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Biomedical subjects

D Ron

Publications and source records attributed to D Ron.

At least 73 records · Page 4Linked to original sources
PubMed1996

Involvement of heparan sulfate and related molecules in sequestration and growth promoting activity of fibroblast growth factor.

Heparan sulfate proteoglycans (HSPGs) are ubiquitous macromolecules associated with the cell surface and extracellular matrix (ECM) of a wide range of cells of vertebrate and invertebrate tissues [1, 2]. The basic HSPG structure consists of a protein core to which several linear heparan sulfate (HS) chains are covalently attached. The polysaccharide chains are typically composed of repeating hexuronic and D-glucosamine disaccharide units that are substituted to a varying extent with N- and O-linked sulfate moieties and N-linked acetyl groups [1, 2]. Beside serving as a scaffold for the attachment of various ECM components (e.g., collagen, laminin, fibronectin), the binding of HS to certain proteins has been suggested to induce a conformational change which may lead to the exposure of novel reactive determinants or conversely stabilize an inert protein configuration [1-4]. Of particular significance is the interaction of HS with fibroblast growth factors (FGFs), mediating their sequestration, stabilization and high affinity receptor binding and signaling [3-7]. Cellular responses to FGFs may hence be modulated by metabolic inhibitors of HS synthesis and sulfation, HS-degrading enzymes, and synthetic mimetics of heparin/HS. In the present review we focus on the involvement of HS in basic FGF (bFGF) receptor binding and mitogenic activity and its modulation by species of heparin, HS, and synthetic polyanionic 'heparin-mimicking' compounds. The results are discussed in relation to the current thoughts on the dual involvement of low and high affinity receptor sites in the growth promoting and angiogenic activities of bFGF and other heparin-binding growth factors.

Animals↗
PubMed1996

Stress-induced binding of the transcriptional factor CHOP to a novel DNA control element.

CHOP (GADD153) is a mammalian nuclear protein that dimerizes with members of the C/EBP family of transcriptional factors. Absent under normal conditions, CHOP is induced by the stress encountered during nutrient deprivation, the acute-phase response, and treatment of cells with certain toxins. The basic region of CHOP deviates considerably in sequence from that of other C/EBP proteins, and CHOP-C/EBP heterodimers are incapable of binding to a common class of C/EBP sites. With respect to such sites, CHOP serves as an inhibitor of the activity of C/EBP proteins. However, recent studies indicate that certain functions of CHOP, such as the induction of growth arrest by overexpression of the wild-type protein and oncogenic transformation by the TLS-CHOP fusion protein, require an intact basic region, suggesting that DNA binding by CHOP may be implicated in these activities. In this study an in vitro PCR-based selection assay was used to identify sequences bound by CHOP-C/EBP dimers. These sequences were found to contain a unique core element PuPuPuTGCAAT(A/C)CCC. Competition in DNA-binding assays, DNase 1 footprint analysis, and methylation interference demonstrate that the binding is sequence specific. Deletions in the basic region of CHOP lead to a loss of DNA binding, suggesting that CHOP participates in this process. Stress induction in NIH 3T3 cells leads to the appearance of CHOP-containing DNA-binding activity. CHOP is found to contain a transcriptional activation domain which is inducible by cellular stress, lending further support to the notion that the protein can function as a positively acting transcription factor. We conclude that CHOP may serve a dual role both as an inhibitor of the ability of C/EBP proteins to activate some target genes and as a direct activator of others.

3T3 Cells↗
PubMed1996

Signals from the stressed endoplasmic reticulum induce C/EBP-homologous protein (CHOP/GADD153).

