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V Nurcombe

Publications and source records attributed to V Nurcombe.

At least 19 recordsLinked to original sources
PubMed2006

Temporal expression of proteoglycans in the rat limb during bone healing.

Proteoglycans found in the bone extracellular matrix and on the cell surface can complex with HBGFs such as the FGFs, TGFs and BMPs which are known to play key roles in regulating fracture healing. Here we have studied the expression of key PGs during the bone repair process in order to determine the relationship between PG expression and healing status. We created non-critical sized trephine defects just proximal to the distal end of the tibial crest of adult male Wistar rats and examined the healing process histologically as well as by monitoring the temporal expression of mRNA transcripts for ALP, OP and OC, together with HSPG, CSPG and FGF-FGF receptor expression. Following surgery, animals were allowed to recover, and then euthanized after 7, 14, 21 and 28 days post-surgery, at which time tissue was harvested for histological examination and total RNA extracted and the mRNA transcripts examined by quantitative real-time PCR. HS and CSPG expression was generally observed to increase in the days immediately following injury, reaching peak expression two weeks post-surgery. This was followed by a gradual return to basal levels by day 28. The expression patterns of PGs were broadly similar with those of ALP, OP and FGFRs. The increase of mRNA expression for many key PGs detected during bone healing coincided with the elevation of bone markers and FGFRs, and provides further evidence that PGs involved in bone repair act in part through susceptible growth factors, including the FGF/FGFR system. The data presented here indicates that increased proteoglycan expression is involved in the early stages of bone healing at a time when previous studies have shown that the levels of HBGFs are maximal. Hence there exists a rationale for an exploration of the use of exogenous PGs as an adjunct therapy to potentiate the powerful effects of these factors and to augment the natural healing response.

Animals↗
PubMed2001

Nerve growth factor stimulates proliferation and survival of human breast cancer cells through two distinct signaling pathways.

We show here that the neurotrophin nerve growth factor (NGF), which has been shown to be a mitogen for breast cancer cells, also stimulates cell survival through a distinct signaling pathway. Breast cancer cell lines (MCF-7, T47-D, BT-20, and MDA-MB-231) were found to express both types of NGF receptors: p140(trkA) and p75(NTR). The two other tyrosine kinase receptors for neurotrophins, TrkB and TrkC, were not expressed. The mitogenic effect of NGF on breast cancer cells required the tyrosine kinase activity of p140(trkA) as well as the mitogen-activated protein kinase (MAPK) cascade, but was independent of p75(NTR). In contrast, the anti-apoptotic effect of NGF (studied using the ceramide analogue C2) required p75(NTR) as well as the activation of the transcription factor NF-kB, but neither p140(trkA) nor MAPK was necessary. Other neurotrophins (BDNF, NT-3, NT-4/5) also induced cell survival, although not proliferation, emphasizing the importance of p75(NTR) in NGF-mediated survival. Both the pharmacological NF-kappaB inhibitor SN50, and cell transfection with IkBm, resulted in a diminution of NGF anti-apoptotic effect. These data show that two distinct signaling pathways are required for NGF activity and confirm the roles played by p75(NTR) and NF-kappaB in the activation of the survival pathway in breast cancer cells.

Breast Neoplasms↗
PubMed2001

Proteomic analysis reveals that 14-3-3sigma is down-regulated in human breast cancer cells.

The class of molecular chaperones known as 14-3-3 is involved in the control of cellular growth by virtue of its apparent regulation of various signaling pathways, including the Raf/mitogen-activated protein kinase pathway. In breast cancer cells, the sigma form of 14-3-3 has been shown to interact with cyclin-dependent kinases and to control the rate of entry into mitosis. To test for a direct role for 14-3-3 in breast epithelial cell neoplasia, we have quantitated 14-3-3 protein levels using a proteomic approach based on two-dimensional electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF). We show here that 14-3-3sigma protein is strongly down-regulated in the prototypic breast cancer cell lines MCF-7 and MDA-MB-231 and in primary breast carcinomas as compared with normal breast epithelial cells. In contrast, levels of the alpha, beta, delta, or zeta isoforms of 14-3-3 were the same in both normal and transformed cells. The data support the idea that 14-3-3sigma is involved in the neoplastic transition of breast epithelial cells by virtue of its role as a tumor suppressor; as such, it may constitute a robust marker with clinical efficacy for this pathology.

14-3-3 Proteins↗
PubMed2001

Proteomics of breast cancer for marker discovery and signal pathway profiling.

