Graded activation of fibroblast growth factor receptor 3 by mutations causing achondroplasia and thanatophoric dysplasia.

The longitudinal growth of the skeleton arises from the continuous process of endochondral ossification occurring at the ends of growing long bones. Dwarfism results when this process is disrupted, as in the autosomal dominant human skeletal diseases hypochondroplasia (HCH), achondroplasia (ACH) and thanatophoric dysplasia (TD). Interestingly, these disorders display a graded spectrum of phenotypic severity and are the result of distinct missense mutations in the fibroblast growth factor receptor 3 gene (FGFR3). TD, characterized by neonatal lethality and profound dwarfism, is the result of FGFR3 mutations, including an R248C substitution in the extracellular domain or a K650E substitution in the tyrosine kinase (TK) domain. ACH, which is non-lethal and presents less severe dwarfism, results almost exclusively from a G380R substitution in the transmembrane domain. Homozygous achondroplasia resembles the phenotype of TD. In this report the effect of the ACH and TD mutations on the activity and regulation of FGFR3 are analysed. We showed that each of the mutations constitutively activate the receptor, as evidenced by ligand-independent receptor tyrosine phosphorylation and cell proliferation. Moreover, the mutations that are responsible for TD were more strongly activating than the mutation causing ACH, providing a biochemical explanation for the observation that the phenotype of TD is more severe than that of ACH.

Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3.

Fibroblast growth factor receptor 3 (Fgfr3) is a tyrosine kinase receptor expressed in developing bone, cochlea, brain and spinal cord. Achondroplasia, the most common genetic form of dwarfism, is caused by mutations in FGFR3. Here we show that mice homozygous for a targeted disruption of Fgfr3 exhibit skeletal and inner ear defects. Skeletal defects include kyphosis, scoliosis, crooked tails and curvature and overgrowth of long bones and vertebrae. Contrasts between the skeletal phenotype and achondroplasia suggest that activation of FGFR3 causes achondroplasia. Inner ear defects include failure of pillar cell differentiation and tunnel of Corti formation and result in profound deafness. Our results demonstrate that Fgfr3 is essential for normal endochondral ossification and inner ear development.

Physiological degradation converts the soluble syndecan-1 ectodomain from an inhibitor to a potent activator of FGF-2.

The activity of fibroblast growth factor 2 (FGF-2) is stringently controlled. Inactive in undisturbed tissues, it is activated during injury and is critical for tissue repair. We find that this control can be imposed by the soluble syndecan-1 ectodomain, a heparan sulfate proteoglycan shed from cell surfaces into wound fluids. The ectodomain potently inhibits heparin-mediated FGF-2 mitogenicity because of the poorly sulfated domains in its heparin sulfate chains. Degradation of these regions by platelet heparanase produces heparin-like heparin sulfate fragments that markedly activate FGF-2 mitogenicity and are found in wound fluids. These results establish a novel physiological control for FGF-2 and suggest new ways to modulate FGF activity.

Pancreatic neoplasia induced by SV40 T-antigen expression in acinar cells of transgenic mice.

Three lines of transgenic mice were produced that develop pancreatic neoplasms as a consequence of expression of an elastase I-SV40 T-antigen fusion gene in the acinar cells. A developmental analysis suggests at least a two-stage process in the ontogeny of this disease. The first stage is a T antigen-induced, preneoplastic state characterized by a progression from hyperplasia to dysplasia of the exocrine pancreas, by an increased percentage of tetraploid cells, and by an arrest in acinar cell differentiation. The second stage is characterized by the formation of tumor nodules that appear to be monoclonal, because they have discrete aneuploid DNA contents. The cells within the nodules as compared to normal pancreatic tissue have less total RNA by a factor of 5, less pancreas-specific messenger RNA by a factor of about 50, and increased levels of T-antigen messenger RNA. A tumor cell line has been derived that retains both pancreatic and neoplastic properties.

FGF binding and FGF receptor activation by synthetic heparan-derived di- and trisaccharides.

Fibroblast growth factors (FGFs) require a polysaccharide cofactor, heparin or heparan sulfate (HS), for receptor binding and activation. To probe the molecular mechanism by which heparin or HS (heparin/HS) activates FGF, small nonsulfated oligosaccharides found within heparin/HS were assayed for activity. These synthetic and isomerically pure compounds can activate the FGF signaling pathway. The crystal structures of complexes between FGF and these heparin/HS oligosaccharides reveal several binding sites on FGF and constrain possible mechanisms by which heparin/HS can activate the FGF receptor. These studies establish a framework for the molecular design of compounds capable of modulating FGF activity.