The gene encoding C/EBP-homologous protein (CHOP), also known as growth arrest and DNA-damage-inducible gene 153 (GADD153), is activated by agents that adversely affect the function of the endoplasmic reticulum (ER). Because of the pleiotropic effects of such agents on other cellular processes, the role of ER stress in inducing CHOP gene expression has remained unclear. We find that cells with conditional (temperature-sensitive) defects in protein glycosylation (CHO K12 and BHK tsBN7) induce CHOP when cultured at the nonpermissive temperature. In addition, cells that are defective in initiating the ER stress response, because of overexpression of an exogenous ER chaperone, BiP/GRP78, exhibit attenuated inducibility of CHOP. Surprisingly, attenuated induction of CHOP was also noted in BiP-overexpressing cells treated with methyl methanesulfonate, an agent thought to activate CHOP by causing DNA damage. The roles of DNA damage and growth arrest in the induction of CHOP were therefore reexamined. Induction of growth arrest by culture to confluence or treatment with the enzymatic inhibitor N-(phosphonacetyl)-L-aspartate did not induce CHOP. Furthermore, both a DNA-damage-causing nucleoside analog (5-hydroxymethyl-2'-deoxyuridine) and UV light alone did not induce CHOP. These results suggest that CHOP is more responsive to ER stress than to growth arrest or DNA damage and indicate a potential role for CHOP in linking stress in the ER to alterations in gene expression.

3T3 Cells↗
PubMed1995

Chimeric molecules between keratinocyte growth factor and basic fibroblast growth factor define domains that confer receptor binding specificities.

Basic fibroblast growth factor (FGF) and keratinocyte growth factor (KGF) are structurally related fibroblast growth factors, yet they exhibit distinct receptor binding specificity. Basic FGF binds with high affinity to FGFR1, FGFR2, and FGFR4, whereas KGF does not interact with these receptors and can only bind an isoform of FGFR2 known as the KGFR. Basic GFG binds KGFR but with lower affinity than KGF. In order to identify domains that confer this specificity, four reciprocal chimeras were generated between the two growth factors and were analyzed for receptor recognition and biological activity. The chimeras are designated BK1 (bFGF1-54:KGF91-194), BK2 (bFGF1-74:KGF111-194), KB1 (KGF31-90:bFGF55-155), and KB2 (KGF31-110:bFGF75-155). The two BK chimera similarly interacted with FGFR1 and FGFR4 but differed from each other with respect to KGFR recognition. BK1 displayed a slightly better affinity for KGFR than BK2 and induced a higher level of DNA synthesis in keratinocytes compared with bFGF and BK2. A neutralizing monoclonal antibody directed against bFGF specifically neutralized the biological activity of the BK chimeras. The reciprocal chimeras, KB1 and KB2, exhibited KGF-like receptor binding and activation properties. However, KB2 displayed higher affinity for KGFR and was significantly more potent mitogen that KB1. Altogether, our results suggest that the amino-terminal part of KGF and bFGF plays an important role in determining their receptor binding specificity. In addition, the results point to the contribution of a segment from the middle part of KGF (residues 91-110) for recognition and activation of the KGFR, as the two chimeras containing these residues (BK1 and KB2) displayed an enhanced interaction with the KGFR.

3T3 Cells↗
PubMed1995

C2 region-derived peptides inhibit translocation and function of beta protein kinase C in vivo.

RACK1 is a protein kinase C (PKC)-binding protein that fulfills the criteria previously established for a receptor for activated C-kinase (RACK). If binding of PKC to RACK anchors the activated enzyme near its protein substrates, then inhibition of this binding should inhibit translocation and function of the enzyme in vivo. Here, we have identified such inhibitors that mimic the RACK1-binding site on beta PKC. We first found that a C2-containing fragment, but not a C1-containing fragment of beta PKC, bound to RACK1 and inhibited subsequent beta PKC binding. The RACK1-binding site was further mapped; peptides beta C2-1 (beta PKC(209-216), beta C2-2 (beta PKC(186-198)), and beta C2-4 (beta PKC(218-226), but not a number of control peptides, bound to RACK1 and inhibited the C2 fragment binding to RACK1. Peptides beta C2-1, beta C2-2, and beta C2-4 specifically inhibited phorbol ester-induced translocation of the C2-containing isozymes in cardiac myocytes and insulin-induced beta PKC translocation and function in Xenopus oocytes. Therefore, peptides corresponding to amino acids 186-198, and 209-226 within the C2 region of the beta PKC are specific inhibitors for functions mediated by beta PKC.

Amino Acid Sequence↗
PubMed1995

Inhibition of adipogenesis by the stress-induced protein CHOP (Gadd153).

