TGFβ overcomes FGF-induced transinhibition of EGFR in lens cells to enable fibrotic secondary cataract.

To cause vision-disrupting fibrotic secondary cataract (PCO), lens epithelial cells that survive cataract surgery must migrate to the posterior of the lens capsule and differentiate into myofibroblasts. During this process, the cells become exposed to the FGF that diffuses out of the vitreous body. In normal development, such relatively high levels of FGF induce lens epithelial cells to differentiate into lens fiber cells.

Tonic ErbB signaling underlies TGFβ-induced activation of ERK and is required for lens cell epithelial to myofibroblast transition.

Fibrosis is a major, but incompletely understood, component of many diseases. The most common vision-disrupting complication of cataract surgery involves differentiation of residual lens cells into myofibroblasts. In serum-free primary cultures of lens epithelial cells (DCDMLs), inhibitors of either ERK or of ErbB signaling prevent TGFβ from upregulating both early (fibronectin) and late (αSMA) markers of myofibroblast differentiation.

ErbBs in Lens Cell Fibrosis and Secondary Cataract.

Purpose: TGFβ-induced epithelial-to-myofibroblast transition (EMyT) of lens cells has been linked to the most common vision-disrupting complication of cataract surgery-namely, posterior capsule opacification (PCO; secondary cataract). Although inhibitors of the ErbB family of receptor tyrosine kinases have been shown to block some PCO-associated processes in model systems, our knowledge of ErbB signaling in the lens is very limited. Here, we investigate the expression of ErbBs and their ligands in primary cultures of chick lens epithelial cells (dissociated cell-derived monolayer cultures [DCDMLs]) and how TGFβ affects ErbB function.

Fibronectin regulates growth factor signaling and cell differentiation in primary lens cells.

Lens epithelial cells are bound to the lens extracellular matrix capsule, of which laminin is a major component. After cataract surgery, surviving lens epithelial cells are exposed to increased levels of fibronectin, and so we addressed whether fibronectin influences lens cell fate, using DCDML cells as a serum-free primary lens epithelial cell culture system. We found that culturing DCDMLs with plasma-derived fibronectin upregulated canonical TGFβ signaling relative to cells plated on laminin.

Identification of Novel Gata3 Distal Enhancers Active in Mouse Embryonic Lens.

Background: The tissue-specific transcriptional programs during normal development require tight control by distal cis-regulatory elements, such as enhancers, with specific DNA sequences recognized by transcription factors, coactivators, and chromatin remodeling enzymes. Gata3 is a sequence-specific DNA-binding transcription factor that regulates formation of multiple tissues and organs, including inner ear, lens, mammary gland, T-cells, urogenital system, and thyroid gland. In the eye, Gata3 has a highly restricted expression domain in the posterior part of the lens vesicle; however, the underlying regulatory mechanisms are unknown.

LMTK3 is essential for oncogenic KIT expression in KIT-mutant GIST and melanoma.

Certain cancers, including gastrointestinal stromal tumor (GIST) and subsets of melanoma, are caused by somatic KIT mutations that result in KIT receptor tyrosine kinase constitutive activity, which drives proliferation. The treatment of KIT-mutant GIST has been revolutionized with the advent of KIT-directed cancer therapies. KIT tyrosine kinase inhibitors (TKI) are superior to conventional chemotherapy in their ability to control advanced KIT-mutant disease.

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

The most common vision-disrupting complication of cataract surgery is posterior capsule opacification (PCO; secondary cataract). PCO is caused by residual lens cells undergoing one of two very different cell fates: either transdifferentiating into myofibroblasts or maturing into lens fiber cells. Although TGFβ has been strongly implicated in lens cell fibrosis, the factors responsible for the latter process have not been identified.

Regulation of c-Maf and αA-Crystallin in Ocular Lens by Fibroblast Growth Factor Signaling.

Fibroblast growth factor (FGF) signaling regulates a multitude of cellular processes, including cell proliferation, survival, migration, and differentiation. In the vertebrate lens, FGF signaling regulates fiber cell differentiation characterized by high expression of crystallin proteins. However, a direct link between FGF signaling and crystallin gene transcriptional machinery remains to be established.

