Nidogen is a prosurvival and promigratory factor for adult Schwann cells

Schwann cells provide a favorable microenvironment for successful regeneration of the injured peripheral nerve. Even though the roles of extracellular matrix proteins in the Schwann cell physiology have long been studied, the precise function of nidogen, a ubiquitous component of the basal lamina, in Schwann cells is unknown. In this study, we show that the protein and mRNA messages for nidogens are up-regulated in the sciatic nerve after sciatic nerve transection. We demonstrate that recombinant nidogen-1 increased the process formation of Schwann cells cultured from adult rat sciatic nerves and that nidogen-1 prevented Schwann cells from serum-deprivation-induced death. In addition, nidogen-1 promoted spontaneous migration of Schwann cells in two-independent migration assays. The Schwann cell responses to the recombinant nidogen-1 were specific because the nidogen-binding ectodomain of tumor endothelial marker 7 inhibited the nidogen responses without affecting Schwann cell response to laminin. Finally, we found that beta1 subunit-containing integrins play a key role in the nidogen-induced process formation, survival, and migration of Schwann cells. Altogether, these results indicate that nidogen has a prosurvival and promigratory activity on Schwann cells in the peripheral nerve.     

High expression of tumor endothelial marker 7 is associated with metastasis and poor survival of patients with osteogenic sarcoma

Our objective is to identify genes regulating metastasis of osteogenic sarcoma (OGS) since metastasis is the primary cause of mortality among patients with OGS. To identify such genes, we first created a database of differentially expressed genes between six low-grade and six high-grade OGS tumors, and between a normal immortalized osteoblast cell line (FOB) and four commercially available OGS-derived cell lines. We specifically searched for surface proteins over-expressed in high-grade OGS, since we hypothesize that tumor-cell specific surface markers are key to metastasis. A gene encoding Tumor Endothelial Marker7 (TEM7) was selected as a candidate for further study. TEM7 expression pattern was assessed by RT-PCR, Western blotting and immunostaining. TEM7 mRNA was abundantly expressed in SAOS cells (derived from high-grade OGS), but not in FOB or MG63 cells (derived from low-grade OGS). Virtually no expression of TEM7 protein was observed in FOB cells but abundant expression was noted in SAOS and TE85 cells. Employing immunostaining of 92 human OGS specimens (50 high-grade and 42 low-grade) collected before chemotherapy show 97% (37 of 38) of high-grade OGS specimens with metastasis have high TEM7 staining. Further, we found that elevated expression of TEM7 correlated with poor survival (p<0.04) of affected patients. Inhibiting TEM7 function by siRNA inhibited invasion and migration of OGS cells with metastatic potential. Our results suggest TEM7 expression level closely parallels histology-based prognostication of OGS metastasis and, therefore, it is a therapeutic target. This is the first demonstration of a link between TEM7 and cancer metastasis.     

Basement-membrane heparan sulfate proteoglycan binds to laminin by its heparan sulfate chains and to nidogen by sites in the protein core.

A large, low-density form of heparan sulfate proteoglycan was isolated from the Engelbreth-Holm-Swarm (EHS) tumor and demonstrated to bind in immobilized-ligand assays to laminin fragment E3, collagen type IV, fibronectin and nidogen. The first three ligands mainly recognize the heparan sulfate chains, as shown by inhibition with heparin and heparan sulfate and by the failure to bind to the proteoglycan protein core. Nidogen, obtained from the EHS tumor or in recombinant form, binds exclusively to the protein core in a heparin-insensitive manner. Studies with other laminin fragments indicate that the fragment E3 possesses a unique binding site of laminin for the proteoglycan. A major binding site of nidogen was localized to its central globular domain G2 by using overlapping fragments. This allows for the formation of ternary complexes between laminin, nidogen and proteoglycan, suggesting a key role for nidogen in basement-membrane assembly. Evidence is provided for a second proteoglycan-binding site in the C-terminal globule G3 of nidogen, but this interaction prevents the formation of such ternary complexes. Therefore, the G3-mediated nidogen binding to laminin and proteoglycan are mutually exclusive.     