Mice with conditional inactivation of fibroblast growth factor receptor-2 signaling in oligodendrocytes have normal myelin but display dramatic hyperactivity when combined with Cnp1 inactivation.

Fibroblast growth factor receptors (Fgfr) comprise a widely expressed family of developmental regulators implicated in oligodendrocyte (OL) maturation of the CNS. Fgfr2 is expressed by OLs in myelinated fiber tracks. In vitro, Fgfr2 is highly upregulated during OL terminal differentiation, and its activation leads to enhanced growth of OL processes and the formation of myelin-like membranes. To investigate the in vivo function of Fgfr2 signaling by myelinating glial cells, we inactivated the floxed Fgfr2 gene in mice that coexpress Cre recombinase (cre) as a knock-in gene into the OL-specific 2',3'-cyclic nucleotide phosphodiesterase (Cnp1) locus. Surprisingly, no obvious defects were detected in brain development of these conditional mutants, including the number of OLs, the onset and extent of myelination, the ultrastructure of myelin, and the expression level of myelin proteins. However, unexpectedly, a subset of these conditional Fgfr2 knock-out mice that are homozygous for cre and therefore are also Cnp1 null, displayed a dramatic hyperactive behavior starting at approximately 2 weeks of age. This hyperactivity was abolished by treatment with dopamine receptor antagonists or catecholamine biosynthesis inhibitors, suggesting that the symptoms involve a dysregulation of the dopaminergic system. Although the molecular mechanisms are presently unknown, this novel mouse model of hyperactivity demonstrates the potential involvement of OLs in neuropsychiatric disorders, as well as the nonpredictable role of genetic interactions in the behavioral phenotype of mice.

Modulation of fibroblast growth factor-2 receptor binding, dimerization, signaling, and angiogenic activity by a synthetic heparin-mimicking polyanionic compound.

Heparan sulfate (HS) proteoglycans play a key role in cell proliferation induced by basic fibroblast growth factor (FGF-2) and other heparin-binding growth factors. To modulate the involvement of HS, we have used a synthetic, nonsulfated polyanionic aromatic compound (RG-13577) that mimics functional features of heparin/HS. FGF-2-stimulated proliferation of vascular endothelial cells was markedly inhibited in the presence of 5-10 microg/ml compound RG-13577 (poly-4-hydroxyphenoxy acetic acid; Mr approximately 5 kD). Direct interaction between RG-13577 and FGF-2 was demonstrated by the ability of the former to compete with heparin on binding to FGF-2. RG-13577 inhibited FGF-2 binding to soluble- and cell surface-FGF receptor 1 (FGFR1). Unlike heparin, RG-13577 alone failed to mediate dimerization of FGF-2. Moreover, it abrogated heparin-mediated dimerization of FGF-2 and FGFR1, as well as FGF-2 mitogenic activity in HS-deficient F32 lymphoid cells. The antiproliferative effect of compound RG-13577 was associated with abrogation of FGF-2-induced tyrosine phosphorylation of FGFR1 and of cytoplasmic proteins involved in FGF-2 signal transduction, such as p90 and mitogen-activated protein kinase. A more effective inhibition of tyrosine phosphorylation was obtained after removal of the cell surface HS by heparinase. In contrast, tyrosine phosphorylation of an approximately 200-kD protein was stimulated by RG-13577, but not by heparin or FGF-2. RG-13577 prevented microvessel outgrowth from rat aortic rings embedded in a collagen gel. Development of nontoxic polyanionic compounds may provide an effective strategy to inhibit FGF-2-induced cell proliferation associated with angiogenesis, arteriosclerosis, and restenosis.

Promoter and enhancer elements from the rat elastase I gene function independently of each other and of heterologous enhancers.

An elastase-human growth hormone (hGH) fusion gene containing 205 base pairs of elastase 5' flanking region is expressed exclusively in pancreatic acinar cells of transgenic mice. This paper shows that the promoter region (-72 to +8) and the enhancer (-205 to -73) function independently of each other. The elastase enhancer can activate the heterologous mouse metallothionein gene and the hGH gene promoters; conversely, enhancers from the thymocyte-specific murine leukemia virus MCF13 and the metal regulatory elements from the metallothionein gene can activate the elastase promoter in a variety of cell types. Combinations of immunoglobulin and elastase enhancers with a heterologous promoter and the hGH gene result in expression in all of the tissues predicted by the sum of each enhancer acting alone. Thus these enhancer elements act independently of each other, suggesting that they do not have silencing activity in cells in which they are normally inactive.

