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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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FGF-induced activation of ERK is higher in the equatorial region than in polar and core fibers. Intact lenses from embryonic day 13 (E13) chicks were incubated overnight in M199 medium and then treated for either 2 or 8 h at 37°C with no additions (control) or with 25 ng/ml FGF-2. The lenses were then snap-frozen in liquid nitrogen and dissected into three domains: the central epithelium, the equatorial region, and the polar and core fibers (see diagram; core fibers are defined as those in the lens inner cortex and nucleus). The samples were solubilized in SDS and whole cell lysates assessed for expression of NCAM and for activated ERK (pERK) as described in Materials and methods. The accuracy of the dissection was verified by the virtual absence in the polar and core fiber fraction of NCAM, which has previously been demonstrated to be expressed at high levels in equatorial region, but not polar or core, fiber cells in the embryonic chick lens (Watanabe et al., 1989). pERK immunoreactivity was quantitated by densitometry; the numbers in parentheses are the fold increase in pERK levels in FGF-treated lens fractions relative to pERK in the corresponding fraction from untreated control lenses (shown to the left of each FGF-treated blot). The pERK blots were subsequently stripped and reprobed for total (active + inactive) ERK protein (totERK). The experiment was repeated three times, with similar results. When expressed on a per lens basis, the polar/core fibers contained ∼1/10 as much FGF-activated pERK as the equatorial region. Even if the polar fibers contained all of the FGF-induced ERK activity in the polar/core fraction, their response would still be much less than that of equatorial region cells.
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fig10: FGF-induced activation of ERK is higher in the equatorial region than in polar and core fibers. Intact lenses from embryonic day 13 (E13) chicks were incubated overnight in M199 medium and then treated for either 2 or 8 h at 37°C with no additions (control) or with 25 ng/ml FGF-2. The lenses were then snap-frozen in liquid nitrogen and dissected into three domains: the central epithelium, the equatorial region, and the polar and core fibers (see diagram; core fibers are defined as those in the lens inner cortex and nucleus). The samples were solubilized in SDS and whole cell lysates assessed for expression of NCAM and for activated ERK (pERK) as described in Materials and methods. The accuracy of the dissection was verified by the virtual absence in the polar and core fiber fraction of NCAM, which has previously been demonstrated to be expressed at high levels in equatorial region, but not polar or core, fiber cells in the embryonic chick lens (Watanabe et al., 1989). pERK immunoreactivity was quantitated by densitometry; the numbers in parentheses are the fold increase in pERK levels in FGF-treated lens fractions relative to pERK in the corresponding fraction from untreated control lenses (shown to the left of each FGF-treated blot). The pERK blots were subsequently stripped and reprobed for total (active + inactive) ERK protein (totERK). The experiment was repeated three times, with similar results. When expressed on a per lens basis, the polar/core fibers contained ∼1/10 as much FGF-activated pERK as the equatorial region. Even if the polar fibers contained all of the FGF-induced ERK activity in the polar/core fraction, their response would still be much less than that of equatorial region cells.

Mentions: In the species examined (Baldo and Mathias, 1992; Mathias et al., 1997), gap junction–mediated intercellular communication between lens fiber cells is not uniform, but is instead higher in the equatorial region than at either the anterior or posterior poles of the organ (see Fig. 10 for a diagram of lens anatomy). Based largely on vibrating probe current measurements (Robinson and Patterson, 1983; Parmelee et al., 1985) and impedance studies (Mathias et al., 1985; Baldo and Mathias, 1992), a model has been developed in which ionic current (carried mainly by sodium) enters the lens predominantly through the extracellular spaces between fiber cells at the poles, crosses the fiber cell membranes, and is then transferred from cell to cell via gap junctions back to the lens surface at the equator. Water and dissolved solutes are thought to follow, establishing a nonvascular microcirculatory system that brings nutrients deep into the lens and flushes out waste products. The asymmetric distribution of open fiber-to-fiber gap junctional channels is believed to dictate the direction of this flow and to therefore play an essential role in the maintenance of lens transparency (for review see Goodenough, 1992; Mathias et al., 1997).


