A novel role for FGF and extracellular signal-regulated kinase in gap junction-mediated intercellular communication in the lens.

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.

Normal differentiation of cultured lens cells after inhibition of gap junction-mediated intercellular communication.

The cells of the vertebrate lens are linked to each other by gap junctions, clusters of intercellular channels that mediate the direct transfer of low-molecular-weight substances between the cytosols of adjoining cells. Although gap junctions are detectable in the unspecialized epithelial cells that comprise the anterior face of the organ, both their number and size are greatly increased in the secondary fiber cells that differentiate from them at the lens equator. In other organs, gap junctions have been shown to play an important role in tissue development and differentiation. It has been proposed, although not experimentally tested, that this may be true in the lens as well. To investigate the function of gap junctions in the development of the lens, we have examined the effect of the gap junction blocker 18beta-glycyrrhetinic acid (betaGA) on the differentiation of primary cultures (both dissociated cell-derived monolayers and central epithelium explants) of embryonic chick lens epithelial cells. We found that betaGA greatly reduced gap junction-mediated intercellular transfer of Lucifer yellow and biocytin throughout the 8-day culture period. betaGA did not, however, affect the differentiation of these cells into MP28-expressing secondary fibers. Furthermore, inhibition of gap junctions had no apparent effect on either of the two other types of intercellular (adherens and tight) junctions present in the lens. We conclude that the high level of gap junctional intercellular communication characteristic of the lens equator in vivo is not required for secondary fiber formation as assayed in culture. Up-regulation of gap junctions is therefore likely to be a consequence rather than a cause of lens fiber differentiation and may primarily play a role in lens physiology.

FGF signaling in chick lens development.

The prevailing concept has been that an FGF induces epithelial-to-fiber differentiation in the mammalian lens, whereas chick lens cells are unresponsive to FGF and are instead induced to differentiate by IGF/insulin-type factors. We show here that when treated for periods in excess of those used in previous investigations (>5 h), purified recombinant FGFs stimulate proliferation of primary cultures of embryonic chick lens epithelial cells and (at higher concentrations) expression of the fiber differentiation markers delta-crystallin and CP49. Surprisingly, upregulation of proliferation and delta-crystallin synthesis by FGF does not require activation of ERK kinases. ERK function is, however, essential for stimulation of delta-crystallin expression in response to insulin or IGF-1. Vitreous humor, the presumptive source of differentiation-promoting activity in vivo, contains a factor capable of diffusing out of the vitreous body and inducing delta-crystallin and CP49 expression in chick lens cultures. This factor binds heparin with high affinity and increases delta-crystallin expression in an ERK-insensitive manner, properties consistent with an FGF but not insulin or IGF. Our findings indicate that differentiation in the chick lens is likely to be mediated by an FGF and provide the first insights into the role of the ERK pathway in growth factor-induced signal transduction in the lens.

Regulation of connexin degradation as a mechanism to increase gap junction assembly and function.

Connexins, the integral membrane protein constituents of gap junctions, are degraded at a rate (t(12) = 1.5-5 h) much faster than most other cell surface proteins. Although the turnover of connexins has been shown to be sensitive to inhibitors of either the lysosome or of the proteasome, how connexins are targeted for degradation and whether this process can be regulated to affect intercellular communication is unknown. We show here that reducing connexin degradation with inhibitors of the proteasome (but not with lysosomal blockers) is associated with a striking increase in gap junction assembly and intercellular dye transfer in cells inefficient in both processes under basal conditions. The effect of proteasome inhibitors on wild-type connexin stability, assembly, and function was mimicked by treatment of assembly-inefficient cells with inhibitors of protein synthesis such as cycloheximide. Sensitivity of connexin degradation to cycloheximide, but not to proteasome inhibitors, was abolished when connexins were rendered structurally abnormal by perturbation of essential disulfide bonds or by mutation. Our findings provide the first evidence that intercellular communication can be up-regulated at the level of connexin turnover and that a short-lived protein may be required for conformationally mature connexins to become substrates of proteasomal degradation.

Synthesis of basic fibroblast growth factor by murine mast cells. Regulation by transforming growth factor beta, tumor necrosis factor alpha, and stem cell factor.

BACKGROUND: Mast cells (MC) are involved in a wide spectrum of disorders characterized by neovascularization and fibroproliferation. We and others recently reported that human MC are a source of basic fibroblast growth factor (b FGF-2), a potent angiogenic and mitogenic polypeptide, in several disease conditions, such as chronic inflammation, hemangioma, and benign cutaneous mastocytosis. These findings suggest that FGF-2 may be an important mediator of cell proliferation and angiogenesis associated with MC. Since MC are heterogeneous across species, it is unknown whether FGF-2 expression is a feature common to all MC, or whether FGF-2 expression by MC can be regulated. We therefore examined FGF-2 expression by MC in mouse tissue and MC lines. METHODS: Immunostaining, RT-PCR, ELISA, immunoblot and Northern blot analyses were employed to study four murine MC lines for FGF-2 expression and its regulation by transforming growth factor-beta (TGF-beta), stem cell factor (SCF), and tumor necrosis factor-alpha (TNF-alpha). RESULTS: Mouse tissue MC and three of four murine MC lines (CFTL-12, CFTL-15, ABFTL-3) express FGF-2 as judged by immunostaining, ELISA, Western blot and Northern blot analyses, and reverse transcription-polymerase chain reaction. While TNF-alpha appeared to downregulate FGF-2 mRNA levels, treatment with SCF or TGF-beta resulted in an increase in the expression of FGF-2 at mRNA level which can be attenuated by TNF-alpha. However, the concurrent increase in FGF-2 protein was negligible, possibly due to immaturity of these cell lines. CONCLUSION: Expression of FGF-2 may be a ubiquitous feature of MC in other species in addition to humans, and can be selectively regulated by SCF, TGF-beta and TNF-alpha.