Heparin is required for cell-free binding of basic fibroblast growth factor to a soluble receptor and for mitogenesis in whole cells.

Heparin is required for the binding of basic fibroblast growth factor (bFGF) to high-affinity receptors on cells deficient in cell surface heparan sulfate proteoglycan. So that this heparin requirement could be evaluated in the absence of other cell surface molecules, we designed a simple assay based on a genetically engineered soluble form of murine FGF receptor 1 (mFR1) tagged with placental alkaline phosphatase. Using this assay, we showed that FGF-receptor binding has an absolute requirement for heparin. By using a cytokine-dependent lymphoid cell line engineered to express mFR1, we also showed that FGF-induced mitogenic activity is heparin dependent. Furthermore, we tested a series of small heparin oligosaccharides of defined lengths for their abilities to support bFGF-receptor binding and biologic activity. We found that a heparin oligosaccharide with as few as eight sugar residues is sufficient to support these activities. We also demonstrated that heparin facilitates FGF dimerization, a property that may be important for receptor activation.

Identification of the cytoplasmic regions of fibroblast growth factor (FGF) receptor 1 which play important roles in induction of neurite outgrowth in PC12 cells by FGF-1.

Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. Recently, we have shown that the FGF receptor 1 (FGFR-1) is much more potent than FGFR-3 in induction of neurite outgrowth. To identify the cytoplasmic regions of FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells, we took advantage of this difference and prepared receptor chimeras containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions of FGFR-1 were identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to FGFR-3. Analysis of FGF-stimulated activation of signal transduction revealed that the JM region of FGFR-1 conferred strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2 protein was demonstrated to be one of the cellular substrates preferentially phosphorylated by chimeras containing the JM domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involved the tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM region functioned primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis revealed that the JM region did not play a role in this cell type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling pathways for neurite outgrowth and mitogenesis use different regions of the FGFR.

The rat elastase I regulatory element is an enhancer that directs correct cell specificity and developmental onset of expression in transgenic mice.

A total of 134 base pairs of the 5' flanking sequence of the elastase I gene is sufficient and necessary to direct expression of the passive human growth hormone gene (hGH) to the exocrine pancreas. We demonstrate that this elastase I regulatory region contains a transcriptional enhancer which directs acinar cell-specific expression in transgenic animals. The elastase I enhancer specifies correct expression of the linked hGH gene in an orientation- and position-independent manner and can activate a heterologous promoter. The enhancer also directs the appropriate temporal activation of the hGH gene in the developing pancreas. Transcription is initiated correctly for the elastase I or hGH promoter, and the transcripts are correctly processed regardless of the enhancer position within or outside the fusion gene. The elastase I enhancer generates coincident DNase I-hypersensitive sites in pancreatic chromatin when moved 3 kilobases upstream or within the first intron of the hGH gene and when associated with the hGH promoter.

Int-2, an autocrine and/or ultra-short-range effector in transgenic mammary tissue transplants.

BACKGROUND: Previous studies have shown associations between overexpression of int-2 messenger RNA (mRNA) and murine mammary tumors and between amplification of the int-2 genomic locus and human breast cancers. The Int-2 protein (fibroblast growth factor 3) is a member of the heparin-binding growth factor family of proteins. The export of these growth factors from cells may depend on the presence of amino terminal sequences containing hydrophobic signal peptides. Although Int-2 has a putative signal sequence, it is not known whether or how this protein is secreted from cells. PURPOSE: Assuming that the Int-2 protein is secreted from mammary epithelial cells in a basolateral direction so that it is available to affect adjacent cells, we investigated whether it acts in a paracrine manner, exerting its effect externally on adjacent cells, or in an autocrine manner, exerting its effect internally within the same cell. METHODS: Using in situ hybridization with 35S-labeled RNA antisense probes that specifically detect mRNA coding for the Int-2 protein, we determined the cell-specific localization of int-2 mRNA expression in the mammary gland of transgenic FVB/N mice overexpressing int-2 mRNA. Then we transplanted pieces of mammary epithelial tissue expressing int-2 mRNA into the mammary fat-pad of wild-type, syngeneic animals. The mammary glands of host animals were examined as whole-mounts and as histologic sections 2-6 months after transplantation. In situ hybridization was used to confirm which cells continued to express int-2 mRNA following transplantation. RESULTS: Int-2 mRNA expression in transgenic mice was localized to the mammary epithelial cells. Transplants expressing int-2 mRNA were similar to wild-type transplants in that they had no observable effect on either the growth or the morphology of host mammary epithelium. Abnormal growth occurred only in transplanted tissue expressing int-2 mRNA but not in adjacent host mammary epithelium. CONCLUSION: Given the limitations of our experimental system and the limited information available to date on the secretion of Int-2 protein, these results suggest that, although the Int-2 protein contains a putative signal peptide, it may act primarily as an autocrine or as an ultra-short-range paracrine growth factor in mammary epithelium.

