Receptor for hyaluronan mediated motility (RHAMM/HMMR) is a novel target for promoting subcutaneous adipogenesis.

Hyaluronan, CD44 and the Receptor for Hyaluronan-Mediated Motility (RHAMM, gene name HMMR) regulate stem cell differentiation including mesenchymal progenitor differentiation. Here, we show that CD44 expression is required for subcutaneous adipogenesis, whereas RHAMM expression suppresses this process. We designed RHAMM function blocking peptides to promote subcutaneous adipogenesis as a clinical and tissue engineering tool. Adipogenic RHAMM peptides were identified by screening for their ability to promote adipogenesis in culture assays using rat bone marrow mesenchymal stem cells, mouse pre-adipocyte cell lines and primary human subcutaneous pre-adipocytes. Oil red O uptake into fat droplets and adiponectin production were used as biomarkers of adipogenesis. Positive peptides were formulated in either collagen I or hyaluronan (Orthovisc) gels then assessed for their adipogenic potential in vivo following injection into dorsal rat skin and mammary fat pads. Fat content was quantified and characterized using micro CT imaging, morphometry, histology, RT-PCR and ELISA analyses of adipogenic gene expression. Injection of screened peptides increased dorsal back subcutaneous fat pad area (208.3 ± 10.4 mm2versus control 84.11 ± 4.2 mm2; p < 0.05) and mammary fat pad size (45 ± 11 mg above control background, p = 0.002) in female rats. This effect lasted >5 weeks as detected by micro CT imaging and perilipin 1 mRNA expression. RHAMM expression suppresses while blocking peptides promote expression of PPARγ, C/EBP and their target genes. Blocking RHAMM function by peptide injection or topical application is a novel and minimally invasive method for potentially promoting subcutaneous adipogenesis in lipodystrophic diseases and a complementary tool to subcutaneous fat augmentation techniques.

CSPG4 in cancer: multiple roles.

Chondroitin sulfate proteoglycan 4 (CSPG4), also known as High Molecular Weight-Melanoma Associated Antigen, is a cell surface proteoglycan which has been recently shown to be expressed not only by melanoma cells, but also by various types of human carcinoma and sarcoma. Furthermore, at least in squamous cell carcinoma of head and neck and in basal breast carcinoma, CSPG4 is expressed by cancer stem cells. CSPG4 plays an important role in tumor cell growth and survival. These CSPG4-associated functional properties of tumor cells are inhibited by CSPG4-specific monoclonal antibodies (mAb) in vitro. Moreover, CSPG4-specific mAb can also inhibit tumor growth and metastasis in vivo. The anti-tumor effects of CSPG4-specific mAb are likely to reflect the blocking of important migratory, mitogenic and survival signaling pathways in tumor cells. These results indicate that CSPG4 is a promising new target to implement mAb-based immunotherapy of various types of cancer.

Cyclin D1 promotes anchorage-independent cell survival by inhibiting FOXO-mediated anoikis.

O-class forkhead box (FOXO) transcription factors are critical regulators of diverse cellular processes, including apoptosis, cell-cycle arrest, DNA damage repair and oxidative stress resistance. Here, we show that FOXO1 and FOXO3a have an essential function in promoting cell detachment-induced anoikis, resistance to which is implicated in cancer development and metastasis. In contrast, the oncoprotein cyclin D1 inhibits anoikis. We further show that cyclin D1 interacts with FOXO proteins and impedes their transcriptional regulatory and anoikis-promoting functions. This effect of cyclin D1 requires its transcription repression domain but is independent of cyclin-dependent kinases CDK4 and CDK6. Moreover, we show that cancer-derived mutants of cyclin D1 are much more stable than wild-type cyclin D1 under anchorage-independent conditions and possess a greater antagonistic effect on FOXO-regulated anoikis and anchorage-independent growth of cancer cells. These data suggest that cyclin D1 may have a critical function in tumorigenesis and cancer metastasis by inhibiting the anoikis-promoting function of FOXO proteins.

