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Collagenase-3 (MMP-13) is a novel matrix metalloproteinase, the expression of which has so far only been documented in human breast carcinomas and osteoarthritic cartilage. In this study we have examined the expression of MMP-13 during human fetal development. Northern blot hybridizations revealed abundant expression of MMP-13 mRNAs in total RNA from fetal cartilage and calvaria at gestational age of 15 weeks. By in situ hybridization MMP-13 transcripts were detected in chondrocytes of hypertrophic cartilage in vertebrae of the spinal column and in the dorsal end of ribs undergoing ossification, as well as in osteoblasts and periosteal cells below the inner periosteal region of ossified ribs. In contrast, no expression of MMP-13 could be detected in osteoclasts. Furthermore, expression of MMP-13 mRNA was detected in osteoblasts and fibroblasts primarily on the inner side of calvarial bone of the skull at 16 weeks of gestation. Expression of MMP-13 mRNA by primary human fetal chondrocytes in culture was enhanced by transforming growth factor-beta (TGF-beta) and inhibited by bone morphogenetic protein-2 (BMP-2). No expression of MMP-13 mRNA could be noted in other fetal tissues, including the skin, lungs, neural tissue, muscle, and liver. These results suggest that MMP-13 plays an important role in the extracellular matrix remodeling during fetal bone development both via endochondral and intramembranous ossification.

Bone morphogenetic proteins (BMPs) are a group of peptide growth factors closely related to transforming growth factors-beta. The BMPs are suggested to play an essential role in bone development and they are strong candidate molecules to be used clinically to improve fracture healing. BMPs are also involved in the differentiation of several other tissues during embryogenesis. Here, we show that human recombinant BMP-2 regulates cell-matrix interactions by modifying the expression of integrin-type receptors. The synthesis of alpha3 integrin was down-regulated by BMP-2 in two osteogenic sarcoma-derived cell lines, Saos-2 and HOS, and also in human fetal chondrocytes. BMP-2 had no effect on the expression of alpha1, alpha2, alpha5, alpha6, and alphaV integrins. BMP-2 reduced the expression of alpha3 integrin subunit at mRNA level. Laminin-5 was shown to be the ligand for alpha3beta1 integrin on Saos cells and BMP-2 decreased the ability of Saos cells to attach to it. These results suggest that BMP-2 has a specific effect on the alpha3beta1 integrin-mediated cell adhesion to laminin-5. Given the fact that BMP-2 is expressed in osteosarcomas, in addition to in bone, this mechanism is putatively important especially in bone development and tumors. We also studied the effect of BMP-2 on a human keratinocyte cell line, HaCaT. In HaCaT cells, the expression of alpha2 integrin was strongly down-regulated by BMP-2, whereas its effect on the expression of alpha3 integrin was smaller. We suggest that the effects of BMP-2 may be partially mediated by specifically altered cell adhesion.

A cell culture inside a three-dimensional gel of fibrillar collagen is an experimental model used to study the response of cells to the extracellular matrix. Many cell types induce the contraction of gel and simultaneously decrease their production of type I collagen, whereas the expression of interstitial collagenase (matrix metalloproteinase-1; MMP-1) is enhanced. We have previously shown that in osteogenic cells the collagen receptor alpha2beta1 integrin is a positive regulator of MMP-1 and that the number of alpha2beta1 integrins on the cell surface also regulates the magnitude of contraction. However, the downregulation of collagen mRNA levels is not initiated by alpha2beta1 integrin. Here, we have studied in human KHOS-240 and MG-63 osteosarcoma cells and in human skin fibroblasts the effects of tyrosine kinase inhibitors on collagen gel contraction and on the regulation of MMP-1 and collagen alpha1(I) genes by extracellular collagen. The induction of MMP-1 could be inhibited by all tyrosine kinase inhibitors tested with the exception of genistein. None of them could prevent the downregulation of collagen expression. Thus, the collagen-induced alterations in the expression of MMP-1 and collagen alpha1(I) seem to be dependent on distinct signal transduction pathways. Many of the inhibitors, including genistein, could prevent the contraction of collagen gels. The effect was not related to their ability to inhibit cell growth, because an inhibitor specific for DNA synthesis and cell division did not have the same effect. Thus, we suggest that the process of collagen gel contraction requires protein-tyrosine phosphorylation and that the ability of cells to contract collagen gels is not related to the induction of MMP-1 or to the level of collagen alpha1(I) expression. Finally, we propose that the tyrosine kinase inhibitors might be considered as candidate molecules in the treatment of pathological scar contraction.

