Heparin-binding Growth-associated Molecule Research Articles

Low-Molecular Weight Protamine Overcomes Chondroitin Sulfate Inhibition of Neural Regeneration.

Protamine is an arginine-rich peptide that replaces histones in the DNA-protein complex during spermatogenesis. Protamine is clinically used in cardiopulmonary bypass surgery to neutralize the effects of heparin that is required during the treatment. Here we demonstrate that protamine and its 14–22 amino acid long fragments overcome the neurite outgrowth inhibition by chondroitin sulfate proteoglycans (CSPGs) that are generally regarded as major inhibitors of regenerative neurite growth after injuries of the adult central nervous system (CNS). Since the full-length protamine was found to have toxic effects on neuronal cells we used the in vitro neurite outgrowth assay to select a protamine fragment that retains the activity to overcome the neurite outgrowth inhibition on CSPG substrate and ended up in the 14 amino acid fragment, low-molecular weight protamine (LMWP). In contrast to the full-length protamine, LMWP displays very low or no toxicity in our assays in vitro and in vivo. We therefore started studies on LMWP as a possible drug lead in treatment of CNS injuries, such as the spinal cord injury (SCI). LMWP mimicks HB-GAM (heparin-binding growth-associated molecule; pleiotrophin) in that it overcomes the CSPG inhibition on neurite outgrowth in primary CNS neurons in vitro and inhibits binding of protein tyrosine phosphatase (PTP) sigma, an inhibitory receptor in neurite outgrowth, to its CSPG ligand. Furthermore, the chondroitin sulfate (CS) chains of the cell matrix even enhance the LMWP-induced neurite outgrowth on CSPG substrate. In vivo studies using the hemisection and hemicontusion SCI models in mice at the cervical level C5 revealed that LMWP enhances recovery when administered through intracerebroventricular or systemic route. We suggest that LMWP is a promising drug lead to develop therapies for CNS injuries.

Heparin-Binding Growth-Associated Molecule (Pleiotrophin) Affects Sensory Signaling and Selected Motor Functions in Mouse Model of Anatomically Incomplete Cervical Spinal Cord Injury.

Heparin-binding growth-associated molecule (pleiotrophin) is a neurite outgrowth-promoting secretory protein that lines developing fiber tracts in juvenile CNS (central nervous system). Previously, we have shown that heparin-binding growth-associated molecule (HB-GAM) reverses the CSPG (chondroitin sulfate proteoglycan) inhibition on neurite outgrowth in the culture medium of primary CNS neurons and enhances axon growth through the injured spinal cord in mice demonstrated by two-photon imaging. In this study, we have started studies on the possible role of HB-GAM in enhancing functional recovery after incomplete spinal cord injury (SCI) using cervical lateral hemisection and hemicontusion mouse models. In vivo imaging of blood-oxygen-level-dependent (BOLD) signals associated with functional activity in the somatosensory cortex was used to assess the sensory functions during vibrotactile hind paw stimulation. The signal displays an exaggerated response in animals with lateral hemisection that recovers to the level seen in the sham-operated mice by injection of HB-GAM to the trauma site. The effect of HB-GAM treatment on sensory-motor functions was assessed by performance in demanding behavioral tests requiring integration of afferent and efferent signaling with central coordination. Administration of HB-GAM either by direct injection into the trauma site or by intrathecal injection improves the climbing abilities in animals with cervical hemisection and in addition enhances the grip strength in animals with lateral hemicontusion without affecting the spontaneous locomotor activity. Recovery of sensory signaling in the sensorimotor cortex by HB-GAM to the level of sham-operated mice may contribute to the improvement of skilled locomotion requiring integration of spatiotemporal signals in the somatosensory cortex.

