Bruce A. Boswell

Essential Role of BMPs in FGF-Induced Secondary Lens Fiber Differentiation

It is widely accepted that vitreous humor-derived FGFs are required for the differentiation of anterior lens epithelial cells into crystallin-rich fibers. We show that BMP2, 4, and 7 can induce the expression of markers of fiber differentiation in primary lens cell cultures to an extent equivalent to FGF or medium conditioned by intact vitreous bodies (VBCM). Abolishing BMP2/4/7 signaling with noggin inhibited VBCM from upregulating fiber marker expression. Remarkably, noggin and anti-BMP antibodies also prevented purified FGF (but not unrelated stimuli) from upregulating the same fiber-specific proteins. This effect is attributable to inhibition of BMPs produced by the lens cells themselves. Although BMP signaling is required for FGF to enhance fiber differentiation, the converse is not true. Expression of noggin in the lenses of transgenic mice resulted in a postnatal block of epithelial-to-secondary fiber differentiation, with extension of the epithelial monolayer to the posterior pole of the organ. These results reveal the central importance of BMP in secondary fiber formation and show that although FGF may be necessary for this process, it is not sufficient. Differentiation of fiber cells, and thus proper vision, is dependent on cross-talk between the FGF and BMP signaling pathways.

Cross-Talk between Fibroblast Growth Factor and Bone Morphogenetic Proteins Regulates Gap Junction-mediated Intercellular Communication in Lens Cells

Homeostasis in the lens is dependent on an extensive network of cell-to-cell gap junctional channels. Gap junction-mediated intercellular coupling (GJIC) is higher in the equatorial region of the lens than at either pole, an asymmetry believed essential for lens transparency. Primary cultures of embryonic chick lens epithelial cells up-regulate GJIC in response to purified fibroblast growth factor (FGF)1/2 or to medium conditioned by vitreous bodies, the major reservoir of factors (including FGF) for the lens equator. We show that purified bone morphogenetic protein (BMP)2, -4, and -7 also up-regulate GJIC in these cultures. BMP2, -4, or both are present in vitreous body conditioned medium, and BMP4 and -7 are endogenously expressed by lens cells. Remarkably, lens-derived BMP signaling is required for up-regulation of GJIC by purified FGF, and sufficient for up-regulation by vitreous humor. This is the first demonstration of an obligatory interaction between FGF and BMPs in postplacode lens cells, and of a role for FGF/BMP cross-talk in regulating GJIC in any cell type. Our results support a model in which the angular gradient in GJIC in the lens, and thus proper lens function, is dependent on signaling between the FGF and BMP pathways.

Upregulation and maintenance of gap junctional communication in lens cells.

The cells of the lens are joined by an extensive network of gap junction intercellular channels consisting of connexins 43, 46, and 50. We have proposed, and experimentally supported, the hypothesis that fibroblast growth factor (FGF) signaling is required for upregulation of gap junction-mediated intercellular coupling (GJIC) at the lens equator. The ability of FGF to increase GJIC in cultured lens cells requires sustained activation of extracellular signal-regulated kinase (ERK). In other cell types, activation of ERK has been shown to block GJIC mediated by connexin43 (Cx43). Why ERK signaling does not block lens cell coupling is not known. Another unresolved issue in lens gap junction regulation is how connexins, synthesized before the loss of biosynthetic organelles in mature lens fiber cells, avoid degradation during formation of the organelle-free zone. We have addressed these questions using serum-free cultures (termed DCDMLs) of primary embryonic chick lens epithelial cells. We show that FGF stimulates ERK in DCDMLs via the canonical Ras/Raf1 pathway, and that the reason that neither basal nor growth factor-stimulated GJIC is blocked by activation of ERK is because it is not mediated by Cx43. In fibroblastic cells, the normally rapid rate of degradation of Cx43 after its transport to the plasma membrane is reduced by treatments that either directly (ALLN; epoxomicin) or indirectly (generation of oxidatively un/mis-folded proteins by arsenic compounds) prevent the ubiquitin/proteasome system (UPS) from acting on its normal substrates. We show here that Cx45.6 and Cx56, the chick orthologs of mammalian Cx50 and Cx46, behave similarly in DCDMLs. When organelles lyse during the maturation of fiber cells, they release into the cytosol a large amount of new proteins that have the potential to saturate the capacity, and/or compromise the function, of the UPS. This would serve to spare gap junctions from degradation during formation of the organelle-free zone, thereby preserving GJIC between mature fiber cells despite the lack of de novo connexin synthesis.

Role of a membrane glycoprotein in friend virus-induced erythroleukemia: Studies of mutant and revertant viruses

We previously reported the isolation and characterization of spontaneous, transmissible mutants of Friend spleen focus-forming virus (SFFV) that are nonpathogenic in adult NIH/Swiss mice and that contain abnormalities in nonoverlapping regions of their envelope glycoprotein (env) genes (M. Ruta, R. Bestwick, C. Machida, and D. Kabat, 1983, Proc. Natl. Acad. Sci. USA 80, 4704-4708). In newborn NIH/Swiss mice, these mutant SFFVs form revertants that are pathogenic in mice of all ages. At least two of three studied revertants contain second site env mutations which affect the sizes and proteolytic fragmentation patterns of their encoded glycoproteins. A variety of structural and genetic evidence suggests that the xenotropic- and ecotropic-related regions of the SFFV glycoprotein fold into separate globular domains that are connected by a flexible proline-rich joint. A glutamyl peptide bond within this joint is exceptionally susceptible to cleavage with Staphylococcus aureus V8 protease. Moreover, disulfide bonds occur within the xenotropic-related domain, but not between the globular domains. These results provide strong additional evidence that the env gene is required for SFFV pathogenesis, and they provide a new system for identifying the features of glycoprotein structure and localization which are essential for its leukemogenic activity.

Synergistic interaction between the fibroblast growth factor and bone morphogenetic protein signaling pathways in lens cells

Relatively little is known about how receptor tyrosine kinase ligands can positively cooperate with BMP signaling. Primary cultures of lens cells were used to reveal an unprecedented type of cross-talk between the canonical FGF and BMP signaling pathways that regulates lens cell differentiation and intercellular coupling.

Regulation of Lens Gap Junctions by Transforming Growth Factor Beta

Using cultured lens epithelial cells, we discovered a new type of cross-talk between the FGF and TGF-β pathways, as well as a novel role for TGF-β and p38 kinase in the regulation of gap junctional intercellular communication. Our findings provide an explanation for how pathologically increased TGF-β signaling could contribute to cataract formation.

Dual function of TGFβ in lens epithelial cell fate: implications for secondary cataract

TGFβ signaling is linked to posterior capsule opacification (PCO), the most common complication of cataract surgery. TGFβ can induce primary lens epithelial cells to differentiate into two disparate, PCO-causing abnormal cell phenotypes, a variation of the TGFβ paradox. Analysis of the signaling pathways downstream of TGFβ reveals novel therapeutic targets for PCO.