Invasion of malignant cells requires altered cellular interaction with extracellular matrix. Integrin-type cell adhesion receptors play an important role in this process. Integrin-related cell biological phenomena explain cancer cell migration, and recent developments in the field have made it possible to propose that integrins are also involved in the penetration through basement membranes and other molecular barriers. Finally, malignant melanoma has been used here as an example to speculate on the function of each integrin in light of information from different experimental models.

In HeLa cells beta 1 integrin forms heterodimers with alpha 1, alpha 2, alpha 3, alpha 5 and alpha 6 integrin subunits. Integrin alpha v beta 5 can also be detected. A monoclonal antibody SR-84 identified the alpha 1 integrin subunit in immunoprecipitation assays and inhibited alpha 1-related cell adhesion to different matrix proteins, laminin-1 and type I, IV, and V collagens, whereas its effect on adhesion to type II collagen was marginal. HeLa cells do not attach to type VI collagen. The presence of magnesium was essential for HeLa cell adhesion, whereas calcium alone was not sufficient and high concentrations of calcium even counteracted the effect of magnesium. Cell adhesion to type I collagen was sensitive to changes in pH, unlike cell adhesion to type IV collagen. We conclude that SR-84 is a valuable tool to study alpha 1 integrin-related functions, and that in HeLa cells alpha 1 beta 1 integrin is a magnesium-dependent receptor for type I, IV, and V collagens but not for type II and VI collagens.

The contraction of floating collagen gels is suggested to mimic the reorganization of collagenous matrix during development and tissue healing. Here, we have studied two osteogenic cell lines, namely MG-63 and HOS, and a chemically transformed subclone of HOS cells, HOS-MNNG. Transforming growth factor-beta (TGF-beta), a putative regulator of bone fracture healing, increased collagen gel contraction by MG-63 and HOS-MNNG, but not by HOS cells. Our data show that TGF-beta-induced fibronectin synthesis is not sufficient for the process. Instead, anti-beta 1 integrin antibodies could prevent the contraction. There are three different integrin heterodimers that are known to mediate the cell-collagen interaction, namely alpha 1 beta 1, alpha 2 beta 1, and alpha 3 beta 1. In MG-63 cells TGF-beta increased the expression of alpha 2 beta 1 integrin and decreased the expression of alpha 3 beta 1 integrin, whereas alpha 1 beta 1 integrin is not expressed. HOS cells had no alpha 2 beta 1 integrin, neither did TGF-beta induce its expression. However, HOS-MNNG cells expressed more alpha 2 beta 1 integrin when treated with TGF-beta. Thus, we suggest that the mechanism of the enhanced collagen gel contraction by TGF-beta is the increased expression of alpha 2 beta 1 integrin heterodimer. To further test this hypothesis, we expressed a full-length alpha 2 integrin cDNA in HOS cells and in MG-63 cells. We obtained HOS cell clones that expressed alpha 2 beta 1 heterodimer, and the ability of these cells to contract collagen gels was greatly enhanced. Furthermore, the contraction by MG-63 cells transfected with alpha 2 integrin cDNA was enhanced, and the contraction by cells transfected with antisense oriented alpha 2 integrin cDNA was decreased. Thus, both in MG-63 and HOS cells the increased alpha 2 integrin expression alone was sufficient for the enhanced contraction of collagen gels. Furthermore, the amount of alpha 2 integrin is critical for the process, and its decrease leads to diminished ability to contract gels.

