Caenorhabditis elegans genes sma-2, sma-3, and sma-4 define a conserved family of transforming growth factor beta pathway components.

Although transforming growth factor beta (TGF-beta) superfamily ligands play critical roles in diverse developmental processes, how cells transduce signals from these ligands is still poorly understood. Cell surface receptors for these ligands have been identified, but their cytoplasmic targets are unknown. We have identified three Caenorhabditis elegans genes, sma-2, sma-3, and sma-4, that have mutant phenotypes similar to those of the TGF-beta-like receptor gene daf-4, indicating that they are required for daf-4-mediated developmental processes. We show that sma-2 functions in the same cells as daf-4, consistent with a role in transducing signals from the receptor. These three genes define a protein family, the dwarfins, that includes the Mad gene product, which participates in the decapentaplegic TGF-beta-like pathway in Drosophila [Sekelsky, J. J., Newfeld, S. J., Raftery, L. A., Chartoff, E. H. & Gelbart, W. M. (1995) Genetics 139, 1347-1358]. The identification of homologous components of these pathways in distantly related organisms suggests that dwarfins may be universally required for TGF-beta-like signal transduction. In fact, we have isolated highly conserved dwarfins from vertebrates, indicating that these components are not idiosyncratic to invertebrates. These analyses suggest that dwarfins are conserved cytoplasmic signal transducers.

Activation of a serine/threonine kinase signaling pathway by transforming growth factor type beta.

Transforming growth factor type beta (TGF-beta) is a multifunctional factor that regulates proliferation and differentiation of many cell types. TGF-beta mediates its effects by binding to and activating cell surface receptors that possess serine/threonine kinase activity. However, the intracellular signaling pathways through which TGF-beta receptors act remain largely unknown. Here we show that TGF-beta activates a 78-kDa protein (p78) serine/threonine kinase as evidenced by an in-gel kinase assay. Ligand-induced activation of the kinase was near-maximal 5 min after TGF-beta addition to the cells and occurred exclusively on serine and threonine residues. This kinase is distinct from TGF-beta receptor type II, as well as several cytoplasmic serine/threonine kinases of similar size, including protein kinase C, Raf, mitogen-activated protein kinase kinase kinase, and ribosomal S6 kinase. Indeed, these kinases can be separated almost completely from p78 kinase by immunoprecipitation with specific antibodies. Furthermore, using different cell lines, we demonstrate that p78 kinase is activated only in cells for which TGF-beta can act as a growth inhibitory factor. These data raise the interesting possibility that protein serine/threonine kinases contribute to the intracellular relay of biological signals originating from receptor serine/threonine kinases such as the TGF-beta receptors.

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development.

Opposing early and late effects of insulin-like growth factor I on differentiation and the cell cycle regulatory retinoblastoma protein in skeletal myoblasts.

The mechanisms by which insulin-like growth factors (IGFs) can be both mitogenic and differentiation-promoting in skeletal myoblasts are unclear because these two processes are believed to be mutually exclusive in this tissue. The phosphorylation state of the ubiquitous nuclear retinoblastoma protein (Rb) plays an important role in determining whether myoblasts proliferate or differentiate: Phosphorylated Rb promotes mitogenesis, whereas un- (or hypo-) phosphorylated Rb promotes cell cycle exit and differentiation. We hypothesized that IGFs might affect the fate of myoblasts by regulating the phosphorylation of Rb. Although long-term IGF treatment is known to stimulate differentiation, we find that IGFs act initially to inhibit differentiation and are exclusively mitogenic. These early effects of IGFs are associated with maintenance of Rb phosphorylation typical of proliferating cells; upregulation of the gene expression of cyclin-dependent kinase 4 and cyclin D1, components of a holoenzyme that plays a principal role in mediating Rb phosphorylation; and marked inhibition of the gene expression of myogenin, a member of the MyoD family of skeletal muscle-specific transcription factors that is essential in muscle differentiation. We also find that IGF-induced inhibition of differentiation occurs through a process that is independent of its mitogenic effects. We demonstrate, thus, that IGFs regulate Rb phosphorylation and cyclin D1 and cyclin-dependent kinase 4 gene expression; together with their biphasic effects on myogenin expression, these results suggest a mechanism by which IGFs are initially mitogenic and subsequently differentiation-promoting in skeletal muscle.