Breast cancer is the most common form of cancer among women and the identification of markers to discriminate tumorigenic from normal cells, as well as the different stages of this pathology, is of critical importance. Two-dimensional electrophoresis has been used before for studying breast cancer, but the progressive completion of human genomic sequencing and the introduction of mass spectrometry, combined with advanced bioinformatics for protein identification, have considerably increased the possibilities for characterizing new markers and therapeutic targets. Breast cancer proteomics has already identified markers of potential clinical interest (such as the molecular chaperone 14-3-3 sigma) and technological innovations such as large scale and high throughput analysis are now driving the field. Methods in functional proteomics have also been developed to study the intracellular signaling pathways that underlie the development of breast cancer. As illustrated with fibroblast growth factor-2, a mitogen and motogen factor for breast cancer cells, proteomics is a powerful approach to identify signaling proteins and to decipher the complex signaling circuitry involved in tumor growth. Together with genomics, proteomics is well on the way to molecularly characterizing the different types of breast tumor, and thus defining new therapeutic targets for future treatment.

14-3-3 Proteins↗
PubMed2000

The proliferative and migratory activities of breast cancer cells can be differentially regulated by heparan sulfates.

To explore how heparan sulfate (HS) controls the responsiveness of the breast cancer cell lines MCF-7 and MDA-MB-231 to fibroblast growth factors (FGFs), we have exposed them to HS preparations known to have specificity for FGF-1 (HS glycosaminoglycan (HSGAG A)) or FGF-2 (HSGAGB). Proliferation assays confirmed that MCF-7 cells were highly responsive to FGF-2 complexed with GAGB, whereas migration assays indicated that FGF-1/HSGAGA combinations were stimulatory for the highly invasive MDA-MB-231 cells. Quantitative polymerase chain reaction for the levels of FGF receptor (FGFR) isoforms revealed that MCF-7 cells have greater levels of FGFR1 and that MDA-MB-231 cells have greater relative levels of FGFR2. Cross-linking demonstrated that FGF-2/HSGAGB primarily activated FGFR1, which in turn up-regulated the activity of mitogen-activated protein kinase; in contrast, FGF-1/HSGAGA led to the phosphorylation of equal proportions of both FGFR1 and FGFR2, which in turn led to the up-regulation of Src and p125(FAK). MDA-MB-231 cells were particularly responsive to vitronectin substrates in the presence of FGF-1/HSGAGA, and blocking antibodies established that they used the alpha(v)beta(3) integrin to bind to it. These results suggest that the clustering of particular FGFR configurations on breast cancer cells induced by different HS chains leads to distinct phenotypic behaviors.

Breast Neoplasms↗
PubMed2000

The mitogenic signaling pathway for fibroblast growth factor-2 involves the tyrosine phosphorylation of cyclin D2 in MCF-7 human breast cancer cells.

Fibroblast growth factor-2 (FGF-2) is mitogenic for the human breast cancer cell line MCF-7; here we investigate some of the signaling pathways subserving this activity. FGF-2 stimulation of MCF-7 cells resulted in a global increase of intracellular tyrosine phosphorylation of proteins, particularly FGF receptor substrate-2, the protooncogene product Src and the mitogen-activated protein kinase (MAP kinase) cascade. A major increase in the tyrosine phosphorylation of a 30-kDa protein species was also found. This protein was identified as cyclin D2 by mass spectrometry after trypsin digestion. Immunoprecipitation of cyclin D2 and immunoblotting with anti-phosphotyrosine antibodies confirmed that the tyrosine phosphorylation of cyclin D2 was indeed induced by FGF-2 stimulation. In addition, pharmacological inhibition of Src (with herbimycin A and PP2), and of the MAP kinase cascade (with PD98059), confirmed that Src activity is required for the FGF-2-induced phosphorylation of cyclin D2 whereas MAP kinase activity is not. Thus, tyrosine phosphorylation of cyclin D2 may be a key regulatory target for FGF-2 signaling.

Adaptor Proteins, Signal Transducing↗
PubMed2000

FGF signals for cell proliferation and migration through different pathways.