Basement membrane deposition of nidogen 1 but not nidogen 2 requires the nidogen binding module of the laminin gamma1 chain

The nidogen-laminin interaction is proposed to play a key role in basement membrane (BM) assembly. However, though there are similarities, the phenotypes in mice lacking nidogen 1 and 2 (nidogen double null) differ to those of mice lacking the nidogen binding module (γ1III4) of the laminin γ1 chain. This indicates different cell- and tissue-specific functions for nidogens and their interaction with laminin and poses the question of whether the phenotypes in nidogen double null mice are caused by the loss of the laminin-nidogen interaction or rather by other unknown nidogen functions. To investigate this, we analyzed BMs, in particular those in the skin of mice lacking the nidogen binding module. In contrast to nidogen double null mice, all skin BMs in γ1III4-deficient mice appeared normal. Furthermore, although nidogen 1 deposition was strongly reduced, nidogen 2 appeared unchanged. Mice with additional deletion of the laminin γ3 chain, which contains a γ1-like nidogen binding module, showed a further reduction of nidogen 1 in the dermoepidermal BM; however, this again did not affect nidogen 2. This demonstrates that in vivo only nidogen 1 deposition is critically dependent on the nidogen binding modules of the laminin γ1 and γ3 chains, whereas nidogen 2 is independently recruited either by binding to an alternative site on laminin or to other BM proteins.     

Chicken gizzard 5'-nucleotidase functions as a binding protein for the laminin/nidogen complex.

Soluble and reconstituted 5'-nucleotidase were used in the binding assays to the laminin/nidogen complex. They both are shown to interact specifically and in a saturable manner with the laminin/nidogen complex using a solid-phase binding assay. Dissociation constants in the region of 10(-8) M were determined for the association of soluble and membrane-bound 5'-nucleotidase. Scatchard analysis of the binding data indicate a stoichiometry of about 2.7 of the homodimeric soluble 5'-nucleotidase to the laminin/nidogen complex. The association of 5'-nucleotidase with laminin/nidogen occurs in the absence of divalent metal ions and does not require N-linked carbohydrate moieties of both laminin/nidogen and 5'-nucleotidase. 5'-Nucleotidase also associates with isolated laminin although with reduced affinity. No binding to isolated nidogen was observed. Peptides containing the RGD sequence did not influence the binding reaction. Monoclonal and polyclonal antibodies directed against 5'-nucleotidase and laminin specifically perturb the association of the reconstituted enzyme to laminin/nidogen. Sulfated polysaccharides such as heparinsulfate and dermatansulfate modulate the interaction of 5'-nucleotidase and laminin/nidogen in a complex biphasic manner and might also regulate the binding reaction in vivo. Immunohistochemistry shows a close spatial correlation of 5'-nucleotidase and laminin also in the epithelium of the small intestine pointing to an in vivo interaction of both glycoproteins.     

Urokinase binding to laminin-nidogen. Structural requirements and interactions with heparin.

Recently we have shown that heparin and related sulfated polyanions are low-affinity ligands of the kringle domain in the amino-terminal region (ATF) of human urokinase (u-PA), and proposed that this may facilitate loading of u-PA onto its receptor at the focal contacts between adherent cells and their matrix. We have now tested other components of the cell matrix (fibronectin, vitronectin, thrombospondin and laminin-nidogen) for u-PA binding, and found that laminin-nidogen is also a ligand of the u-PA ATF. Direct binding assays and competition binding assays with defined fragments of laminin-nidogen showed that there are u-PA binding sites in fragment E4 of laminin as well as in nidogen. The long-arm terminal domain of laminin (fragment E3), which contains a heparin-binding site, competed for binding of u-PA to immobilised heparin. However nidogen, which does not bind to heparin, also inhibited binding of u-PA to heparin, and this effect was also observed with recombinant nidogen and with a fragment of nidogen lacking the carboxy-terminal domain. Direct binding assays confirmed that u-PA binds to nidogen through a site in the u-PA ATF. We conclude that u-PA binds to laminin-nidogen by interactions involving the ATF region of u-PA, the E4 domain of laminin and the rod or amino-terminal regions of nidogen. Since nidogen is suggested to be an important bridging molecule in the maintenance of the supramolecular organization in basement membranes, the presence of a binding site for u-PA in nidogen indicates a role for plasminogen activation in basement membrane remodelling.     

Recombinant nidogen consists of three globular domains and mediates binding of laminin to collagen type IV.

