MT1-MMP is a membrane-bound matrix metalloproteinase (MT-MMP) capable of mediating pericellular proteolysis of extracellular matrix components. MT1-MMP is therefore thought to be an important molecular tool for cellular remodeling of the surrounding matrix. To establish the biological role of this membrane proteinase we generated MT1-MMP-deficient mice by gene targeting. MT1-MMP deficiency causes craniofacial dysmorphism, arthritis, osteopenia, dwarfism, and fibrosis of soft tissues due to ablation of a collagenolytic activity that is essential for modeling of skeletal and extraskeletal connective tissues. Our findings demonstrate the pivotal function of MT1-MMP in connective tissue metabolism, and illustrate that modeling of the soft connective tissue matrix by resident cells is essential for the development and maintenance of the hard tissues of the skeleton.

Abstract. We have developed two rat mAbs that recognize different subunits of the human fibroblast fibronectin receptor complex and have used them to probe the function of this cell surface heterodimer. mAb 13 recognizes the integrin class 1 beta polypeptide and mAb 16 recognizes the fibronectin receptor alpha polypeptide. We tested these mAbs for their inhibitory activities in cell adhesion, spreading, migration, and matrix assembly assays using WI38 human lung fibroblasts, mAb 13 inhibited the initial attachment as well as the spreading of WI38 cells on fibronectin and laminin substrates but not on vitronectin. Laminin-mediated adhesion was particularly sensitive to mAb 13. In contrast, mAb 16 inhibited initial cell attachment to fibronectin substrates but had no effect on attachment to either laminin or vitronectin substrates. When coated on plastic, both mAbs promoted WI38 cell spreading. However, mAb 13 (but not mAb 16) inhibited the radial outgrowth of cells from an explant on fibronectin substrates, mAb 16 also did not inhibit the motility of individual fibroblasts on fibronectin in low density culture and, in fact, substantially accelerated migration rates. In assays of the assembly of an extracellular fibronectin matrix by WI38 fibroblasts, both mAbs produced substantial inhibition in a concentration-dependent manner. The inhibition of matrix assembly resulted from impaired retention of fibronectin on the cell surface. Treatment of cells with mAb 16 also resulted in a striking redistribution of cell surface fibronectin receptors from a streak-like pattern to a relatively diffuse distribution. Concomitant morphological changes included decreases in thick microfilament bundle formation and reduced adhesive contacts of the streak-like and focal contact type. Our results indicate that the fibrol~last fibronectin receptor (a) functions in initial fibroblast attachment and in certain types of adhesive contact, but not in the later steps of cell spreading; (b) is not required for fibroblast motility but instead retards migration; and (c) is critically involved in fibronectin retention and matrix assembly. These findings suggest a central role for the fibronectin receptor in regulating cell adhesion and migration.T HE interaction of cells with solid substrates is important for their anchorage, proliferation, migration, and differentiation. Cells can attach, spread, and migrate on a variety of extracellular glycoproteins including fibronectin, laminin, vitronectin, and collagen. These interactions occur through specific cell surface receptors. Many of these cell adhesion receptors belong to a large superfamily of related cell surface complexes called the integrins or the cytoadhesins (for reviews see 3,11,26,30,33,37,52). The integrins are noncovalent, heterodimeric glycoprotein complexes that consist of a 140,000-180,000-D alpha subunit and a 105,000-125,000-D beta subunit. The alpha subunit often consists of a small, transmembrane polypeptide of 20,000 D disulfide bonded to a larger, extracellular polypeptide ...

Mannose receptor–mediated uptake of collagen by M2-like macrophages is a major mechanism of collagen turnover in mice.