Pleiotropin enhances the osteo/dentinogenic differentiation potential of dental pulp stem cells

ABSTRACT Purpose: Pleiotrophin (PTN) is a heparin-binding growth-associated molecule and expressed in ameloblasts and odontoblasts throughout tooth maturation. Our previous study has shown that PTN expressed more than 20-fold higher in dental tissue than dental stem cells. However, the role of PTN on proliferation and osteo/dentinogenesis of dental pulp stem cells (DPSCs) is unclear. The purpose of the present study was to investigate the role of PTN on the DPSCs’ function. Methods: DPSCs were isolated from human third molars. Short hairpin RNAs (shRNAs) was used to knock down the PTN expression in DPSCs. Real-time RT-PCR, alizarin red staining, quantitative calcium analysis, in vivo transplantation and cell counting kit-8 (CCK8) assay were used to study the function of DPSCs. Possible mechanism was studied by RNA sequencing. Results: After PTN depletion, ALP activity and mineralization ability of DPSCs decreased. Expression of DMP-1 and BSP weakened. Proliferation of DPSCs at 48 h and 72 h was inhibited. Furthermore, 50 pg/mL PTN recombinant protein rescued the impaired osteo/dentinogenic differentiation potential and proliferation ability caused by PTN depletion. In addition, RNA sequencing showed 221 genes were downregulated and 233 genes upregulated in PTN depleted DPSCs. Several genes including BMP2 and IGFBP5 might be associated with PTN function on the DPSCs. P53 and the AMPK signaling pathways were involved. LncRNA analysis displayed 47 significantly upregulated lncRNA and 31 downregulated lncRNA comparing PTN depleted DPSCs with the control. Conclusion: Our research demonstrated that PTN has a positive role in maintaining DPSCs proliferation and osteo/dentinogenic differentiation potential.

Inhibition and enhancement of neural regeneration by chondroitin sulfate proteoglycans.

The current dogma in neural regeneration research implies that chondroitin sulfate proteoglycans (CSPGs) inhibit plasticity and regeneration in the adult central nervous system (CNS). We argue that the role of the CSPGs can be reversed from inhibition to activation by developmentally expressed CSPG-binding factors. Heparin-binding growth-associated molecule (HB-GAM; also designated as pleiotrophin) has been studied as a candidate molecule that might modulate the role of CSPG matrices in plasticity and regeneration. Studies in vitro show that in the presence of soluble HB-GAM chondroitin sulfate (CS) chains of CSPGs display an enhancing effect on neurite outgrowth. Based on the in vitro studies, we suggest a model according to which the HB-GAM/CS complex binds to the neuron surface receptor glypican-2, which induces neurite growth. Furthermore, HB-GAM masks the CS binding sites of the neurite outgrowth inhibiting receptor protein tyrosine phosphatase sigma (PTPσ), which may contribute to the HB-GAM-induced regenerative effect. In vivo studies using two-photon imaging after local HB-GAM injection into prick-injury of the cerebral cortex reveal regeneration of dendrites that has not been previously demonstrated after injuries of the mammalian nervous system. In the spinal cord, two-photon imaging displays HB-GAM-induced axonal regeneration. Studies on the HB-GAM/CS mechanism in vitro and in vivo are expected to pave the way for drug development for injuries of brain and spinal cord.

HB-GAM (pleiotrophin) reverses inhibition of neural regeneration by the CNS extracellular matrix.

Chondroitin sulfate (CS) glycosaminoglycans inhibit regeneration in the adult central nervous system (CNS). We report here that HB-GAM (heparin-binding growth-associated molecule; also known as pleiotrophin), a CS-binding protein expressed at high levels in the developing CNS, reverses the role of the CS chains in neurite growth of CNS neurons in vitro from inhibition to activation. The CS-bound HB-GAM promotes neurite growth through binding to the cell surface proteoglycan glypican-2; furthermore, HB-GAM abrogates the CS ligand binding to the inhibitory receptor PTPσ (protein tyrosine phosphatase sigma). Our in vivo studies using two-photon imaging of CNS injuries support the in vitro studies and show that HB-GAM increases dendrite regeneration in the adult cerebral cortex and axonal regeneration in the adult spinal cord. Our findings may enable the development of novel therapies for CNS injuries.

Activation of Mechanosensitive Transcription Factors in Murine C2C12 Mesenchymal Precursors by Focused Low-Intensity Pulsed Ultrasound (FLIPUS).

