Biomedical subjects

L F Lau

Publications and source records attributed to L F Lau.

At least 37 records · Page 2Linked to original sources

PubMed1999

Fisp12/mouse connective tissue growth factor mediates endothelial cell adhesion and migration through integrin alphavbeta3, promotes endothelial cell survival, and induces angiogenesis in vivo.

Fisp12 was first identified as a secreted protein encoded by a growth factor-inducible immediate-early gene in mouse fibroblasts, whereas its human ortholog, CTGF (connective tissue growth factor), was identified as a mitogenic activity in conditioned media of human umbilical vein endothelial cells. Fisp12/CTGF is a member of a family of secreted proteins that includes CYR61, Nov, Elm-1, Cop-1/WISP-2, and WISP-3. Fisp12/CTGF has been shown to promote cell adhesion and mitogenesis in both fibroblasts and endothelial cells and to stimulate cell migration in fibroblasts. These findings, together with the localization of Fisp12/CTGF in angiogenic tissues, as well as in atherosclerotic plaques, suggest a possible role for Fisp12/CTGF in the regulation of vessel growth during development, wound healing, and vascular disease. In this study, we show that purified Fisp12 (mCTGF) protein promotes the adhesion of microvascular endothelial cells through the integrin receptor alphavbeta3. Furthermore, Fisp12 stimulates the migration of microvascular endothelial cells in culture, also through an integrin-alphavbeta3-dependent mechanism. In addition, the presence of Fisp12 promotes endothelial cell survival when cells are plated on laminin and deprived of growth factors, a condition that otherwise induces apoptosis. In vivo, Fisp12 induces neovascularization in rat corneal micropocket implants. These results demonstrate that Fisp12 is a novel angiogenic inducer and suggest a direct role for Fisp12 in the adhesion, migration, and survival of endothelial cells during blood vessel growth. Taken together with the recent finding that the related protein CYR61 also induces angiogenesis, we suggest that Fisp12/mCTGF and CYR61 comprise prototypes of a new family of angiogenic regulators that function, at least in part, through integrin-alphavbeta3-dependent pathways.

Animals↗

PubMed1999

Flow cytometric analysis of the cell cycle in transfected cells without cell fixation.

We describe a simple and rapid protocol for the flow cytometric analysis of the cell cycle in transfected cells using a green fluorescent protein anchored in the intracellular membranes of the endoplasmic reticulum (ER-GFP) as a transfection marker. The transfected cells are analyzed by dual-parameter flow cytometry after a brief incubation with digitonin, followed by staining with propidium iodide. Treatment of cells with digitonin efficiently preserves the ER-GFP fluorescence and allows reproducible and quantitative DNA staining, thus obviating the need for cell fixation before flow cytometry. The digitonin-based protocol is faster and easier to perform than conventional cell fixation and is illustrated herein by cell cycle analyses of U20S and NIH 3T3 cells.

3T3 Cells↗

PubMed1998

Isolation of growth suppressors from a cDNA expression library.

We describe an experimental procedure for the isolation of growth inhibitory sequences from a complex cDNA library. This approach first takes advantage of the SETGAP technique (selectable expression of transient growth arrest phenotype) to enrich for growth inhibitory sequences, followed by a screening procedure to identify individual cDNAs that inhibit cell proliferation. Here we provide a detailed description of the experimental protocol and report the characterization of two cDNA sequences isolated in our initial screen of a mouse cDNA library. One of these cDNAs encodes the mouse ubiquitin-conjugation enzyme UbcM2. The other encodes a truncated form of a novel WD40 repeat protein, named Bopl, which is conserved from yeast to human. Together, these results demonstrate a new approach for the isolation of growth suppressors from cDNA libraries, and identify a previously unknown gene likely to be involved in growth control.

3T3 Cells↗

PubMed1998

CYR61, a product of a growth factor-inducible immediate early gene, promotes angiogenesis and tumor growth.

