Linda S. Musil

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 1 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.

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. Dislocation of endogenously expressed connexin from the ER was reduced 50–80% when the cytosolic heat shock response was induced by mild oxidative or thermal stress, but not by treatments that instead upregulate the ER unfolded protein response. Cytosolic but not ER stresses slowed the normally rapid degradation of connexins, and led to a striking increase in gap junction formation and function in otherwise assembly-inefficient cell types. These treatments also inhibited the dislocation and turnover of a connexin-unrelated ERAD substrate, unassembled major histocompatibility complex class I heavy chain. Our findings demonstrate that dislocation is negatively regulated by physiologically relevant, nonlethal stress. They also reveal a previously unrecognized relationship between cytosolic stress and intercellular communication.

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.

Point: A critical appraisal of the lens circulation model--an experimental paradigm for understanding the maintenance of lens transparency?

Cataract is the leading cause of blindness in the world, accounting for approximately 42% of all blindness.1 Surgical treatment of cataracts imposes a substantial economic burden on health systems. Since cataract is primarily a disease of old age, we are facing a looming cataract epidemic in which the demand for cataract surgery will place greater demands on the resources available for treatment. An alternative approach to surgery is the development of therapies designed to prevent or delay the onset of cataract. It is therefore not surprising that the ultimate goal of many international lens research groups is to determine the causes of lens cataract, with a view toward developing novel anticataract therapies. A major obstacle to achieving this laudable goal is our current understanding of how the normal lens maintains its transparency. It has been proposed that the lens operates an internal microcirculation system that contributes to lens transparency by delivering nutrients to, and removing metabolic wastes from, the deep fiber cells while maintaining steady state lens volume (the lens fluid circulation model [FCM]).2–4 Key features of the model remain to be tested. Such scientific debate is a normal and healthy component of the research discovery process, but the lack of an accepted understanding of lens physiology is compromising progress toward the ultimate goal of developing targeted anticataract therapies. The purpose of the two perspectives presented in Point/Counterpoint is to formalize this debate. Evidence for and against the FCM will be presented, with the goal of identifying areas of future experimentation that are needed to test its validity. A general overview of the model is provided, followed by a summary of the evidence supporting it by Richard Mathias, Paul Donaldson, and Linda Musil. In the Counterpoint, David Beebe and Roger Truscott present a critique of the model. These articles are followed by brief rebuttals that summarize the critical experiments needed to test the model. It is important to acknowledge that our understanding of lens physiology has evolved from an initial view of the lens as inert tissue to one that recognizes it as a complex and dynamic organ. This evolution in understanding was initially driven by advances in histologic and electrophysiological recording techniques and then by our ability to determine the molecular identity and cellular localization of key transport proteins associated with the circulation system. Most recently, the ability to combine whole lens electrophysiological recording with transgenic animal models has enabled us to study the physiological roles that specific lens proteins play in the maintenance of lens transparency. It is highly likely that the application of new technologies to the lens will cause us to further modify our current understanding of lens structure and function, a summary of which is provided herein.

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. We show that Cx43 synthesized at physiologically relevant levels undergoes a multistep conformational maturation process in which folding of connexin monomers within the ER is a prerequisite for multisubunit assembly in the TGN. Similar results were obtained with Cx32, disproving the widely reported contention that the site of endogenous beta connexin assembly is the ER. Exogenous overexpression of Cx43, Cx32, or Cx26 allows these events to take place within the ER, the first example of the TGN and ER as alternative sites for oligomeric assembly. Our findings also constitute the first biochemical evidence that defective connexin folding is a cause of the human disorder X-linked Charcot-Marie-Tooth disease.

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.

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. Previously, we have shown that the bZIP proto-oncogene c-Maf regulates expression of αA-crystallin (Cryaa) through binding to its promoter and distal enhancer, DCR1, both activated by FGF2 in cell culture. Herein, we identified and characterized a novel FGF2-responsive region in the c-Maf promoter (−272/−70, FRE). Both c-Maf and Cryaa regulatory regions contain arrays of AP-1 and Ets-binding sites. Chromatin immunoprecipitation (ChIP) assays established binding of c-Jun (an AP-1 factor) and Etv5/ERM (an Ets factor) to these regions in lens chromatin. Analysis of temporal and spatial expression of c-Jun, phospho-c-Jun, and Etv5/ERM in wild type and ERK1/2 deficient lenses supports their roles as nuclear effectors of FGF signaling in mouse embryonic lens. Collectively, these studies show that FGF signaling up-regulates expression of αA-crystallin both directly and indirectly via up-regulation of c-Maf. These molecular mechanisms are applicable for other crystallins and genes highly expressed in terminally differentiated lens fibers.

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. A key advance in the field was the development of a primary embryonic chick lens cell culture system by Drs. Sue Menko and Ross Johnson. Unlike central epithelial explants, these cultures also include cells from the peripheral (preequatorial and equatorial) epithelium, which is the most physiologically relevant population for the study of fiber-type gap junction formation. We have modified the Menko/Johnson system and refer to our cultures as dissociated cell-derived monolayer cultures (DCDMLs). We culture DCDMLs without serum to mimic the avascular lens environment and on laminin, the major matrix component of the lens capsule. Here, I review the features of the DCDML system and how we have used it to study lens gap junctions and fiber cell differentiation. Our results demonstrate the power of DCDMLs to generate new findings germane to the mammalian lens and how these cultures can be exploited to conduct experiments that would be impossible, prohibitively expensive and/or difficult to interpret using transgenic animals in vivo.

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.