Embryonic growth factors.

The role of growth factors in development is under analysis on three main fronts: examination of patterns of growth factor expression in embryogenesis, studies of biological activity in vitro, and mutational analysis in vivo. Recent findings indicate that growth factors control developmental decisions, are strictly controlled in their delivery to responding cells, and act in conjunction to create tissue-specific regulatory networks.

Defective gp130-mediated signal transducer and activator of transcription (STAT) signaling results in degenerative joint disease, gastrointestinal ulceration, and failure of uterine implantation.

The receptor subunit gp130 transduces multiple cell type-specific activities of the leukemia inhibitory factor (LIF)/interleukin (IL)-6 family of cytokines through the signal transducer and activator of transcription (STAT) and src homology 2 domain-bearing protein tyrosine phosphatase (SHP)-2/ras/Erk pathways. To define STAT-dependent physiological responses, we generated mice with a COOH-terminal gp130(DeltaSTAT) "knock-in" mutation which deleted all STAT-binding sites. gp130(DeltaSTAT) mice phenocopyed mice deficient for IL-6 (impaired humoral and mucosal immune and hepatic acute phase responses) and LIF (failure of blastocyst implantation). However, unlike mice with null mutations in any of the components in the gp130 signaling pathway, gp130(DeltaSTAT) mice also displayed gastrointestinal ulceration and a severe joint disease with features of chronic synovitis, cartilaginous metaplasia, and degradation of the articular cartilage. Mitogenic hyperresponsiveness of synovial cells to the LIF/IL-6 family of cyto-kines was caused by sustained gp130-mediated SHP-2/ras/Erk activation due to impaired STAT-mediated induction of suppressor of cytokine signaling (SOCS) proteins which normally limits gp130 signaling. Therefore, the joint pathology in gp130(DeltaSTAT) mice is likely to arise from the disturbance of the otherwise balanced activation of the SHP-2/ras/Erk and STAT signaling cascades emanating from gp130.

Determining the LIF-sensitive period for implantation using a LIF-receptor antagonist.

Uteri of Lif null mice do not support embryo implantation. Since deletion of some genes often prevents the survival of null mice to adulthood, we have used a proven inhibitor of leukaemia inhibitory factor (LIF) signalling to identify the precise window of time during which LIF is required in vivo, and assessed the cellular expression of several LIF-associated targets. On day 4 of pregnancy, mice were injected with hLIF-05 (inhibitor) into the uterine lumen, with corresponding volumes of PBS (vehicle) injected into the contralateral horn. On days 5 and 6, the number of implantation sites was recorded and the uteri processed for immunohistochemistry. Blockade of LIF on day 4 reduced embryo implantation by 50% (P

Characterization of a binding protein for leukemia inhibitory factor localized in extracellular matrix.

Leukemia Inhibitory Factor (LIF) interacts with two classes of high affinity binding sites on rat UMR cells cultured in monolayer. One class of binding sites was found to be localized in the extracellular matrix (ECM) after removal of cells from the culture dish. The interaction of LIF with ECM-localized binding sites is not dependent upon either glycosylation of LIF or the presence of extracellular glycosyaminoglycans. Chemical cross-linking studies demonstrate that LIF interacts with a 200-kD cell-associated protein and a 140-kD ECM-localized protein. A 140-kD protein could also be specifically precipitated from solubilised metabolically radiolabeled UMR ECM by antibodies directed against LIF by virtue of its ability to form a stable complex with unlabeled LIF. In addition, soluble LIF associated with this ECM-localized protein is biologically active in terms of inhibition of ES cell differentiation. The properties of ECM-localized 140-kD species are very similar to those of the secreted form of the LIF receptor suggesting that the ECM localization of LIF and LIF signal transduction may be closely coupled.

Developmental expression of 2ar (osteopontin) and SPARC (osteonectin) RNA as revealed by in situ hybridization.