Adipocytic conversion of 3T3-L1 cells is dependent on induction of transcription factors from the C/EBP family that activate promoters of adipogenic genes. We find that expression of CHOP, a nuclear protein that dimerizes avidly with C/EBP isoforms alpha and beta and directs the resulting heterodimer away from classic C/EBP-binding sites, markedly inhibits this differentiation process. Surprisingly, the presence of CHOP early in the differentiation process inhibits C/EBP alpha and beta gene expression. Ectopic expression of C/EBP alpha bypasses the inhibitory effect of CHOP on differentiation, providing further evidence that CHOP action is mediated by inhibition of C/EBP alpha gene expression rather than merely inhibiting the encoded protein's DNA-binding activity. A similar pattern of attenuated expression of C/EBP alpha and beta is also observed in cells induced to differentiate in media with low glucose concentration. This stressed culture condition is associated with induction of endogenous CHOP and marked attenuation of the differentiation process. Our data suggest that CHOP functions as an inducible inhibitor of adipocytic differentiation in response to metabolic stress. It does so by interfering with the accumulation of adipogenic C/EBP isoforms.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Fibroblast growth factor receptors display both common and distinct signaling pathways.

We compared the mitogenic and signaling pathways of three Fibroblast Growth Factor Receptors (FGFRs), FGFR1, KGFR and FGFR4 in the same cell line. Each receptor was expressed in L6E9 rat myoblasts that do not normally express detectable levels of FGFRs and clones that express comparable levels of each receptor were selected. Our results show that FGFs induce an effective survival and growth of FGFR1 and KGFR expressing cells. In addition, these cells exhibit a morphology that is reminiscent of that of malignantly transformed cells and display anchorage independent growth in a ligand dependent manner. Unlike KGFR and FGFR1, FGFR4 mediates a less effective growth, and cells overexpressing this receptor do not undergo any morphological changes nor do they display an anchorage independent growth in response to FGFs. All three receptors exhibit both quantitative and qualitative differences in their ability to induce tyrosine phosphorylation of cellular substrates. Both FGFR1 and KGFR induce strong phosphorylation of phospholipase C-gamma and a 90 kDa protein, while FGFR4 induces a relatively weak phosphorylation of phospholipase C-gamma and completely fails to induce phosphorylation of the 90 kDa. The three receptors also induce phosphorylation of the mitogen activated protein kinases (MAPK) but the effect of FGFR1 is far stronger than that of the other two receptors. Since FGFR4 is expressed in myoblasts in vivo, we examined whether this receptor can function in the differentiation pathway of myoblasts. Contrary to its weak mitogenic activity, FGFR4 effectively mediates the inhibition of myogenic differentiation in L6E9 cells and also suppresses the expression of the myogenic regulatory protein myogenin. Taken together, our results suggest that the signaling mechanism of FGFR4 differs from that of FGFR1 and KGFR, and that the primary role of FGFR4 in myoblasts may be the maintenance of their non differentiated state.

Animals↗
PubMed1995

An autoregulatory region in protein kinase C: the pseudoanchoring site.

We have previously identified receptors for activated C kinase (RACKs) as components of protein kinase C (PKC) signaling. RACK1, a recently cloned 36-kDa RACK, has short sequences of homology to PKC. A possible explanation for the homologous sequences between the ligand (PKC) and its intracellular receptor (RACK1) may be that, similar to the pseudosubstrate autoregulatory sequence on PKC, there is also a pseudo-RACK1 binding site on the enzyme. If this is the case, peptides with these sequences (derived from either RACK1 or PKC) are expected to affect PKC binding to RACK1 in vitro and PKC-mediated functions in vivo. Here, we show that the PKC-derived peptide (pseudo-RACK1 peptide), but not its RACK1 homologue, modulated PKC function both in vitro and in vivo. Our data suggest that the pseudo-RACK1 peptide binds and activates PKC in the absence of PKC activators and thereby acts as an agonist of PKC function in vivo. Therefore, the pseudo-RACK1 sequence in PKC appears to be another autoregulatory site; when PKC is in an inactive conformation, the pseudo-RACK1 site interacts with the RACK-binding site. Activation of PKC exposes the RACK-binding site, enabling the association of the enzyme with its anchoring RACK. Similar pseudoanchoring sites may regulate the function of other protein kinases.

