An essential role for FGF receptor signaling in lens development

Since the days of Hans Spemann, the ocular lens has served as one of the most important developmental systems for elucidating fundamental processes of induction and differentiation. More recently, studies in the lens have contributed significantly to our understanding of cell cycle regulation and apoptosis. Over 20 years of accumulated evidence using several different vertebrate species has suggested that fibroblast growth factors (FGFs) and/or fibroblast growth factor receptors (FGFRs) play a key role in lens development. FGFR signaling has been implicated in lens induction, lens cell proliferation and survival, lens fiber differentiation and lens regeneration. Here we will review and discuss historical and recent evidence suggesting that (FGFR) signaling plays a vital and universal role in multiple aspects of lens development.     

Fibroblast growth factor receptor signaling is essential for lens fiber cell differentiation

The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens development, while mice possessing only a single Fgfr1 allele developed cataracts and microphthalmia. Profound defects were observed in lenses lacking all three Fgfrs. These included lack of fiber cell elongation, abnormal proliferation in prospective lens fiber cells, reduced expression of the cell cycle inhibitors p27^kip1 and p57^kip2, increased apoptosis and aberrant or reduced expression of Prox1, Pax6, c-Maf, E-cadherin and α-, β- and γ-crystallins. Therefore, while signaling by FGF receptors is essential for lens fiber differentiation, different FGF receptors function redundantly.     

Extracellular matrix and integrin signaling in lens development and cataract

During development of the vertebrate lens there are dynamic interactions between the extracellular matrix (ECM) of the lens capsule and lens cells. Disruption of the ECM causes perturbation of lens development and cataract. Similarly, changes in cell signaling can result in abnormal ECM and cataract. Integrins are key mediators of ECM signals and recent studies have documented distinct repertoires of integrin expression during lens development, and in anterior subcapsular cataract (ASC) and posterior caspsule opacification (PCO). Increasingly, studies are being directed to investigating the signaling pathways that integrins modulate and have identified Src, focal adhesion kinase (FAK) and integrin-linked kinase (ILK) as downstream kinases that mediate proliferation, differentiation and morphological changes in the lens during development and cataract formation.     

Genetic epistasis between heparan sulfate and FGF-Ras signaling controls lens development

Vertebrate lens development depends on a complex network of signaling molecules to coordinate cell proliferation, migration and differentiation. In this study, we have investigated the role of heparan sulfate in lens specific signaling by generating a conditional ablation of heparan sulfate modification genes, Ndst1 and Ndst2. In this mutant, N-sulfation of heparan sulfate was disrupted after the lens induction stage, resulting in reduced lens cell proliferation, increased cell death and defective lens fiber differentiation in later lens development. The loss of Ndst function also prevented the assembly of Fgf/Fgfr complexes on the lens cell surface and disrupted ERK signaling within the lens. We further demonstrated that Ndst mutation completely inhibited the FGF1 and Fgf3 overexpression phenotypes, but Kras reactivation was sufficient to reverse the Ndst deficient lens differentiation defect. The epistatic relationship between Ndst and FGF–Ras signaling demonstrates that FGF signaling is the predominant signaling pathway controlled by Ndst in lens development.     

Wnt signaling is required for organization of the lens fiber cell cytoskeleton and development of lens three-dimensional architecture

How an organ develops its characteristic shape is a major issue. This is particularly critical for the eye lens as its function depends on having appropriately ordered three-dimensional cellular architecture. Recent in vitro studies indicate that Wnt signaling plays key roles in regulating morphological events in FGF-induced fiber cell differentiation in the mammalian lens. To further investigate this the Wnt signaling antagonist, secreted frizzled-related protein 2 (Sfrp2), was overexpressed in lens fiber cells of transgenic mice. In these mice fiber cell elongation was attenuated and individual fibers exhibited irregular shapes and consequently did not align or pack regularly; microtubules, microfilaments and intermediate filaments were clearly disordered in these fibers. Furthermore, a striking feature of transgenic lenses was that fibers did not develop the convex curvature typically seen in normal lenses. This appears to be related to a lack of protrusive processes that are required for directed migratory activity at their apical and basal tips as well as for the formation of interlocking processes along their lateral margins. Components of the Wnt/Planar Cell Polarity (PCP) pathway were downregulated or inhibited. Taken together this supports a role for Wnt/PCP signaling in orchestrating the complex organization and dynamics of the fiber cell cytoskeleton.     

