Collagen XVII, a type II transmembrane protein and epithelial adhesion molecule, can be proteolytically shed from the cell surface to generate a soluble collagen. Here we investigated the release of the ectodomain and identi®ed the enzymes involved. After surface biotinylation of keratinocytes, the ectodomain was detectable in the medium within minutes and remained stable for >48 h. Shedding was enhanced by phorbol esters and inhibited by metalloprotease inhibitors, including hydroxamates and TIMP-3, but not by inhibitors of other protease classes or by TIMP-2. This pro®le implicated MMPs or ADAMs as candidate sheddases. MMP-2, MMP-9 and MT1-MMP were excluded, but TACE, ADAM-10 and ADAM-9 were shown to be expressed in keratinocytes and to be actively involved. Transfection with cDNAs for the three ADAMs resulted in increased shedding and, vice versa, in TACE-de®cient cells shedding was signi®cantly reduced, indicating that transmembrane collagen XVII represents a novel class of substrates for ADAMs. Functionally, release of the ectodomain of collagen XVII from the cell surface was associated with altered keratinocyte motility in vitro.

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The fibroblast integrin ␣11␤1 is a key receptor for fibrillar collagens. To study the potential function of ␣11 in vivo, we generated a null allele of the ␣11 gene. Integrin ␣11 ؊/؊ mice are viable and fertile but display dwarfism with increased mortality, most probably due to severely defective incisors. Mutant incisors are characterized by disorganized periodontal ligaments, whereas molar ligaments appear normal. The primary defect in the incisor ligament leads to halted tooth eruption. ␣11␤1-defective embryonic fibroblasts displayed severe defects in vitro, characterized by (i) greatly reduced cell adhesion and spreading on collagen I, (ii) reduced ability to retract collagen lattices, and (iii) reduced cell proliferation. Analysis of matrix metalloproteinase in vitro and in vivo revealed disturbed MMP13 and MMP14 synthesis in ␣11 ؊/؊ cells. We show that ␣11␤1 is the major receptor for collagen I on mouse embryonic fibroblasts and suggest that ␣11␤1 integrin is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate the forces needed for axial tooth movement. Our data show a unique role for ␣11␤1 integrin during tooth eruption.Integrins constitute a family of multifunctional cell adhesion receptors involved in a variety of biological processes. In higher vertebrates the integrin family is composed of 18 ␣ subunits and 8 ␤ subunits. Out of the 24 integrin heterodimers, ␣1␤1, ␣2␤1, ␣10␤1, and ␣11␤1 act as primary receptors for native collagens. The ␣3␤1 integrin, initially described as a collagen receptor, was later shown to act as a receptor for laminin-5 and does not display any measurable affinity for collagen I (21). The observed role of ␣v␤3 integrin in collagen lattice remodeling in vitro (13, 33) and in vivo (33) may be related to indirect binding to RGD ligands which are locally deposited in the collagen matrix.Collagen-binding integrins bind native collagens via their ␣I domain, recognizing a GFOGER motif (30, 52) or similar sequences with varying specificities and affinities depending on the collagen type and fibrillar status (27). In addition to mediating cell adhesion, cell spreading, and cell migration (22), collagen-binding integrins regulate collagen turnover (32) and take part in assembling (31, 49) and reorganizing three-dimensional collagen matrices (23).Fibroblasts express characteristic collagen receptor repertoires in a tissue-specific manner, partly reflecting their different embryonic origins. We have previously demonstrated that ␣11␤1 is the only detectable collagen-binding integrin in the incisor periodontal ligament (PDL) fibroblasts of mice (38). The PDL in the mouse incisor is composed of a relatively dense connective tissue on the side facing the tooth and a loose connective tissue region rich in blood vessels on the side facing the bone. The odontogenic ectomesenchyme is the source of the cells populating these two domains in the PDL, which are sometimes referred to as tooth-associated fibroblasts and bone-associated...

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Activation of pro-matrix metalloproteinase (MMP)-2 on the surface of malignant cells by membrane-bound MT1-MMP is believed to play a critical role during tumor progression and metastasis. In this study we present evidence that MT1-MMP plays a key role for the in vitro invasiveness of malignant melanoma. Melanoma cell lines secreted latent MMP-2 when cultured on plastic. However, when cells were grown in floating type I collagen lattices, only high invasive melanoma cells activated proMMP-2. Activation could be inhibited by antibodies against MT1-MMP, by addition of recombinant tissue inhibitor of metalloproteinases (TIMP)-2 and by inhibition of MT1-MMP cleavage. MT1-MMP protein was detected as an inactive protein in all cell lines cultured as monolayers, whereas in collagen gels, active MT1-MMP protein was detected in the membranes of both high and low invasive melanoma cells. Production of TIMP-2 was about 10-fold higher in low invasive cells as compared with high invasive melanoma cells and was further increased in the low invasive cells upon contact to collagen. Thus, in melanoma cells TIMP-2 expression levels might regulate MT1-MMP-mediated activation of proMMP-2. High invasive melanoma cells displayed increased in vitro invasiveness, which was inhibited by TIMP-2. These data indicate the importance of these enzymes for the invasion processes and support a role for MT1-MMP as an activator of proMMP-2 in malignant melanoma.Local degradation of connective tissue in the vicinity of the cell surface is thought to be essential for tumor cell invasion and metastasis. Neoplastic cells that invade surrounding tissues and metastasize through the blood stream or lymphatic system must penetrate several barriers, including basement membranes and the interstitial connective tissue (1, 2). Degradation of extracellular matrix components is accomplished through the combined action of different proteases, primarily of the matrix metalloproteinase (MMP) 1 (3) and serine protease families (4).Because of the importance of MMP activity for initiating efficient matrix degradation, MMP expression and activity is tightly regulated and is subject to several levels of control, including gene transcription, post-translational extracellular activation, and inhibition by soluble inhibitors (3, 5). Basal production of metalloproteinases is typically low or absent and requires an inducing factor, such as a cytokine, phorbol ester, or contact with components of the extracellular matrix to increase their expression (3). Enzyme activation is achieved by removal of the N-terminal propeptide domain through exogenous or autocatalytic cleavage. In vitro, serine proteases, such as plasmin or trypsin, have been shown to activate most MMPs (3). However, MMP-2 is unique among the MMPs in that its expression is constitutive and its activation can be achieved in a membrane-associated manner as has been shown for fibroblasts (6), for endothelial cells (7,8), and for tumor cells (9 -11). A novel membrane-type matrix metalloproteinase (MT1-MMP) was origi...