Recombinant mouse nidogen and two fragments were produced in mammalian cells and purified from culture medium without resorting to denaturing conditions. The truncated products were fragments Nd-I (positions 1-905) comprising the N-terminal globule and rod-like domain and Nd-II corresponding mainly to the C-terminal globule (position 906-1217). Recombinant nidogen was indistinguishable from authentic nidogen obtained by guanidine dissociation from tumor tissue with respect to size, N-terminal sequence, CD spectra and immunochemical properties. They differed in protease stability and shape indicating that the N-terminal domain of the more native, recombinant protein consists of two globules connected by a flexible segment. This established a new model for the shape of nidogen consisting of three globes of variable mass (31-56 kDa) connected by either a rod-like or a thin segment. Recombinant nidogen formed stable complexes (Kd less than or equal to 1 nM) with laminin and collagen IV in binding assays with soluble and immobilized ligands and as shown by electron microscopy. Inhibition assays demonstrated different binding sites on nidogen for both ligands with different specificities. This was confirmed in studies with fragment Nd-I binding to collagen IV and fragment Nd-II binding to laminin fragment P1. In addition, recombinant nidogen but not Nd-I was able to bridge between laminin or P1 and collagen IV. Formation of such ternary complexes implicates a similar role for nidogen in the supramolecular organization of basement membranes.     

huCdc7激酶及其与肿瘤的关系

Cdc7在人类细胞里的同源蛋白huCdc7几乎在人体所有组织中均有表达,在细胞周期中huCdc7可以通过磷酸化作用行使调控DNA复制起始的功能,同时也可通过参与ATR/Chk1途径调控细胞周期中DNA损伤的S期检验点．而近来研究发现,huCdc7表达水平的改变也参与恶性肿瘤的发生发展,在肿瘤细胞里huCdc7能加快肿瘤细胞的异常增殖同时对抗化疗药物也起到关键性的作用．目前,huCdc7已成为治疗肿瘤的一个重要靶标,最新发现的huCdc7有效抑制剂——PHA-767491在癌症治疗中显示出了独特的抑制肿瘤的作用,并且对正常细胞无毒副作用．因此,对huCdc7激酶及其抑制剂的进一步深入研究有望为临床治疗肿瘤开辟新的途径．     

The plant limonoid 7-oxo-deacetoxygedunin inhibits RANKL-induced osteoclastogenesis by suppressing activation of the NF-κB and MAPK pathways

Osteoclasts together with osteoblasts play pivotal roles in bone remodeling. Aberrations in osteoclast differentiation and activity contribute to osteopenic disease. Osteoclasts differentiate from monocyte/macrophage progenitors, a process that is initiated by the interaction between receptor activator of NF-κB (RANK) and its ligand, RANKL. In this study, we identified 7-oxo-7-deacetoxygedunin (7-OG), a gedunin type limonoid from seeds of the mangrove Xylocarpus moluccensis, as a potent inhibitor of osteoclastogenesis. Additionally, 7-OG showed strong anti-osteoclastogenic activity with low cytotoxicity against the monocyte/macrophage progenitor cell line, RAW264.7. The IC50 for anti-osteoclastogenic activity was 4.14 μM. Treatment with 7-OG completely abolished the appearance of multinucleated giant cells with tartrate-resistant acid phosphatase activity in RAW264.7 cells stimulated with RANKL. When the expression of genes related to osteoclastogenesis was investigated, a complete downregulation of NFATc1 and cathepsin K and a delayed downregulation of irf8 were observed upon 7-OG treatment in the presence of RANKL. Furthermore, treatment with this limonoid suppressed RANKL-induced activation of p38, MAPK and Erk and nuclear localization of NF-κB p65. Taken together, we present evidence indicating a plant limonoid as a novel osteoclastogenic inhibitor that could be used for osteoporosis and related conditions.     

A single EGF-like motif of laminin is responsible for high affinity nidogen binding.

A major nidogen binding site of mouse laminin was previously localized to about three EGF-like repeats (Nos 3-5) of its B2 chain domain III [M. Gerl et al. (1991) Eur. J. Biochem., 202, 167]. The corresponding cDNA was amplified by polymerase chain reaction and inserted into a eukaryotic expression vector tagged with a signal peptide. Stably transfected human kidney cell clones were shown to process and secrete the resulting fragment B2III3-5 in substantial quantities. It possessed high binding activity for recombinant nidogen in ligand assays, with an affinity comparable with that of authentic laminin fragments. In addition, complexes of B2III3-5 and nidogen could be efficiently converted into a covalent complex by cross-linking reagents. Proteolytic degradation of the covalent complex demonstrated the association of B2III3-5 with a approximately 80 residue segment of nidogen domain G3 to which laminin binding has previously been attributed. The correct formation of most of the 12 disulfide bridges in B2III3-5 was indicated from its protease resistance and the complete loss of cross-reacting epitopes as well as of nidogen-binding activity after reduction and alkylation. Smaller fragments were prepared by the same recombinant procedure and showed that combinations of EGF-like repeats 3-4 and 4-5 and the single repeat 4 but not repeats 3 or 5 possess full nidogen-binding activity. This identifies repeat 4 as the only binding structure. The sequence of repeat 4 is well conserved in the human and in part in the Drosophila laminin B2 chain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spatial and temporal expression pattern of a novel gene in the frog Xenopus laevis: correlations with adult intestinal epithelial differentiation during metamorphosis