In this paper, we investigated the mechanoresponse of C2C12 mesenchymal precursor cells to focused low-intensity pulsed ultrasound (FLIPUS). The setup has been developed for in vitro stimulation of adherent cells in the defocused far field of the ultrasound propagating through the bottom of the well plate. Twenty-four-well tissue culture plates, carrying the cell monolayers, were incubated in a temperature-controlled water tank. The ultrasound was applied at 3.6-MHz frequency, pulsed at 100-Hz repetition frequency with a 27.8% duty cycle, and calibrated at an output intensity of ISATA = 44.5 ±7.1 mW/cm2. Numerical sound propagation simulations showed no generation of standing waves in the well plate. The response of murine C2C12 cells to FLIPUS was evaluated by measuring activation of mechanosensitive transcription factors, i.e., activator protein-1 (AP-1), specificity protein 1 (Sp1), and transcriptional enhancer factor (TEAD), and expression of mechanosensitive genes, i.e., c-fos, c-jun, heparin binding growth associated molecule (HB-GAM), and Cyr-61. FLIPUS induced 50% ( p ≤ 0.05 ) and 70% ( p ≤ 0.05 ) increases in AP-1 and TEAD promoter activities, respectively, when stimulated for 5 min. The Sp1 activity was enhanced by about 20% ( p ≤ 0.05 ) after 5-min FLIPUS exposure and the trend persisted for 30-min ( p ≤ 0.05 ) and 1-h ( p ≤ 0.05 ) stimulation times. Expressions of mechanosensitive genes c-fos ( p ≤ 0.05 ), c-jun ( p ≤ 0.05 ), HB-GAM ( p ≤ 0.05 ), and cystein-rich protein 61 ( p ≤ 0.05 ) were enhanced in response to 5-min FLIPUS stimulation. The increase in proliferation of C2C12s occurred after the FLIPUS stimulation ( p ≤ 0.05 ), with AP-1, Sp1, and TEAD possibly regulating the observed cellular activities.

Ligand-induced dimerization of syndecan-3 at the cell surface

Syndecan-3 (N-syndecan) is a transmembrane heparan sulfate proteoglycan abundantly expressed in developing brain. In addition to acting as a coreceptor, syndecan-3 acts as a signaling receptor upon binding of its ligand HB-GAM (heparin-binding growth-associated molecule; pleiotrophin), which activates the cortactin-src kinase signaling pathway. This leads to rapid neurite extension in neuronal cells, which makes syndecan-3 as an interesting transmembrane receptor in neuronal development and regeneration. However, little is known about the signaling mechanism of syndecan-3. Here we have analyzed formation of ligand-N-syndecan signaling complexes at the cell surface using fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET). We show that ligand binding leads to dimerization of syndecan-3 at the cell surface. The dimerized syndecan-3 colocalizes with actin in the filopodia of cells. Several amino acid residues (K383, G392 and G396) in the transmembrane domain are shown to be important for the ligand-induced dimerization, whereas the cytosolic domain is not required for the dimerization.

Generation and analysis of expressed sequence tags from the bone marrow of Chinese Sika deer

Sika deer is one of the best-known and highly valued animals of China. Despite its economic, cultural, and biological importance, there has not been a large-scale sequencing project for Sika deer to date. With the ultimate goal of sequencing the complete genome of this organism, we first established a bone marrow cDNA library for Sika deer and generated a total of 2,025 reads. After processing the sequences, 2,017 high-quality expressed sequence tags (ESTs) were obtained. These ESTs were assembled into 1,157 unigenes, including 238 contigs and 919 singletons. Comparative analyses indicated that 888 (76.75%) of the unigenes had significant matches to sequences in the non-redundant protein database, In addition to highly expressed genes, such as stearoyl-CoA desaturase, cytochrome c oxidase, adipocyte-type fatty acid-binding protein, adiponectin and thymosin beta-4, we also obtained vascular endothelial growth factor-A and heparin-binding growth-associated molecule, both of which are of great importance for angiogenesis research. There were 244 (21.09%) unigenes with no significant match to any sequence in current protein or nucleotide databases, and these sequences may represent genes with unknown function in Sika deer. Open reading frame analysis of the sequences was performed using the getorf program. In addition, the sequences were functionally classified using the gene ontology hierarchy, clusters of orthologous groups of proteins and Kyoto encyclopedia of genes and genomes databases. Analysis of ESTs described in this paper provides an important resource for the transcriptome exploration of Sika deer, and will also facilitate further studies on functional genomics, gene discovery and genome annotation of Sika deer.