Coordinating cell proliferation and migration in the lens and cornea

Migration is a complex process for epithelial tissues, because the epithelium must move as an intact sheet to preserve its barrier function. The requirement for structural integrity is met by coupling cell-to-matrix and cell-to-cell adhesion at the cellular level, and by coordinating cell proliferation and cell migration in the tissue as a whole. Proliferation is suppressed at the migrating cell front, allowing cells in this region to remain tightly packed while advancing rapidly. At the same time, proliferation is enhanced in a region behind the advancing cell front to expand the epithelial cell sheet. This review considers the extracellular signals and intracellular signaling pathways that regulate these processes in the lens and corneal epithelium, with emphasis on the commonalities that link these tissues.     

Low proliferation and high apoptosis of osteoblastic cells on hydrophobic surface are associated with defective Ras signaling

The hydrophobic (HPB) nature of most polymeric biomaterials has been a major obstacle in using those materials in vivo due to low compatibility with cells. However, there is little knowledge of the molecular detail to explain how surface hydrophobicity affects cell responses. In this study, we compared the proliferation and apoptosis of human osteoblastic MG63 cells adhered to hydrophilic (HPL) and hydrophobic surfaces. On the hydrophobic surface, less formation of focal contacts and actin stress fibers, a delay in cell cycle progression, and an increase in apoptosis were observed. By using fibroblast growth factor 1 (FGF1) as a model growth factor, we also investigated intracellular signaling pathways on hydrophilic and hydrophobic surfaces. The activation of Ras, Akt, and ERK by FGF1 was impaired in MG63 cells on the hydrophobic surface. The overexpression of constitutively active form of Ras and Akt rescued those cells from apoptosis and recovered cell cycle progression. Furthermore, their overexpression also restored the actin cytoskeletal organization on the hydrophobic surface. Finally, the proliferative, antiapoptotic, and cytoskeletal effects of constitutively active Ras in MG63 cells on the hydrophobic surface were blocked by wortmannin and PD98059 that inhibit Akt and ERK activation, respectively. Therefore, our results suggest that the activation of Ras and its downstream molecules Akt and ERK to an appropriate level is one of crucial elements in the determination of osteoblast cell responses. The Ras pathway may represent a cell biological target that should be considered for successful surface modification of biomaterials to induce adequate cell responses in the bone tissue.     

RCAN1-4 knockdown attenuates cell growth through the inhibition of Ras signaling

Forced changes in the expression of regulator of calcineurin 1 (RCAN1) affects cell growth. This has been linked to the suppression of calcineurin-nuclear factor of activated T cells signaling by RCAN1. Here, we describe a novel role of RCAN1 isoform 4 in proper expression of Ras protein and its signaling. RCAN1 isoform 4 knockdown attenuated growth factor-induced extracellular signal-regulated kinase activation and cell growth; reduced Ras levels and its translation rate; and led to a reduction of eukaryotic initiation factor 4E in the initiation complex and a slight repression of global protein synthesis. Experiments utilizing activity-modified mutants of calcineurin A demonstrated that these effects were calcineurin-independent. Our findings reveal a previously unknown role of RCAN1-4 in protein synthesis, which may be relevant to cell growth.     

TIMP-1 stimulates proliferation of human aortic smooth muscle cells and Ras effector pathways

Tissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein, which is found in most tissues and body fluids. Here, we demonstrated that recombinant TIMP-1 but not the synthetic matrix metalloproteinase inhibitor, GM6001, stimulated proliferation of human aortic smooth muscle cells (AoSMC) in a dose-dependent manner. The mitogenic effect was associated with activation of Ras, increased phosphorylation of ERK, and stimulation of cyclin D1 expression. The phosphatidylinositol 3-kinase (PI3K) signaling pathway was also involved since the PI3K inhibitor, LY294002, abolished the TIMP-1-mediated growth stimulation. These data suggest that TIMP-1 activates Ras, which then turns on the ERK and PI3K signaling pathways to promote cell cycle progression of the AoSMC.     