The aim of the present study was to examine the expression of β1 integrin subunit after peripheral nerve transection. After sciatic nerve transection two experimental procedures were used; changes in the freely regenerating rat sciatic nerve were compared to a situation in which spontaneous regeneration was prevented by suturing both ends of the nerve to the muscle next to the point of transection. Specimens for morphological analysis were collected 6 h, 1, 3, 5, 7 days and 2, 4, 6 and 8 weeks after the axotomy. Sections from the proximal (two zones) and distal (three zones) stumps next to the point of transection were stained with antibodies against β1 integrin subunit, macrophages, collagen types I and III, and S-100 protein. The control nerves showed β1 integrin-stained cells in the perineurium and vasa nervorum but the endoncurium was negative. Positively stained endoneurial fibroblast-like cells could be seen in the proximal part of the nerve already at 24 h after transection. The number of these positively stained cells increased steadily; they were most numerous 4 weeks after transection in the distal zone 2. Subsequently, the number of positively stained endoneural cells declined sharply and 8 weeks after transection no positively stained cells could be found. The morphological appearance and the immunohistochemical properties of the cells suggest that the majority of β1 integrin-positive cells are endoneurial fibroblast-like cells. Thus, the process appeared to be dynamic, starting from the proximal part and continuing to the distal parts, and was similar in both experimental groups. The positive staining in perineurial cells indicate that β1 integrin, which is an important mediator of the cell-matrix interaction, may have an essential role in the formation and strengthening of the normal peripheral nerve structures. Furthermore, β1 integrin seems to have an active role in reactions which occur during the early phases of peripheral nerve regeneration.

Syndecan-1, a cell surface proteoglycan, binds many extracellular matrix components via its heparan sulfate side chains. In previous studies syndecan-1 has failed to bind laminin, although both syndecan-1 and laminin are expressed at sites of early matrix accumulation, like in basement membranes of epithelial-mesenchyma boundaries and in condensing mesenchymes during embryonic development. In order to study whether syndecan-1 regulates the adhesion of mesenchymal cells to laminin, syndecan-1 was expressed in NIH-3T3 cells by transfection. Syndecan-1-transfected cells showed increased binding to laminin in comparison to control transfected cells. We then compared the properties of syndecan-1 isolated from transfected NIH-3T3 cells and from NMuMG mammary epithelial cells. In solid-phase binding assays, syndecan-1 from NIH-3T3 cells, but not NMuMG cells, bound to laminin. NIH-3T3-derived syndecan contained more chondroitin sulfate than NMuMG-derived syndecan-1, and our results revealed that both heparan sulfate and chondroitin sulfate mediate syndecan-1 binding to laminin. E3 domain revealed highest binding for syndecan-1 among the elastase-derived fragments of laminin. These results suggest that induction of syndecan-1 in mesenchymal cells may be involved in cellular recognition of laminin during developmental processes.

Granulation-tissue fibroblasts express an unique phenotype distinct from normal fibroblasts. Due to the importance of the cell-matrix interactions in the regulation of cell morphology and behavior, we have compared the cell adhesion apparatus, especially integrin-type receptors, in fibroblasts cultured from healthy human periodontal connective tissues and from chronic and wound granulation tissues. The spreading of granulation-tissue cells on fibronectin, but not on type I collagen or laminin, was slower when compared with the normal fibroblasts. Cell spreading on fibronectin could be inhibited by RGD-containing peptide, suggesting integrin-mediated interaction. Both cell types expressed beta 1 integrin subunit, which associated with several integrin alpha subunits, namely alpha 1, alpha 2, alpha 3, alpha 5 and alpha v. In addition to beta 1 subunit, alpha v chain formed heterodimers with beta 3 and beta 5 subunits. Thus, these cells have multiple putative fibronectin, laminin, collagen, and vitronectin receptors. Cell spreading of both cell types on fibronectin was inhibited with anti-beta 1 and anti-alpha 5 antibodies, but antibodies against other putative FN-binding integrins (alpha 3, alpha v, and alpha v beta 3) had no effects. Furthermore, granulation-tissue fibroblasts showed delayed spreading on substrates coated with anti-beta 1 or anti-alpha 5 integrin antibodies. On substrates coated with anti-alpha 3 antibody, both cell types spread equally well. By FACS analysis, the amount of beta 1 and alpha 5 integrin subunits expressed on the cell surfaces was slightly elevated in GTFs compared with HGFs. Thus, the findings in this study indicate that the weakened interaction of granulation-tissue fibroblasts with fibronectin is regulated by altered function of alpha 5 beta 1 integrin.

