factors or other stimuli has been causally linked to an inhibition of Cx43-mediated intercellular communication in several nonlenticular cell types (Hossain et al., 1998; Warn-Cramer et al., 1998; Zhou et al., 1999) but not, to our knowledge, to a posttranslational increase in the function of any connexin species. The ability of FGF to upregulate gap junctional coupling in cultured lens cells could therefore either be due to an ERK-independent activity of the growth factor or be the result of a previously unknown effect of the MAP kinase cascade on gap junction function. To distinguish between these two possibilities, we used UO126, a potent, cell permeable, nontoxic, and highly specific inhibitor of the kinases (MEK 1/2) immediately upstream of ERK in the MAP kinase cascade (Favata et al., 1998; Le and Musil, 2001). As assayed by Western blotting with an antibody specific for the dually phosphorylated, active form of ERK (Khokhlatchev et al., 1997), UO126 completely blocked the ability of FGF to activate the ERK pathway in lens cells (Fig. 4 A). This effect persisted for at least a week provided the cells were refed with fresh UO126-containing medium every 2 d. Scrape-load dye transfer analysis demonstrated that UO126 pretreatment completely prevented FGF from increasing intercellular coupling in lens cultures. In contrast, upregulation of junctional communication in response to 15% fetal calf serum was insensitive to inhibition of the ERK pathway (Fig. 4 B). The latter finding demonstrated that UO126 is not a general inhibitor of gap junction function and reveals the existence of ERK-independent (fetal calf serum–mediated) in addition to ERK-dependent (FGF-mediated) pathways of gap junction upregulation in lens epithelial cells. Because ocular fluids contain only very low levels of serum proteins (Beebe et al., 1986), the effect of fetal calf serum on lens cell junctional coupling is unlikely to be of physiological relevance.


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)

ERK activation is required for FGF to increase gap junctional intercellular communication in chick lens cells. (A) 3-d-old DCDMLs, cultured in M199/BOTS, were incubated for 15 min at 37°C with no additions (control), with 15 ng/ml FGF-2, or with 15 μM UO126 plus 15 ng/ml FGF-2 after a 30 min pretreatment with 15 μM UO126 alone. The samples were then immediately solubilized in SDS and whole cell lysates assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Molecular mass markers are indicated on the right. Note that chick lens cells express only the 42 kD (ERK2) MAP kinase species. (B) 1-d-old DCDMLs were incubated for 30 min with or without 15 μM UO126, after which they were incubated for an additional 48 h with no additions (control), 15 ng/ml FGF-2, or 15% fetal calf serum, in the absence or presence of 15 μM UO126 as indicated. 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 (see Fig. 1). 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. P for UO126 +FGF-2 compared with +FGF-2 was 0.0004; UO126 +FCS was not significantly different from +FCS (P = 0.55). UO126 did not affect the amount of total (activated plus inactive) ERK protein, nor did it influence the basal level of intercellular dye transfer (not shown). Bar, 50 μm.
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fig4: ERK activation is required for FGF to increase gap junctional intercellular communication in chick lens cells. (A) 3-d-old DCDMLs, cultured in M199/BOTS, were incubated for 15 min at 37°C with no additions (control), with 15 ng/ml FGF-2, or with 15 μM UO126 plus 15 ng/ml FGF-2 after a 30 min pretreatment with 15 μM UO126 alone. The samples were then immediately solubilized in SDS and whole cell lysates assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Molecular mass markers are indicated on the right. Note that chick lens cells express only the 42 kD (ERK2) MAP kinase species. (B) 1-d-old DCDMLs were incubated for 30 min with or without 15 μM UO126, after which they were incubated for an additional 48 h with no additions (control), 15 ng/ml FGF-2, or 15% fetal calf serum, in the absence or presence of 15 μM UO126 as indicated. 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 (see Fig. 1). 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. P for UO126 +FGF-2 compared with +FGF-2 was 0.0004; UO126 +FCS was not significantly different from +FCS (P = 0.55). UO126 did not affect the amount of total (activated plus inactive) ERK protein, nor did it influence the basal level of intercellular dye transfer (not shown). Bar, 50 μm.
Mentions: In the lens, as in many other tissues, FGF activates the ERK subclass of MAP kinases (Chow et al., 1995; Le and Musil, 2001). Activation of ERKs by growth factors or other stimuli has been causally linked to an inhibition of Cx43-mediated intercellular communication in several nonlenticular cell types (Hossain et al., 1998; Warn-Cramer et al., 1998; Zhou et al., 1999) but not, to our knowledge, to a posttranslational increase in the function of any connexin species. The ability of FGF to upregulate gap junctional coupling in cultured lens cells could therefore either be due to an ERK-independent activity of the growth factor or be the result of a previously unknown effect of the MAP kinase cascade on gap junction function. To distinguish between these two possibilities, we used UO126, a potent, cell permeable, nontoxic, and highly specific inhibitor of the kinases (MEK 1/2) immediately upstream of ERK in the MAP kinase cascade (Favata et al., 1998; Le and Musil, 2001). As assayed by Western blotting with an antibody specific for the dually phosphorylated, active form of ERK (Khokhlatchev et al., 1997), UO126 completely blocked the ability of FGF to activate the ERK pathway in lens cells (Fig. 4 A). This effect persisted for at least a week provided the cells were refed with fresh UO126-containing medium every 2 d. Scrape-load dye transfer analysis demonstrated that UO126 pretreatment completely prevented FGF from increasing intercellular coupling in lens cultures. In contrast, upregulation of junctional communication in response to 15% fetal calf serum was insensitive to inhibition of the ERK pathway (Fig. 4 B). The latter finding demonstrated that UO126 is not a general inhibitor of gap junction function and reveals the existence of ERK-independent (fetal calf serum–mediated) in addition to ERK-dependent (FGF-mediated) pathways of gap junction upregulation in lens epithelial cells. Because ocular fluids contain only very low levels of serum proteins (Beebe et al., 1986), the effect of fetal calf serum on lens cell junctional coupling is unlikely to be of physiological relevance.

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