MMP25 (MT6-MMP) is highly expressed in human colon cancer, promotes tumor growth, and exhibits unique biochemical properties

MMP25 (MT6-MMP) is one of the two glycosylphosphatidylinositol-anchored matrix metalloproteinases (MMPs) that have been suggested to play a role in pericellular proteolysis. However, its role in cancer is unknown, and its biochemical properties are not well established. Here we found a marked increase in MT6-MMP expression within in situ dysplasia and invasive cancer in 61 samples of human colon cancer. Expression of MT6-MMP in HCT-116 human colon cancer cells promoted tumori-genesis in nude mice. Histologically, the MT6-MMP-expressing tumors demonstrated an infiltrative leading edge in contrast to a rounded leading edge in vector control tumors. Biochemical and biosynthesis analyses revealed that MT6-MMP displayed on the cell surface exists as a major form of 120 kDa that likely represents enzyme homodimers linked by disulfide bonds. Upon reduction, a single 57-kDa active MT6-MMP was detected. Interestingly, neither membrane-anchored nor phosphatidylinositol-specific phospholipase C-released MT6-MMPs were found to be associated with tissue inhibitor of metalloproteinases (TIMPs) and did not activate pro-gelatinases (pro-MMP-2 and pro-MMP-9) even in the presence of exogenous TIMP-2 or TIMP-1. A catalytic domain of MT6-MMP was inhibited preferentially by TIMP-1 (K(i) = 0.2 nm) over TIMP-2 (K(i) = 2.0 nm), because of a slower association rate. These results show that MT6-MMP may play a role in colon cancer and exhibit unique biochemical and structural properties that may regulate proteolytic function at the cell surface.     

Functional expression of recombinant human stefin A in mammalian and bacterial cells

Recombinant human cysteine protease inhibitor, stefin A, was expressed in both Escherichia coli and BS-C-1 monkey kidney cells utilizing pET and recombinant vaccinia virus systems, respectively. The expressed protein was purified and analyzed by SDS-PAGE and Western blot analysis utilizing a polyclonal antibody against rat cystatin alpha. In both cases the purified protein appeared as a single band corresponding to the molecular weight of stefin A ( approximately 10kDa). Viability of the expressed stefin A was determined by the inhibition of the plant cysteine protease, papain. Recombinant human stefin A expressed in both E. coli and BS-C-1 cells, was shown to almost completely inhibit papain. The expression of a fully functional recombinant human stefin A in the bacterial system provides a highly efficient tool for the production of large quantities of the protein. This can be an important tool in kinetic studies as well as in production of antibodies for other analytical studies (immunoblot, immunohistochemical studies, etc.). Expression in the mammalian cells, on the other hand, can provide a significant research tool to study the functional roles of stefin A in mammalian systems such as regulation of cysteine proteases.     

Metabolomics, genomics, proteomics, and the identification of enzymes and their substrates and products

A large proportion of the genes in any plant genome encode enzymes of primary and specialized (secondary) metabolism. Not all plant primary metabolites, those that are found in all or most species, have been identified. Moreover, only a small portion of the estimated hundreds of thousand specialized metabolites, those found only in restricted lineages, have been studied in any species. The correlative analysis of extensive metabolic profiling and gene expression profiling has proven a powerful approach for the identification of candidate genes and enzymes, particularly those in secondary metabolism. The final characterization of substrates, enzymatic activities, and products requires biochemical analysis, which has been most successful when candidate proteins have homology to other enzymes of known function. The challenges are to identify new types of enzymes and to develop biochemical techniques that are suitable for large-scale analysis.     

