The alpha 2 chain of collagen type VI sequesters latent proforms of matrix-metalloproteinases and modulates their activation and activity

The extracellular matrix (ECM) attracts increasing attention as a store of biologically active molecules and as a reservoir of potent cell signalling molecules released by proteolytic action. Both, cytokines and proteases mediating such release are sequestered in the ECM. Here, we found matrix metalloproteinase (MMP) proforms closely associated with collagenous septae in fibrotic liver tissue, and we screened immobilized human placenta-derived collagen chains and other ECM proteins for MMP-binding activity. Following the establishment of a novel highly-efficient two-step chromatography procedure for the isolation of the purified α-chains of the pepsin-resistant triple-helical CVI fragment (CVI/PR) solid phase and surface plasmon resonance binding studies were performed. We identified the triple-helical domain of the α2 chain of microfilamentous CVI α2(VI) as having nanomolar affinity for the collagenases proMMP-1, -8 , -13 and stromelysin-1 (MMP-3), thus extending the repertoire of pericellular and substrate-based interactions of MMPs. Enzymatic activity assays enabled the correlation of MMP activity with CVI binding, in that α2(VI) chain-mediated inhibition of enzymatic activity is accompanied by increased binding. Similar results were shown for the gelatinase proMMP-9, whereas for proMMP-2, the α2(VI) chain at low concentrations seems to interfere with prodomain binding resulting in enhanced activity without scission of the prodomain. Stable complexes of proMMP-2 and α2(VI) chain competed with gelatinase binding to the preferred ligand, collagen type I. In conclusion, the α2(VI) chain modulates MMP availability by sequestering proMMPs in the ECM, blocking proteolytic activity. Therefore, CVI and especially its α2(VI) chain might serve as a lead structure for MMP-based therapeutics which modulates the action of these matrix components, e.g. in fibrosis and cancer.     

Bone marrow stromal cell interaction reduces Syndecan-1 expression and induces kinomic changes in myeloma cells

CD138 (Syndecan 1) is a heparan sulfate proteoglycan that concentrates heparan sulfate-binding growth factors on the surface of normal and malignant plasma cells (multiple myeloma, MMC). Recent studies have shown the presence of a CD138-negative fraction of MMC within myelomatous bone marrow (BM). We employed kinome array technology to characterize this fraction at a molecular level, using a myeloma cell line model. Compared to CD138-positive cells, CD138-negative MMC showed (i) a reduced activity of kinases involved in cell cycle progression, in agreement with a decreased labeling index and (ii) reduced Rho signaling to F-actin. Interestingly, CD138 mRNA and protein expression was reduced upon interaction of MM cells with stromal cell lines and primary mesenchymal cultures, which was accompanied by the acquisition of an increased Bcl6/Blimp1 ratio. Co-culture induced an increased activity of kinases involved in adhesion and a decreased S-phase transition in both CD138-positive and -negative fractions. In addition, CD138-negative MMC demonstrated an increased STAT3 and ERK1/2 activation compared to CD138+ MMC, in agreement with a lower sensitivity to compound exposure. The presence of a less mature, more resistant CD138-negative myeloma cell fraction within bone marrow microniches might contribute to high incidence of relapse of Myeloma patients.     

Wideband MRE and static mechanical indentation of human liver specimen: Sensitivity of viscoelastic constants to the alteration of tissue structure in hepatic fibrosis

Despite the success of elastography in grading hepatic fibrosis by stiffness related noninvasive markers the relationship between viscoelastic constants in the liver and tissue structure remains unclear. We therefore studied the mechanical properties of 16 human liver specimens with different degrees of fibrosis, inflammation and steatosis by wideband magnetic resonance elastography (MRE) and static indentation experiments providing the specimens? static Young?s modulus (E), dynamic storage modulus (G′) and dynamic loss modulus (G″). A frequency-independent shear modulus μ and a powerlaw exponent α were obtained by fitting G′ and G″ using the two-parameter sprinpot model. The mechanical parameters were compared to the specimens? histology derived parameters such as degree of Fibrosis (F), inflammation score and fat score, amount of hydroxyproline (HYP) used for quantification of collagen, blood markers and presurgery in vivo function tests.     

An atomic resolution structure for human fibroblast growth factor 1

A 1.10-A atomic resolution X-ray structure of human fibroblast growth factor 1 (FGF-1), a member of the beta-trefoil superfold, has been determined. The beta-trefoil is one of 10 fundamental protein superfolds and is the only superfold to exhibit 3-fold structural symmetry (comprising 3 "trefoil" units). The quality of the diffraction data permits unambiguous assignment of Asn, Gln, and His rotamers, Pro ring pucker, as well as refinement of atomic anisotropic displacement parameters (ADPs). The FGF-1 structure exhibits numerous core-packing defects, detectable using a 1.0-A probe radius. In addition to contributing to the relatively low thermal stability of FGF-1, these defects may also permit domain motions within the structure. The availability of refined ADPs allows a translation/libration/screw (TLS) analysis of putative rigid body domains. The TLS analysis shows that beta-strands 6-12 together form a rigid body, and there is a clear demarcation in TLS motions between the adjacent carboxyl- and amino-termini. Although separate from beta-strands 6-12, the individual beta-strands 1-5 do not exhibit correlated motions; thus, this region appears to be comparatively flexible. The heparin-binding contacts of FGF-1 are located within beta-strands 6-12; conversely, a significant portion of the receptor-binding contacts are located within beta-strands 1-5. Thus, the observed rigid body motion in FGF-1 appears related to the ligand-binding functionalities.     