Medium-throughput computer aided micro-island method to assay embryonic dopaminergic neuron cultures in vitro

In Parkinson's disease (PD) midbrain dopaminergic (DA) neurons degenerate and die, causing loss of motor function. Currently no therapies exist to ameliorate neurodegeneration or to restore DA neurons, although neurotrophic factors (NTFs) are promising leads. Prior in vivo studies the NTFs are routinely assessed in vitro by quantifying the survival of DA neurons from embryonic rodent midbrain cultures. Current in vitro methods are limited in terms of assay reliability, arduous workflow, low throughput, low statistical power and may obscure detection of molecules with minor yet critically important therapeutic effects. We have developed a medium-throughput, micro-island culture method. It permits analysis of 10–12 data points from a single embryo – several fold more than any previously published method – and enables comparisons of DA neurons from a single gene knockout (KO) embryo. It is computer-aided, improves statistical power and decreases the number of animals and workload per experiment. This method enhances testing capabilities of NTFs and other factors, and enables small scale screening of chemical drug libraries. We have validated the method by confirming the known effects of glial cell line-derived neurotrophic factor (GDNF) and neurturin (NRTN), and demonstrated additive effects via simultaneous addition of GDNF and heparin binding growth associated molecule (HB-GAM). We also show for the first time that DA neurons isolated from GDNF receptor RET-deficient mice are still GDNF responsive, suggesting the presence of an alternative non-RET receptor for GDNF in the DA system. Finally, the method can be adapted for analyses of other low abundance neuronal systems.

Heparin-binding determinants of GDNF reduce its tissue distribution but are beneficial for the protection of nigral dopaminergic neurons

Glial cell line-derived neurotrophic factor (GDNF) protects and repairs dopamine neurons. It binds to GDNF family receptor α1 (GFRα1) and activates receptor tyrosine kinase. Heparan sulphate proteoglycans (HSPGs) also participate in the signalling of GDNF, though binding to HS may hinder the diffusion of infused GDNF. We assessed the importance of heparin-binding determinants in the neuroprotective effects of GDNF in the 6-OHDA rat model of Parkinson's disease. We utilized a truncated, non-heparin-binding Δ38N-GDNF or combined wtGDNF with heparin-binding growth-associated molecule (HB-GAM, pleiotrophin). Tissue diffusion of wtGDNF ± HB-GAM and Δ38N-GDNF was also compared. A protective effect against ipsilateral d-amphetamine-induced turning was seen with 10 μg wtGDNF, 17 μg HB-GAM + 10 μg wtGDNF or 10 μg Δ38N-GDNF at 8 weeks post lesion. This effect was most pronounced with wtGDNF alone. HB-GAM (17 or 50 μg) also reduced rotational behaviour, but did not protect dopaminergic cells. Otherwise, the survival of TH-positive cells in the substantia nigra correlated with the behavioural data. Although Δ38N-GDNF was more widely distributed than wtGDNF (irrespective of its origin), stable in a brain extract, and potent in mitogen-activated kinase assay, it was inferior in vivo. The results imply that GDNF binding to HSs is needed for the optimum neuroprotective effect.

Sensory neurons from N-syndecan-deficient mice are defective in survival

Extracellular matrix is a crucial regulator of development, plasticity and regeneration in the nervous system. We have now found that N-syndecan, the receptor for the extracellular matrix component heparin-binding growth-associated molecule, is required for survival of primary sensory neurons. We demonstrate massive cell death of cultured dorsal root ganglion (DRG) neurons from mice deficient in the N-syndecan gene as compared with wild-type controls. Importantly, this cell death could not be prevented by nerve growth factor - the neurotrophin, which activates multiple antiapoptotic cascades in DRG neurons. The survival deficiency was observed during first postnatal week. In contrast, DRG neurons from young adult N-syndecan knockout mice exhibited normal survival. This study identifies a completely new syndecan-dependent type of signaling that regulates cell death in neurons.

