Intracellular trafficking of LET-756, a fibroblast growth factor of C. elegans, is controlled by a balance of export and nuclear signals

The superfamily of fibroblast growth factors (FGF), which counts 22 members in humans, exerts many functions during animal development and adult life. LET-756 is one of the two FGFs of the nematode C. elegans. Re-introduction of LET-756 in a null mutant strain restores viability, allowing the study of structural requirements for LET-756 trafficking and function. LET-756 protein has several regions and motifs, including a non-classical internal motif required for secretion. We show here that a main difference in the wild-type LET-756 molecule and a truncated molecule that mimics a partial loss-of-function mutant lies on subnuclear expression. Using Cos-1 cells and rescue activity we show that: (i) nuclear localization is due to various redundant NLS, one of them acting as a nucleolar localization signal; (ii) nuclear LET-756 is addressed to the speckles by a stretch of glutamine residues; (iii) nuclear LET-756 is trafficking between speckles and nucleoli; (iv) in the nucleolus, LET-756 is associated with proteins of the rRNA splicing compartment; (v) changing LET-756 secretion signal prevents its nuclear localization. We propose that LET-756 exerts its functions through a balance between secreted and nuclear forms due to two opposite addressing signals, (i) synergy of several NLS and (ii) attenuated secretion signal.     

FGF negatively regulates muscle membrane extension in Caenorhabditis elegans

Striated muscles from Drosophila and several vertebrates extend plasma membrane to facilitate the formation of the neuromuscular junction (NMJ) during development. However, the regulation of these membrane extensions is poorly understood. In C. elegans, the body wall muscles (BWMs) also have plasma membrane extensions called muscle arms that are guided to the motor axons where they form the postsynaptic element of the NMJ. To investigate the regulation of muscle membrane extension, we screened 871 genes by RNAi for ectopic muscle membrane extensions (EMEs) in C. elegans. We discovered that an FGF pathway, including let-756(FGF), egl-15(FGF receptor), sem-5(GRB2) and other genes negatively regulates plasma membrane extension from muscles. Although compromised FGF pathway activity results in EMEs, hyperactivity of the pathway disrupts larval muscle arm extension, a phenotype we call muscle arm extension defective or MAD. We show that expression of egl-15 and sem-5 in the BWMs are each necessary and sufficient to prevent EMEs. Furthermore, we demonstrate that let-756 expression from any one of several tissues can rescue the EMEs of let-756 mutants, suggesting that LET-756 does not guide muscle membrane extensions. Our screen also revealed that loss-of-function in laminin and integrin components results in both MADs and EMEs, the latter of which are suppressed by hyperactive FGF signaling. Our data are consistent with a model in which integrins and laminins are needed for directed muscle arm extension to the nerve cords, while FGF signaling provides a general mechanism to regulate muscle membrane extension.     

Fgf genes in the basal chordate <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

In vertebrates, a number of fibroblast growth factors (FGFs) have been shown to play important roles in developing embryos and adult organisms. However, the molecular relationships of the vertebrate FGFs are not yet completely understood, partly due to the divergence of their amino acid sequences. To solve this problem, we have identified six FGF genes in a basal chordate, the ascidian Ciona intestinalis. A phylogenetic analysis confidently assigned two of them to vertebrate FGF8/17/18 and FGF11/12/13/14, respectively. Based on the presence of the conserved domains within or outside of the FGF domains, we speculate that three of the other genes are orthologous to vertebrate FGF3/7/10/22, FGF4/5/6 and FGF9/16/20, respectively, although we cannot assign the sixth member to any of the vertebrate FGFs. A survey of the raw whole genome shotgun sequences of C. intestinalis demonstrated the presence of no FGF genes other than the six genes in the genome. The identification of these six FGF genes in the basal chordate gave us an insight into the diversification of specific subfamilies of vertebrate FGFs.     