TrkA immunoreactive astrocytes in dendritic fields of the hippocampal formation across estrous.

Neurotrophins are important modulators of structural synaptic plasticity. (Through trophic action (Jordan. J Neurobiol 40:434-445, 1999), astrocytes serve as permissive substrates to support axonal regrowth (Ridet et al. Trends Neurosci 20:570-571, 1997), and are involved in estrogen-induced synaptic structural plasticity (Garcia-Segura et al. Cell Mol Neurobiol 16:225-237, 1996). Previously, we reported that tyrosine kinase A receptor (TrkA) immunoreactivity was present both in presynaptic neuronal processes (axons and terminals) and in select astrocytes of the male rat hippocampal formation (Barker-Gibb et al. J Comp Neurol 430:182-199, 2001). We show that the number of TrkA-immunoreactive astrocytes in female rats fluctuates 16-fold across the estrous cycle in dendritic fields of the hippocampal formation, with the greatest number at estrus after the peak plasma estradiol concentration of proestrus. Few TrkA-labeled astrocytes were found in ovariectomized animals; after estrogen replacement, this number increased by 12-fold in the hippocampal formation, indicating estrogen-mediated induction. Dual-labeling studies showed that TrkA-labeled astrocytes were also immunoreactive for vimentin, a protein expressed by reactive astrocytes. Ultrastructural analysis of the dentate gyrus molecular layer demonstrated that TrkA immunoreactive astrocytes are positioned primarily next to dendrites and unmyelinated axons. Because nerve growth factor (NGF) has been reported to stimulate astrocytes to function as substrates for axon growth (Kawaja and Gage. Neuron 7:1019-1030, 1991), these findings are consistent with the theory that TrkA immunoreactive astrocytes serve a role in structural plasticity, axon guidance, and synaptic regeneration across the estrous cycle in the hippocampal formation.

Adhesion of alpha5beta1 receptors to biomimetic substrates constructed from peptide amphiphiles.

Biomimetic membrane surfaces functionalized with fragments of the extracellular matrix protein, fibronectin, are constructed from mixtures of peptide and polyethylene glycol (PEG) amphiphiles. Peptides from the primary binding loop, GRGDSP, were used in conjunction with the synergy site peptide, PHSRN, in the III(9-10) sites of human fibronectin. These peptides were attached to dialkyl lipid tails to form peptide amphiphiles. PEG amphiphiles were mixed in the layer to minimize non-specific adhesion in the background. GRGDSP and PEG amphiphiles or GRGDSP, PHSRN, and PEG amphiphiles were mixed in various ratios and deposited on solid substrates from the air-water interface using Langmuir-Blodgett techniques. In this method, peptide composition, density, and presentation could be controlled accurately. The effectiveness of these substrates to mimic native fibronectin is evaluated by their ability to generate adhesive forces when they are in contact with purified activated alpha5beta1 integrin receptors that are immobilized on an opposing surface. Adhesion is measured using a contact mechanical approach (JKR experiment). The effects of membrane composition, density, temperature, and peptide conformation on adhesion to activated integrins in this simulated cell adhesion setup were determined. Addition of the synergy site, PHSRN, was found to increase adhesion of alpha5beta1, to biomimetic substrates markedly. Increased peptide mobility (due to increased experimental temperature) increased integrin adhesion markedly at low peptide concentrations. A balance between peptide density and steric accessibility of the receptor binding face to alpha5beta1 integrin was required for highest adhesion.

Melanoma chondroitin sulfate proteoglycan regulates matrix metalloproteinase-dependent human melanoma invasion into type I collagen.