Dynamic interactions of Fc gamma receptor IIB with filamin-bound SHIP1 amplify filamentous actin-dependent negative regulation of Fc epsilon receptor I signaling

The engagement of high affinity receptors for IgE (FcepsilonRI) generates both positive and negative signals whose integration determines the intensity of mast cell responses. FcepsilonRI-positive signals are also negatively regulated by low affinity receptors for IgG (FcgammaRIIB). Although the constitutive negative regulation of FcepsilonRI signaling was shown to depend on the submembranous F-actin skeleton, the role of this compartment in FcgammaRIIB-dependent inhibition is unknown. We show in this study that the F-actin skeleton is essential for FcgammaRIIB-dependent negative regulation. It contains SHIP1, the phosphatase responsible for inhibition, which is constitutively associated with the actin-binding protein, filamin-1. After coaggregation, FcgammaRIIB and FcepsilonRI rapidly interact with the F-actin skeleton and engage SHIP1 and filamin-1. Later, filamin-1 and F-actin dissociate from FcR complexes, whereas SHIP1 remains associated with FcgammaRIIB. Based on these results, we propose a dynamic model in which the submembranous F-actin skeleton forms an inhibitory compartment where filamin-1 functions as a donor of SHIP1 for FcgammaRIIB, which concentrate this phosphatase in the vicinity of FcepsilonRI and thereby extinguish activation signals.     

Novel ganglioside antigen identified by B cells in human medullary breast carcinomas: the proof of principle concerning the tumor-infiltrating B lymphocytes

The potential tumor-recognizing capacity of B cells infiltrating human breast carcinoma is an important aspect of breast cancer biology. As an experimental system, we used human medullary breast carcinoma because of its heavy B lymphocytic infiltration paralleled to a relatively better prognosis. Ig-rearranged V region V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda genes, amplified by RT-PCR of the infiltrating B cells, were cloned, sequenced, and subjected to a comparative DNA analysis. A combinatorial single-chain variable fragment Ab minilibrary was constructed out of randomly selected V(H) and Vkappa clones and tested for binding activity. Our data analysis revealed that some of the V(H)-J(H), Vkappa-Jkappa, and Vlambda-Jlambda region sequences were being assigned to clusters with oligoclonal predominance, while other characteristics of the Ab repertoire were defined also. A tumor-restricted binder clone could be selected out of the single-chain variable fragment kappa minilibrary tested against membrane fractions of primary breast tumor cells and tumor cell lines, the V(H) of which proved to be the overexpressed V(H)3-1 cluster. The specific binding was confirmed by FACS analysis with primary breast carcinoma cells and MDA-MB 231 cell line. ELISA and thin layer chromatography dot-blot experiments showed this target Ag to be a ganglioside D3 (GD3). Our results are a proof of principle about the capacity of B cells infiltrating breast carcinomas to reveal key cancer-related Ags, such as the GD3. GD3-specific Abs may influence tumor cell progression and could be used for further development of diagnostic and/or therapeutic purposes.     

CCL1-CCR8 interactions: an axis mediating the recruitment of T cells and Langerhans-type dendritic cells to sites of atopic skin inflammation

Atopic dermatitis represents a chronically relapsing skin disease with a steadily increasing prevalence of 10-20% in children. Skin-infiltrating T cells, dendritic cells (DC), and mast cells are thought to play a crucial role in its pathogenesis. We report that the expression of the CC chemokine CCL1 (I-309) is significantly and selectively up-regulated in atopic dermatitis in comparison to psoriasis, cutaneous lupus erythematosus, or normal skin. CCL1 serum levels of atopic dermatitis patients are significantly higher than levels in healthy individuals. DC, mast cells, and dermal endothelial cells are abundant sources of CCL1 during atopic skin inflammation and allergen challenge, and Staphylococcus aureus-derived products induce its production. In vitro, binding and cross-linking of IgE on mast cells resulted in a significant up-regulation of this inflammatory chemokine. Its specific receptor, CCR8, is expressed on a small subset of circulating T cells and is abundantly expressed on interstitial DC, Langerhans cells generated in vitro, and their monocytic precursors. Although DC maintain their CCR8+ status during maturation, brief activation of circulating T cells recruits CCR8 from intracytoplamic stores to the cell surface. Moreover, the inflammatory and atopy-associated chemokine CCL1 synergizes with the homeostatic chemokine CXCL12 (SDF-1alpha) resulting in the recruitment of T cell and Langerhans cell-like DC. Taken together, these findings suggest that the axis CCL1-CCR8 links adaptive and innate immune functions that play a role in the initiation and amplification of atopic skin inflammation.     