FGFs are pleiotropic growth factors that control cell proliferation, migration and differentiation. However, FGF transduction studies have so far focused primarily on the mitogenic effect of this growth factor family and it has been difficult to assess if the described intracellular signaling pathways are dedicated solely to cell proliferation, or whether they are equally important for the migratory activity often seen in responsive cells. We review here papers in which the migratory effects of this growth factor family were clearly discriminated from proliferative effects. In toto, these studies suggest that cells use different signaling pathways for migration, such as Src and p38 MAP kinase, from those for proliferation, which tend to upregulate the ERKs. Which signaling pathway a cell uses for proliferation or migration appears to depend on many factors, including the structure and the quantity of available FGF trapped in the basal lamina by heparan sulfate co-factors, the disposition of cognate high affinity receptors and the general environment of the cell. Thus the density of the cell population, the state of the cell cycle, the presence of other factors or receptors will modulate the migratory response of cells to FGF.

Animals↗
PubMed1999

Neurite-outgrowth regulating functions of the amyloid protein precursor of Alzheimer's disease.

Many studies have shown that breakdown of the amyloid protein precursor (APP) to produce the amyloid protein is an important step in the pathogenic mechanism which causes Alzheimer's disease (AD). However, little is known about the normal function of APP. Developmental studies show that APP expression increases during the period of brain development when neurite outgrowth and synaptogenesis is maximal. APP is expressed highly within growing neurites and in growth cones, and purified APP has been shown to stimulate neurite outgrowth from cells in culture. Thus APP may regulate neurite outgrowth or synaptogenesis in vivo. APP is actively secreted from many cells, and the C-terminally secreted APP has been shown to associate with components of the extracellular matrix, such as the heparan sulphate proteoglycans (HSPGs). Two putative heparin-binding domains on APP have been reported. Binding of HSPGs to an N-terminal heparin-binding domain (HBD-1) stimulates the effect of substrate-bound APP on neurite outgrowth. In the mature nervous system, APP may play an important role in the regulation of wound repair. It is highly likely that studies on the normal functions of APP will shed further light on aspects of the pathogenesis of AD.

Journal Article↗
PubMed1998

Structural modification of fibroblast growth factor-binding heparan sulfate at a determinative stage of neural development.

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity and appear to act by coupling particular forms of FGF to appropriate FGF receptors. During neural development, one particular HS proteoglycan is able to rapidly switch its potentiating activity from FGF-2, as neural precursor cell proliferation occurs, to FGF-1, as neuronal differentiation occurs. Using various analytical techniques, including chemical and enzymatic cleavage, low pressure chromatography, and strong anion-exchange high performance liquid chromatography, we have analyzed the different HSs expressed during these crucial developmental stages. There are distinct alterations in patterns of 6-O-sulfation, total chain length, and the number of sulfated domains of the HS from the more mature embryonic brain. These changes correlate with a switch in the ability of the HS to potentiate the actions of FGF-1 in triggering cell differentiation. It thus appears that each HS pool is designed to function in the modulation of an intricate interaction with a specific growth factor and its cognate receptor, and suggests tightly regulated expression of specific, bioactive disaccharide sequences. The data can be used to construct a simple model of controlled variations in HS chain structure which have functional consequences at a crucial stage of neuronal maturation.

Animals↗
PubMed1998

Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line.

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity both in vivo and in vitro, and appear to act by cross-linking particular forms of FGF to appropriate FGF receptors. We have recently isolated and characterized two separate HS pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D cells: one from cells in log growth phase, which greatly potentiates the activity of FGF-2, and the other from cells undergoing contact-inhibition and differentiation, which preferentially activates FGF-1. These two pools of HS have very similar functional activities to those species isolated from primary neuroepithelial cells at corresponding stages of active proliferation or differentiation. We present here a structural comparison between these cell line HS species to establish the nature of the changes that occur in the biosynthesis of HS. A combination of chemical and enzymatic cleavage, low pressure chromatography and strong anion-exchange HPLC were used to generate full chain models of each species. Overall, the HS pools synthesized in the dividing cell line pools possessed less complex sulfation than those derived from more differentiated, growth arrested cells.

Animals↗
PubMed1997

Essential role of heparan sulfates in axon navigation and targeting in the developing visual system.

Heparan sulfate (HS) is abundant in the developing brain and is a required co-factor for many types of fibroblast growth factor (FGF) signaling in vitro. We report that some HSs, when added exogenously to the developing Xenopus optic pathway, severely disrupt target recognition causing axons from the retina to bypass their primary target, the optic tectum. Significantly, HS sidechains from a neuroepithelial perlecan variant that preferentially bind FGF-2, HS(FGF-2), cause aberrant targeting, whereas those that preferentially bind FGF-1 do not. Charge-matched fragments of HS(FGF-2) show that the mistargeting activity associates with the FGF-binding fragments. Heparitinase removal of native HSs at the beginning of optic tract formation retards retinal axon elongation; addition of FGF-2 restores axon extension but axons lose directionality. Late HS removal, after axons have extended through the tract, elicits a tectal bypass phenotype indicating a growth promoting and guidance function for native HSs. Our results demonstrate that different HS sidechains from the same core protein differentially affect axon growth in vivo, possibly due to their distinct FGF-binding preferences, and suggest that growth factors and HSs are important partners in regulating axon growth and guidance in the developing visual system.