Attachment and entry of coxsackievirus A9 (CAV-9) to GMK cells were previously shown to be dependent on an arginine-glycine-aspartic acid (RGD) motif in the capsid protein VP1, suggesting integrins as candidate receptors for the virus. We have pursued the matter further and show that antibodies specific for the alpha v and/or beta 3 integrin subunits protect GMK cells from CAV-9 infection. Affinity purification of radioiodinated cell surface proteins using CAV-9 or virus-specific peptide (RRRGDL) columns confirmed that the alpha v beta 3 heterodimer, known as the vitronectin receptor, is recognized by the virus in GMK cells. Other proteins, of lower molecular weight (less than 40 kDa), were also bound to and specifically eluted from the columns, but their possible role in attachment and entry of CAV-9 remains to be elucidated by further studies. Of several other related viruses studied, only echovirus 22, which also has an RGD motif in the VP1 capsid protein, was found to compete for cell surface binding with CAV-9.

A large pool of precursor beta 1-integrin subunits is frequently found intracellularly. During malignant transformation this pool often disappears. Concomitantly, integrin-mediated cell-adhesion functions are disturbed, even though no change in the number of beta 1-integrin receptors on the cell surface can be observed. Here, we have studied the role of an intracellular pre-beta 1-integrin pool by transfecting human MG-63 osteosarcoma cells with plasmid construction producing an antisense RNA for the beta 1-integrin subunit. Stable cell clones expressing beta 1-integrin antisense RNA were shown to have a reduced intracellular pool of pre-beta 1-integrin subunits. In the antisense-transfected cells, the synthesis of the beta 1-integrin chain was reduced by 65% compared with non-transfected or vector-transfected MG-63 cells. The decreased synthesis of the beta 1-integrin chain was associated with accelerated maturation of the beta 1-integrin chain (half-maturation time about 5 h in antisense-transfected cells compared with about 10.5 h in control cells), whereas maturation of the alpha-integrin chain slowed down. The amount of beta 1-integrins on the cell surface, however, remained unaltered. Cell clones with the largest decrease in the relative amount of the pre-beta 1-integrin subunit also showed altered integrin function. They were found to synthesize fibronectin, but were unable to assemble it into a fibronectin matrix on the cell surface. Thus we conclude that the repression of biosynthesis of the beta 1-integrin chain leads to alterations in receptor maturation and may be connected with altered receptor function.

Integrins are heterodimeric cell-surface receptors with key functions in cell-cell and cell-matrix adhesion. Integrin α and β subunits are present throughout the metazoans, but it is unclear whether the subunits predate the origin of multicellular organisms. Several component domains have been detected in bacteria, one of which, a specific 7-bladed β-propeller domain, is a unique feature of the integrin α subunits. Here, we describe a structure-derived motif, which incorporates key features of each blade from the X-ray structures of human αIIbβ3 and αVβ3, includes elements of the FG-GAP/Cage and Ca(2+)-binding motifs, and is specific only for the metazoan integrin domains. Separately, we searched for the metazoan integrin type β-propeller domains among all available sequences from bacteria and unicellular eukaryotic organisms, which must incorporate seven repeats, corresponding to the seven blades of the β-propeller domain, and so that the newly found structure-derived motif would exist in every repeat. As the result, among 47 available genomes of unicellular eukaryotes we could not find a single instance of seven repeats with the motif. Several sequences contained three repeats, a predicted transmembrane segment, and a short cytoplasmic motif associated with some integrins, but otherwise differ from the metazoan integrin α subunits. Among the available bacterial sequences, we found five examples containing seven sequential metazoan integrin-specific motifs within the seven repeats. The motifs differ in having one Ca(2+)-binding site per repeat, whereas metazoan integrins have three or four sites. The bacterial sequences are more conserved in terms of motif conservation and loop length, suggesting that the structure is more regular and compact than those example structures from human integrins. Although the bacterial examples are not full-length integrins, the full-length metazoan-type 7-bladed β-propeller domains are present, and sometimes two tandem copies are found.