Tumor cell adhesion and proteolysis of the extracellular matrix proteins surrounding the cells are tightly linked processes in tumor invasion. In this study, we sought to identify components of the cell surface of a vertical growth phase melanoma cell line, WM1341D, that mediate invasive cellular behavior. We determined by antisense inhibition that melanoma chondroitin sulfate proteoglycan (MCSP) and membrane-type 3 matrix metalloproteinase (MT3-MMP) expressed on WM1341D are required for invasion of type I collagen and degradation of type I gelatin. MT3-MMP co-immunoprecipitated with MCSP in WM1341D melanoma cells cultured on type I collagen or laminin. The association between MT3-MMP and MCSP was largely disrupted by removing chondroitin sulfate glycosaminoglycan (CS) from the cell surface, suggesting CS could mediate the association between the two cell surface core proteins. Recombinant MT3-MMP and MT3-MMP from whole cell lysates of WM1341D cells were specifically eluted from CS- conjugated affinity columns. The results indicate that MT3-MMP possesses the potential to promote melanoma invasion and proteolysis and that the formation of a complex between MT3-MMP and MCSP may be a crucial step in activating these processes.

Hyaluronan synthase elevation in metastatic prostate carcinoma cells correlates with hyaluronan surface retention, a prerequisite for rapid adhesion to bone marrow endothelial cells.

Bone marrow is the primary site of metastasis in patients with advanced stage prostate cancer. Prostate carcinoma cells metastasizing to bone must initially adhere to endothelial cells in the bone marrow sinusoids. In this report, we have modeled that interaction in vitro using two bone marrow endothelial cell (BMEC) lines and four prostate adenocarcinoma cell lines to investigate the adhesion mechanism. Highly metastatic PC3 and PC3M-LN4 cells were found to adhere rapidly and specifically (70-90%) to BMEC-1 and trHBMEC bone marrow endothelial cells, but not to human umbilical vein endothelial cells (15-25%). Specific adhesion to BMEC-1 and trHBMEC was dependent upon the presence of a hyaluronan (HA) pericellular matrix assembled on the prostate carcinoma cells. DU145 and LNCaP cells were only weakly adherent and retained no cell surface HA. Maximal BMEC adhesion and HA encapsulation were associated with high levels of HA synthesis by the prostate carcinoma cells. Up-regulation of HA synthase isoforms Has2 and Has3 relative to levels expressed by normal prostate corresponded to elevated HA synthesis and avid BMEC adhesion. These results support a model in which tumor cells with up-regulated HA synthase expression assemble a cell surface hyaluronan matrix that promotes adhesion to bone marrow endothelial cells. This interaction could contribute to preferential bone metastasis by prostate carcinoma cells.

Evidence for a satellite secretory pathway in neuronal dendritic spines.

Long-term information storage within the brain requires the synthesis of new proteins and their use in synapse-specific modifications [1]. Recently, we demonstrated that translation sites for the local synthesis of integral membrane and secretory proteins occur within distal dendritic spines [2]. It remains unresolved, however, whether a complete secretory pathway, including Golgi and trans Golgi network-like membranes, exists near synapses for the local transport and processing of newly synthesized proteins. Here, we report evidence of a satellite secretory pathway in distal dendritic spines and distal dendrites of the mammalian brain. Membranes analogous to early (RER and ERGIC), middle (Golgi cisternae), and late (TGN) secretory pathway compartments are present within dendritic spines and in distal dendrites. Local synthesis, processing, and transport of newly translated integral membrane and secretory proteins may thus provide the molecular basis for synapse-specific modifications during long-term information storage in the brain.

The C-terminal tail of the metabotropic glutamate receptor subtype 7 is necessary but not sufficient for cell surface delivery and polarized targeting in neurons and epithelia.