Amino Acid Sequence↗
PubMed1995

Pattern of keratinocyte growth factor and keratinocyte growth factor receptor expression during mouse fetal development suggests a role in mediating morphogenetic mesenchymal-epithelial interactions.

Mesenchymal cells are required for the induction of epithelial development during mammalian organogenesis. Keratinocyte growth factor (KGF) is a mesenchymally derived mitogen with specific activity for epithelial cells, suggesting that it may play a role in mediating these interactions. To further evaluate this hypothesis, in situ hybridization was used to examine the spatial distribution of KGF and KGF receptor (KGFR) transcripts during organogenesis and limb formation in mouse embryos (days 14.5 through 16.5). To facilitate this aim, mouse KGF cDNA clones were isolated. There was extensive identity between the deduced mouse KGF protein sequence and that of its human and rat cognates, indicating that this gene has been highly conserved during mammalian evolution. In addition, mouse KGF protein was purified from fibroblasts and demonstrated to be structurally and functionally similar to human KGF protein. For organs within the integumental, respiratory, gastrointestinal, and urogenital systems, whose development is dependent upon mesenchymal-epithelial interactions, KGF mRNA was detected in mesenchymal cells, while epithelial cells expressed transcripts for the KGFR, KGF and KGFR mRNA was also expressed in certain other tissues such as perichondrium, cartilage of developing bones, developing skeletal muscle, and visceral smooth muscle whose development is not regulated by mesenchymal-epithelial interactions. KGF expression was also detected in tissues isolated from human embryos, suggesting similar functions for KGF in human development. Taken together, our results suggest that KGF plays an important role in mediating mesenchymal-epithelial interactions during organogenesis, but may also have other developmental functions in tissues not governed by such interactions.

Adult↗
PubMed1995

Keratinocyte growth factor.

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, albeit from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and studies of KGF regulation by sex steroid hormones reinforced the idea that KGF acts predominantly on epithelial cells to elicit a variety of responses including proliferation, migration and morphogenesis.

Animals↗
PubMed1995

Keratinocyte growth factor as a cytokine that mediates mesenchymal-epithelial interaction.

Keratinocyte growth factor (KGF) is a member of the heparin-binding fibroblast growth factor family (FGF-7) with a distinctive pattern of target-cell specificity. Studies performed in cell culture suggested that KGF was mitogenically active only on epithelial cells, though from a variety of tissues. In contrast, KGF was produced solely by cells of mesenchymal origin, leading to the hypothesis that it might function as a paracrine mediator of mesenchymal-epithelial communication. Biochemical analysis and molecular cloning established that the KGF receptor (KGFR) was a tyrosine kinase isoform encoded by the fgfr-2 gene. Many detailed investigations of KGF and KGFR expression in whole tissue and cell lines largely substantiated the pattern initially perceived in vitro of mesenchymal and epithelial distribution, respectively. Moreover, functional assays in organ culture and in vivo and analysis of agents regulating KGF expression reinforced the idea that KGF acts predominantly on epithelial cells. While the data do not implicate a KGF autocrine loop in neoplasia, paracrine sources of factor or ligand-independent signaling by the KGFR might contribute to malignancy. Alternatively, because of its differentiation-promoting effects, KGF may retard processes that culminate in uncontrolled cell growth.

Animals↗
PubMed1995

Association of SARFH (sarcoma-associated RNA-binding fly homolog) with regions of chromatin transcribed by RNA polymerase II.