We report the cloning of a novel gene (ID14) and its expression pattern in tadpoles and adults of Xenopus laevis. ID14 encodes a 315-amino acid protein that has a signal peptide and a nidogen domain. Even though several genes have a nidogen domain, ID14 is not the homolog of any known gene. ID14 is a late thyroid hormone (TH)-regulated gene in the tadpole intestine, and its expression in the intestine does not begin until the climax of metamorphosis, correlating with adult intestinal epithelial differentiation. In contrast, ID14 is expressed in tadpole skin and tail and is not regulated by TH. In situ hybridization revealed that this putative extracellular matrix protein is expressed in the epithelia of the tadpole skin and tail and in the intestinal epithelium after metamorphosis. In the adult, ID14 is found predominantly in the intestine with weak expression in the stomach, lung, and testis. Its exclusive expression in the adult intestinal epithelial cells makes it a useful marker for developmental studies and may give insights into cell/cell interactions in intestinal metamorphosis and adult intestinal stem cell maintenance.     

MDA‐7/IL‐24 regulates proliferation,invasion and tumor cell radiosensitivity: A new cancer therapy?

The novel cytokine MDA-7/IL-24 was identified by subtractive hybridization in the mid-1990s as a cytokine whose expression increased during the induction of terminal differentiation, and that was either not expressed or was present at low levels in tumor cells compared to non-transformed cells. Multiple studies from several laboratories have subsequently demonstrated that expression of IL-24 in tumor cells, but not in non-transformed cells, causes their growth arrest and ultimately cell death. In addition, IL-24 has been noted to be a radiosensitizing cytokine, which in part is due to the generation of reactive oxygen species (ROS) and causing endoplasmic reticulum stress. Recent publications of Phase I trial data have shown that a recombinant adenovirus to express MDA-7/IL-24 (Ad.mda-7 (INGN 241)) was safe and had tumoricidal effects in patients, which argues that IL-24 may have therapeutic value. This review describes what is known about the impact of IL-24 on tumor cell biology in addition to approaches that may enhance the toxicity of this novel cytokine.     

Analysis of degradation of the basement membrane protein nidogen, using a specific monoclonal antibody

A monoclonal antibody was produced against purified nidogen extracted from a mouse basement-membrane-producing tumor. This antibody reacted with a determinant on Nd-40, a rod which separates the globular domains of nidogen. Antigenicity depends on intrachain disulfide bonds within this rod. The monoclonal antibody was used to detect nidogen fragments after proteolytic cleavage of isolated nidogen, and nidogen complexed to laminin. The data indicate that thrombin and thermolysin generated very different patterns of degradation, but in both cases no differences were found between isolated and complexed nidogen. In contrast, nidogen in the laminin-nidogen complex was much less degraded by trypsin than isolated nidogen, indicating that an interaction between these basement membrane components reduces the susceptibility of nidogen to trypsin digestion. Immunofluorescent studies, using the monoclonal antibody on sections of the EHS tumor after proteolytic digestion, showed that the retention or disappearance of the Nd-40 determinant correlated with the in vitro digestion pattern of the laminin-nidogen complex.     

Role of laminin terminal globular domains in basement membrane assembly

Laminins contribute to basement membrane assembly through interactions of their N- and C-terminal globular domains. To further analyze this process, recombinant laminin-111 heterotrimers with deletions and point mutations were generated by recombinant expression and evaluated for their ability to self-assemble, interact with nidogen-1 and type IV collagen, and form extracellular matrices on cultured Schwann cells by immunofluorescence and electron microscopy. Wild-type laminin and laminin without LG domains polymerized in contrast to laminins with deleted alpha1-, beta1-, or gamma1-LN domains or with duplicated beta1- or alpha1-LN domains. Laminins with a full complement of LN and LG domains accumulated on cell surfaces substantially above those lacking either LN or LG domains and formed a lamina densa. Accumulation of type IV collagen onto the cell surface was found to require laminin with separate contributions arising from the presence of laminin LN domains, nidogen-1, and the nidogen-binding site in laminin. Collectively, the data support the hypothesis that basement membrane assembly depends on laminin self-assembly through formation of alpha-, beta-, and gamma-LN domain complexes and LG-mediated cell surface anchorage. Furthermore, type IV collagen recruitment into the laminin extracellular matrices appears to be mediated through a nidogen bridge with a lesser contribution arising from a direct interaction with laminin.     