We have isolated the major cell surface glycoprotein of chick embryo fibroblasts, CSP, and added it to a variety of transformed cells in vitro. The transformed cells become more elongated, often more flattened, and show increased adhesion to the substratum. Several transformed cell lines also align in striking parallel arrays. This alignment is characterized by a decrease in the amount of nuclear overlapping, probably indicating restoration of contact inhibition of movement. The morphological changes are antagonized by antibody to CSP. These effects of CSP are not associated with an elevation of cellular 3':5'-cyclic AMP. Moreover, the morphological reversion is not accompanied by an alteration in growth properties. Our results are consistent with a role for CSP in cell adhesion and morphology but not in growth control.The major cell surface glycoprotein of chick embryo fibroblasts, CSP, is substantially decreased after transformation by oncogenic viruses. Similar proteins, designated "LETS" proteins, are decreased in other cell types after transformation (refs. 1-3; for other references see 4 and 5). We have isolated CSP and found that it will aggregate several cell types, including erythrocytes, embryonic chick cells, and transformed NRK (normal rat kidney) cells (6, 7), suggesting that CSP may play a physiological role in cell:cell adhesion. The agglutinin activity of CSP is destroyed by treatment with proteases and chelating agents and is greatly diminished in transformed chick cells (7). Since transformation is often accompanied by altered growth control, morphology, adhesion, and contact inhibition of movement, we investigated whether re-attaching this glycoprotein to the cell surface could restore a more normal phenotype to transformed cells. We find that CSP partially restores morphology, adhesion, and the alignment characteristic of untransformed fibroblasts.MATERIALS AND METHODS Isolation of CSP. We prepared CSP from secondary cultures of chick embryo fibroblasts as described previously; the protein is essentially homogeneous by electrophoresis in sodium dodecyl sulfate with or without 8 M urea at pH 7 or pH 11 (ref. 7; unpublished). We then added solid ammonium sulfate to 70% saturation and adjusted the pH to 7.4 with NH40H. After 30 min at 4°, the solution was centrifuged at 25,000 X g for 15 min. The pellet was solubilized in 1ko volume of buffer A (0.15 M NaCl, 1 mM CaCl2, 10 mM cyclohexylaminopropane sulfonic acid, pH 11.0) at about 1 mg/ml, dialyzed overnight against two changes of 400 volumes of buffer A, and stored at -70°. These conditions are optimal for maintaining a concentrated, nonaggregated preparation of CSP with high agglutinating activity (unpublished Cytoplasmic Area and Nuclear Overlap Ratio. SVT2 cells were plated at 1 X I04 per cm2 on glass coverslips in CSP-containing or control media. After 48 hr they were fixed and stained with hematoxylin (9). Cytoplasmic area was measured using the intersections of a 10 X 10 microscope eyepiece grid by the formula A = aCF/IN, where ...

Abstract. The ability of single subunit chimeric receptors containing various integrin fl intracellular domains to mimic and/or inhibit endogenous integrin function was examined. Chimeric receptors consisting of the extracellular and transmembrane domains of the small subunit of the human interleukin-2 receptor connected to either the fl~, f13, fl3B, or r5 intracellular domain were transiently expressed in normal human fibroblasts. When expressed at relatively low levels, the r3 and r5 chimeras mimicked endogenous ligandoccupied integrins and, like the fl~ chimera (LaFlamme, S. E., S. K. Akiyama, and K. M. Yamada. 1992. J. Cell Biol. 117:437), concentrated with endogenous integrins in focal adhesions and sites of fibronectin fibril formation. In contrast, the chimeric receptor containing the fl3s intracellular domain (a r3 intracellular domain modified by alternative splicing) was expressed diffusely on the cell surface, indicating that alternative splicing can regulate integrin receptor distribution by an intraceUular mechanism. Furthermore, when expressed at higher levels, the fl~ and r3 chimeric receptors functioned as dominant negative mutants and inhibited endogenous integrin function in localization to fibronectin fibrils, fibronectin matrix assembly, cell spreading, and cell migration. The r5 chimera was a less effective inhibitor, and the fl3B chimera and the reporter lacking an intracellular domain did not inhibit endogenous integrin function. Comparison of the relative levels of expression of the transfected fll chimera and the endogenous fl~ subunit indicated that in 10 to 15h assays, the fll chimera can inhibit cell spreading when expressed at levels approximately equal to to the endogenous/3~ subunit. Levels of chimeric receptor expression that inhibited cell spreading also inhibited cell migration, whereas lower levels were able to inhibit otsfl~ localization to fibrils and matrix assembly.Our results indicate that single subunit chimeric integrins can mimic and/or inhibit endogenous integrin receptor function, presumably by interacting with cytoplasmic components critical for endogenous integrin function. Our results also demonstrate that fl intracellular domains, expressed in this context, display specificity in their abilities to mimic and inhibit endogenous integrin function. Furthermore, the approach that we have used permits the analysis of intracellular domain function in the processes of cell spreading, migration and extracellular matrix assembly independent of effects due to the rest of integrin dimers. This approach should prove valuable in the further analysis of integrin intracellular domain function in these and other integrin-mediated processes requiring the interaction of integrins with cytoplasmic components.

