Connexins, the integral membrane protein constituents of gap junctions, are degraded at a rate (t1 ⁄2 ‫؍‬ 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.

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.

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.