CYR61 is a secreted, cysteine-rich, heparin-binding protein encoded by a growth factor-inducible immediate-early gene. Acting as an extracellular, matrix-associated signaling molecule, CYR61 promotes the adhesion of endothelial cells through interaction with the integrin alphaVbeta3 and augments growth factor-induced DNA synthesis in the same cell type. In this study, we show that purified CYR61 stimulates directed migration of human microvascular endothelial cells in culture through an alphaV beta3-dependent pathway and induces neovascularization in rat corneas. Both the chemotactic and angiogenic activities of CYR61 can be blocked by specific anti-CYR61 antibodies. Whereas most human tumor-derived cell lines tested express CYR61, the gastric adenocarcinoma cell line RF-1 does not. Expression of the CYR61 cDNA under the regulation of a constitutive promoter in RF-1 cells significantly enhances the tumorigenicity of these cells as measured by growth in immunodeficient mice, resulting in tumors that are larger and more vascularized than those produced by control RF-1 cells. Taken together, these results identify CYR61 as an angiogenic inducer that can promote tumor growth and vascularization; the results also suggest potential roles for CYR61 in physiologic and pathologic neovascularization.

Animals↗

PubMed1998

Budding yeast Cdc20: a target of the spindle checkpoint.

The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2, and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitated with Cdc20 at all stages of the cell cycle. The binding of Mad2 depended on Mad1 and that of Mad3 on Mad1 and Mad2. Overexpression of Cdc20 allowed cells with a depolymerized spindle or damaged DNA to leave mitosis but did not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that were resistant to the spindle checkpoint no longer bound Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint.

Amino Acid Sequence↗

PubMed1998

Human CYR61-mediated enhancement of bFGF-induced DNA synthesis in human umbilical vein endothelial cells.

CYR61 is a member of an emerging family of growth regulators that includes the human connective tissue growth factor and the chicken protooncoprotein Nov. Encoded by a growth factor-inducible immediate-early gene, CYR61 is a secreted, cysteine-rich heparin-binding protein that associates with the extracellular matrix and cell surface. In this report, we describe the cloning of the human hCYR61 cDNA and expression and purification of the hCYR61 protein. The hCYR61 mRNA is abundant in the heart, lung, pancreas and placenta, is present at a low level in skeletal muscle and brain and is not detectable in liver and kidney. Purified recombinant hCYR61 protein does not induce thymidine incorporation in human umbilical vein endothelial cells by itself, but enhances bFGF-induced DNA synthesis. We show that hCYR61 acts in this cell system in part by displacement of bFGF bound to the extracellular matrix, thus increasing the effective concentration of bFGF. Possible biological implications of these observations are discussed.

Animals↗

PubMed1998

Adhesion of human umbilical vein endothelial cells to the immediate-early gene product Cyr61 is mediated through integrin alphavbeta3.

Cyr61 is a member of a family of growth factor-inducible immediate-early gene products thought to act cooperatively with the activities of growth factors. Upon synthesis, Cyr61 is secreted and is predominantly incorporated into the extracellular matrix. Recently, we demonstrated that Cyr61 promotes cell adhesion and migration and augments growth factor-induced DNA synthesis (Kireeva, M. L., Mo, F.-E., Yang, G. P., and Lau, L. F. (1996) Mol. Cell. Biol. 16, 1326-1334). In the present study, we investigated possible candidate receptor(s) on human umbilical vein endothelial cells (HUVECs) mediating adhesion to Cyr61. Under both serum-containing and serum-free conditions, adhesion of HUVECs to Cyr61 was dose-dependent, saturable, and abolished by affinity-purified anti-Cyr61 antibodies. Cell adhesion to Cyr61 was divalent cation-dependent and specifically inhibited by the peptide RGDS and LM609, a monoclonal antibody against integrin alphavbeta3. Furthermore, purified alphavbeta3 bound directly to an affinity matrix of Cyr61-coupled Sepharose 4B, and this interaction was specifically blocked by anti-Cyr61 antibodies. Additionally, in a solid phase binding assay, soluble Cyr61 bound to immobilized alphavbeta3 in a dose-dependent manner, and half-saturation binding occurred at approximately 5 nM Cyr61. As expected, the interaction of Cyr61 with immobilized alphavbeta3 was blocked by RGDS and LM609. In sum, these results identified Cyr61 as a novel ligand for alphavbeta3 and indicate that the adhesion of HUVECs to Cyr61 is mediated through interaction with this integrin. The possibility that integrin alphavbeta3 functions as a signaling receptor for Cyr61 accounts for most if not all activities that can be ascribed to Cyr61 to date and suggests a mechanism of action discussed herein.