The osteocyte is the terminally differentiated state of the osteogenic mesenchymal progenitor immobilized in the bone matrix. Despite their numerical prominence, little is known about osteocytes and their formation. Osteocytes are physically separated in the bone matrix but seemingly compensate for their seclusion from other cells by maintaining an elaborate network of cell processes through which they interact with other osteocytes and bone-lining cells at the periosteal and endosteal surfaces of the bone. This highly organized architecture suggests that osteocytes make an active contribution to the structure and maintenance of their environment rather than passively submitting to random embedding during bone growth or repair. The most abundant matrix protein in the osteocyte environment is type-I collagen and we demonstrate here that, in the mouse, osteocyte phenotype and the formation of osteocyte processes is highly dependent on continuous cleavage of type-I collagen. This collagenolytic activity and formation of osteocyte processes is dependent on matrix metalloproteinase activity. Specifically, a deficiency of membrane type-1 matrix metalloproteinase leads to disruption of collagen cleavage in osteocytes and ultimately to the loss of formation of osteocyte processes. Osteocytogenesis is thus an active invasive process requiring cleavage of collagen for maintenance of the osteocyte phenotype.

Abstract. We have examined the expression, localization, and function of/$~ integrins on cultured human epidermal keratinocytes using polyclonal and monoclonal antibodies against the/~,, 0~2, 0~3, and 0~5 integrin subunits. The/3, polypeptide, common to all class 1 integrins, was localized primarily in areas of cell-cell contacts of cultured keratinocytes, as were or2 and t~3 polypeptides, suggesting a possible role in cell-cell adhesion for these integrin polypeptides. In contrast, the fibronectin receptor or5 subunit showed no such accumulations in regions of cell-ceU contact but was more diffusely distributed in the keratinocyte plasma membrane, consistent with the absence of fibronectin at cell-cell contact sites. Colonies of cultured keratinocytes could be dissociated by treatment with monoclonal antibody specific to the ~1 polypeptide. Such dissociation of cell-cell contacts also occurred under conditions where the monoclonal antibody had no effect on cell-substrate adhesion. Therefore,/3~ integrin-dependent cell-cell adhesion can be inhibited without affecting other cell-adhesive interactions. Antibody treatment of keratinocytes maintained in either low (0.15 mM) or high (1.2 mM) CaC12 also resulted in the loss of organization of intracellular F-actin illaments and ~ integrins, even when the anti-~t monoclonal antibody had no dissociating effect on keratinocyte colonies at the higher calcium concentration. Our results indicate that B~ integrins play roles in the maintenance of cell-cell contacts between keratinocytes and in the organization of intracellular microfilaments. They suggest that in epithelial cells integrins can function in cell-cell interactions as well as in cellsubstrate adhesion.

We recently reported that uPARAP/Endo180 can mediate the cellular uptake and lysosomal degradation of collagen by cultured fibroblasts. Here, we show that uPARAP/Endo180 has a key role in the degradation of collagen during mammary carcinoma progression. In the normal murine mammary gland, uPARAP/Endo180 is widely expressed in periductal fibroblast-like mesenchymal cells that line mammary epithelial cells. This pattern of uPARAP/Endo180 expression is preserved during polyomavirus middle T–induced mammary carcinogenesis, with strong uPARAP/Endo180 expression by mesenchymal cells embedded within the collagenous stroma surrounding nests of uPARAP/Endo180-negative tumor cells. Genetic ablation of uPARAP/Endo180 impaired collagen turnover that is critical to tumor expansion, as evidenced by the abrogation of cellular collagen uptake, tumor fibrosis, and blunted tumor growth. These studies identify uPARAP/Endo180 as a key mediator of collagen turnover in a pathophysiological context.