2ar has been identified as a gene inducible by tumor promoters and growth factors in a variety of cultured mouse cell lines (Smith, J. H., and D. T. Denhardt. 1987. J. Cell. Biochem. 34:13-22). Sequence analysis shows that it codes for mouse osteopontin, an RGDS-containing, phosphorylated, sialic acid-rich Ca++-binding protein originally isolated from bone (Oldberg, A., A. Franzen, and D. Heinegard. 1986. Proc. Natl. Acad. Sci. USA. 83:8819-8823; Prince, C. W., T. Oosawa, W. T. Butler, M. Tomana, A. S. Brown, and R. E. Schrohenloer. 1987. J. Biol. Chem. 262:2900-3907.). In this paper we use Northern blot analysis and in situ hybridization to localize expression of 2ar during mouse embryogenesis. 2ar RNA is first detected in developing limb bones and calvaria at 14.5 d p.c., in a population of cells distinct from those expressing SPARC (osteonectin). High levels of 2ar expression are also seen in the bone marrow-derived granulated metrial gland cells of the deciduum and placenta, and in a number of epithelial tissues, including embryonic and postnatal kidney tubules, uterine epithelium and sensory epithelium of the embryonic ear. The temporal and spatial pattern of 2ar expression seen in vivo suggests that the protein plays a wider role than previously realized, in processes which are not confined to bone development.

Oligodendrocyte population dynamics and the role of PDGF in vivo.

Oligodendrocyte progenitors originate near the floor plate of the spinal cord, then proliferate and migrate throughout the cord before giving rise to oligodendrocytes. Progenitor cell proliferation stops before birth because the cell cycle slows down, linked to an increase in differentiation and death. Experiments with transgenic mice show that platelet-derived growth factor (PDGF) drives progenitor cell division and suggest that slowing of and exit from the cycle reflects a decline in PDGF signaling. Overexpressing PDGF induces hyperproliferation of progenitor cells and excessive, ectopic production of oligodendrocytes. However, the superfluous oligodendrocytes die at an immature stage of differentiation, leaving a normal complement of myelin-forming cells. Therefore, cell survival controls override proliferation controls for determining the final number and distribution of mature oligodendrocytes.

A mass action model of a Fibroblast Growth Factor signaling pathway and its simplification.

We consider a kinetic law of mass action model for Fibroblast Growth Factor (FGF) signaling, focusing on the induction of the RAS-MAP kinase pathway via GRB2 binding. Our biologically simple model suffers a combinatorial explosion in the number of differential equations required to simulate the system. In addition to numerically solving the full model, we show that it can be accurately simplified. This requires combining matched asymptotics, the quasi-steady state hypothesis, and the fact subsets of the equations decouple asymptotically. Both the full and simplified models reproduce the qualitative dynamics observed experimentally and in previous stochastic models. The simplified model also elucidates both the qualitative features of GRB2 binding and the complex relationship between SHP2 levels, the rate SHP2 induces dephosphorylation and levels of bound GRB2. In addition to providing insight into the important and redundant features of FGF signaling, such work further highlights the usefulness of numerous simplification techniques in the study of mass action models of signal transduction, as also illustrated recently by Borisov and co-workers (Borisov et al. in Biophys. J. 89, 951-966, 2005, Biosystems 83, 152-166, 2006; Kiyatkin et al. in J. Biol. Chem. 281, 19925-19938, 2006). These developments will facilitate the construction of tractable models of FGF signaling, incorporating further biological realism, such as spatial effects or realistic binding stoichiometries, despite a more severe combinatorial explosion associated with the latter.

Functions of fibroblast growth factors and their receptors.

Fibroblast growth factors were first characterized twenty years ago as mitogens of cultured fibroblasts. Despite a wealth of data from experiments in vitro, insights have begun to emerge only recently on the normal function of these growth factors in mice and humans, as a result of studies of natural and experimental mutations in the factors and their receptors.

Most highly repeated dispersed DNA families in the mouse genome.