Promotion of cell growth and differentiation by growth factors during early development and organ formation are both temporally and spatially very precise. Syndecan is a well characterized integral membrane proteoglycan that binds several extracellular matrix components via its heparan sulfate chains and is therefore suggested to participate in cell regulation. Syndecan-like molecules, as low affinity receptors for heparin-binding growth factors, have been recently suggested to also regulate growth factor activity. Heparin/heparan sulfate interaction is required before, e.g. basic fibroblast growth factor (bFGF) can associate with its high affinity cell surface receptors and trigger signal transduction. In this paper we show that syndecan, but not free heparan sulfate chains, can simultaneously bind both bFGF and extracellular matrix molecules. Moreover, increased DNA synthesis of 3T3 cells was observed when the 3T3 cells were exposed to beads coated with the fibronectin-syndecan-bFGF complex, indicating that bFGF remains biologically active even when immobilized to matrix via the heparan sulfate chains of syndecan. Finally, when bFGF was bound to the surface of another cell type (epithelial), co-culture with 3T3 cells stimulated 3T3 cell growth. Therefore, we suggest that syndecan-like molecules may determine sites of growth factor action at cell-matrix and cell-cell interfaces.

The granulated nickel slag is the biggest unexploited solid side stream formed in the area of the Harjavalta industrial ecopark in south-west Finland. Nowadays one-quarter of the granulated nickel slag is utilised in the sand blasting and roofing felt industries and three-quarters are still dumped in landfills. At the moment the main obstacles to a wider utilisation of granulated nickel slag are the environmental properties of slag and fluctuations in its composition, which lead to changes in the slag's stability. Despite the obstacles, there are possibilities to promote slag utilisation. A possible way to affect slag properties is through slag modification by immobilising harmful elements in the slag. Also, the determination of environmental and technical properties of slag plays an important role in the utilisation aims. The utilisation of slag is one way to support the eco-efficiency of the Harjavalta industrial ecopark. Slag can be used in other applications as a raw material or as a product instead of natural raw materials.

The attachment and spreading of cultured fibroblasts on potentially bioactive glasses (bioglasses) of ten different compositions were studied. Human gingival fibroblasts were allowed to attach and spread on bio-glasses for 1-72 h. Unreactive silica glass and cell culture polystyrene served as controls. The attachment and spreading of cells were examined by 3H-thymidine labeling of cells, planimetric analysis, cytological staining, immunocytochemistry, and scanning electron microscopy. The cell attachment to bioglasses and silica glass and the cell spreading on bioglasses were slower and cell morphology more elongated compared to control plastic. In spite of great differences in bioglass compositions no great differences in cell behavior on these surfaces were detected. Thus the initial events in the tissue-implant interface might be independent on the bioglass composition, and furthermore the differences in the organization of the tissue-implant interface in vivo might depend on the nature of the surrounding tissues and subsequent changes of the implant surface and the extracellular environment.

– Degradation of fibronectin (FN) by subgingival and supragingival plaque and Bacteroides gingivalis (Bg) was studied in vitro. The degradation of FN by both types of plaque was relatively rapid, continuous but incomplete. Some differences were found between supra-and subgingival samples. Supragingival plaque extracts produced several FN fragments of 110–180 kd during short incubations, of 15–60 min. The predominant fragment after overnight incubation was a 110 kd polypeptide. With subgingival plaque extract a more extensive degradation of FN was noted. The main degradation product was a 120 kd fragment after overnight incubation. Several peptide fragments were released from fibronectin by Bg extracts. Their molecular size was different from those produced by trypsin, elastase or dental plaque. When cell extracts of Bg were fractionated by high performance liquid chromatography, three separate peaks of fibronectin degrading activity were obtained. Two of those peaks also contained trypsin-like enzyme activity. The degradation of fibronectin and the subsequent formation of biologically active peptides may have many effects in periodontal pockets. These may include modifying effects on plaque growth and wound healing.