Many oncogenes associated with human sarcomas are composed of a fusion between transcription factors and the N-terminal portions of two similar RNA-binding proteins, TLS and EWS. Though the oncogenic fusion proteins lack the RNA-binding domain and do not bind RNA, the contribution from the N-terminal portion of the RNA-binding protein is essential for their transforming activity. TLS and EWS associate in vivo with RNA polymerase II (Pol II) transcripts. To learn more about the target gene specificity of this interaction, the localization of a Drosophila melanogaster protein that has extensive sequence identity to the C-terminal RNA-binding portions of TLS and EWS was studied in preparations of Drosophila polytene nuclei. cDNA clones encoding the full-length Drosophila TLS-EWS homolog, SARFH (stands for sarcoma-associated RNA-binding fly homolog), were isolated. Functional similarity to TLS and EWS was revealed by the association of SARFH with Pol II transcripts in mammalian cells and by the ability of SARFH to elicit homologous down-regulation of the levels of the mammalian proteins. The SARFH gene is expressed in the developing Drosophila embryo from the earliest stages of cellularization and is subsequently found in many cell types. In preparations of polytene chromosomes from salivary gland nuclei, SARFH antibodies recognize their target associated with the majority of active transcription units, revealed by colocalization with the phosphorylated form of RNA Pol II. We conclude that SARFH and, by homology, EWS and TLS participate in a function common to the expression of most genes transcribed by RNA Pol II.

3T3 Cells↗
PubMed1994

Differential effect of cell-associated heparan sulfates on the binding of keratinocyte growth factor (KGF) and acidic fibroblast growth factor to the KGF receptor.

The fibroblast growth factors (FGFs) act through high affinity tyrosine kinase receptors and, in addition, interact with lower affinity receptors that represent cell- or matrix-associated heparan sulfate proteoglycans. These lower affinity receptors modulate the biological activities of FGFs, but the mechanism by which they exert these effects is rather controversial. We have previously shown (Ron, D., Bottaro, D. P., Finch, P. W., Morris, D., Rubin, J. S., and Aaronson, S. A. (1993) J. Biol. Chem. 268, 2984-2988) that heparin potentiates the mitogenic activity of acidic FGF (aFGF) but inhibits that of the keratinocyte growth factor (KGF) in cells that express the KGF receptor (KGFR). Both growth factors bind the KGFR with high affinity. To gain an insight into the mechanism by which heparin modulates the biological activity of aFGF and KGF, we studied the effect of heparin and cell-associated heparan sulfates on the binding of these two growth factors to the KGFR. To work in a well defined system, we expressed functional KGFR in L6E9 myoblasts that lack detectable high affinity binding sites for FGFs. Low concentrations of heparin inhibited the binding of KGF to the KGFR. By contrast, similar concentrations of heparin enhanced the binding of aFGF to this receptor. The effect of heparin was not unique to L6E9 cells expressing the KGFR; it was also observed in Balb/MK cells that naturally express KGFR. Treatment of cells with sodium chlorate, which blocks sulfation of proteoglycans, reduced the binding of aFGF to its low and high affinity binding sites by 95 and 80%, respectively. In contrast, the binding of KGF to its high affinity binding sites was enhanced about 2-fold. Similar results were obtained after degradation of cell-associated heparan sulfates by heparinase and heparitinase. Heparin restored the high affinity binding of aFGF to chlorate-treated cells and completely abolished the high affinity binding of KGF. Binding competition experiments suggest that aFGF and KGF bind to the same population of cell-associated heparan sulfates. In addition, KGF is apparently interacting with an as yet unidentified type of low affinity binding site that is not affected by chlorate or heparan sulfate-degrading enzymes. An important property of the FGF high affinity receptors is their ability to bind more than one ligand with high affinity. Based on the differential effect of cell-associated heparan sulfates on the binding of KGF and aFGF to the KGFR, we propose a regulatory role for cell-associated heparan sulfates as coordinators of the interaction of aFGF and KGF with the KGFR.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Characterization of the CHOP breakpoints and fusion transcripts in myxoid liposarcomas with the 12;16 translocation.