Improving endothelial healing with novel chimeric mitogens

Background Chimeric proteins may be used to direct cell-specific activity. Heparin-binding growth-associated molecule (HBGAM) binds to cell receptors that are relatively more robust on endothelial cells, and it may confer endothelial cell selectivity to potent angiogens such as fibroblast growth factor-1 (FGF-1). Methods By ligating fibroblast growth factor or its potent mutant, S130K, to HBGAM, we tested their effect on re-endothelialization after angioplasty injury by using a canine model. Results Both HBGAM/S130K- and HBGAM/FGF-1–treated arteries had increased neointimal mitotic index and re-endothelialization rates at 30 days compared with control arteries without inducing a significant increase in the neointimal thickness or the ratio of neointimal to medial thickness between treatment and control groups. Conclusion HBGAM/S130K and HBGAM/FGF-1 facilitates endothelial healing without myointimal thickening after canine carotid artery balloon angioplasty injury. Application of these growth factors in fibrin glue may improve endothelialization clinically after angioplasty or endarterectomy.

Neuroglycan C Is a Novel Midkine Receptor Involved in Process Elongation of Oligodendroglial Precursor-like Cells

Midkine is a heparin-binding growth factor that promotes cell attachment and process extension in undifferentiated bipolar CG-4 cells, an oligodendroglial precursor cell line. We found that CG-4 cells expressed a non-proteoglycan form of neuroglycan C, known as a part-time transmembrane proteoglycan. We demonstrated that neuroglycan C before or after chondroitinase ABC treatment bound to a midkine affinity column. Neuroglycan C lacking chondroitin sulfate chains was eluted with 0.5 m NaCl as a major fraction from the column. We confirmed that CG-4 cells expressed two isoforms of neuroglycan C, I, and III, by isolating cDNA. Among three functional domains of the extracellular part of neuroglycan C, the chondroitin sulfate attachment domain and acidic amino acid cluster box domain showed affinity for midkine, but the epidermal growth factor domain did not. Furthermore, cell surface neuroglycan C could be cross-linked with soluble midkine. Process extension on midkine-coated dishes was inhibited by either a monoclonal anti-neuroglycan C antibody C1 or a glutathione S-transferase-neuroglycan C fusion protein. Finally, stable transfectants of B104 neuroblastoma cells overexpressing neuroglycan C-I or neuroglycan C-III attached to the midkine substrate, spread well, and gave rise to cytoskeletal changes. Based on these results, we conclude that neuroglycan C is a novel component of midkine receptors involved in process elongation.

N-syndecan deficiency impairs neural migration in brain

N-syndecan (syndecan-3) is a transmembrane proteoglycan that is abundantly expressed in the major axonal pathways and in the migratory routes of the developing brain. When ligated by heparin-binding (HB) growth-associated molecule (GAM; pleiotrophin), N-syndecan mediates cortactin–Src kinase-dependent neurite outgrowth. However, the functional role of N-syndecan in brain development remains unexplored. In this study, we show that N-syndecan deficiency perturbs the laminar structure of the cerebral cortex as a result of impaired radial migration. In addition, neural migration in the rostral migratory stream is impaired in the N-syndecan–null mice. We suggest that the migration defect depends on impaired HB-GAM–induced Src kinase activation and haptotactic migration. Furthermore, we show that N-syndecan interacts with EGF receptor (EGFR) at the plasma membrane and is required in EGFR-induced neuronal migration.

Enhanced hippocampal GABAergic inhibition in mice overexpressing heparin-binding growth-associated molecule

Heparin-binding growth-associated molecule is a developmentally regulated extracellular matrix protein promoting neurite outgrowth, axonal guidance and synaptogenesis. In the hippocampus, heparin-binding growth-associated molecule is expressed in an activity-dependent manner, and has been shown to suppress long-term potentiation of glutamatergic synapses in the area CA1, but the mechanisms underlying this action are unknown. One of the mechanisms by which extracellular matrix proteins might modulate fast synaptic transmission is by altering GABAergic function. Therefore, we have studied the properties of GABA A receptor-mediated inhibition in hippocampus of mutant mice overexpressing heparin-binding growth-associated molecule (heparin-binding growth-associated molecule transgenics). Under control conditions the wild-type mice have much higher level of long-term potentiation than the transgenics. However, in the absence of the GABA A receptor-mediated-inhibition a similar level of long-term potentiation is seen in both strains. In field potential recordings blockade of GABA A receptors by picrotoxin resulted in more accentuated increase in the CA1 population spike in the transgenics than in the wild-type animals. Whole-cell patch-clamp recordings revealed that when compared with the wild-type animals the transgenic mice had higher frequency of spontaneous inhibitory postsynaptic currents in CA1 pyramidal neurons. However, the frequency of action potential-independent miniature inhibitory postsynaptic currents was similar in both strains. Further, the transgenics had reduced paired-pulse depression of inhibitory postsynaptic currents, which was insensitive to the blockade of GABA B receptors in contrast to wild-type mice. The results demonstrate that the mice overexpressing heparin-binding growth-associated molecule have accentuated hippocampal GABA A receptor-mediated inhibition, which in turn may explain the lowered predisposition of glutamatergic synapses to undergo plastic changes in these animals. Thus, our findings suggest a mechanism by which heparin-binding growth-associated molecule can regulate synaptic plasticity.