Human granulation-tissue fibroblasts were cultured from oral chronic inflammatory lesions and compared with fibroblasts of healthy gingival connective tissue with respect to cell-surface sialoglycoproteins, and the synthesis of extracellular matrix components. Granulation-tissue fibroblasts exhibited a slower growth rate and larger size than their controls. Their cell-surface sialoglycoproteins resembled those of the control cells, except that the relative amount of glycoproteins in the 140-kd region was lower. The ratio of mRNAs for proαl(I) and proαl(III) collagen chains was decreased in granulation-tissue fibroblasts, although electrophoretic fractionation of the proteins did not reveal consistent differences in type I/type III collagen ratio. Granulation-tissue fibroblasts secreted into the culture medium a dermatan sulfate proteoglycan with a lower molecular weight. After digestion with chondroitinase ABC, the molecular weight of the core protein appeared to be identical with that of the control fibroblasts, suggesting a difference in the glycosylation of the core protein. These results support the theory that granulation-tissue fibroblasts represent a distinct phenotype of fibroblastic cells.

A female patient with congenital ectodermal dysplasia is described, who developed xanthoma tendinosum in her hands simulating rheumatoid arthritis. The serum lipids of this patient were normal. Both ectodermal dysplasia and xanthoma tendinosum in a normolipemic patient are rather uncommon and to our knowledge have not been previously reported in one and the same patient.

We have examined the ability of transforming growth factor-β1 (TGF-β1) to regulate the expression of members of the αβ2 and αβ3 families of integrins. TGF-β1 elevates the expression of vitronectin receptors (αvβ3 integrin) in all cells examined including WI-38 human lung fibroblasts, 3T3-L1 mouse fibroblasts, and MG-63 human osteogenic sarcoma cells. TGF-β1 action increases the level of mRNA and the synthesis of vitronectin receptor subunits with t½of 3–4 h and 6 h, respectively. TGF-β1 up-regulates expression of the intercellular adhesion receptor, LFA-1 (αLβ2), in THP-1 human monocytic leukemia cells by increasing the synthesis of αL subunit but not β2 subunit. The increase in αl synthesis and assembly into LFA-1 complexes induced by TGF-β1 occurs in parallel with elevated fibronectin receptor synthesis in THP-1 cells. These responses to TGF-β1 are lost upon phorbol esterinduced differentiation of THP-1 cells into the macrophage phenotype. The results suggest a role of TGF-β in the regulation of cell-matrix interactions mediated by vitronectin receptors and cell-cell interactions mediated by LFA-1 in the immune system.

Transforming growth factor-beta (TGF-beta) induces a marked decrease in adhesion of MG-63 human osteosarcoma cells to laminin-coated surfaces, but does not significantly alter adhesion to fibronectin- or collagen-coated surfaces. We provide evidence that this effect is due to a switch in the repertoire of cell adhesion receptors in response to TGF-beta. MG-63 cells express high levels of alpha 3 beta 1-integrin, which is a polyspecific laminin/collagen/fibronectin receptor, and low levels of alpha 2 beta 1- and alpha 5 beta 1-integrins, which are collagen and fibronectin receptors, respectively. No other integrins of the beta 1-class could be detected in MG-63 cells. Treatment with TGF-beta 1 induces a marked (approximately 60%) decrease in the level of expression of alpha 3-integrin subunit mRNA and protein and a concomitant 8-fold increase in alpha 2-subunit expression. These responses become maximal 7-12 h after addition of TGF-beta 1 to the cells. Expression of alpha 5- and beta 1-integrin subunits also increases in response to TGF-beta 1, but to a lesser extent than alpha 2-subunit expression. Thus, as a result of TGF-beta action, the alpha 2 beta 1-collagen and alpha 5 beta 1-fibronectin receptors replace the alpha 3 beta 1-laminin/collagen/fibronectin receptor as the predominant integrins of the beta 1-class in MG-63 cells. These results suggest that one of the effects of TGF-beta is to modify the adhesive behavior of certain tumor cells by changing the binding specificity of the complement of integrins that they express.