Complex neuronal functions rely upon the precise sorting, targeting, and restriction of receptors to specific synaptic microdomains. Little is known, however, of the molecular signals responsible for mediating these selective distributions. Here we report that metabotropic glutamate receptor subtype 7a (mGluR7a) is polarized at the basolateral surface when expressed in Madin-Darby canine kidney (MDCK) epithelial cells but is not polarized when expressed in cultured hippocampal neurons. Truncation of the mGluR7 cytoplasmic tail produces a protein that is restricted to a perinuclear intracellular compartment in both neurons and MDCK cells, where this protein colocalizes with a trans-Golgi network antigen. The mGluR7 cytoplasmic domain appended to the transmembrane portion of the vesicular stomatitis virus G protein and the ectodomain of human placental alkaline phosphatase is distributed over the entire cell surface in cultured neurons. When expressed in MDCK cells, this construct remains in an intracellular compartment distinct from endosomes or lysosomes. Thus, the cytoplasmic tail domain of mGluR7 is necessary but not sufficient for polarized targeting in MDCK monolayers, whereas in neurons the cytoplasmic tail is sufficient for cell surface expression but not polarization. Additional mechanisms are likely required to mediate mGluR7 neuronal polarization and synaptic clustering.

Enhancement of nigral graft survival in rat brain with the systemic administration of synthetic fibronectin peptide V.

A major obstacle in neural transplantation is a severe loss of neurons in grafts soon after implantation. In the present study, we have investigated whether the systemic administration of synthetic fibronectin peptide V can increase the survival of neural grafts. Synthetic fibronectin peptide V is derived from the 33,000 mol. wt carboxyl-terminal heparin-binding domain of fibronectin. Previous studies have shown that these polypeptides possess anti-inflammatory properties. However, it is currently unknown whether this peptide has anti-apoptotic properties. Dissociated neural grafts were prepared from the ventral mesencephalon of pregnant Sprague-Dawley rats and were stereotaxically injected as a cell suspension into the striatum of adult Sprague-Dawley rats. A group of recipient rats received i.v. injections of peptide V (5mg/kg, dissolved in saline) at 24 and 4h prior to transplantation, at the time of transplantation, and 24, 48 and 72h post-transplantation. Saline-treated rats served as controls. The rats were killed at two, four and 42 days post-grafting and the brain tissue was immunologically processed for tyrosine-hydroxylase, major histocompatibility complex class I and class II antigens, complement receptor type 3 and leukocyte common antigen immunocytochemistry, and terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. We found a significant increase (approximately twofold) in the number of dopamine neurons in the grafts for the peptide-treated group at four and 42 days compared with the controls. In contrast, there was no significant difference in the patterns of inflammation using different immunocytochemical markers in the two different groups. The levels of expression for these markers, however, were reduced over time. Interestingly, the number of apoptotic cells in the graft areas was significantly smaller in the peptide-treated group than in the control group two days after grafting. The results demonstrate that the systemic administration of synthetic fibronectin peptide V can dramatically increase the survival of nigral grafts in the brain and substantially reduce the number of apoptotic cells in the graft site, suggesting that this peptide may exert a beneficial effect on survival of nigral grafts through an anti-apoptotic mechanism.

Ligand accessibility as means to control cell response to bioactive bilayer membranes.

We report a new method to create a biofunctional surface in which the accessibility of a ligand is used as a means to influence the cell behavior. Supported bioactive bilayer membranes were created by Langmuir-Blodgett (LB) deposition of either a pure poly(ethylene glycol) (PEG) lipid, having PEG head groups of various lengths, or 50 mol % binary mixtures of a PEG lipid and a novel collagen-like peptide amphiphile on a hydrophobic surface. The peptide amphiphile contains a peptide synthetically lipidated by covalent linkage to hydrophobic dialkyl tails. The amphiphile head group lengths were determined using neutron reflectivity. Cell adhesion and spreading assays showed that the cell response to the membranes depends on the length difference between head groups of the membrane components. Cells adhere and spread on mixtures of the peptide amphiphile with the PEG lipids having PEG chains of 120 and 750 molecular weight (MW). In contrast, cells adhered but did not spread on the mixture containing the 2000 MW PEG. Cells did not adhere to any of the pure PEG lipid membranes or to the mixture containing the 5000 MW PEG. Selective masking of a ligand on a surface is one method of controlling the surface bioactivity.

Correlation of CD44S expression in renal clear cell carcinomas with subsequent tumor progression or recurrence.