The site of action of neuronal acidic fibroblast growth factor is the organ of Corti of the rat cochlea.

Here we show that the mature cochlear neurons are a rich source of acidic fibroblast growth factor (aFGF), which is expressed in the neuronal circuitry consisting of afferent and efferent innervation. The site of action of neuronal aFGF is likely to reside in the organ of Corti, where one of the four known FGF receptor (FGFR) tyrosine kinases--namely, FGFR-3 mRNA--is expressed. Following acoustic overstimulation, known to cause damage to the organ of Corti, a rapid up-regulation of FGFR-3 is evident in this sensory epithelium, at both mRNA and protein levels. The present results provide in vivo evidence for aFGF being a sensory neuron-derived, anterogradely transported factor that may exert trophic effects on a peripheral target tissue. In this sensory system, aFGF, rather than being a neurotrophic factor, seems to promote maintenance of the integrity of the organ of Corti. In addition, aFGF, released from the traumatized nerve endings, may be one of the first signals initiating protective recovery and repair processes following damaging auditory stimuli.

Identification of a viability domain in the granulocyte/macrophage colony-stimulating factor receptor beta-chain involving tyrosine-750.

The granulocyte/macrophage colony-stimulating factor (GM-CSF) receptor (GMR) is a heterodimeric receptor expressed by myeloid lineage cells. In this study we have investigated domains of the GMR beta-chain (GMR beta) involved in maintaining cellular viability. Using a series of nested GMR beta deletion mutants, we demonstrate that there are at least two domains of GMR beta that contribute to viability signals. Deletion of amino acid residues 626-763 causes a viability defect that can be rescued with fetal calf serum (FCS). Deletion of residues 518-626, in contrast, causes a further decrement in viability that can be only partially compensated by the addition of FCS. GMR beta truncated proximal to amino acid 517 will not support long-term growth under any conditions. Site-directed mutagenesis of tyrosine-750 (Y750), which is contained within the distal viability domain, to phenylalanine eliminates all demonstrable tyrosine phosphorylation of GMR beta. Cell lines transfected with mutant GMR beta (Y750-->F) have a viability disadvantage when compared to cell lines containing wild-type GMR that is partially rescued by the addition of FCS. We studied signal transduction in mutant cell lines in an effort to identify pathways that might participate in the viability signal. Although tyrosine phosphorylation of JAK2, SHPTP2, and Vav is intact in Y750-->F mutant cell lines, Shc tyrosine phosphorylation is reduced. This suggests a potential role for Y750 and potentially Shc in a GM-CSF-induced signaling pathway that helps maintain cellular viability.

Transforming growth factor beta mediates the progesterone suppression of an epithelial metalloproteinase by adjacent stroma in the human endometrium.

Unlike most normal adult tissues, cyclic growth and tissue remodeling occur within the uterine endometrium throughout the reproductive years. The matrix metalloproteinases (MMPs), a family of structurally related enzymes that degrade specific components of the extracellular matrix are thought to be the physiologically relevant mediators of extracellular matrix composition and turnover. Our laboratory has identified MMPs of the stromelysin family in the cycling human endometrium, implicating these enzymes in mediating the extensive remodeling that occurs in this tissue. While the stromelysins are expressed in vivo during proliferation-associated remodeling and menstruation-associated endometrial breakdown, none of the stromelysins are expressed during the progesterone-dominated secretory phase of the cycle. Our in vitro studies of isolated cell types have confirmed progesterone suppression of stromal MMPs, but a stromal-derived paracrine factor was found necessary for suppression of the epithelial-specific MMP matrilysin. In this report, we demonstrate that transforming growth factor beta (TGF-beta) is produced by endometrial stroma in response to progesterone and can suppress expression of epithelial matrilysin independent of progesterone. Additionally, we find that an antibody directed against the mammalian isoforms of TGF-beta abolishes progesterone suppression of matrilysin in stromal-epithelial cocultures, implicating TGF-beta as the principal mediator of matrilysin suppression in the human endometrium.

Activation of mitogen-activated protein kinases by vascular endothelial growth factor and basic fibroblast growth factor in capillary endothelial cells is inhibited by the antiangiogenic factor 16-kDa N-terminal fragment of prolactin.