Recombinant CD63/ME491/neuroglandular/NKI/C-3 antigen inhibits growth of established tumors in transgenic mice

Attempts to vaccinate against tumors can be hindered by the induction of immunological tolerance to the target Ag as a result of Ag expression on normal tissues. In this study, we find that transgenic mice expressing the melanoma-associated Ag CD63/ME491/neuroglandular/NKI/C-3 on their normal tissues do, in fact, exhibit immunological tolerance to the Ag, recapitulating the conditions in cancer patients. In these mice, growth of murine melanoma cells expressing the Ag after gene transfer was inhibited by immunization with Ag-expressing recombinant vaccinia virus combined with IL-2, but not by immunization with the protein alone, anti-idiotypic Abs, or irradiated tumor cells. The effect of the recombinant virus was demonstrated both for nonestablished and established tumors. Infiltration with both CD4(+) and CD8(+) T lymphocytes was significantly more extensive in tumors from experimental mice than in tumors from control mice. MHC class I-positive, but not class I-negative, tumors were inhibited by the vaccine, suggesting that MHC class I-restricted T lymphocytes play a role in the antitumor effects. Abs did not appear to be involved in the vaccine effects. CD63 was immunogenic in 2 of 13 melanoma patients, pointing to the potential of this Ag, combined with IL-2, as a vaccine for melanoma patients.     

Molecular cloning and characterization of a rat sensory nerve Ca2+-sensing receptor

A full-length cDNA encoding a Ca2+-sensing receptor (CaSR) expressed in rat dorsal root ganglia (DRG) was identified using rapid amplification of 5'-cDNA ends and primer extension and then cloned into the plasmid vector pCR3.1. The DNA sequence of the DRG CaSR was 99.9% homologous with published rat kidney CaSR in the coding region and 247 bp upstream of the start site but showed little homology 5' to this site, which maps to exonic junction I/II, supporting the hypothesis that CaSR message arises as a splice variant and showing tissue-to-tissue heterogeneity. Western blot revealed a doublet of 140 and 160 kDa in a thyroparathyroid preparation and a single 140-kDa band in DRG. Deglycosylation using N-glycanase increased the mobility of CaSR protein from both DRG and thyroparathyroid, whereas endo-H was without effect, indicating that the DGR CaSR is a mature form of the receptor. A DRG CaSR-pEGFP fusion product was constructed, and when transfected into HEK-293 cells, it was distributed at the cell membrane and resulted in extracellular Ca2+ (0.5-3 mM)-evoked increases in intracellular Ca2+, which in some instances exhibited oscillatory behavior. We conclude that DRG CaSR cDNA arises from tissue-specific alternative splicing of a single gene, that the amino acid sequence of DRG CaSR is homologous to other known CaSRs, and that the DRG CaSR undergoes differential posttranslational processing relative to the thyroparathyroid CaSR and is functionally active when transfected into a human-derived cell line.     

CXC chemokine ligand 12 (stromal cell-derived factor 1 alpha) and CXCR4-dependent migration of CTLs toward melanoma cells in organotypic culture

Studies in experimental animal models have demonstrated that chemokines produced by tumor cells attract chemokine receptor-positive T lymphocytes into the tumor area, which may lead to tumor growth inhibition in vitro and in vivo. However, in cancer patients, the role of chemokines in T lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a melanoma patient's CTL toward autologous tumor cells has been studied in a novel organotypic melanoma culture, consisting of a bottom layer of collagen type I with embedded fibroblasts followed successively by a tumor cell layer, collagen/fibroblast separating layer, and, finally, a top layer of collagen with embedded fibroblasts and T cells. In this model, CTL migrated from the top layer through the separating layer toward tumor cells, resulting in tumor cell apoptosis. CTL migration was mediated by chemokine receptor CXCR4 expressed by the CTL and CXCL12 (stromal cell-derived factor 1alpha) secreted by tumor cells, as evidenced by blockage of CTL migration by Abs to CXCL12 or CXCR4, high concentrations of CXCL12 or small molecule CXCR4 antagonist. These studies, together with studies in mice indicating regression of CXCL12-transduced tumor cells, followed by regression of nontransduced challenge tumor cells, suggest that CXCL12 may be useful as an immunotherapeutic agent for cancer patients, when transduced into tumor cells, or fused to anti-tumor Ag Ab or tumor Ag.     

Alterations in tumour suppressor gene p53 in human gliomas from Indian patients

Alterations in the tumour suppressorp53 gene are among the most common defects seen in a variety of human cancers. In order to study the significance of thep53 gene in the genesis and development of human glioma from Indian patients, we checked 44 untreated primary gliomas for mutations in exons 5–9 of thep53 gene by PCR-SSCP and DNA sequencing. Sequencing analysis revealed six missense mutations. The incidence of p53 mutations was 13⋅6% (6 of 44). All the six mutations were found to be located in the central core domain of p53, which carries the sequence-specific DNA-binding domain. These results suggest a rather low incidence but a definite involvement of p53 mutations in the gliomas of Indian patients.     

A revised structure for caulerpicin from Caulerpa racemosa

Caulerpicin, from the green alga Caulerpa racemosa, is shown to be a mixture of N-acylsphingosines.