BACKGROUND: Recent reports have shown altered expression of CD44 in renal cell carcinomas. However, to the authors' knowledge there are no data correlating CD44 expression in renal cell carcinomas with subsequent tumor progression or recurrence, nor is there information about the presence of particular splice variants of CD44 in these tumors. METHODS: The authors examined the immunohistochemical expression of CD44S, the standard isoform of CD44, in renal cell carcinomas from 43 patients using 2 different monoclonal antibodies, Mab2137 and Hermes-3. In addition, they stained the renal cell carcinomas with antibodies to 2 splice variants of CD44, CD44v3 and CD44v6. RESULTS: Increased staining of renal clear cell carcinomas with Mab2137 was observed in high grade versus low grade tumors (45% vs. 0%, P = 0.013), whereas increased staining of clear cell carcinomas with Hermes-3 was noted in high stage versus low stage tumors (40% vs. 0%, P = 0.006). Few tumors stained with antibodies to CD44v3. Although increased expression of the splice variant CD44v6 was noted in papillary versus clear cell carcinomas, and increased staining of papillary carcinomas with Mab2137 and with antibodies to CD44v6 was noted for low stage versus high stage tumors, these differences did not achieve statistical significance. Clinical follow-up of at least 43 months was available for 26 patients. Six of these patients (five with clear cell carcinoma and one with papillary carcinoma) developed progressive or recurrent disease. The primary tumors from all 5 patients with progressive or recurrent clear cell carcinoma showed staining with Mab2137, whereas the primary tumors from only 2 of the 15 patients with at least 43 months follow-up and no evidence of progressive or recurrent clear cell carcinoma (13%) showed staining with Mab2137 (P = 0.001). Alternatively, 5 of 7 clear cell carcinomas (71%) that stained with Mab2137 were from patients who subsequently developed recurrence or progression, compared with 0 of 13 clear cell carcinomas that did not stain. Similar findings were not observed for papillary carcinomas, which appeared to be biologically distinct from clear cell carcinomas. CONCLUSIONS: CD44S staining with Mab2137 correlates with progression or recurrence of clear cell renal cell carcinoma. CD44S may, therefore, play a pathogenetic role in tumor progression.

Cellular recognition of synthetic peptide amphiphiles in self-assembled monolayer films.

The incorporation of lipidated cell adhesion peptides into self-assembled structures such as films provides the opportunity to develop unique biomimetic materials with well-organized interfaces. Synthetic dialkyl tails have been linked to the amino-terminus, carboxyl-terminus, and both termini of the cell recognition sequence Arg-Gly-Asp (RGD) to produce amino-coupled, carboxyl-coupled, and looped RGD peptide amphiphiles. All three amphiphilic RGD versions self-assembled into fairly stable mixed monolayers that deposited well as Langmuir-Blodgett films on surfaces, except for films containing amino-coupled RGD amphiphiles at high peptide concentrations. FT-IR studies showed that amino-coupled RGD head groups formed the strongest lateral hydrogen bonds. Melanoma cells spread on looped RGD amphiphiles in a concentration-dependent manner, spread indiscriminately on carboxyl-coupled RGD amphiphiles, and did not spread on amino-coupled RGD amphiphiles. Looped RGD amphiphiles promoted the adhesion, spreading, and cytoskeletal reorganization of melanoma and endothelial cells while control looped Arg-Gly-Glu (RGE) amphiphiles inhibited them. Antibody inhibition of the integrin receptor alpha3beta1 blocked melanoma cell adhesion to looped RGD amphiphiles. These results confirm that novel biomolecular materials containing synthetic peptide amphiphiles have the potential to control cellular behavior in a specific manner.

Melanoma chondroitin sulphate proteoglycan regulates cell spreading through Cdc42, Ack-1 and p130cas.