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

Fibroblast growth factors (FGFs) play a central role in two processes essential for lens transparency--fiber cell differentiation and gap junction-mediated intercellular communication (GJIC). Using serum-free primary cultures of chick lens epithelial cells (DCDMLs), we investigated how the FGF and bone morphogenetic protein (BMP) signaling pathways positively cooperate to regulate lens development and function. We found that culturing DCDMLs for 6 d with the BMP blocker noggin inhibits the canonical FGF-to-ERK pathway upstream of FRS2 activation and also prevents FGF from stimulating FRS2- and ERK-independent gene expression, indicating that BMP signaling is required at the level of FGF receptors.

Primary cultures of embryonic chick lens cells as a model system to study lens gap junctions and fiber cell differentiation.

A major limitation in lens gap junction research has been the lack of experimentally tractable ex vivo systems to study the formation and regulation of fiber-type gap junctions. Although immortalized lens-derived cell lines are amenable to both gene transfection and siRNA-mediated knockdown, to our knowledge none are capable of undergoing appreciable epithelial-to-fiber differentiation. Lens central epithelial explants have the converse limitation.

Regulation of lens gap junctions by Transforming Growth Factor beta.

Gap junction-mediated intercellular communication (GJIC) is essential for the proper function of many organs, including the lens. GJIC in lens epithelial cells is increased by FGF in a concentration-dependent process that has been linked to the intralenticular gradient of GJIC required for lens transparency. Unlike FGF, elevated levels of TGF-beta are associated with lens dysfunction.

Conformational maturation and post-ER multisubunit assembly of gap junction proteins.

For all previously well-characterized oligomeric integral membrane proteins, folding, multisubunit assembly, and recognition of conformationally immature molecules for degradation occurs at their organelle of synthesis. This cannot, however, be the case for the gap junction-forming protein connexin43 (Cx43), which when endogenously expressed undergoes multisubunit assembly into connexons only after its transport to the trans-Golgi network. We have developed two novel assays to assess Cx43 folding and assembly: acquisition of resistance of disulfide bonds to reduction by extracellularly added DTT and Triton X-114 detergent phase partitioning.

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).

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.

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.

Regulation of ubiquitin-proteasome system mediated degradation by cytosolic stress.

ER-associated, ubiquitin-proteasome system (UPS)-mediated degradation of the wild-type (WT) gap junction protein connexin32 (Cx32) is inhibited by mild forms of cytosolic stress at a step before its dislocation into the cytosol. We show that the same conditions (a 30-min, 42 degrees C heat shock or oxidative stress induced by arsenite) also reduce the endoplasmic reticulum (ER)-associated turnover of disease-causing mutants of Cx32 and the cystic fibrosis transmembrane conductance regulator (CFTR), as well as that of WT CFTR and unassembled Ig light chain. Stress-stabilized WT Cx32 and CFTR, but not the mutant/unassembled proteins examined, could traverse the secretory pathway.

Cytosolic stress reduces degradation of connexin43 internalized from the cell surface and enhances gap junction formation and function.

The protein constituents of gap junctions, connexins, have a rapid basal rate of degradation even after transport to the cell surface. We have used cell surface biotinylation to label gap junction-unassembled plasma membrane pools of connexin43 (Cx43) and show that their degradation is inhibited by mild hyperthermia, oxidative stress, and proteasome inhibitors. Cytosolic stress does not perturb endocytosis of biotinylated Cx43, but instead it seems to interfere with its targeting and/or transport to the lysosome, possibly by increasing the level of unfolded protein in the cytosol.

Degradation of connexins from the plasma membrane is regulated by inhibitors of protein synthesis.

Little is known about the mechanism and regulation of connexin turnover from the plasma membrane. We have used a combination of cell surface biotinylation, immunofluorescence microscopy, and scrape-load dye transfer assays to investigate the effect of the protein synthesis inhibitor cycloheximide on connexin43 and connexin32 after their transport to the plasmalemma. The results obtained demonstrate that cycloheximide inhibits the turnover of connexins from the surface of both gap junction assembly-deficient and -efficient cells.

Dislocation and degradation from the ER are regulated by cytosolic stress.

Akey step in ER-associated degradation (ERAD) is dislocation of the substrate protein from the ER into the cytosol to gain access to the proteasome. Very little is known about how this process is regulated, especially in the case of polytopic proteins. Using pulse-chase analysis combined with subcellular fractionation, we show that connexins, the four transmembrane structural components of gap junctions, can be chased in an intact form from the ER membrane into the cytosol of proteasome inhibitor-treated cells.