Antibodies, Monoclonal↗

PubMed1998

SynGAP: a synaptic RasGAP that associates with the PSD-95/SAP90 protein family.

The PSD-95/SAP90 family of proteins has recently been implicated in the organization of synaptic structure. Here, we describe the isolation of a novel Ras-GTPase activating protein, SynGAP, that interacts with the PDZ domains of PSD-95 and SAP102 in vitro and in vivo. SynGAP is selectively expressed in brain and is highly enriched at excitatory synapses, where it is present in a large macromolecular complex with PSD-95 and the NMDA receptor. SynGAP stimulates the GTPase activity of Ras, suggesting that it negatively regulates Ras activity at excitatory synapses. Ras signaling at the postsynaptic membrane may be involved in the modulation of excitatory synaptic transmission by NMDA receptors and neurotrophins. These results indicate that SynGAP may play an important role in the modulation of synaptic plasticity.

Amino Acid Sequence↗

PubMed1998

Molecular mechanisms of glutamate receptor clustering at excitatory synapses.

The targeting of AMPA- and NMDA-type glutamate receptors to synapses in the central nervous system is essential for efficient excitatory synaptic transmission. Recent studies have indicated that protein-protein interactions of these receptors with synaptic proteins that contain PDZ domains are crucial for receptor targeting. NMDA receptors have been found to bind to the PSD-95 family of proteins, whereas AMPA receptors interact with the PDZ-domain-containing protein GRIP (glutamate receptor interacting protein). PSD-95 and GRIP contain multiple PDZ domains as well as other protein-protein interaction motifs that help to form large macromolecular complexes that may be important for the formation and plasticity of synapses.

Cloning, Molecular↗

PubMed1998

Raf and fibroblast growth factor phosphorylate Elk1 and activate the serum response element of the immediate early gene pip92 by mitogen-activated protein kinase-independent as well as -dependent signaling pathways.

Previous studies have shown that a mitogen activated protein (MAP) kinase (MEK)-independent signaling pathway is required by activated Raf or fibroblast-derived growth factor (FGF) for the differentiation of rat hippocampal neuronal H19-7 cells. We now demonstrate that both Raf and FGF similarly induce prolonged transcription and translation of the immediate early gene pip92 in the absence of activation of the MAP kinases (MAPKs) ERK1 and ERK2. To determine the mechanism by which this occurs and to identify novel Raf-activated signaling pathways, we investigated the induction of the pip92 promoter by both FGF and an estradiol-activated Raf-1-estrogen receptor fusion protein (deltaRaf-1:ER) in H19-7 cells. Deletion analysis of the pip92 promoter indicated that activation by the MAPK-independent pathway occurs primarily within the region containing a serum response element (SRE). Further analysis of the SRE by using a heterologous thymidine kinase promoter showed that both an Ets and CArG-like site are required. Elk1, which binds to the Ets site, was phosphorylated both in vitro and in vivo by the MAPK-independent pathway, and phosphorylation of an Elk1-GAL4 fusion protein by this pathway was sufficient for transactivation. Finally, at least two Elk1 kinases were fractionated by gel filtration, and analysis by an in-gel kinase assay revealed at least three novel Raf-activated Elk1 kinases. These results indicate that both FGF and Raf activate MAPK-independent kinases that can stimulate Elk1 phosphorylation and immediate early gene transcription.

Animals↗

PubMed1998

The -4 kilobase promoter region of the winged helix transcription factor HNF-3alpha gene elicits transgene expression in mouse embryonic hepatic and intestinal diverticula.