Melanoma chondroitin sulphate proteoglycan (MCSP) is a cell-surface antigen that has been implicated in the growth and invasion of melanoma tumours. Although this antigen is expressed early in melanoma progression, its biological function is unknown. MCSP can stimulate the integrin-alpha4 beta1-mediated adhesion and spreading of melanoma cells. Here we show that stimulated MCSP recruits tyrosine-phosphorylated p130 cas, an adaptor protein important in tumour cell motility and invasion. MCSP stimulation also results in a pronounced activation and recruitment of the Rho-family GTPase Cdc42. MCSP-induced spreading of melanoma cells is dependent upon active Cdc42, a Cdc42-associated tyrosine kinase (Ack-1) and tyrosine phosphorylation of p130cas. Furthermore, vectors inhibiting Ack-1 or Cdc42 expression and/or function abrogate MCSP-induced tyrosine phosphorylation and recruitment of p130cas. Our findings indicate that MCSP may modify tumour growth or invasion by a unique signal-transduction pathway that links Cdc42 activation to downstream tyrosine phosphorylation and subsequent cytoskeletal reorganization.

Stimulation of beta1-integrin function by epidermal growth factor and heregulin-beta has distinct requirements for erbB2 but a similar dependence on phosphoinositide 3-OH kinase.

Integrins and growth factor receptors are important participants in cellular adhesion and migration. The EGF receptor (EGFR) family of tyrosine kinases and the beta1-integrin adhesion receptors are of particular interest, given the implication for their involvement in the initiation and progression of tumorigenesis. We used adhesion and chemotaxis assays to further elucidate the relationship between these two families of transmembrane signaling molecules. Specifically, we examined integrin-mediated adhesive and migratory characteristics of the metastatic breast carcinoma cell line MDA-MB-435 in response to stimulation with growth factors that bind to and activate the EGFR or erbB3 in these cells. Although ligand engagement of the EGFR stimulated modest beta1-dependent increases in cell adhesion and motility, heregulin-beta (HRGbeta) binding to the erbB3 receptor initiated rapid and potent induction of breast carcinoma cell adhesion and migration and required dimerization of erbB3 with erbB2. Pharmacologic inhibitors of phosphoinositide 3-OH kinase (PI 3-K) or transient expression of dominant negative forms of PI 3-K inhibited both EGF- and HRGbeta-mediated adhesion and potently blocked HRGbeta- and EGF-induced cell motility. Our results illustrate the critical role of PI 3-K activity in signaling pathways initiated by the EGFR or erbB3 to up-regulate beta1-integrin function.

CXC-chemokines stimulate invasion and chemotaxis in prostate carcinoma cells through the CXCR2 receptor.

BACKGROUND: Metastasis of prostate carcinoma requires invasion through the basement membrane, a thin extracellular matrix that underlies the epithelial cells, which must be breached by tumor cells invading into surrounding tissue. The CXC-chemokines, which have been shown to promote the migration of neutrophils and carcinoma cells, are candidates to influence prostate carcinoma-cell invasion. METHODS: CXC-chemokines were examined for the ability to stimulate prostate cell line PC3 invasion in vitro through a reconstituted basement membrane and long-term migration and short-term adhesion to laminin, a major component of the basement membrane. RESULTS: PC3 cells responded to IL-8 and GROalpha with a 1. 6-2-fold increase in invasion through reconstituted basement membrane. A corresponding 2-3-fold increase in chemotaxis toward IL-8 and GROa was seen on laminin. Anti-CXCR2 antibody inhibited IL-8-stimulated migration. Expression levels of the beta(1) integrins were not changed by IL-8, and alpha(6beta1) integrin was used for both stimulated and baseline migration. In addition to the increases in migration and invasion, 2-6-fold transient increases in adhesion on laminin were seen with both IL-8 and GROalpha. CONCLUSIONS: These results suggest that the CXC-chemokines stimulate migration and invasion in part by altering the activation state of the beta(1) integrins. The CXC-chemokines act on prostate carcinoma cells through the CXCR2 receptor to promote behavior important for metastasis, and as such may be important in prostate carcinoma progression and metastasis.

Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function.

Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.

Biosynthesis and metabolism of native and oxidized neuropeptide Y in the hippocampal mossy fiber system.

Neuropeptide Y (NPY) gene expression is known to be modulated in the mossy fiber projection of hippocampal granule cells following seizure. We investigated NPY biosynthesis and metabolism in an attempt to characterize NPY biochemically as a neurotransmitter in the granule cell mossy fiber projection. NPY biosynthesis was compared in normal control animals and in animals that had experienced a single pentylenetetrazole-induced seizure. In situ hybridization analysis established the postseizure time course of preproNPY mRNA expression in the hippocampal formation, localizing the majority of increased preproNPY mRNA content to the hilus of the dentate gyrus. Radioimmunoassay analysis of the CA3/mossy fiber terminal subfield confirmed a subsequent increase in NPY peptide content. Biosynthesis of NPY peptide by granule cells and transport to the CA3/mossy fiber subfield was demonstrated by in vivo radiolabel infusion to the dentate gyrus/hilus followed by sequential HPLC purification of identified radiolabeled peptide from the CA3/mossy fiber terminal subfield. Additional in vivo radiolabeling studies revealed a postseizure increase in an unidentified NPY-like immunoreactive (NPY-LI) species. HPLC/radioimmunoassay analyses of CA3 subfield tissue extracts comparing normal control animals and pentylenetetrazole-treated animals confirmed the increased total NPY-LI, and demonstrated that the increased NPY-LI was comprised of a minor increase in native NPY and a major increase in the unknown NPY-LI. Data from subsequent and separate analyses incorporating immunoprecipitation with anti-C-terminal flanking peptide of NPY, further HPLC purification, and matrix-assisted laser desorption/ionization mass spectrometry support the conclusion that the unknown NPY-LI is methionine sulfoxide NPY. NPY and NPY-sulfoxide displayed differential calcium sensitivity for release from mossy fiber synaptosomes. Similar to NPY, NPY sulfoxide displayed high-affinity binding to each of the cloned Y1, Y2, Y4, and Y5 receptor subtypes. Postrelease inactivation of NPY was demonstrated in a mossy fiber synaptosomal preparation. Thus, the present study in combination with previously reported electrophysiological activity of NPY in the CA3 subfield demonstrates that NPY fulfills the classical criteria for a neurotransmitter in the hippocampal granule cell mossy fiber projection, and reveals the presence of two molecular forms of NPY that display differential mechanisms of release while maintaining similar receptor potencies.

A role of chondroitin sulfate glycosaminoglycan binding site in alpha4beta1 integrin-mediated melanoma cell adhesion.

We have previously reported that alpha4beta1 (but not alpha5beta1) integrin-mediated melanoma cell adhesion is inhibited by removal of cell surface chondroitin sulfate glycosaminoglycan (CSGAG), suggesting that melanoma chondroitin sulfate proteoglycan plays a role in modulating the adhesive function of alpha4beta1 integrin. In the current study, we demonstrated that alpha4beta1 integrin binds to CSGAG. We have identified a peptide from within alpha4 integrin termed SG1 (KKEKDIMKKTI) that binds to cell surface melanoma chondroitin sulfate proteoglycan, indicating that SG1 represents a CSGAG binding site within the alpha4 integrin subunit. Soluble SG1 inhibits alpha4beta1 integrin-mediated human melanoma cell adhesion to CS1. Polyclonal antibody generated against the peptide inhibits melanoma cell adhesion to CS1, and the inhibition is reversed by Mn2+ and an activating monoclonal antibody anti-beta1 (8A2). Additionally, pretreatment of cells with anti-SG1 IgG inhibits the expression of the monoclonal antibody 15/7 epitope in the presence of soluble CS1 peptide, suggesting that anti-SG1 IgG prevents ligand binding by alpha4beta1 integrin. These results demonstrate that alpha4beta1 integrin interacts directly with CSGAG through SG1 site, and that this site can affect the ligand binding properties of the integrin.