Differential inhibition of membrane type 3 (MT3)-matrix metalloproteinase (MMP) and MT1-MMP by tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3 rgulates pro-MMP-2 activation

The membrane type (MT)-matrix metalloproteinases (MMPs) constitute a subgroup of membrane-anchored MMPs that are major mediators of pericellular proteolysis and physiological activators of pro-MMP-2. The MT-MMPs also exhibit differential inhibition by members of the tissue inhibitor of metalloproteinase (TIMP) family. Here we investigated the processing, catalytic activity, and TIMP inhibition of MT3-MMP (MMP-16). Inhibitor profile and mutant enzyme studies indicated that MT3-MMP is regulated on the cell surface by autocatalytic processing and ectodomain shedding. Inhibition kinetic studies showed that TIMP-3 is a high affinity inhibitor of MT3-MMP when compared with MT1-MMP (K(i) = 0.008 nm for MT3-MMP versus K(i) = 0.16 nm for MT1-MMP). In contrast, TIMP-2 is a better inhibitor of MT1-MMP. MT3-MMP requires TIMP-2 to accomplish full pro-MMP-2 activation and this process is enhanced in marimastatpretreated cells, consistent with regulation of active enzyme turnover by synthetic MMP inhibitors. TIMP-3 also enhances the activation of pro-MMP-2 by MT3-MMP but not by MT1-MMP. TIMP-4, in contrast, cannot support pro-MMP-2 activation with either enzyme. Affinity chromatography experiments demonstrated that pro-MMP-2 can assemble trimolecular complexes with a catalytic domain of MT3-MMP and TIMP-2 or TIMP-3 suggesting that pro-MMP-2 activation by MT3-MMP involves ternary complex formation on the cell surface. These results demonstrate that TIMP-3 is a major regulator of MT3-MMP activity and further underscores the unique interactions of TIMPs with MT-MMPs in the control of pericellular proteolysis.     

CC chemokine ligand 18, an atopic dermatitis-associated and dendritic cell-derived chemokine, is regulated by staphylococcal products and allergen exposure

Atopic dermatitis is a chronic inflammatory skin disease with a steadily increasing prevalence. Exposure to allergens or bacterial superantigens triggers T and dendritic cell (DC) recruitment and induces atopic skin inflammation. In this study, we report that among all known chemokines CCL18/DC-CK1/PARC represents the most highly expressed ligand in atopic dermatitis. Moreover, CCL18 expression is associated with an atopic dermatitis phenotype when compared with other chronic inflammatory skin diseases. DCs either dispersed within the dermis or clustering at sites showing perivascular infiltrates are abundant sources of CCL18. In vitro, microbial products including LPS, peptidoglycan, and mannan, as well as the T cell-derived activation signal CD40L, induced CCL18 in monocytes. In contrast to monocytes, monocyte-derived, interstitial-type, and Langerhans-type DCs showed a constitutive and abundant expression of CCL18. In comparison to Langerhans cells, interstitial-type DCs produced higher constitutive levels of CCL18. In vivo, topical exposure to the relevant allergen or the superantigen staphylococcal enterotoxin B, resulted in a significant induction of CCL18 in atopic dermatitis patients. Furthermore, in nonatopic NiSO4-sensitized individuals, only relevant allergen but not irritant exposure resulted in the induction of CCL18. Taken together, findings of the present study demonstrate that CCL18 is associated with an atopy/allergy skin phenotype, and is expressed at the interface between the environment and the host by cells constantly screening foreign Ags. Its regulation by allergen exposure and microbial products suggests an important role for CCL18 in the initiation and amplification of atopic skin inflammation.     

Relationship between micro- and minisatellites in the human genome

Micro- and minisatellites constitute an essential part of DNA with a low sequence complexity and carry several important functions. A search for tandem repeats in the human genome with a length of a repeat unit of up to 70 bp, including repeats with a great number of nucleotide substitutions, has been performed using the TaadeaSWAN program. It was shown that, for a considerable number of minisatellites with the length of the repeating unit of less than 25 nt, a shorter repeating motif can be distinguished in the sequence of this repeat, which often is similar to the sequence of minisatellites widely occurring in the human genome. A model of hierarchic origination of minisatellites in the human genome is suggested.