LET-767 is required for the production of branched chain and long chain fatty acids in Caenorhabditis elegans

LET-767 from Caenorhabditis elegans belongs to a family of short chain dehydrogenases/reductases and is homologous to 17beta-hydroxysterol dehydrogenases of type 3 and 3-ketoacyl-CoA reductases. Worms subjected to RNA interference (RNAi) of let-767 displayed multiple growth and developmental defects in the first generation and arrested in the second generation as L1 larvae. To determine the function of LET-767 in vivo, we exploited a biochemical complementation approach, in which let-767 (RNAi)-arrested larvae were rescued by feeding with compounds isolated from wild type worms. The arrest was only rescued by the addition of triacylglycerides extracted from worms but not from various natural sources, such as animal fats and plant oils. The mass spectrometric analyses showed alterations in the fatty acid content of triacylglycerides. Essential for the rescue were odd-numbered fatty acids with monomethyl branched chains. The rescue was improved when worms were additionally supplemented with long chain even-numbered fatty acids. Remarkably, let-767 completely rescued the yeast 3-ketoacyl-CoA reductase mutant (ybr159Delta). Because worm ceramides exclusively contain a monomethyl branched chain sphingoid base, we also investigated ceramides in let-767 (RNAi). Indeed, the amount of ceramides was greatly reduced, and unusual sphingoid bases were observed. Taken together, we conclude that LET-767 is a major 3-ketoacyl-CoA reductase in C. elegans required for the bulk production of monomethyl branched and long chain fatty acids, and the developmental arrest in let-767 (RNAi) worms is caused by the deficiency of the former.     

Extending the family table: Insights from beyond vertebrates into the regulation of embryonic development by FGFs

Since the discovery of fibroblast growth factors (FGFs) much focus has been placed on elucidating the roles for each vertebrate FGF ligand, receptor, and regulating molecules in the context of vertebrate development, human disorders and cancer. Studies in human, mouse, frog, chick, and zebrafish have made great contributions to our understanding of the role of FGFs in specific processes. However, in recent years, as more genomes are sequenced, information is becoming available from many non‐vertebrate models and a more complete picture of the FGF superfamily as a whole is emerging. In some cases, less redundancy in these FGF signaling systems may allow for more mechanistic insights. Studies in sea anemones have highlighted how ancient FGF signaling is and helped provide insight into the evolution of the FGF gene family. Work in nematodes has shown that different splice forms can be used for functional specificity in invertebrate FGF signaling. Comparing FGFs between urochordates and vertebrates as well as between different insect species reveals important clues into the process of gene loss, duplication and subfunctionalization of FGFs throughout evolution. Finally, comparing all members of the FGF ligand superfamily reveals variability in many properties, which may point to a feature of FGFs as being highly adaptable with regards to protein structure and signaling mechanism. Further studies on FGF signaling outside of vertebrates is likely to continue to complement work in vertebrates by contributing additional insights to the FGF field and providing unexpected information that could be used for medical applications. Birth Defects Research (Part C) 90:214–227, 2010. © 2010 Wiley‐Liss, Inc.     

Crystal structure of fibroblast growth factor 9 reveals regions implicated in dimerization and autoinhibition

Fibroblast growth factors (FGFs) constitute a large family of heparin-binding growth factors with diverse biological activities. FGF9 was originally described as glia-activating factor and is expressed in the nervous system as a potent mitogen for glia cells. Unlike most FGFs, FGF9 forms dimers in solution with a K(d) of 680 nm. To elucidate the molecular mechanism of FGF9 dimerization, the crystal structure of FGF9 was determined at 2.2 A resolution. FGF9 adopts a beta-trefoil fold similar to other FGFs. However, unlike other FGFs, the N- and C-terminal regions outside the beta-trefoil core in FGF9 are ordered and involved in the formation of a 2-fold crystallographic dimer. A significant surface area (>2000 A(2)) is buried in the dimer interface that occludes a major receptor binding site of FGF9. Thus, we propose an autoinhibitory mechanism for FGF9 that is dependent on sequences outside of the beta-trefoil core. Moreover, a model is presented providing a molecular basis for the preferential affinity of FGF9 toward FGFR3.     