Murine hepatocyte nuclear factor-3alpha (HNF-3alpha) protein is a member of a large family of developmentally regulated transcription factors that share homology in the winged helix/fork head DNA binding domain and participate in embryonic pattern formation. HNF-3alpha also mediates cell-specific transcription of genes important for the function of hepatocytes, intestinal, pancreatic and bronchiolar epithelium. We have previously determined that -520 nucleotides upstream of the rat HNF-3alpha gene were sufficient to elicit hepatoma-specific expression in transfection assays and reported on a novel HNF-3alpha expression pattern in the renal pelvis urothelium of the embryonic and adult kidney. We also showed that retinoic acid mediated activation of the HNF-3alpha gene required -4 kb of the HNF-3alpha promoter region in F9 teratocarcinoma transfections. In order to determine regulatory sequences mediating the HNF-3alpha cellular expression pattern in developing mouse embryos, we created transgenic mice bearing the -4 kb HNF-3alpha promoter region driving expression of the beta-galactosidase transgene. Embryonic analysis of two transgenic mouse lines demonstrated that the -4 kb HNF-3alpha promoter sequences were sufficient to elicit transgene expression in the developing liver, intestine, esophagus, nasal epithelial cells and floorplate of the neurotube, but not in the mesodermal notochord or in the lung bud. One of the transgenic lines also exhibited proper expression in the mesonephric ducts and metanephric diverticulum, suggesting that the -4 kb HNF-3alpha promoter region contained a subset of the regulatory sequences necessary for HNF-3alpha expression in the developing kidney.

Animals↗

PubMed1997

Cyr61, product of a growth factor-inducible immediate-early gene, regulates chondrogenesis in mouse limb bud mesenchymal cells.

Chondrogenesis during embryonic skeletal development involves the condensation of mesenchymal cells followed by their differentiation into chondrocytes. We describe herein a previously unrecognized regulator of mammalian chondrogenesis encoded by a murine growth factor-inducible immediate-early gene, cyr61. The Cyr61 protein is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The expression pattern of the cyr61 gene during embryogenesis is tissue specific and temporally regulated. Most notably, cyr61 is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins undergoing chondrogenesis, suggesting that Cyr61 may play a role in the development of the embryonic skeleton. In this communication, we demonstrate that the Cyr61 protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cells in vitro and is likely to play a similar role in vivo based on the following observations: (1) Cyr61 is present in the embryonic limb mesenchyme during chondrogenesis in vivo and in vitro; (2) purified recombinant Cyr61 protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr61 enhances cell-cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic differentiation in cultures plated at subthreshold cell densities that are otherwise unable to support differentiation; and (4) neutralization of the endogenous Cyr61 with specific antibodies inhibits chondrogenesis. Taken together, these results identify Cyr61 as a novel player in chondrogenesis that contributes to the development of the mammalian embryonic skeleton.

Animals↗

PubMed1997

Cyr61 and Fisp12 are both ECM-associated signaling molecules: activities, metabolism, and localization during development.

cyr61 and fisp12 are homologous immediate-early genes that are transcriptionally activated upon growth factor stimulation in fibroblasts. Their gene products belong to an emerging family of secreted proteins with a high degree of sequence homology, including conservation of all 38 cysteine residues in their secreted portions. We have recently shown that Cyr61 is an extracellular matrix (ECM) signaling molecule that promotes cell proliferation, migration, and adhesion. We describe herein the first purification of the Fisp12 protein and we compare the activities of purified Cyr61 and Fisp12, their metabolism, targeting, and their localization during development. Although Fisp12 is the mouse homolog of the human connective tissue growth factor (CTGF), it has no detectable mitogenic activity by itself. Rather, Fisp12 enhances fibroblast growth factor-induced DNA synthesis. The activities of Fisp12 and Cyr61 are nearly indistinguishable in three cell types tested: fibroblasts, endothelial, and epithelial cells. Both proteins are found in the ECM, although Cyr61 associates with the ECM more strongly and binds heparin with higher affinity. Fisp12, but not Cyr61, is also found in the culture medium, suggesting that Fisp12 might be able to act at a distance from its site of secretion, whereas Cyr61 might act more locally. Both secreted proteins are internalized and degraded through the lysosomal pathway, suggesting interaction with cell surface receptors. Both Cyr61 and Fisp12 are found in the placenta and the circulatory system as detected by immunohistochemistry, whereas Cyr61, but not Fisp12, is found in the skeletal and nervous systems. Fisp12, but not Cyr61, is found in secretory organs. Taken together, we propose that Cyr61 and Fisp12 are both signaling cell adhesion molecules that have similar or overlapping activities, and their differential sites of localization and targeting may dictate specificity in their biological roles.