Animals↗
PubMed1996

Secreted glypican binds to the amyloid precursor protein of Alzheimer's disease (APP) and inhibits APP-induced neurite outgrowth.

The amyloid precursor protein (APP) of Alzheimer's disease has been shown to stimulate neurite outgrowth in vitro. The effect of APP on neurite outgrowth can be enhanced if APP is presented to neurons in substrate-bound form, in the presence of heparan sulfate proteoglycans. To identify specific heparan sulfate proteoglycans that bind to APP, conditioned medium from neonatal mouse brain cells was subjected to affinity chromatography with recombinant APP695 as a ligand. Glypican bound strongly to the APP affinity column. Purified glypican bound to APP with an equilibrium dissociation constant of 2.8 nM and inhibited APP-induced neurite outgrowth from chick sympathetic neurons. The effect of glypican was specific for APP, as glypican did not inhibit laminin-induced neurite outgrowth. Furthermore, treatment of cultures with 4-methylumbelliferyl-beta-D-xyloside, a competitive inhibitor of proteoglycan glycanation, inhibited APP-induced neurite outgrowth but did not inhibit laminin-induced neurite outgrowth. This result suggests that endogenous proteoglycans are required for substrate-bound APP to stimulate neurite outgrowth. Secreted glypican may act to inhibit APP-induced neurite outgrowth in vivo by competing with endogenous proteoglycans for binding to APP.

Alzheimer Disease↗
PubMed1996

Expression and localization of FGF-1 in the developing rat olfactory system.

Primary olfactory axons project from the nasal olfactory neuroepithelium to glomeruli in the olfactory bulb where they synapse with mitral cells, the second-order olfactory neurons. We have shown that the heparin-binding growth factor FGF-1 is expressed by olfactory nerve ensheathing cells which surround fascicles of primary olfactory axons en route to the olfactory bulb. These cells are believed to modulate olfactory axon growth between the olfactory neuroepithelium and the olfactory bulb. During late embryogenesis, FGF-1 expression is turned on in the mitral cells, and the FGF-1 peptide becomes confined to layers of synaptic neuropil in the postnatal olfactory bulb. FGF-1 is selectively present in glomeruli and the external plexiform layer. In cultures of olfactory neuroepithelial cells, complexes between FGF-1 and an appropriate activating heparan sulfate proteoglycan stimulated morphological differentiation of both olfactory nerve ensheathing cells and primary sensory olfactory neurons. Thus, the spatiotemporal expression and the functional properties of FGF-1 in this system suggest that this molecule plays an important regulatory role in the formation of the olfactory pathway.

Animals↗
PubMed1996

The role of heparan sulfate proteoglycans in the pathogenesis of Alzheimer's disease.

The hallmark of Alzheimer's disease (AD) is the deposition of amyloid plaques and neurofibrillary tangles in the brain. The relationship between amyloid deposition and the cognitive deficit is still unclear. The amyloid beta A4 protein is produced by proteolytic cleavage of the amyloid protein precursor (APP). Very little is known about the normal function of APP and the role the protein may play in pathogenesis. Several studies have shown that APP is important for the regulation of neurite outgrowth. Our studies support these findings and indicate that the neurite outgrowth-promoting effects of APP are stimulated by an interaction between APP and specific proteoglycans. Using site-directed mutagenesis, a heparan sulfate binding site which mediates this effect has been mapped to the N-terminus of APP (residues 96-110, HBD-1). A peptide homologous to HBD-1 blocks the trophic effects of APP in cell culture. To purify specific proteoglycans which stimulate the action of APP, an affinity column was constructed using a biotinylated peptide homologous to HBD-1 coupled to streptavidin-agarose. Two proteoglycans were isolated from a crude brain cell conditioned medium by affinity chromatography. The purified proteoglycans bound APP saturably with high affinity and stimulated the action of APP on neurite outgrowth from chick sympathetic neurons. Digestion of the proteoglycan fraction with heparitinase I or chondroitinase ABC demonstrated the presence of two major proteins, a heparan sulfate proteoglycan with a core protein of 63-67 kD molecular mass and a chondroitin sulfate proteoglycan with a core protein of 100-110 kD molecular mass. The results demonstrate that APP binds to at least two proteoglycans and that this interaction may regulate the trophic effects of the protein. The interaction of specific APP-binding proteoglycans with amyloid plaques may disturb the normal function of APP and contribute to the neuritic degeneration that is commonly seen around the amyloid plaque cores.