An evolutionary history of the FGF superfamily

Fibroblast growth factors (FGF) are associated with multiple developmental and metabolic processes in triploblasts, and perhaps also in diploblasts. The evolution of the FGF superfamily has accompanied the major morphological and functional innovations of metazoan species. The study of FGFs throughout species shows that the FGF superfamily can be subdivided in eight families in present-day organisms and has evolved through phases of gene duplications and gene losses. At least two major expansions of the superfamily can be recognized: a first expansion increased the number of FGFs from one or few archeo-FGFs to eight proto-FGFs, prototypic of the eight families. A second expansion, which took place during euchordate evolution, is associated with genome duplications. It increased the number of members in the families. Subsequent losses reduced that number to the present-day figures.     

Sli-1, a Negative Regulator of Let-23-Mediated Signaling in C. Elegans

By screening for suppressors of hypomorphic mutations of let-23, a receptor tyrosine kinase necessary for vulval induction in Caenorhabditis elegans, we recovered >/=12 mutations defining the sli-1 (suppressor of lineage defect) locus. sli-1 mutations suppress four of five phenotypes associated with hypomorphic alleles of let-23 but do not suppress let-23 null alleles. Thus, a sli-1 mutation does not bypass the requirement for functional let-23 but rather allows more potent LET-23-dependent signaling. Mutations at the sli-1 locus are otherwise silent with respect to vulval differentiation and cause only a low-penetrance abnormal head phenotype. Mutations at sli-1 also suppress the vulval defects but not other defects associated with mutations of sem-5, whose product likely interacts with LET-23 protein during vulval induction. Mutations at sli-1 suppress lin-2, lin-7 and lin-10 mutations but only partially suppress lin-3 and let-60 mutations and do not suppress a lin-45 mutation. The sli-1 locus displays dosage sensitivity: severe reduction of function alleles of sli-1 are semidominant suppressors; a duplication of the sli-1 (+) region enhances the vulvaless phenotype of hypomorphic mutations of let-23. We propose that sli-1 is a negative regulator that acts at or near the LET-23-mediated step of the vulval induction pathway. Our analysis suggests that let-23 can activate distinct signaling pathways in different tissues: one pathway is required for vulval induction; another pathway is involved in hermaphrodite fertilty and is not regulated by sli-1.     

FGF9 is an autocrine and paracrine prostatic growth factor expressed by prostatic stromal cells

Polypeptide growth factors, including members of the fibroblast growth factor (FGF) family, play an important role in the growth and maintenance of the normal prostate. We have found that FGF9 is expressed at high levels in the normal peripheral and transition zone of the human prostate. Analysis of FGF9 production by primary cultures of prostatic epithelial and stromal cells has shown that FGF9 is produced and secreted by the prostatic stromal cells. Neither of these processes appears to be modulated by androgens. Production of FGF9 by stromal cells in vivo was confirmed by immunohistochemistry. FGF9 is a potent mitogen for both prostatic epithelial and stromal cells in culture and is a more potent mitogen for these cells than either FGF2 or FGF7, two other FGFs expressed in the human prostate. FGF9 is an abundant secreted growth factor that can act as both a paracrine mitogen for epithelial cells and an autocrine mitogen for stromal cells. Western blot analysis of tissue extracts from the normal and hyperplastic transition zone shows that FGF9 is present at two to threefold higher levels in the hyperplastic transition zone. Overexpression of this paracrine and autocrine growth factor may play an important role in the epithelial and stromal proliferation in benign prostatic hyperplasia. J. Cell. Physiol. 180:53–60, 1999. © 1999 Wiley-Liss, Inc.     