Drosophila liquid facets-Related encodes Golgi epsin and is an essential gene required for cell proliferation, growth, and patterning

Epsin and epsin-Related (epsinR) are multi-modular proteins that stimulate clathrin-coated vesicle formation. Epsin promotes endocytosis at the plasma membrane, and epsinR functions at the Golgi and early endosomes for trans-Golgi network/endosome vesicle trafficking. In Drosophila, endocytic epsin is known as Liquid facets, and it is essential specifically for Notch signaling. Here, by generating and analyzing loss-of-function mutants in the liquid facets-Related (lqfR) gene of Drosophila, we investigated the function of Golgi epsin in a multicellular context. We found that LqfR is indeed a Golgi protein, and that like liquid facets, lqfR is essential for Drosophila viability. In addition, primarily by analyzing mutant eye discs, we found that lqfR is required for cell proliferation, insulin-independent cell growth, and cell patterning, consistent with a role in one or several signaling pathways. Epsins in all organisms share an ENTH (epsin N-terminal homology) domain, which binds phosphoinositides enriched at the plasma membrane or the Golgi membrane. The epsinR ENTH domain is also the recognition element for particular cargos. By generating wild-type and mutant lqfR transgenes, we found that all apparent LqfR functions are independent of its ENTH domain. These results suggest that LqfR transports specific cargo critical to one or more signaling pathways, and lays the foundation for identifying those proteins.     

The role of Ras guanine nucleotide releasing protein 4 in Fc epsilonRI-mediated signaling, mast cell function, and T cell development

The RasGRP (Ras guanine nucleotide-releasing protein) family proteins are guanine nucleotide exchange factors that activate Ras GTPases, ultimately leading to MAPK activation and many cellular processes. The RasGRP family has four members. Published studies demonstrate that RasGRP1, RasGRP2, and RasGRP3 play critical roles in T cells, platelets, and B cells, respectively. RasGRP4 is highly expressed in mast cells. Although previous data suggest that it is important in mast cell development and function, the role of RasGRP4 in mast cells and allergic responses has not been clearly demonstrated. In this study, we generated RasGRP4(-/-) mice to examine the function of RasGRP4. Analyses of these mice showed that mast cells were able to develop normally in vivo and in vitro. Despite high levels of RasGRP4 expression in mast cells, RasGRP4 deficiency led to only a modest reduction in FcεRI-mediated degranulation and cytokine production. Interestingly, mast cells deficient in both RasGRP1 and RasGRP4 had a much more severe block in FcεRI-mediated signaling and mast cell function. We also made the unexpected finding that RasGRP4 functions during thymocyte development. Our data suggest that after the engagement of immunoreceptors, immune cells likely employ multiple members of the RasGRP family to transduce critical signals.     

Progranulin A-mediated MET signaling is essential for liver morphogenesis in zebrafish

The mechanism that regulates embryonic liver morphogenesis remains elusive. Progranulin (PGRN) is postulated to play a critical role in regulating pathological liver growth. Nevertheless, the exact regulatory mechanism of PGRN in relation to its functional role in embryonic liver development remains to be elucidated. In our study, the knockdown of progranulin A (GrnA), an orthologue of mammalian PGRN, using antisense morpholinos resulted in impaired liver morphogenesis in zebrafish (Danio rerio). The vital role of GrnA in hepatic outgrowth and not in liver bud formation was further confirmed using whole-mount in situ hybridization markers. In addition, a GrnA deficiency was also found to be associated with the deregulation of MET-related genes in the neonatal liver using a microarray analysis. In contrast, the decrease in liver size that was observed in grnA morphants was avoided when ectopic MET expression was produced by co-injecting met mRNA and grnA morpholinos. This phenomenon suggests that GrnA might play a role in liver growth regulation via MET signaling. Furthermore, our study has shown that GrnA positively modulates hepatic MET expression both in vivo and in vitro. Therefore, our data have indicated that GrnA plays a vital role in embryonic liver morphogenesis in zebrafish. As a result, a novel link between PGRN and MET signaling is proposed.     

Co-operative roles for E-cadherin and N-cadherin during lens vesicle separation and lens epithelial cell survival

The classical cadherins are known to have both adhesive and signaling functions. It has also been proposed that localized regulation of cadherin activity may be important in cell assortment during development. In the context of eye development, it has been suggested that cadherins are important for separation of the invaginated lens vesicle from the surface ectoderm. To test this hypothesis, we conditionally deleted N-cadherin or E-cadherin from the presumptive lens ectoderm of the mouse. Conditional deletion of either cadherin alone did not produce a lens vesicle separation defect. However, these conditional mutants did exhibit common structural deficits, including microphthalmia, severe iris hyperplasia, persistent vacuolization within the fibre cell region, and eventual lens epithelial cell deterioration. To assess the co-operative roles of E-cadherin and N-cadherin within the developing lens, double conditional knockout embryos were generated. These mice displayed distinct defects in lens vesicle separation and persistent expression of another classical cadherin, P-cadherin, within the cells of the persistent lens stalk. Double mutant lenses also exhibited severe defects in lens epithelial cell adhesion and survival. Finally, the severity of the lens phenotype was shown to be sensitive to the number of wild-type E- and N-cadherin alleles. These data suggest that the co-operative expression of both E- and N-cadherin during lens development is essential for normal cell sorting and subsequent lens vesicle separation.     