The construction of a small library of mouse repetitive DNA has been previously reported (Pietras et al., Nucleic Acids Res. 11:6965-6983, 1983). Here we report that the 35 plasmids in this library corresponding to highly repeated (greater than 30,000 copies per genome) dispersed DNA sequences can be grouped into no more than 5 distinct families. These families together comprise 8 to 10% of the mouse genome. They include the previously described small elements B1, B2, and R and the large MIF-1 element. Twelve of the 35 clones contain evolutionarily conserved (EC) sequences. One EC clone in our library mostly consists of alternating dCdT residues; another consists of tandem repeats of the sequence CCTCT. The majority of B1s and B2s in the genome appear to be homogeneous, whereas R sequences, ECs, and MIF-1s are heterogeneous. Two earlier reports showed highly repeated mammalian DNA sequences in the herpesvirus genome (Peden et al., Cell 31:71-80, 1982; Puga et al., Cell 31:81-87, 1982). We show that sequences homologous to our EC clones are present in the herpesvirus genome, although these polypyrimidine stretches are not detected in poxvirus, adenovirus, and simian virus 40 genomes. We detect transcripts containing homology to all of these sequences in a nuclear transcription assay. Also, we show that small, polyadenylated RNA molecules homologous to B2 sequences are expressed in undifferentiated embryonal carcinoma cells but not in their differentiated derivatives. The significance of these findings is discussed.

Crystal structure and functional dissection of the cytostatic cytokine oncostatin M.

BACKGROUND: The cytokine oncostatin M (OSM) inhibits growth of certain tumour-derived cells, induces proliferation in other cell types (e.g. haemangioblasts) and is a mediator of inflammatory responses. Its mechanism of action is via specific binding to gp130 and either the leukaemia inhibitory factor receptor (LIFR) or oncostatin M receptor (OSMR) systems at the cell surface to form an active signalling complex. RESULTS: We report here the crystal structure of human oncostatin M (hOSM) along with mutagenesis data which map the receptor-binding epitopes of the molecule. The structure was determined to a resolution of 2.2 A and conforms to the haematopoietin cytokine up-up-down-down four-helix bundle topology. The site 2 epitope, responsible for gp130 binding, is centred around Gly120 which forms a 'dimple' on the surface of the molecule located on helices A and C. The site 3 motif, responsible for LIFR and OSMR binding, consists of a protruding Phe160/Lys163 pair located at the start of helix D. CONCLUSIONS: The data presented allow functional dissection of the receptor-binding interfaces to atomic resolution. Modelling suggests that the gp130 residue Phe169 packs into the site 2 dimple in an analogous fashion to structurally equivalent residues at the growth hormone-growth hormone receptor interface, implying that certain key features may underlie recognition across the whole cytokine/receptor superfamily. Conversely, detailed comparison of the available structures suggests that variations on a common theme dictate the specificity of receptor-ligand interactions within the gp130 family of cytokines.

Experimental approaches to studying the nature and impact of splicing variation in zebrafish.

From a fixed number of genes carried in all cells, organisms create considerable diversity in cellular phenotype through differential regulation of gene expression. One prevalent source of transcriptome diversity is alternative pre-mRNA splicing, which is manifested in many different forms. Zebrafish models of splicing dysfunction due to mutated spliceosome components provide opportunity to link biochemical analyses of spliceosome structure and function with whole organism phenotypic outcomes. Drawing from experience with two zebrafish mutants: cephalophonus (a prpf8 mutant, isolated for defects in granulopoiesis) and caliban (a rnpc3 mutant, isolated for defects in digestive organ development), we describe the use of glycerol gradient sedimentation and native gel electrophoresis to resolve components of aberrant splicing complexes. We also describe how RNAseq can be employed to examine relatively rare alternative splicing events including intron retention. Such experimental approaches in zebrafish can promote understanding of how splicing variation and dysfunction contribute to phenotypic diversity and disease pathogenesis.

2-Aminopurine abolishes epidermal growth factor-stimulated phosphorylation of complexed and chromatin-associated forms of a 33 kDa phosphoprotein.