Myxoid liposarcomas are cytogenetically characterized by t(12;16)(q13;p11). The translocation results in rearrangements of the CHOP gene in 12q13 and the FUS gene in 16p11, creating a fusion gene where the RNA-binding domain of FUS is replaced by the DNA-binding and leucine zipper dimerization domain of CHOP. In the present study, we have mapped 16 genomic breakpoints in the region of the CHOP gene and isolated and sequenced a new variant (type II) of the chimeric FUS/CHOP transcript. The genomic breakpoints were dispersed along a 7.50-kilobase pair region from a SstI cleavage site upstream of the promoter of CHOP to a PstI cleavage site within intron 1. Reverse transcriptase-polymerase chain reaction analysis of tumor samples demonstrated the presence of two variant fragments, 654 base pairs (type I) and 378 base pairs (type II) in size. Of the 13 samples analyzed, 7 showed the smaller, 3 showed the larger, and 3 showed both types of transcripts. We cloned and sequenced the two fragments and found in type II a novel fusion point in the FUS mRNA 275 base pairs upstream of that present in the type I transcript. In both types of transcripts the interrupted FUS is followed by the entire exon 2 of CHOP. As a consequence the normally nontranslated exon 2 is translated and in both types there is in the junction between FUS and CHOP a shift from a FUS glycine codon to a valine codon in the chimeric mRNA.

Base Sequence↗
PubMed1994

A novel effector domain from the RNA-binding protein TLS or EWS is required for oncogenic transformation by CHOP.

In human myxoid liposarcoma, a chromosomal rearrangement leads to fusion of the growth-arresting and DNA-damage-inducible transcription factor CHOP (GADD153) to a peptide fragment encoded by the TLS gene. We have found that wild-type TLS and a closely related sarcoma-associated protein, EWS, are both abundant nuclear proteins that associate in vivo with products of RNA polymerase II transcription. This association leads to the formation of a ternary complex with other heterogeneous RNA-binding proteins (hnRNPs), such as A1 and C1/C2. An NIH-3T3-based transformation assay was used to study the oncogenic role of the sarcoma-associated domain of these RNA-binding proteins. Transduction of the TLS-CHOP oncogene into cells by means of a retroviral expression vector leads to loss of contact inhibition, acquisition of the ability to grow as colonies in soft agar, and tumor formation in nude mice. Mutations that interfere with the function of the leucine zipper dimerization domain or the adjacent basic region of CHOP abolish transformation. The essential role of the TLS component was revealed by the inability of truncated forms to fully transform cells. Domain swap between TLS- and EWS-associated oncogenes demonstrated that the component contributed by the RNA-binding proteins are functionally interchangeable, whereas the transcription factor component specifies tumor phenotype. The sarcoma-associated component of TLS and EWS contribute a strong transcriptional activation domain to the fusion proteins; however, transforming activity cannot be fully substituted by fusion of CHOP to other strong trans-activators. The juxtaposition of a novel effector domain from sarcoma-associated RNA-binding proteins to the targeting domain of transcription factors such as CHOP leads to the creation of a potent oncogene.

3T3 Cells↗
PubMed1994

Agonists and antagonists of protein kinase C function, derived from its binding proteins.

Physical association between proteins involved in signal transduction is required for their functions. Therefore, identification of the interacting sites in the signaling molecules can lead to the development of means to modulate these interactions. We applied this approach to study signal transduction by protein kinase C (PKC). We have previously identified potential PKC binding sites in two PKC binding proteins (annexin I and RACK1). Peptides derived from these sequences inhibit PKC binding to RACK1 in vitro. Here, we tested the ability of two of these peptides, I (KGDYEKILVALCGGN) and rVI (DIINALCF), to affect PKC-mediated function in vivo. The peptides were microinjected into Xenopus oocytes, and insulin-induced beta PKC translocation and oocyte maturation were examined. The peptides had opposite activities on oocyte; peptide I inhibited whereas peptide rVI stimulated insulin-induced Xenopus oocyte maturation. As expected, beta PKC translocation from the cytosol to the particulate fraction of the Xenopus oocytes was inhibited after microinjection of peptide I and induced after microinjection of peptide rVI. Moreover, peptide rVI caused translocation of beta PKC and oocyte maturation without hormone stimulation. In the absence of PKC activators, peptide rVI but not peptide I, activated PKC in vitro as demonstrated in three assays: increased sensitivity to Arg-C endopeptidase, PKC autophosphorylation, and histone phosphorylation. Therefore, although peptides I and rVI have sequence homology, one mimicked hormone-induced PKC-mediated function whereas the other inhibited this hormone-induced function. The molecular mechanisms underlying these opposing effects of the peptides are discussed.

Amino Acid Sequence↗