The Two Thrombospondin Type I Repeat Domains of HB-GAM Display a Cooperative Function in N-syndecan Binding and Regulation of Synaptic Plasticity

: syndecan, TSR, HB-GAM, brain plasticity The TSR (thrombospondin type I repeat) is defined by a conserved tryptophan/cysteine motif and found in a large family of extracellular matrix and cell surface proteins (for review, see [1]). These proteins include thrombospondins 1 and 2, F-spondin, mindin, and semaphorins F and G. HB-GAM (heparin-binding growth-associated molecule, also designated as pleiotrophin) and MK (midgestation-kidney protein) form a subfamily within this type of proteins and have very similar structures (see Fig. 1 for essential structural features of HB-GAM and MK). Structures of the TSR domains of the thrombospondins have been recently resolved using X-ray crystallography[2], and secondary structures of MK[3] and HB-GAM[4] are currently known based on NMR spectroscopy. HB-GAM and MK fold in a very similar manner, but their TSR domains apparently display significant differences from the TSR structures resolved for the thrombospondins. In general, TSR proteins are implicated in cell surface/matrix binding and regulate migratory responses in many types of cells (for reviews, see [1,5]). A wide variety of TSR proteins are highly expressed in the nervous system, but their functional roles and possible significance of the TSRs in these proteins remain elusive. Structure-function studies of HB-GAM[6] have recently shown that the two TSRs of the protein mediate binding to heparin/HS (heparan sulfate). In hippocampal neurons, N-syndecan (neural syndecan, syndecan-3) is the carrier of the HS chains interacting with HB-GAM. Furthermore, studies using hippocampal neurons and slices implicate the di-TSR domain in the regulation of neurite outgrowth/plasticity in the developing and adult hippocampus. The rationale of the structure-function studies using neurite outgrowth/plasticity as a readout derives from previous studies where HB-GAM was shown to enhance neurite extension in hippocampal neurons when presented as a matrix-bound form[7,8] and to inhibit it when an excess of HB-GAM occurs in the culture medium of cells[8]. Furthermore, HB-GAM was shown to be a neuronal activity-induced gene in the hippocampus[9] which, together with the effects on hippocampal neurons

The Two Thrombospondin Type I Repeat Domains of the Heparin-binding Growth-associated Molecule Bind to Heparin/Heparan Sulfate and Regulate Neurite Extension and Plasticity in Hippocampal Neurons

HB-GAM (heparin-binding growth-associated molecule, also designated as pleiotrophin) and midkine form a two-member family of extracellular matrix proteins that bind tightly to sulfated carbohydrate structures such as heparan sulfate. These proteins are used by developing neurons as extracellular cues in axonal growth and guidance. HB-GAM was recently reported to enhance differentiation of neural stem cells. Based on the solution structure of HB-GAM, we have recently shown that HB-GAM consists of two beta-sheet domains flanked by flexible lysine-rich N- and C-terminal tails with no apparent structure. These domains are homologous to thrombospondin type I repeats present in numerous extracellular proteins that interact with the cell surface. Our findings showed that the two beta-sheet domains fold independently. We showed that the domains (but not the lysine-rich tails) in HB-GAM are required and sufficient for interaction with hippocampal neurons. The individual domains bind heparan sulfate weakly and fail to produce significant biological effects in neurite outgrowth and long term potentiation assays. The amino acids in the linker region joining the two domains may be replaced with glycines with no effect on protein function. These results suggest a co-operative action of the two beta-sheet domains in the biologically relevant interaction with neuron surface heparan sulfate.

