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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 sufficient to upregulate gap junctional intercellular communication in chick lens cells. DCDMLs were transfected the day after plating with plasmids encoding either a mutant form of MEK1 that constitutively activate ERKs (CA-MEK), or wild-type MEK1 (WT-MEK). Where indicated, the MEK inhibitor UO126 (15 μM) was added 3 h after transfection. Top, whole cell lysates were prepared 48 h after transfection and assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Phospho-ERK immunoreactivity is lower in UO126-treated transfectants than in untransfected controls because UO126 reduces the level of basal ERK activity in DCDML cultures after long-term (>4 h) treatment (Le and Musil, 2001). Bottom, gap junctional intercellular communication was assessed 48 h after transfection 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 under the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untransfected controls within the same experiment; n, number of independent experiments. Only the value obtained for CA-MEK was significantly higher than control (P > 0.05). Bar, 50 μm.
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fig5: ERK activation is sufficient to upregulate gap junctional intercellular communication in chick lens cells. DCDMLs were transfected the day after plating with plasmids encoding either a mutant form of MEK1 that constitutively activate ERKs (CA-MEK), or wild-type MEK1 (WT-MEK). Where indicated, the MEK inhibitor UO126 (15 μM) was added 3 h after transfection. Top, whole cell lysates were prepared 48 h after transfection and assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Phospho-ERK immunoreactivity is lower in UO126-treated transfectants than in untransfected controls because UO126 reduces the level of basal ERK activity in DCDML cultures after long-term (>4 h) treatment (Le and Musil, 2001). Bottom, gap junctional intercellular communication was assessed 48 h after transfection 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 under the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untransfected controls within the same experiment; n, number of independent experiments. Only the value obtained for CA-MEK was significantly higher than control (P > 0.05). Bar, 50 μm.

Mentions: To determine whether ERK activation was sufficient to increase gap junctional coupling in lens cells, DCDML cultures were transiently transfected with a plasmid encoding a constitutively active form of MEK1 (CA-MEK) (Cowley et al., 1994; Le and Musil, 2001). Immunocytochemistry with antiphosphoERK and anticonnexin antibodies revealed that the CA-MEK construct activated endogenous ERK in 60–70% of the cells in the culture without detectably altering the immunostaining pattern of Cx43, Cx45.6, or Cx56 (data not shown). Expression of CA-MEK1 induced a large increase in the amount of phosphorylated ERK detectable by immunoblotting (Fig. 5 , top) and increased the intercellular transfer of Lucifer yellow (Fig. 5, bottom) in cells cultured in the absence of FGF or other added growth factors by an average of 2.3-fold. Both effects were completely blocked by the MEK inhibitor UO126. Neither wild-type MEK1 (Fig. 5; WT-MEK) nor the irrelevant transfection control β-galactosidase (not shown) stimulated ERK activation (Fig. 5, top) or gap junctional coupling (Fig. 5, bottom). Culture medium conditioned by CA-MEK–expressing cells failed to increase Lucifer yellow transfer in untransfected cells, suggesting that the effect of the activated kinase was cell autonomous (data not shown). We conclude that ERK family kinases are positive effectors of gap junctional intercellular communication in lens cells.


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 sufficient to upregulate gap junctional intercellular communication in chick lens cells. DCDMLs were transfected the day after plating with plasmids encoding either a mutant form of MEK1 that constitutively activate ERKs (CA-MEK), or wild-type MEK1 (WT-MEK). Where indicated, the MEK inhibitor UO126 (15 μM) was added 3 h after transfection. Top, whole cell lysates were prepared 48 h after transfection and assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Phospho-ERK immunoreactivity is lower in UO126-treated transfectants than in untransfected controls because UO126 reduces the level of basal ERK activity in DCDML cultures after long-term (>4 h) treatment (Le and Musil, 2001). Bottom, gap junctional intercellular communication was assessed 48 h after transfection 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 under the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untransfected controls within the same experiment; n, number of independent experiments. Only the value obtained for CA-MEK was significantly higher than control (P > 0.05). Bar, 50 μm.
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Related In: Results  -  Collection

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fig5: ERK activation is sufficient to upregulate gap junctional intercellular communication in chick lens cells. DCDMLs were transfected the day after plating with plasmids encoding either a mutant form of MEK1 that constitutively activate ERKs (CA-MEK), or wild-type MEK1 (WT-MEK). Where indicated, the MEK inhibitor UO126 (15 μM) was added 3 h after transfection. Top, whole cell lysates were prepared 48 h after transfection and assessed for activation of ERK by immunoblotting with the phospho-specific anti-p44/42 MAP kinase E10 monoclonal antibody. Phospho-ERK immunoreactivity is lower in UO126-treated transfectants than in untransfected controls because UO126 reduces the level of basal ERK activity in DCDML cultures after long-term (>4 h) treatment (Le and Musil, 2001). Bottom, gap junctional intercellular communication was assessed 48 h after transfection 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 under the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untransfected controls within the same experiment; n, number of independent experiments. Only the value obtained for CA-MEK was significantly higher than control (P > 0.05). Bar, 50 μm.
Mentions: To determine whether ERK activation was sufficient to increase gap junctional coupling in lens cells, DCDML cultures were transiently transfected with a plasmid encoding a constitutively active form of MEK1 (CA-MEK) (Cowley et al., 1994; Le and Musil, 2001). Immunocytochemistry with antiphosphoERK and anticonnexin antibodies revealed that the CA-MEK construct activated endogenous ERK in 60–70% of the cells in the culture without detectably altering the immunostaining pattern of Cx43, Cx45.6, or Cx56 (data not shown). Expression of CA-MEK1 induced a large increase in the amount of phosphorylated ERK detectable by immunoblotting (Fig. 5 , top) and increased the intercellular transfer of Lucifer yellow (Fig. 5, bottom) in cells cultured in the absence of FGF or other added growth factors by an average of 2.3-fold. Both effects were completely blocked by the MEK inhibitor UO126. Neither wild-type MEK1 (Fig. 5; WT-MEK) nor the irrelevant transfection control β-galactosidase (not shown) stimulated ERK activation (Fig. 5, top) or gap junctional coupling (Fig. 5, bottom). Culture medium conditioned by CA-MEK–expressing cells failed to increase Lucifer yellow transfer in untransfected cells, suggesting that the effect of the activated kinase was cell autonomous (data not shown). We conclude that ERK family kinases are positive effectors of gap junctional intercellular communication in lens cells.

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