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-induced activation of ERK is higher in the equatorial region than in polar and core fibers. Intact lenses from embryonic day 13 (E13) chicks were incubated overnight in M199 medium and then treated for either 2 or 8 h at 37°C with no additions (control) or with 25 ng/ml FGF-2. The lenses were then snap-frozen in liquid nitrogen and dissected into three domains: the central epithelium, the equatorial region, and the polar and core fibers (see diagram; core fibers are defined as those in the lens inner cortex and nucleus). The samples were solubilized in SDS and whole cell lysates assessed for expression of NCAM and for activated ERK (pERK) as described in Materials and methods. The accuracy of the dissection was verified by the virtual absence in the polar and core fiber fraction of NCAM, which has previously been demonstrated to be expressed at high levels in equatorial region, but not polar or core, fiber cells in the embryonic chick lens (Watanabe et al., 1989). pERK immunoreactivity was quantitated by densitometry; the numbers in parentheses are the fold increase in pERK levels in FGF-treated lens fractions relative to pERK in the corresponding fraction from untreated control lenses (shown to the left of each FGF-treated blot). The pERK blots were subsequently stripped and reprobed for total (active + inactive) ERK protein (totERK). The experiment was repeated three times, with similar results. When expressed on a per lens basis, the polar/core fibers contained ∼1/10 as much FGF-activated pERK as the equatorial region. Even if the polar fibers contained all of the FGF-induced ERK activity in the polar/core fraction, their response would still be much less than that of equatorial region cells.
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Related In: Results  -  Collection

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fig10: FGF-induced activation of ERK is higher in the equatorial region than in polar and core fibers. Intact lenses from embryonic day 13 (E13) chicks were incubated overnight in M199 medium and then treated for either 2 or 8 h at 37°C with no additions (control) or with 25 ng/ml FGF-2. The lenses were then snap-frozen in liquid nitrogen and dissected into three domains: the central epithelium, the equatorial region, and the polar and core fibers (see diagram; core fibers are defined as those in the lens inner cortex and nucleus). The samples were solubilized in SDS and whole cell lysates assessed for expression of NCAM and for activated ERK (pERK) as described in Materials and methods. The accuracy of the dissection was verified by the virtual absence in the polar and core fiber fraction of NCAM, which has previously been demonstrated to be expressed at high levels in equatorial region, but not polar or core, fiber cells in the embryonic chick lens (Watanabe et al., 1989). pERK immunoreactivity was quantitated by densitometry; the numbers in parentheses are the fold increase in pERK levels in FGF-treated lens fractions relative to pERK in the corresponding fraction from untreated control lenses (shown to the left of each FGF-treated blot). The pERK blots were subsequently stripped and reprobed for total (active + inactive) ERK protein (totERK). The experiment was repeated three times, with similar results. When expressed on a per lens basis, the polar/core fibers contained ∼1/10 as much FGF-activated pERK as the equatorial region. Even if the polar fibers contained all of the FGF-induced ERK activity in the polar/core fraction, their response would still be much less than that of equatorial region cells.
Mentions: In the species examined (Baldo and Mathias, 1992; Mathias et al., 1997), gap junction–mediated intercellular communication between lens fiber cells is not uniform, but is instead higher in the equatorial region than at either the anterior or posterior poles of the organ (see Fig. 10 for a diagram of lens anatomy). Based largely on vibrating probe current measurements (Robinson and Patterson, 1983; Parmelee et al., 1985) and impedance studies (Mathias et al., 1985; Baldo and Mathias, 1992), a model has been developed in which ionic current (carried mainly by sodium) enters the lens predominantly through the extracellular spaces between fiber cells at the poles, crosses the fiber cell membranes, and is then transferred from cell to cell via gap junctions back to the lens surface at the equator. Water and dissolved solutes are thought to follow, establishing a nonvascular microcirculatory system that brings nutrients deep into the lens and flushes out waste products. The asymmetric distribution of open fiber-to-fiber gap junctional channels is believed to dictate the direction of this flow and to therefore play an essential role in the maintenance of lens transparency (for review see Goodenough, 1992; Mathias et al., 1997).

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