MMTV-Fgf8 transgenic mice develop mammary and salivary gland neoplasia and ovarian stromal hyperplasia.

Prior studies have identified Fibroblast Growth Factor-8 (Fgf8) as a possible proto-oncogene in mouse mammary tumorigenesis. We now report on the generation of two types of Fgf8 transgenic mice that each utilize the mouse mammary tumor virus (MMTV) promoter. The first transgene (MMTV-Fgf8b) results in the overexpression of the FGF8b isoform exclusively. Male and female MMTV-Fgf8b transgenic mice are viable and fertile. RNA for FGF8b is detected in mammary gland and salivary gland tissues of transgenic mice by Northern blot analysis. Nearly 85% of breeding transgenic female mice developed mammary lobular adenocarcinomas by 12 months of age, while no tumors developed in non-transgenic littermates. Salivary gland tumors occurred in some animals, always in association with mammary tumors. Several MMTV-Fgf8b transgenic mice had lung metastases at necropsy. The second transgene (MMTV-Fgf8) uses the entire Fgf8 gene and potentially encodes all FGF8 isoforms. Fgf8 is expressed by this transgene in several tissues in addition to those described above, notably the ovaries. The two MMTV-Fgf8 founders developed mammary ductal adenocarcinomas at five and eight months of age, and both displayed ovarian stromal hyperplasia. The founders expressing either transgene did not successfully nurse their pups. These results demonstrate that production of FGF8b, and possibly other FGF8 isoforms, in the mammary and salivary glands contributes to oncogenesis, and that ovarian expression results in stromal hyperplasia.

Brain tumours and lymphomas in transgenic mice that carry HTLV-I LTR/c-myc and Ig/tax genes.

The human T-cell leukemia virus type 1 (HTLV-I) is associated with adult CD4+ T-cell leukemia (ATL) and tropical spastic paraparesis (TSP). In as much as only a small percentage of individuals infected with HTLV-I develop either disease, we set out to model a genetic partner for this virus in an effort to understand and possibly reproduce its pathophysiology. To this end we have developed a binary set of transgenic mice, one bearing the relatively inactive HTLV-I long terminal repeat (LTR) positioned to drive the c-myc oncogene and another bearing a fusion transgene consisting of the immunoglobulin promoter/enhancer driving the gene for the HTLV-I transcription activator, tax. Alone, the tax construct, though expressed in the thymus, spleen, lung and brain, has no deleterious effect. Alone, the HTLV-I LTR/c-myc construct is expressed at very low levels in lymphoid cells and occasionally induces lymphomas in older animals. When these two transgenic lines are mated, bigenic offspring harboring both transgenes exhibit dramatic tumor formation. As in the human, these animals develop CD4+ T-cell lymphomas, but they also develop central nervous system tumors by 25-90 days of age. The syndrome, which is 100% penetrant and lethal, provides an animal model for adult T-cell lymphoma and a source of cultured cells of neurogenic origin.

Subcellular and developmental expression of alternatively spliced forms of fibroblast growth factor 14.

Fibroblast growth factors (FGFs) 11-14 comprise a subfamily of FGFs with poorly defined biological function. Here we characterize two isoforms of FGF14 (FGF14-1a and FGF14-1b) that result from the alternative usage of two different first exons. We demonstrate that these isoforms have differential subcellular localization and that they are differentially expressed in various adult tissues. Using in situ hybridization we show that Fgf14 is widely expressed in brain, spinal cord, major arteries and thymus between 12.5 and 14.5 days of mouse embryonic development. We also show that during cerebellar development, Fgf14 is first observed at postnatal day 1 in post mitotic granule cells, and later in development, in migrating and post migratory granule cells. The developmental expression pattern of Fgf14 in the cerebellum is complementary to that of Math1, a marker for proliferating granule cells in the external germinal layer.

Genomic structure, mapping, activity and expression of fibroblast growth factor 17.