We have recently reported that several purified polypeptide mitogens such as epidermal growth factor, embryonal carcinoma-derived growth factor, basic fibroblast growth factor and Bombesin induce the rapid appearance of a 33 kDa chromatin-associated phosphoprotein in the nuclei of murine fibroblasts. We show here that in both mouse and human cell lines, a second form of the 33 kDa phosphoprotein exists in a detergent-extractable complexed form which may be pelleted by ultracentrifugation. When quiescent [32P]-labelled cells are treated with EGF, both complexed and chromatin-associated forms of the labelled phosphoprotein are detectable within 10 min, the response peaking at about 1 h and being substantially over 3 h after EGF stimulation. By chymotryptic and cyanogen bromide phosphopeptide mapping studies, the two forms of the 33 kDa phosphoprotein are indistinguishable, as are the mouse and human forms of the protein. The protein kinase inhibitor 2-aminopurine, which has recently been shown to block growth factor-stimulated c-fos and c-myc induction, specifically abolishes the mitogen-stimulated appearance of both forms of the 33 kDa phosphoprotein without affecting the phosphorylation of other cellular proteins. The 33 kDa protein has been purified from Hela cells by a combination of sucrose density gradient centrifugation, preparative electrophoresis and reverse-phase HPLC during which the protein is resolved into two closely-eluting peaks which differ markedly in their specific activity. These results are discussed in relation to the possible role of these events in coupling growth factor-receptor interaction at the cell surface to the early responses of transcriptional activation in the nucleus.

2-Aminopurine abolishes EGF- and TPA-stimulated pp33 phosphorylation and c-fos induction without affecting the activation of protein kinase C.

Epidermal Growth Factor (EGF) and Tetradecanoyl Phorbol Acetate (TPA) initiate signalling cascades in C3H 10T1/2 fibroblasts by primarily activating distinct protein kinases, the EGF receptor tyrosine kinase and protein kinase C respectively; there is no signal crossover at the initiation of signalling. Nevertheless, we show here that both agents rapidly elicit common intracellular responses, including the phosphorylation of complexed and chromatin-associated forms of a 33 kDa phosphoprotein (pp33), that of a 15 kDa chromatin-associated phosphoprotein (pp15), as well as the transcriptional activation of a common subset of genes including the c-fos proto-oncogene. 2-aminopurine specifically abolishes complexed and chromatin-associated pp33 phosphorylation in response to EGF and TPA, as well as the induction of c-fos by both agents. The activation of protein kinase C and the levels of transcription factors that bind to the serum response element (SRE), TPA response element (TRE) or NFkB sites in stimulated cells are relatively unaffected by 2-aminopurine. This, to our knowledge, is the first demonstration that it is possible, by using 2-aminopurine which selectively blocks TPA-stimulated pp33 phosphorylation, to block c-fos induction in TPA-treated cells although protein kinase C remains fully active. Further, we show here that although EGF- and TPA-stimulated induction of c-fos is abolished by 2-aminopurine, the appearance of TRE-binding activity in nuclear extracts of stimulated cells is unaffected, suggesting that EGF- and TPA-stimulated induction of TRE-binding activity utilises existing proteins and is not dependent on fresh c-FOS synthesis. These results imply that 2-aminopurine-sensitive complexed and chromatin-associated pp33 phosphorylation may be crucial to c-fos induction in response to EGF and TPA.

Rapid appearance of novel phosphoproteins in the nuclei of mitogen-stimulated fibroblasts.