The skin water barrier, essential for terrestrial life, is formed by a multilayered stratifying epithelium, which shows a polarized distribution of both differentiation and intercellular junction markers. Recently, several reports showed the crucial importance of tight junctions for the in vivo water barrier function of the skin. In simple epithelial cells, intercellular junction formation is closely coupled to the establishment of polarity. However, if and how polarity proteins contribute to epidermal differentiation and junction formation is not yet known. Here, we have characterized the localization and isoform expression of the polarity protein atypical PKC (aPKC) and its binding partners Par3 and Par6 in epidermis and primary keratinocytes of mice. Their distribution is only partially overlapping in the granular layer, the site of functional tight junctions, suggesting that next to a common Par3/Par6/aPKC function they also may have functions independent of each other. Both aPKCzeta and aPKCiota/lambda, are expressed in the epidermis but only aPKCiota/lambda showed a strong enrichment in the junctions, suggesting that this aPKC isoform is important for epidermal tight junction function. Indeed, inhibition of aPKC function showed that endogenous aPKC is crucial for in vitro barrier function and this required the presence of both the Par3 and Par6 binding sites.

Proceeding on the assumption that all cancer cells have equal malignant capacities, current regimens in cancer therapy attempt to eradicate all malignant cells of a tumor lesion. Using in vivo targeting of tumor cell subsets, we demonstrate that selective elimination of a definite, minor tumor cell subpopulation is particularly effective in eradicating established melanoma lesions irrespective of the bulk of cancer cells. Tumor cell subsets were specifically eliminated in a tumor lesion by adoptive transfer of engineered cytotoxic T cells redirected in an antigen-restricted manner via a chimeric antigen receptor. Targeted elimination of less than 2% of the tumor cells that coexpress high molecular weight melanoma-associated antigen (HMW-MAA) (melanomaassociated chondroitin sulfate proteoglycan, MCSP) and CD20 lastingly eradicated melanoma lesions, whereas targeting of any random 10% tumor cell subset was not effective. Our data challenge the biological therapy and current drug development paradigms in the treatment of cancer.

Abstract. Osteocalcin, also called Bone Gla Protein (BGP), is the most abundant of the non-collagenous proteins of bone produced by osteoblasts. It consists of a single chain of 46-50 amino acids, according to the species, and contains three vitamin K-dependent gamma-carboxyglutamic acid residues (GLA), involved in its binding to calcium and hydroxylapatite. Accumulating evidences suggest its involvement in bone remodeling, its physiological role, however, is still unclear. In this study the adhesion properties and the biological effects of osteocalcin on osteoclasts have been analyzed using as an experimental model, human osteoclast-like cells derived from giant cell tumors of bone (GCT). Osteocalcin promoted adhesion and spreading of these cells, triggering the release of bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN), that in turn induced the clustering in focal adhesions of fl~ and/33 integrin chains. Spreading was dependent upon the synthesis of these proteins. In fact, when the cells were incubated in the presence of monensin during the adhesion assay, they still adhered but spreading did not occur, focal adhesions disappeared and BSP, OPN, and FN were accumulated in intracellular granules. Furthermore osteocalcin induced chemotaxis in a dose-dependent manner. The action of BGP on osteoclasts was mediated by an intracellular calcium increase due to release from thapsigargin-sensitive stores. These results provide evidences that BGP exerts a role in the resorption process, inducing intracellular signaling, migration and adhesion, followed by synthesis and secretion of endogenous proteins. STEOCALCIN, alSO called Bone Gla Protein (BGP), ~is the most abundant of the non-collagenous proteins of bone produced by osteoblasts, and has several interesting features. It consists of a single chain of 46-50 amino acids, according to the species, and contains three vitamin K-dependent gamma-carboxyglutamic acid residues (GLA), involved in its binding to calcium and hydroxylapatite (19,33). So far, BGP has been considered highly specific for bone, secreted by mature osteoblasts, and incorporated into the extracellular matrix. Recently, it has been demonstrated that the mouse genome contains an osteocalcin cluster composed of three genes (34, 13). The first two, named OG1 and OG2, are expressed only in bone, while the third