A number of factors both stimulating and inhibiting angiogenesis have been described. In the current work, we demonstrate that the angiogenic factor vascular endothelial growth factor (VEGF) activates mitogen-activated protein kinase (MAPK) as has been previously shown for basic fibroblast growth factor. The antiagiogenic factor 16-kDa N-terminal fragment of human prolactin inhibits activation of MAPK distal to autophosphorylation of the putative VEGF receptor, Flk-1, and phospholipase C-gamma. These data show that activation and inhibition of MAPK may play a central role in the control of angiogenesis.

Growth factors can enhance lymphocyte survival without committing the cell to undergo cell division.

Growth factors have been defined by their ability to promote the proliferative expansion of receptor-bearing cells. For example, antigen-activated T cells expressing the alpha beta gamma form of the interleukin 2 (IL-2) receptor will proliferate in response to IL-2. In contrast, resting T cells, which express the IL-2 receptor beta and gamma chains, do not proliferate in response to IL-2. We demonstrate that the survival of resting T cells following gamma irradiation is greatly enhanced by pretreatment with IL-2. The radioprotective effect of IL-2 is dose dependent, does not result from the induction of cell proliferation, and does not require expression of the IL-2 receptor alpha chain. Thus, the beta gamma IL-2 receptor expressed on resting T cells can transduce signals that promote cell survival without committing the T cell to undergo cell division. IL-4 and IL-7, but not IL-1, IL-3, or IL-6, were also found to enhance the survival of quiescent T cells following gamma irradiation. Thus, certain growth factor-receptor interactions can serve to maintain cell viability in a manner that is independent of their ability to initiate or maintain cell proliferation. These data may have important implications for the use of growth factors in patients being treated with radiation and/or chemotherapy.

Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.

The Wsh and Ph mutations affect the c-kit expression profile: c-kit misexpression in embryogenesis impairs melanogenesis in Wsh and Ph mutant mice.

The receptor tyrosine kinases (RTKs) c-kit and platelet-derived growth factor receptor alpha chain (PDG-FRa) are encoded at the white spotting (W) and patch (Ph) loci on mouse chromosome 5. While W mutations affect melanogenesis, gametogenesis, and hematopoiesis, the Ph mutation affects melanogenesis and causes early lethality in homozygotes. W-sash (Wsh) is an expression mutation and blocks c-kit expression in certain cell types and enhances c-kit expression in others, including at sites important for early melanogenesis. We have determined the effect of Ph on c-kit expression during embryogenesis in Ph heterozygotes. Immunohistochemical analysis revealed enhanced c-kit expression in several cell types, including sites important for early melanogenesis. We propose that in both Wsh and Ph mutant mice c-kit misexpression affects early melanogenesis and is responsible for the pigment deficiency. Moreover, we have defined the organization of the RTKs in the W/Ph region on chromosome 5 and characterized the Wsh mutation by using pulsed-field gel electrophoresis. Whereas the order of the RTK genes was determined as Pdgfra-c-kit-flk1, analysis of the Wsh mutation revealed that the c-kit and Pdgfra genes are unlinked in Wsh, presumably because of an inversion of a small segment of chromosome 5. The Ph mutation consists of a deletion including Pdgfra and the 3' deletion endpoint of Ph lies between Pdgfra and c-kit. Therefore, positive 5' upstream elements controlling c-kit expression in mast cells and some other cell types are affected by the Wsh mutation and negative elements are affected by both the Wsh and the Ph mutation.

Identification of cyclins A1, E1 and vimentin as downstream targets of heme oxygenase-1 in vascular endothelial growth factor-mediated angiogenesis