A sequential extraction procedure has been used to prepare three protein fractions from control and mitogen-stimulated [32P]-PO4-labelled fibroblasts, the first containing soluble and membrane-bound proteins, the second mainly the cytoskeletal proteins vimentin and actin, and the third, a chromatin-derived fraction enriched in histones. We describe here an analysis of the mitogen-stimulated changes in the [32P] labelled phosphoprotein composition of these fractions. The most obvious changes in response to epidermal growth factor, embryonal carcinoma-derived growth factor, basic fibroblast growth factor and bombesin were the rapid appearance of 33 and 15 kDa phosphoproteins in the chromatin-derived fraction. The epidermal growth factor- and basic fibroblast growth factor-stimulated 33 kDa phosphoprotein produced similar chymotryptic peptides and was phosphorylated on serine residues. DNAse/RNAse treatment of the lysates was essential for the extraction of the 33 kDa phosphoprotein. Further, its presence could be demonstrated in preparations of conventionally purified nuclei. An in situ extraction procedure has been used to provide morphological verification of the sequential extraction data. The final structure containing these phosphoproteins is clearly derived from nuclei, enriched in histones, stains for DNA and appears by electron microscopy, to be homogenously composed of chromatin-like material. Thus, we describe here the rapid mitogen-induced appearance of novel phosphoproteins in the nucleus, raising the possibility that they may be involved in orchestrating early nuclear responses to polypeptide growth factors.

Leukemia inhibitory factor determines the growth status of injured adult sensory neurons.

Conditioning injury to adult mammalian sensory neurons enhances their regeneration potential. Here we show that leukemia inhibitory factor (LIF) is a fundamental component of the conditioning response. Conditioning injury in vivo significantly increases the intrinsic growth capacity of sensory neurons in vitro from LIF+/+ mice. This conditioning effect is significantly blunted in sensory neurons from LIF-/- mice. Enhanced growth is rescued in vitro in LIF-/- mice by the addition of exogenous LIF, and the effect blocked by human LIF-05, an LIF receptor antagonist. Furthermore, we demonstrate that LIF promotes elongating but not arborizing neurite outgrowth in vitro and is required for normal regeneration of injured adult sensory neurons in vivo. LIF is also functionally protective to peptidergic sensory neurons after nerve damage in vivo. Our results indicate that the alteration in intrinsic growth status of injured sensory neurons depends, at least in part, on LIF.

Mouse osteoblasts synthesize collagenase in response to bone resorbing agents.

Bone cells isolated from mouse calvariae by a sequential digestion procedure have many osteoblast characteristics: they respond to PTH and prostaglandin E2 by activation of adenylate cyclase but not to calcitonin, they stain for alkaline phosphatase and they make only type I collagen. In confluent monolayer culture, they do not secrete collagenase in appreciable quantities, unless stimulated with resorptive substances such as PTH, prostaglandin E2, 1,25(OH)2 vitamin D-3 and monocyte-conditioned medium. This suggests they play a direct role in bone resorption.

A factor synthesized by rabbit periosteal fibroblasts stimulates bone resorption and collagenase production by connective tissue cells in vitro.

Unstimulated monolayer cultures of confluent rabbit periosteal fibroblasts synthesize a factor that stimulates bone resorption in vitro. Furthermore it stimulates rabbit chondrocytes and mouse osteoblasts to synthesize collagenase. The factor has no effect on dead bone in culture, and its activity on live bone is mediated principally by osteoclasts, since it is 75% inhibited by salmon calcitonin. Characterization of the factor by gel filtration and isoelectric focusing indicates an Mr in the range 15000-25000 and a pI corresponding to approx. pH 4.7. These biological and physiochemical properties are similar to those reported for a factor released by peripheral blood monocytes. However, whereas human monocyte factor in both the crude and partially-purified state exhibits interleukin-1 activity, crude and fractionated periosteal fibroblast-conditioned medium does not. This is the first report of a conditioned medium containing a molecule like the monocyte-factor which appears to have no interleukin 1 activity. The factor may be synthesized by a wide range of cell types, and could have an important role in mediating connective tissue degradation during both physiological and pathological resorption.

Involvement of AP1 and PEA3 binding sites in the regulation of murine tissue inhibitor of metalloproteinases-1 (TIMP-1) transcription.