Fibroblast growth factor 16 and 18 are expressed in human cardiovascular tissues and induce on endothelial cells migration but not proliferation

Endothelial cells line the blood vessel and precursor endothelial cells appear to have a pivotal effect on the organ formation of the heart, the embryonic development of the kidney, and the liver. Several growth factors including the fibroblast growth factors (FGF) seem to be involved in these processes. Ligands such as basic FGF produced and secreted by endothelial cells may also coordinate cellular migration, differentiation, and proliferation under pathological conditions including wound healing, tumorgenesis, and fibrogenesis in the adult. Recently we demonstrated the expression of two secreted FGFs, FGF16, and FGF18, in HUVEC and in rat aortic tissue. In the present report, we confirmed by RT-PCR analysis that FGF18 is wildly expressed in the cardiovascular tissue, while FGF16 showed a more restricted expression pattern. HUVEC clearly demonstrated chemotaxis towards FGF16 and FGF18. Both FGFs also enhanced cell migration in response to mechanical damage. However, recombinant FGF16 and FGF18 failed to induce endothelial cell proliferation or sprouting in a three-dimensional in vitro angiogenesis assay. Fgf18 expression was earlier reported in the liver, and we detected FGF18 expression in liver vascular and liver sinusoidal endothelial cells (LSECs), but not in hepatic parenchymal cells. Recombinant FGF18 stimulated DNA synthesis in primary hepatocytes, suggesting, that endothelial FGF18 might have a paracrine function in promoting growth of the parenchymal tissue. Interestingly, FGF2, which is mitogenic on endothelial cells and hepatocytes stimulates a sustained MAPK activation in both cell types, while FGF18 causes a short transient activation of the MAPK pathway in endothelial cells but a sustained activation in hepatocytes. Therefore, the difference in the time course of MAPK activation by the different FGFs appears to be the cause for the different cellular responses.     

Soluble dominant-negative receptor uncovers essential roles for fibroblast growth factors in multi-organ induction and patterning.

Despite a wealth of experimental data implicating fibroblast growth factor (FGF) signaling in various developmental processes, genetic inactivation of individual genes encoding specific FGFs or their receptors (FGFRs) has generally failed to demonstrate their role in vertebrate organogenesis due to early embryonic lethality or functional redundancy. Here we show that broad mid-gestational expression of a novel secreted kinase-deficient receptor, specific for a defined subset of the FGF superfamily, caused agenesis or severe dysgenesis of kidney, lung, specific cutaneous structures, exocrine and endocrine glands, and craniofacial and limb abnormalities reminiscent of human skeletal disorders associated with FGFR mutations. Analysis of diagnostic molecular markers revealed that this soluble dominant-negative mutant disrupted early inductive signaling in affected tissues, indicating that FGF signaling is required for growth and patterning in a broad array of organs and in limbs. In contrast, transgenic mice expressing a membrane-tethered kinase-deficient FGFR were viable. Our results demonstrate that secreted FGFR mutants are uniquely effective as dominant-negative agents in vivo, and suggest that related soluble receptor isoforms expressed in wild-type mouse embryos may help regulate FGF activity during normal development.     

Investigating the mechanism of the assembly of FGF1-binding heparan sulfate motifs

Heparan sulfate (HS) chains play crucial biological roles by binding to various signaling molecules including fibroblast growth factors (FGFs). Distinct sulfation patterns of HS chains are required for their binding to FGFs/FGF receptors (FGFRs). These sulfation patterns are putatively regulated by biosynthetic enzyme complexes, called GAGOSOMES, in the Golgi. While the structural requirements of HS-FGF interactions have been described previously, it is still unclear how the FGF-binding motif is assembled in vivo. In this study, we generated HS structures using biosynthetic enzymes in a sequential or concurrent manner to elucidate the potential mechanism by which the FGF1-binding HS motif is assembled. Our results indicate that the HS chains form ternary complexes with FGF1/FGFR when enzymes carry out modifications in a specific manner.     