3T3 Cells↗

PubMed1997

p27Kip1 induces an accumulation of the repressor complexes of E2F and inhibits expression of the E2F-regulated genes.

p27Kip1 is an inhibitor of the cyclin-dependent kinases and it plays an inhibitory role in the progression of cell cycle through G1 phase. To investigate the mechanism of cell cycle inhibition by p27Kip1, we constructed a cell line that inducibly expresses p27Kip1 upon addition of isopropyl-1-thio-beta-D-galactopyranoside in the culture medium. Isopropyl-1-thio-beta-D-galactopyranoside-induced expression of p27Kip1 in these cells causes a specific reduction in the expression of the E2F-regulated genes such as cyclin E, cyclin A, and dihydrofolate reductase. The reduction in the expression of these genes correlates with the p27Kip1-induced accumulation of the repressor complexes of the E2F family of factors (E2Fs). Our previous studies indicated that p21WAF1 could disrupt the interaction between cyclin/cyclin-dependent kinase 2 (cdk2) and the E2F repressor complexes E2F-p130 and E2F-p107. We show that p27Kip1, like p21WAF1, disrupts cyclin/cdk2-containing complexes of E2F-p130 leading to the accumulation of the E2F-p130 complexes, which is found in growth-arrested cells. In transient transfection assays, expression of p27Kip1 specifically inhibits transcription of a promoter containing E2F-binding sites. Mutants of p27Kip1 harboring changes in the cyclin- and cdk2-binding motifs are deficient in inhibiting transcription from the E2F sites containing reporter gene. Moreover, these mutants of p27Kip1 are also impaired in disrupting the interaction between cyclin/cdk2 and the repressor complexes of E2Fs. Taken together, these observations suggest that p27Kip1 reduces expression of the E2F-regulated genes by generating repressor complexes of E2Fs. Furthermore, the results also demonstrate that p27Kip1 inhibits expression of cyclin A and cyclin E, which are critical for progression through the G1-S phases.

Animals↗

PubMed1997

Adrenocorticotropic hormone regulates the activities of the orphan nuclear receptor Nur77 through modulation of phosphorylation.

ACTH treatment of Y1 adrenocortical cells induces the synthesis of Nur77, an orphan nuclear receptor that can act as a potent transactivator for such genes as 21-hydroxylase (CYP21). Nur77 has thus been proposed to be a mediator of ACTH action in activating the expression of genes that encode steroidogenic enzymes. Here we show that ACTH regulates the activity of Nur77 at the level of phosphorylation. ACTH induces the synthesis of transcriptionally active, DNA-binding Nur77 that is unphosphorylated at Ser354, which resides within the DNA-binding domain. By contrast, the Nur77 population that is constitutively present in Y1 cells is phosphorylated at Ser354 and does not bind DNA. Substitutions of Ser354 with negatively charged amino acids, such as Asp or Glu, dramatically decreased Nur77 DNA-binding and trans-activation activities, whereas mutation to the neutral Ala had no effect. Aside from phosphorylation within the DNA-binding domain, ACTH treatment does not induce modifications in the N- and C-terminal domains of Nur77 that significantly affect activity. Although the specific kinases that phosphorylate Nur77 in vivo are not known, the mitogen-activated protein kinase/pp90RSK pathway is not critical to Nur77 regulation. We propose that ACTH treatment of Y1 cells results in modulation of the activities of both kinases and phosphatases, which, in turn, regulate the activities of such transcription factors as Nur77.