DJ-1 enhances cell survival through the binding of Cezanne, a negative regulator of NF-kappaB

Heightened DJ-1 (Park7) expression is associated with a reduction in chemotherapeutic-induced cell death and poor prognosis in several cancers, whereas the loss of DJ-1 function is found in a subgroup of Parkinson disease associated with neuronal death. This study describes a novel pathway by which DJ-1 modulates cell survival. Mass spectrometry shows that DJ-1 interacts with BBS1, CLCF1, MTREF, and Cezanne/OTUD7B/Za20d1. Among these, Cezanne is a known deubiquitination enzyme that inhibits NF-κB activity. DJ-1/Cezanne interaction is confirmed by co-immunoprecipitation of overexpressed and endogenous proteins, maps to the amino-terminal 70 residues of DJ-1, and leads to the inhibition of the deubiquitinating activity of Cezanne. Microarray profiling of shRNA-transduced cells shows that DJ-1 and Cezanne regulate IL-8 and ICAM-1 expression in opposing directions. Similarly, DJ-1 enhances NF-κB nuclear translocation and cell survival, whereas Cezanne reduces these outcomes. Analysis of mouse Park7(-/-) primary cells confirms the regulation of ICAM-1 by DJ-1 and Cezanne. As NF-κB is important in cellular survival and transformation, IL-8 functions as an angiogenic factor and pro-survival signal, and ICAM-1 has been implicated in tumor progression, invasion, and metastasis; these data provide an additional modality by which DJ-1 controls cell survival and possibly tumor progression via interaction with Cezanne.     

Interactome Analysis of KIN (Kin17) Shows New Functions of This Protein

KIN (Kin17) protein is overexpressed in a number of cancerous cell lines, and is therefore considered a possible cancer biomarker. It is a well-conserved protein across eukaryotes and is ubiquitously expressed in all cell types studied, suggesting an important role in the maintenance of basic cellular function which is yet to be well determined. Early studies on KIN suggested that this nuclear protein plays a role in cellular mechanisms such as DNA replication and/or repair; however, its association with chromatin depends on its methylation state. In order to provide a better understanding of the cellular role of this protein, we investigated its interactome by proximity-dependent biotin identification coupled to mass spectrometry (BioID-MS), used for identification of protein–protein interactions. Our analyses detected interaction with a novel set of proteins and reinforced previous observations linking KIN to factors involved in RNA processing, notably pre-mRNA splicing and ribosome biogenesis. However, little evidence supports that this protein is directly coupled to DNA replication and/or repair processes, as previously suggested. Furthermore, a novel interaction was observed with PRMT7 (protein arginine methyltransferase 7) and we demonstrated that KIN is modified by this enzyme. This interactome analysis indicates that KIN is associated with several cell metabolism functions, and shows for the first time an association with ribosome biogenesis, suggesting that KIN is likely a moonlight protein.     

Endosomal recycling regulates anthrax toxin receptor 1/tumor endothelial marker 8-dependent cell spreading

Mechanisms for receptor-mediated anthrax toxin internalization and delivery to the cytosol are well understood. However, far less is known about the fate followed by anthrax toxin receptors prior and after cell exposure to the toxin. We report that Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8 (TEM8) localized at steady state in Rab11a-positive and transferrin receptor-containing recycling endosomes. TEM8 followed a slow constitutive recycling route of ∼ 30 min as determined by pulsed surface biotinylation and chase experiments. A Rab11a dominant negative mutant and Myosin Vb tail expression impaired TEM8 recycling by sequestering TEM8 in intracellular compartments. Sequestration of TEM8 in intracellular compartments with monensin coincided with increased TEM8 association with a multi-protein complex isolated with antibodies against transferrin receptor. Addition of the cell-binding component of anthrax toxin, Protective Antigen, reduced TEM8 half-life from 7 to 3 hours, without preventing receptor recycling. Pharmacological and molecular perturbation of recycling endosome function using monensin, dominant negative Rab11a, or myosin Vb tail, reduced PA binding efficiency and TEM8-dependent cell spreading on PA-coated surfaces without affecting toxin delivery to the cytosol. These results indicate that the intracellular fate of TEM8 differentially affect its cell adhesion and cell intoxication functions.