LET-413 is a basolateral protein required for the assembly of adherens junctions in Caenorhabditis elegans

Epithelial cells are polarized, with apical and basal compartments demarcated by tight and adherens junctions. Proper establishment of these subapical junctions is critical for normal development and histogenesis. We report the characterization of the gene let-413 which has a critical role in assembling adherens junctions in Caenorhabditis elegans. In let-413 mutants, adherens junctions are abnormal and mislocalized to more basolateral positions, epithelial cell polarity is affected and the actin cytoskeleton is disorganized. The LET-413 protein contains one PDZ domain and 16 leucine-rich repeats with high homology to proteins known to interact with small GTPases. Strikingly, LET-413 localizes to the basolateral membrane. We suggest that LET-413 acts as an adaptor protein involved in polarizing protein trafficking in epithelial cells.     

Basolateral Localization of the Caenorhabditis elegans Epidermal Growth Factor Receptor in Epithelial Cells by the PDZ Protein LIN-10

In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.     

Role of fibroblast growth factors in elicitation of cell responses

Fibroblast growth factors (FGFs) are signalling peptides that control important cell processes such as proliferation, differentiation, migration, adhesion and survival. Through binding to different types of receptor on the cell surface, these peptides can have different effects on a target cell, the effect achieved depending on many features. Thus, each of the known FGFs elicits specific biological responses. FGF receptors (FGFR 1–5) initiate diverse intracellular pathways, which in turn lead to a variety of results. FGFs also bind the range of FGFRs with a series of affinities and each type of cells expresses FGFRs in different qualitative and quantitative patterns, which also affect responses. To summarize, cell response to binding of an FGF ligand depends on type of FGF, FGF receptor and target cell, all interacting in concert. This review aims to examine properties of the FGF family and its members receptors. It also aims to summarize features of intracellular signalling and highlight differential effects of the various FGFs in different circumstances.     

An In Vivo C. elegans Model System for Screening EGFR-Inhibiting Anti-Cancer Drugs

The epidermal growth factor receptor (EGFR) is a well-established target for cancer treatment. EGFR tyrosine kinase (TK) inhibitors, such as gefinitib and erlotinib, have been developed as anti-cancer drugs. Although non-small cell lung carcinoma with an activating EGFR mutation, L858R, responds well to gefinitib and erlotinib, tumors with a doubly mutated EGFR, T790M-L858R, acquire resistance to these drugs. The C. elegans EGFR homolog LET-23 and its downstream signaling pathway have been studied extensively to provide insight into regulatory mechanisms conserved from C. elegans to humans. To develop an in vivo screening system for potential cancer drugs targeting specific EGFR mutants, we expressed three LET-23 chimeras in which the TK domain was replaced with either the human wild-type TK domain (LET-23::hEGFR-TK), a TK domain with the L858R mutation (LET-23::hEGFR-TK[L858R]), or a TK domain with the T790M-L858R mutations (LET-23::hEGFR-TK[T790M-L858R]) in C. elegans vulval cells using the let-23 promoter. The wild-type hEGFR-TK chimeric protein rescued the let-23 mutant phenotype, and the activating mutant hEGFR-TK chimeras induced a multivulva (Muv) phenotype in a wild-type C. elegans background. The anti-cancer drugs gefitinib and erlotinib suppressed the Muv phenotype in LET-23::hEGFR-TK[L858R]-expressing transgenic animals, but not in LET-23::hEGFR-TK[T790M-L858R] transgenic animals. As a pilot screen, 8,960 small chemicals were tested for Muv suppression, and AG1478 (an EGFR-TK inhibitor) and U0126 (a MEK inhibitor) were identified as potential inhibitors of EGFR-mediated biological function. In conclusion, transgenic C. elegans expressing chimeric LET-23::hEGFR-TK proteins are a model system that can be used in mutation-specific screens for new anti-cancer drugs.