Alzheimer Disease↗
PubMed1996

Expression of extracellular matrix molecules in the embryonic rat olfactory pathway.

Primary olfactory neurons arise from placodal neuroepithelium that is separate from the neuroepithelial plate that forms the neural tube and crest. The axons of these neurons course along a stereotypical pathway and invade the rostral telencephalic vesicle where they induce the formation of the olfactory bulb. In the present study we examined the expression of several extracellular matrix constituents during formation of the olfactory nerve pathway in order to identify putative developmentally significant molecules. Double-label immunofluorescence was used to simultaneously map the trajectory of growing primary olfactory axons by expression of growth associated protein 43 (GAP-43) and the distribution of either laminin, heparan sulfate proteoglycans (HSPG), or chondroitin sulfate proteoglycans (CSPG). At embryonic day 12.5 (E12.5) primary olfactory axons have exited the olfactory neuroepithelium of the nasal pit and formed a rudimentary olfactory nerve. These axons together with migrating neural cells form a large mass outside the rostral surface of the telencephalon. This nerve pathway is clearly defined by a punctate distribution of laminin and HSPG. CSPG is selectively present in the mesenchyme between the olfactory nerve pathway and the nasal pit and in the marginal zone of the telencephalon. At E14.5 primary olfactory axons pierce the telencephalon through gaps that have emerged in the basement membrane. At this age both laminin and HSPG are colocalized with the primary olfactory axons that have entered the marginal zone of the telencephalon. CSPG expression becomes downregulated in this same region while it remains highly expressed in the marginal zone adjacent to the presumptive olfactory bulb. By E16.5 most of the basement membrane separating the olfactory nerve from the telencephalon has degraded, and there is direct continuity between the olfactory nerve pathway and the central nervous system. This strict spatiotemporal regulation of extracellular matrix constituents in the olfactory nerve pathway supports an important role of these molecules in axon guidance. We propose that laminin and HSPG are expressed by migrating olfactory Schwann cells in the developing olfactory nerve pathway and that these molecules provide a conducive substrate for axon growth between the olfactory neuroepithelium and the brain. CSPG in the surrounding mesenchyme may act to restrict axon growth to within this pathway. The regional degradation of the basement membrane of the telencephalon and the downregulation of CSPG within the marginal zone probably facilitates the passage of primary olfactory axons into the brain to form the presumptive nerve fiber layer of the olfactory bulb.

Animals↗
PubMed1996

Light-microscopic immunolocalization of fibroblast growth factor-1 and -2 in adult rat kidney.

The fibroblast growth factors (FGFs) are a family of conserved polypeptides known to regulate cell differentiation and proliferation. We have used avidin-biotin-enhanced indirect immunohistochemistry to localize FGF-1 and FGF-2 in the rat kidney. The most consistent specific immunostaining pattern is found in paraffin sections from kidneys perfusion-fixed with 4% paraformaldehyde in 0.1 M phosphate buffer. Intracellular immunoreactivity for FGF-1 and FGF-2 is co-localized in visceral (podocytes) and parietal (Bowman's capsule) glomerular epithelial cells, S3 segments of proximal tubules, distal tubules and collecting ducts in the cortex, and thick ascending limbs and collecting ducts in the medulla. Immunoreactivity is also observed within urothelium and the tunica adventitia of large blood vessels. No immunostaining is found in cortical S1 or S2 segments of proximal tubules, in frozen sections prepared from unfixed or 4% paraformaldehyde perfusion-fixed kidneys, or in paraffin sections from Bouin-fixed kidneys. Immersion fixation with 4% paraformaldehyde gives a similar staining pattern in paraffin sections to that achieved with perfusion fixation. However, in paraffin sections fixed with methyl Carnoy's fixative, immunoreactivity is primarily localized to the tunica media of blood vessels, with little tubular or glomerular immunostaining. Thus, variation in immunolocalization patterns for FGFs can be partially attributed to differences in fixative, preparative technique and antibody specificity.

Animals↗