Hydrogen Peroxide Stimulates Proliferation and Migration of Human Prostate Cancer Cells through Activation of Activator Protein-1 and Up-regulation of the Heparin Affin Regulatory Peptide Gene

It is becoming increasingly recognized that hydrogen peroxide (HP) plays a role in cell proliferation and migration. In the present study we found that exogenous HP significantly induced human prostate cancer LNCaP cell proliferation and migration. Heparin affin regulatory peptide (HARP) seems to be involved in the stimulatory effect of HP, because the latter had no effect on stably transfected LNCaP cells that did not express HARP. Moreover, HP significantly increased HARP mRNA and protein amounts in a concentration- and time-dependent manner. Curcumin and activator protein-1 (AP-1) decoy oligonucleotides abrogated both HP-induced HARP expression and LNCaP cell proliferation and migration. HP increased luciferase activity of the 5'-flanking region of the HARP gene introduced in a reporter gene vector, an effect that was abolished when even one of the two putative AP-1 binding sites of the HARP promoter was mutated. The effect of HP seems to be due to the binding of Fra-1, JunD, and phospho-c-Jun to the HARP promoter. These results support the notion that HARP is important for human prostate cancer cell proliferation and migration, establish the role of AP-1 in the up-regulation of HARP expression by low concentrations of HP, and characterize the AP-1 dimers involved.

Specific Structural Features of Heparan Sulfate Proteoglycans Potentiate Neuregulin-1 Signaling

Neuregulins are a family of growth and differentiation factors that act through activation of cell-surface erbB receptor tyrosine kinases and have essential functions both during development and on the growth of cancer cells. One alternatively spliced neuregulin-1 form has a distinct heparin-binding immunoglobulin-like domain that enables it to adhere to heparan sulfate proteoglycans at key locations during development and substantially potentiates its activity. We examined the structural specificity needed for neuregulin-1-heparin interactions using a gel mobility shift assay together with an assay that measures the ability of specific oligosaccharides to block erbB receptor phosphorylation in L6 muscle cells. Whereas the N-sulfate group of heparin was most important, the 2-O-sulfate and 6-O-sulfate groups also contributed to neuregulin-1 binding in these two assays. Optimal binding to neuregulin-1 required eight or more heparin disaccharides; however, as few as two disaccharides were still able to bind neuregulin-1 to a lesser extent. The physiological importance of this specificity was shown both by chemical and siRNA treatment of cultured muscle cells. Pretreatment of muscle cells with chlorate that blocks all sulfation or with an siRNA that selectively blocks N-sulfation significantly reduced erbB receptor activation by neuregulin-1 but had no effect on the activity of neuregulin-1 that lacks the heparin-binding domain. These results suggest that the regulation of glycosaminoglycan sulfation is an important biological mechanism that can modulate both the localization and potentiation of neuregulin-1 signaling.

Heparin-independent mitogenicity in an endothelial and smooth muscle cell chimeric growth factor (S130K-HBGAM)

Background Through site-directed mutagenesis we have created a favorable fibroblast growth factor-1 (FGF-1) mutant (S130K) and linked it to a heparin-binding growth-associated molecule (HBGAM) to form the chimera S130K-HBGAM creating a heparin-independent, endothelial cell (EC)-specific mitogen. Methods The proliferative responses of primary canine carotid artery smooth muscle cells (SMC) and jugular vein EC to FGF-1, S130K, or S130K-HBGAM, with and without heparin (5 U/mL), was quantitated by measuring tritiated thymidine uptake over 24 hours and expressing the results as percent of positive control (20% fetal bovine serum [FBS]) for group comparison. Results Unlike FGF-1, both S130K and S130K-HBGAM are heparin-independent mitogens for EC and SMC. S130K-HBGAM was equivalent to FGF-1 with heparin at 6 nmol/L. S130K-HBGAM did not demonstrate relative EC specificity in this assay. Conclusions At higher concentrations, S130K-HBGAM is a potent, heparin-independent EC and SMC mitogen. Co-culture assays and in vivo delivery models may demonstrate EC specificity not identified in this single cell type proliferation assay.