Angiogenesis is an essential physiological process and an important factor in disease pathogenesis. However, its exploitation as a clinical target has achieved limited success and novel molecular targets are required. Although heme oxygenase-1 (HO-1) acts downstream of vascular endothelial growth factor (VEGF) to modulate angiogenesis, knowledge of the mechanisms involved remains limited. We set out identify novel HO-1 targets involved in angiogenesis. HO-1 depletion attenuated VEGF-induced human endothelial cell (EC) proliferation and tube formation. The latter response suggested a role for HO-1 in EC migration, and indeed HO-1 siRNA negatively affected directional migration of EC towards VEGF; a phenotype reversed by HO-1 over-expression. EC from Hmox1(-/-) mice behaved similarly. Microarray analysis of HO-1-depleted and control EC exposed to VEGF identified cyclins A1 and E1 as HO-1 targets. Migrating HO-1-deficient EC showed increased p27, reduced cyclin A1 and attenuated cyclin-dependent kinase 2 activity. In vivo, cyclin A1 siRNA inhibited VEGF-driven angiogenesis, a response reversed by Ad-HO-1. Proteomics identified structural protein vimentin as an additional VEGF-HO-1 target. HO-1 depletion inhibited VEGF-induced calpain activity and vimentin cleavage, while vimentin silencing attenuated HO-1-driven proliferation. Thus, vimentin and cyclins A1 and E1 represent VEGF-activated HO-1-dependent targets important for VEGF-driven angiogenesis.

Growth, survival, gene expression, microbiota and tryptic activity during feeding of Scophthalmus maximus first feeding larvae with beta-glucan (MacroGard)

Reflecting the natural biology of mass spawning fish aquaculture production of fish larvae is often hampered by high and unpredictable mortality rates. The present study aimed to enhance larval performance and immunity via the oral administration of an immunomodulator, beta-glucan (MacroGard®) in turbot (Scophthalmus maximus). Rotifers (Brachionus plicatilis) were incubated with or without yeast beta-1,3/1,6-glucan in form of MacroGard® at a concentration of 0.5 g/L. Rotifers were fed to first feeding turbot larvae once a day. From day 13 dph onwards all tanks were additionally fed untreated Artemia sp. nauplii (1 nauplius ml/L). Daily mortality was monitored and larvae were sampled at 11 and 24 dph for expression of 30 genes, trypsin activity and size measurements. Along with the feeding of beta-glucan daily mortality was significantly reduced by ca. 15% and an alteration of the larval microbiota was observed. At 11 dph gene expression of trypsin and chymotrypsin was elevated in the MacroGard® fed fish, which resulted in heightened tryptic enzyme activity. No effect on genes encoding antioxidative proteins was observed, whilst the immune response was clearly modulated by beta-glucan. At 11 dph complement component c3 was elevated whilst cytokines, antimicrobial peptides, toll like receptor 3 and heat shock protein 70 were not affected. At the later time point (24 dph) an anti-inflammatory effect in form of a down-regulation of hsp 70, tnf-alpha and il-1beta was observed. We conclude that the administration of beta-glucan induced an immunomodulatory response and could be used as an effective measure to increase survival in rearing of turbot.

Vascular endothelial growth factor-B and retinal vascular development in the mouse

Purpose: Vascular endothelial growth factor-A (VEGF-A) is crucial to retinal vascular growth, both normal and pathological. VEGF-B, recently characterized, is reported to be expressed in retinal tissues, but the importance of VEGF-B to retinal vascular development remained unknown. The aim of this study was to analyse retinal vascular growth in the Vegfb (-/-) knockout mouse. Methods: Retinal vascular growth was measured in Vegfb (-/-) knockout mice raised under normal conditions, and Vegfb (-/-) knockout mice with an oxygen-induced proliferative retinopathy. Wild type Vegfb (+/+) mice served as controls. Vessels were perfused with ink and retinal flatmounts secondarily labelled with FITC-lectin (BS-1, Griffonia simplicifolia ). Area and diameter of retinal growth and retinal vascular growth were recorded over days 0-20, and capillary density and mean diameter recorded from day 17 pups. Results: A variety of techniques confirmed that Vegfb (+/+) mice expressed VEGF-B and that VEGF-B expression was absent in Vegfb (-/-) mice. Vegfb (-/-) mice raised in room air showed no significant differences from Vegfb (+/+) controls. No differences were found in oxygen-induced retinopathy between Vegfb (-/-) and Vegfb (+/+) pups in either the extent of the initial oxygen-induced ablation, or in the regrowth of retinal vessels or vitreal (neovascular) sprouts; vitreal sprouts are important markers of the abnormal proliferative response, and are maximally expressed on day 17 in this model of oxygen-induced retinopathy. Conclusions: These results indicate that a lack of VEGF-B does not significantly affect development of the retinal vasculature under normal conditions, nor does it appear to affect the proliferative retinal responses seen in oxygen-induced retinopathy.