Transcription of tissue inhibitor of metalloproteinases-1 (TIMP-1), a secreted protein that regulates the activities of the metalloproteinases, collagenase and stromelysin, is activated by serum growth factors. Transient transfection experiments have revealed several regions of cis-acting regulatory sequences involved in the response of the murine TIMP-1 gene to serum. One area is in the vicinity of the promoter, consisting of a non-consensus binding site (5'-TGAGTAA-3' at -59/-53) for transcription factor AP1 and an adjacent 24 bp region of dyad symmetry that contains a PEA3-binding site. A second is an upstream region (-1020 to -780) that acts as an enhancer when linked to a heterologous promoter, and contains a consensus AP1 binding site (at -803/ -797). Gel retardation assays revealed differences between nuclear factors in mouse C3H10T1/2 cells that bound to the TIMP(-59/ -53)AP1 site and a consensus collagenase TRE (TPA-response element). The TIMP(-59/ -53)AP1 site is a promiscuous motif that binds c-Fos/c-Jun AP1 translated in vitro and is an effective competitor for binding of nuclear AP1 factors to the consensus TRE, but in addition it binds factors that do not associate with the consensus TRE. The TIMP(-59/ -53)AP1 motif and the dyad symmetry region stimulated expression from a thymidine kinase promoter in an additive fashion, and competition experiments showed that excess copies of these factor binding sites reduced expression from a reporter plasmid driven by the TIMP-1 promoter. Our data show that binding sites for AP1 and PEA3 transcription factors are involved in the regulation of TIMP-1 transcription, which suggests that the coordinated induction of TIMP-1, collagenase and stromelysin may be achieved through the actions of a shared set of nuclear transcription factors. However, the properties of the TIMP-1(-59/ -53)AP1 motif likely reflect an additional type of transcriptional regulation restricted to TIMP-1.

The murine A33 antigen is expressed at two distinct sites during development, the ICM of the blastocyst and the intestinal epithelium.

The expression pattern of the murine A33 antigen has been defined during development using wholemount immunohistochemistry. Two temporally and spatially distinct sites of expression were identified: the inner cell mass of the blastocyst and the endoderm cell layer of the intestinal tract where expression is initiated at E14.5 in the hindgut and subsequently extends throughout the length of the intestine. The onset of mA33 antigen expression in the gut occurs at the beginning of an extensive phase of cell movement involved in the conversion of the endoderm cell layer to a single cell layer of polarized epithelium. Expression of mA33 antigen is then maintained into adulthood, where it is a definitive marker of intestinal epithelium.

Retinoic acid stimulates transcriptional activity from the alkaline phosphatase promoter in the immortalized rat calvarial cell line, RCT-1.

The immortalized rat calvarial bone cell line RCT-1 responds to treatment with retinoic acid (RA) by increased expression of osteoblast phenotype-related features, including the induction of liver/bone/kidney alkaline phosphatase (ALP) activity. ALP mRNA could not be demonstrated in unstimulated cells, but was first detected in cells treated for 6 h with 1 microM RA. Cycloheximide failed to block the RA induction of ALP mRNA, indicating that de novo protein synthesis was not a requirement for the RA effect and that the ALP gene may be a direct target for RA action. This was confirmed by nuclear run-on assays, which demonstrated a 2.5-fold increase in the abundance of ALP transcripts after 6 h of RA treatment. To determine whether the RA responsiveness was mediated by a specific segment of the ALP promoter, RCT-1 cells were transfected with a series of plasmids containing deletions of the 5'-flanking sequence of the human ALP gene fused to the chloramphenicol acetyl transferase (CAT) gene. CAT activity was measured in cells cultured in the presence of RA or vehicle. All but the smallest construct, which contained 44 basepairs up-stream of the initiation of transcription, were found to mediate a 2- to 3-fold increase in the expression of CAT activity in response to RA. Furthermore, when the region -108 to -45 of the human ALP gene was inserted into the expression vector pBLcat2, in a position immediately up-stream of the herpes simplex virus thymidine kinase promoter, the construct was found to mediate a 2-fold enhancement of CAT activity in response to RA. In gel retardation assays, a major band was present corresponding to the formation of a complex between the 32P-labeled probe containing the -108 to -45 sequence and proteins present in nuclear extracts of RCT-1 cells stimulated for 3 h with RA. These data suggest that the sequence of 64 basepairs (-108 to -45) 5' to the transcription start site is involved in the RA inducibility of the human ALP gene.