Notch signaling regulates growth and differentiation in the mammalian lens

The Notch signal transduction pathway regulates the decision to proliferate versus differentiate. Although there are a myriad of mouse models for the Notch pathway, surprisingly little is known about how these genes regulate early eye development, particularly in the anterior lens. We employed both gain-of-function and loss-of-function approaches to determine the role of Notch signaling in lens development. Here we analyzed mice containing conditional deletion of the Notch effector Rbpj or overexpression of the activated Notch1 intracellular domain during lens formation. We demonstrate distinct functions for Notch signaling in progenitor cell growth, fiber cell differentiation and maintenance of the transition zone. In particular, Notch signaling controls the timing of primary fiber cell differentiation and is essential for secondary fiber cell differentiation. Either gain or loss of Notch signaling leads to formation of a dysgenic lens, which in loss-of-function mice undergoes a profound postnatal degeneration. Our data suggest both Cyclin D1 and Cyclin D2, and the p27^Kip1 cyclin-dependent kinase inhibitor act downstream of Notch signaling, and define multiple critical functions for this pathway during lens development.     

Notch signaling is required for lateral induction of Jagged1 during FGF-induced lens fiber differentiation

Previous studies of the developing lens have shown that Notch signaling regulates differentiation of lens fiber cells by maintaining a proliferating precursor pool in the anterior epithelium. However, whether Notch signaling is further required after the onset of fiber cell differentiation is not clear. This work investigates the role of Notch2 and Jagged1 (Jag1) in secondary fiber cell differentiation using rat lens epithelial explants undergoing FGF-2 dependent differentiation in vitro. FGF induced Jag1 expression and Notch2 signaling (as judged by the appearance of activated Notch2 Intracellular Domain (N2ICD)) within 12-24 h. These changes were correlated with induction of the Notch effector, Hes5, upregulation of N-cadherin (N-cad), and downregulation of E-cadherin (E-cad), a cadherin switch characteristic of fiber cell differentiation. Induction of Jag1 was efficiently blocked by U0126, a specific inhibitor of MAPK/ERK signaling, indicating a requirement for signaling through this pathway downstream of the FGF receptor. Other growth factors that activate MAPK/ERK signaling (EGF, PDGF, IGF) did not induce Jag1. Inhibition of Notch signaling using gamma secretase inhibitors DAPT and L-685,458 or anti-Jag1 antibody markedly decreased FGF-dependent expression of Jag1 demonstrating Notch-dependent lateral induction. In addition, inhibition of Notch signaling reduced expression of N-cad, and the cyclin dependent kinase inhibitor, p57Kip2, indicating a direct role for Notch signaling in secondary fiber cell differentiation. These results demonstrate that Notch-mediated lateral induction of Jag1 is an essential component of FGF-dependent lens fiber cell differentiation.     

SH3‐class Ras guanine nucleotide exchange factors are essential for Aspergillus fumigatus invasive growth

Proper hyphal morphogenesis is essential for the establishment and progression of invasive disease caused by filamentous fungi. In the human pathogen Aspergillus fumigatus, signalling cascades driven by Ras and Ras‐like proteins orchestrate a wide variety of cellular processes required for hyphal growth. For activation, these proteins require interactions with Ras‐subfamily‐specific guanine nucleotide exchange factors (RasGEFs). Although Ras‐protein networks are essential for virulence in all pathogenic fungi, the importance of RasGEF proteins is largely unexplored. A. fumigatus encodes four putative RasGEFs that represent three separate classes of RasGEF proteins (SH3‐, Ras guanyl nucleotide‐releasing protein [RasGRP]–, and LTE‐class), each with fungus‐specific attributes. Here, we show that the SH3‐class and RasGRP‐class RasGEFs are required for properly timed polarity establishment during early growth and branch emergence as well as for cell wall stability. Further, we show that SH3‐class RasGEF activity is essential for polarity establishment and maintenance, a phenotype that is, at least, partially independent of the major A. fumigatus Ras proteins, RasA and RasB. Finally, loss of both SH3‐class RasGEFs resulted in avirulence in multiple models of invasive aspergillosis. Together, our findings suggest that RasGEF activity is essential for the integration of multiple signalling networks to drive invasive growth in A. fumigatus.