Adrenal Cortex Neoplasms↗

PubMed1996

Interaction of the N-methyl-D-aspartate receptor complex with a novel synapse-associated protein, SAP102.

Ionotropic glutamate receptors are known to cluster at high concentration on the postsynaptic membrane of excitatory synapses, but the mechanism by which this occurs is poorly understood. Studies on the neuromuscular junction and central inhibitory synapses suggest that clustering of neurotransmitter receptors requires its interaction with a cytoplasmic protein. Recently, in vitro studies have shown that members of the N-methyl--aspartate (NMDA) class of glutamate receptors interact with a synapse-associated protein, SAP90 (PSD-95). However, evidence for the in vivo interaction of NMDA receptors with SAPs is still lacking. In the present study, we demonstrate the specific interaction between SAP102, a novel synapse-associated protein, and the NMDA receptor complex from the rat cortical synaptic plasma membranes using co-immunoprecipitation techniques. No association was observed between SAP102 and GluR1, a member of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate class of glutamate receptors. To identify the domain on the NMDA receptor responsible for this interaction, we constructed hexahistidine fusion proteins from different regions of the NR1a and NR2 subunits of the NMDA receptor. Immunoblot overlay experiments showed that while the C-terminal domain of the NR2 subunit displayed strong binding, the NR1a intracellular C-terminal tail did not interact with SAP102. The site of interaction was more precisely located to the last 20 amino acids of the NR2 subunit as indicated by the interaction of the synthetic peptide with SAP102. In summary, we demonstrate here for the first time an in vivo interaction between the native NMDA receptor complex and a synapse-associated protein. These results suggest that SAP102 may play an important role in NMDA receptor clustering and immobilization at excitatory synapses.

Animals↗

PubMed1996

Elk-1 can recruit SRF to form a ternary complex upon the serum response element.

The initial genomic response to serum growth factors is the transcriptional activation of a set of immediate-early genes. Serum-induced transcriptional activation of several of these genes involves the formation of a ternary complex that includes the serum response factor (SRF), a 62 kDa ternary complex factor (TCF) and a serum response element (SRE). TCF alone does not bind the SRE of the protooncogene c-fos, but requires the prior assembly of the SRF-SRE binary complex for it to be recruited into a ternary complex. Here we show that this SRF-SRE binary complex is not an obligatory prerequisite for the formation of a serum responsive ternary complex. We demonstrate that Elk-1, which has properties of TCF can recruit SRF into a ternary complex on elements that do not support formation of the SRF-DNA binary complex. We also show that for two immediate-early genes, pip92 and nur77, formation of the ternary complex may occur without the prior assembly of SRF-DNA binary complex. Finally, we show that the ability of different sequences to support formation of Elk-l-SRF-DNA ternary complex in vitro correlates with their ability to respond to serum growth factors in vivo. Our results suggest that a much broader range of DNA sequences than the consensus SRF and TCF binding sites can support ternary complex formation, and by inference, serum induction. Possible implications of these results are discussed.

3T3 Cells↗

PubMed1996

SAP102, a novel postsynaptic protein that interacts with NMDA receptor complexes in vivo.

Synapse-associated proteins (SAPs) are constituents of the pre- and postsynaptic submembraneous cytomatrix. Here, we present SAP102, a novel 102kDa SAP detected in dendritic shafts and spines of asymmetric type 1 synapses. SAP102 is enriched in preparations of synaptic junctions, where it biochemically behaves as a component of the cortical cytoskeleton. Antibodies directed against NMDA receptors coimmunoprecipitate SAP102 from rat brain synaptosomes. Recombinant proteins containing the carboxy-terminal tail of NMDA receptor subunit NR2B interact with SAP102 from rat brain homogenates. All three PDZ domains in SAP102 bind the cytoplasmic tail of NR2B in vitro. These data represent direct evidence that in vivo SAP102 is involved in linking NMDA receptors to the submembraneous cytomatrix associated with postsynaptic densities at excitatory synapses.

Amino Acid Sequence↗