Limits...
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.

Show MeSH

Related in: MedlinePlus

Vitreous humor induces sustained ERK activation and increases gap junctional intercellular communication in chick lens cells. (A) 3-d-old DCDMLs cultured in M199/BOTS were incubated at 37°C in fresh M199/BOTS medium without additions (control), in fresh M199/BOTS with 15 ng/ml FGF-2, in chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH), in M199/BOTS conditioned with intact E10 chick vitreous bodies (VBCM), or in 30% vitreous humor prepared from adult mouse (30% MVH) as indicated. After either 15 min or 8 h, the cells were 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. The numbers under the blots are the fold increase in pERK immunoreactivity in treated cells at the indicated time relative to the amount of pERK in control cells at time 0. (B) Gap junctional intercellular communication in DCDMLs cultured for 2 d in the absence (control) or presence of the agents described in A was assessed as in Fig. 1 A. Only Lucifer yellow immunofluorescence is presented; rhodamine-dextran was confined to a single row of cells immediately bordering the wound. The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment; n, number of independent experiments. All treatments significantly elevated dye coupling (P ≤ 0.05). Bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2196873&req=5

fig8: Vitreous humor induces sustained ERK activation and increases gap junctional intercellular communication in chick lens cells. (A) 3-d-old DCDMLs cultured in M199/BOTS were incubated at 37°C in fresh M199/BOTS medium without additions (control), in fresh M199/BOTS with 15 ng/ml FGF-2, in chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH), in M199/BOTS conditioned with intact E10 chick vitreous bodies (VBCM), or in 30% vitreous humor prepared from adult mouse (30% MVH) as indicated. After either 15 min or 8 h, the cells were 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. The numbers under the blots are the fold increase in pERK immunoreactivity in treated cells at the indicated time relative to the amount of pERK in control cells at time 0. (B) Gap junctional intercellular communication in DCDMLs cultured for 2 d in the absence (control) or presence of the agents described in A was assessed as in Fig. 1 A. Only Lucifer yellow immunofluorescence is presented; rhodamine-dextran was confined to a single row of cells immediately bordering the wound. The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment; n, number of independent experiments. All treatments significantly elevated dye coupling (P ≤ 0.05). Bar, 50 μm.

Mentions: For FGF to play a physiologically important role in the regulation of gap junction–mediated intercellular communication in the lens, it must be present in an appropriate location in the ocular environment. In both the mammalian and avian eye, FGFs (derived largely from the retina) are concentrated in vitreous humor, the liquid component of the gel-like vitreous body that occupies most of the posterior chamber (Mascarelli et al., 1987; Caruelle et al., 1989; Schulz et al., 1993). We have recently shown that crude vitreous humor diluted 1:2.3 in M199/BOTS medium (termed 30% vitreous humor [VH]) increases the expression of fiber differentiation markers including δ-crystallin in embryonic chick lens DCDML cultures. Moreover, M199/BOTS conditioned overnight with whole vitreous bodies has a similar effect, demonstrating that the active factor in vitreous humor is capable of diffusing out of the vitreous body and affecting lens cell fate (Le and Musil, 2001). Both 30% VH (from either embryonic chick or adult mouse) and vitreous body conditioned medium (VBCM) increased intercellular transfer of Lucifer yellow to an extent comparable to 15 ng/ml purified recombinant FGF (Fig. 8 B). Moreover, they also induced sustained activation of ERKs as assessed by antiphosphoERK immunoblotting (Fig. 8 A). The effect of vitreous on junctional communication, like that of recombinant FGF, was sensitive to the gap junction blocker 18β-GA, required a minimum of 12–24 h of treatment, and did not detectably alter the immunostaining pattern of either Cx43, Cx45.6, Cx56, NCAM, or N-cadherin (data not shown).


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 induces sustained ERK activation and increases gap junctional intercellular communication in chick lens cells. (A) 3-d-old DCDMLs cultured in M199/BOTS were incubated at 37°C in fresh M199/BOTS medium without additions (control), in fresh M199/BOTS with 15 ng/ml FGF-2, in chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH), in M199/BOTS conditioned with intact E10 chick vitreous bodies (VBCM), or in 30% vitreous humor prepared from adult mouse (30% MVH) as indicated. After either 15 min or 8 h, the cells were 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. The numbers under the blots are the fold increase in pERK immunoreactivity in treated cells at the indicated time relative to the amount of pERK in control cells at time 0. (B) Gap junctional intercellular communication in DCDMLs cultured for 2 d in the absence (control) or presence of the agents described in A was assessed as in Fig. 1 A. Only Lucifer yellow immunofluorescence is presented; rhodamine-dextran was confined to a single row of cells immediately bordering the wound. The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment; n, number of independent experiments. All treatments significantly elevated dye coupling (P ≤ 0.05). Bar, 50 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196873&req=5

fig8: Vitreous humor induces sustained ERK activation and increases gap junctional intercellular communication in chick lens cells. (A) 3-d-old DCDMLs cultured in M199/BOTS were incubated at 37°C in fresh M199/BOTS medium without additions (control), in fresh M199/BOTS with 15 ng/ml FGF-2, in chick vitreous humor diluted with 2.3 vol of M199/BOTS (30% VH), in M199/BOTS conditioned with intact E10 chick vitreous bodies (VBCM), or in 30% vitreous humor prepared from adult mouse (30% MVH) as indicated. After either 15 min or 8 h, the cells were 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. The numbers under the blots are the fold increase in pERK immunoreactivity in treated cells at the indicated time relative to the amount of pERK in control cells at time 0. (B) Gap junctional intercellular communication in DCDMLs cultured for 2 d in the absence (control) or presence of the agents described in A was assessed as in Fig. 1 A. Only Lucifer yellow immunofluorescence is presented; rhodamine-dextran was confined to a single row of cells immediately bordering the wound. The values given underneath the micrographs represent the fold Lucifer yellow transfer (± standard deviation) relative to untreated controls within the same experiment; n, number of independent experiments. All treatments significantly elevated dye coupling (P ≤ 0.05). Bar, 50 μm.
Mentions: For FGF to play a physiologically important role in the regulation of gap junction–mediated intercellular communication in the lens, it must be present in an appropriate location in the ocular environment. In both the mammalian and avian eye, FGFs (derived largely from the retina) are concentrated in vitreous humor, the liquid component of the gel-like vitreous body that occupies most of the posterior chamber (Mascarelli et al., 1987; Caruelle et al., 1989; Schulz et al., 1993). We have recently shown that crude vitreous humor diluted 1:2.3 in M199/BOTS medium (termed 30% vitreous humor [VH]) increases the expression of fiber differentiation markers including δ-crystallin in embryonic chick lens DCDML cultures. Moreover, M199/BOTS conditioned overnight with whole vitreous bodies has a similar effect, demonstrating that the active factor in vitreous humor is capable of diffusing out of the vitreous body and affecting lens cell fate (Le and Musil, 2001). Both 30% VH (from either embryonic chick or adult mouse) and vitreous body conditioned medium (VBCM) increased intercellular transfer of Lucifer yellow to an extent comparable to 15 ng/ml purified recombinant FGF (Fig. 8 B). Moreover, they also induced sustained activation of ERKs as assessed by antiphosphoERK immunoblotting (Fig. 8 A). The effect of vitreous on junctional communication, like that of recombinant FGF, was sensitive to the gap junction blocker 18β-GA, required a minimum of 12–24 h of treatment, and did not detectably alter the immunostaining pattern of either Cx43, Cx45.6, Cx56, NCAM, or N-cadherin (data not shown).

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