Fibroblast growth factors are essential molecules for development. Here we characterize Fgfl7, a new member of the fibroblast growth factor (FGF) family. The Fgfl7 gene maps to mouse chromosome 14 and is highly conserved between mouse and human (93% identity). It exhibits 60% amino acid identity with Fgf8 and 50% identity with Fgf8. Both Fgf8 and Fgf17 have a similar structure and a similar pattern of alternative splicing in the 5' coding region. When expressed in 3T3 fibroblasts, mouse FGF17 is transforming, indicating that it can activate the 'c' splice form of either FGF receptor (FGFR) one or two. During midgestation embryogenesis, in situ hybridization analysis localized Fgf17 expression to specific sites in the midline structures of the forebrain, the midbrain-hindbrain junction, the developing skeleton and in developing arteries. Comparison to Fgf8 revealed a striking similarity in expression patterns, especially in the central nervous system (CNS), suggesting that both genes may be important for CNS development, although Fgf17 is expressed somewhat later than Fgf8. In the developing skeleton, both genes are expressed in costal cartilage while Fgf8 is preferentially expressed in long bones. In the developing great vessels Fgfl7 is preferentially expressed, suggesting that it may have a more prominent role in vascular growth.

Expression of FGFR3 with the G380R achondroplasia mutation inhibits proliferation and maturation of CFK2 chondrocytic cells.

A G380R substitution in the transmembrane-spanning region of FGFR3 (FGFR3Ach) results in constitutive receptor kinase activity and is the most common cause of achondroplastic dwarfism in humans. The epiphyseal growth plates of affected individuals are disorganized and hypocellular and show aberrant chondrocyte maturation. To examine the molecular basis of these abnormalities, we used a chondrocytic cell line, CFK2, to stably express the b variant of wild-type FGFR3 or the the constitutively active FGFR3Ach. Overexpression of FGFR3 had minimal effects on CFK2 proliferation and maturation compared with the severe growth retardation found in cells expressing FGFR3Ach. Cells expressing the mutant receptor also showed an abnormal apoptotic response to serum deprivation and failed to undergo differentiation under appropriate culture conditions. These changes were associated with altered expression of integrin subunits, which effectively led to a switch in substrate preference of the immature cell from fibronectin to type II collagen. These in vitro observations support those from in vivo studies indicating that FGFR3 mediates an inhibitory influence on chondrocyte proliferation. We now suggest that the mechanism is related to altered integrin expression.

FGF signaling in skeletal development.

The fibroblast growth factor receptor family consists of four receptor tyrosine kinases which bind with varying affinity and specificity to a family of at least fifteen polypeptide ligands. The receptors and ligands perform many essential functions during growth, development and repair. Recent discoveries show that a growing number of skeletal abnormalities result from mutations in the fibroblast growth factor receptors. These findings have led to a greater understanding of the role of fibroblast growth factor signaling during skeletogenesis and have focused research interests on the effects of fibroblast growth factors on endochondral and intramembranous bone development.

Regulation of chondrocyte growth and differentiation by fibroblast growth factor receptor 3.

Both gain-of-function and loss-of-function mutations in fibroblast growth factor receptor 3 (Fgfr3) have revealed unique roles for this receptor during skeletal development. Loss-of-function alleles of Fgfr3 lead to an increase in the size of the hypertrophic zone, delayed closure of the growth plate and the subsequent overgrowth of long bones. Gain-of-function mutations in Fgfr3 have been genetically linked to autosomal dominant dwarfing chondrodysplasia syndromes where both the size and architecture of the epiphyseal growth plate are altered. Analysis of these phenotypes and the biochemical consequences of the mutations in FGFR3 demonstrate that FGFR3-mediated signalling is an essential negative regulator of endochondral ossification.

Development and maintenance of otoconia: biochemical considerations.

The first part of this review deals with recent advances in the understanding of biochemical mechanisms of otoconial morphogenesis. Most important in this regard is the molecular characterization of otoconin 90, the principal matrix protein of mammalian calcitic otoconia, which was found to be a homologue of the phospholytic enzyme PLA2. The unique and unexpected expression pattern of this protein required radical rethinking of traditional concepts. The new data, when integrated with existing information, provide a rational basis for an explanation of the mechanisms leading to crystal nucleation and growth. Based on this information, a hypothetical model is presented that posits interaction of otoconin 90 with microvesicles derived from the supporting cells as a key event in the formation of otoconia. The second part of the review is directed at the controversial subject of maintenance of mature otoconia and systematically analyzes the available indirect information on this topic. A synthesis of these theoretical considerations is viewed in relation to the pathogenesis of the important otoneurologic entities of BPPN and senile otoconial degeneration. The last part of the review deals with several animal models that promise to help elucidate normal and abnormal mechanisms of otoconial morphogenesis, including mineral deficiencies, mutations with selective otoconial agenesis, as well as targeted disruption of essential genes.