Changes in the structure and organization of collagen fibrils were recently described in the skin of aspartylglycosaminuria patients1. The skin of the patients contained a normal amount and distribution of glycosaminoglycans. but the dermatan sulfate of aspartylglycosaminuria skin was more sensitive to chondroitinase AC digestion, resulting in unsaturated 4-sulfated disaccharides which were not detected in controls. Isolated dermatan sulfate chains as well as the chains present in the intact core protein synthesized by skin fibroblasts from an aspartylglycosaminuria patient were also digestible with chondroitinase AC. while those of a control fibroblast culture could be digested with chondroitinase ABC only. This is indirect evidence for abnormal epimerization of dermatan sulfate in the skin of aspartylglycosaminuria patients, which may be associated with the changes in collagen fibril formation.

Cell surface sialoglycoproteins of human mononuclear phagocytes in different maturation stages were labelled by the periodate/borohydride method and separated by SDS-polyacrylamide gel electrophoresis. The main surface glycoproteins of peripheral blood monocytes had molecular masses of 115 and 95 kDa. During in vitro transition into adherent macrophages, the monocyte-characteristic surface glycoproteins disappeared. Most of the changes in the surface glycoprotein pattern occurred during the first 24 h and after 96 h the changes were completed. The major sialoglycoproteins of the macrophage cell surface had molecular masses of 130 and 55 kDa. The macrophage cell surface showed further changes when cultured in the presence of synovial fluid (10%). These results may reflect the in vivo maturation of monocytes into tissue macrophages. In synovium, tissue-derived factors may also take part in differentiatíon.

Cell adhesion to extracellular matrices is mediated by a set of heterodimeric cell surface receptors called integrins that might be the subject of regulation by growth and differentiation factors. We have examined the effect of transforming growth factor-beta 1 (TGF-beta 1) on the expression of the very late antigens or alpha beta 1 group of integrins in human cell lines. The six known members of this family share a common beta 1 subunit but have distinct alpha subunits that confer selective affinity toward type I collagen, fibronectin, laminin, and other as yet unknown cell adhesion proteins. Using a panel of specific antibodies and cDNA probes, we show that in WI-38 lung fibroblasts TGF-beta 1 elevates concomitantly the expression of alpha 1, alpha 2, alpha 3, alpha 5, and beta 1 integrin subunits at the protein and/or mRNA level, their assembly into the corresponding alpha beta 1 complexes, and their exposure on the cell surface. The rate of synthesis of total alpha subunits relative to beta 1 subunit is higher in TGF-beta 1-treated cells than in control cells. The characteristically slow (t1/2 approximately 10 h) rate of beta 1 conversion from precursor form to mature glycoprotein in untreated cells increases markedly (to t1/2 approximately 3 h) in response to TGF-beta 1. The results suggest that in WI-38 fibroblasts the beta 1 subunit is synthesized in excess over alpha subunits, and assembly of beta 1 subunits with rate-limiting alpha subunits is required for transit through the Golgi and exposure of alpha beta 1 complex on the cell surface. TGF-beta 1 does not induce the synthesis of integrin subunits that are not expressed in unstimulated cells, such as alpha 4 and alpha 6 subunits in WI-38 fibroblasts. However, alpha 4 and alpha 6 subunits can be regulated by TGF-beta in those cells that express them. The results suggest that TGF-beta regulates the expression of individual integrin subunits by parallel but independent mechanisms. By modifying the balance of individual alpha beta 1 integrins, TGF-beta 1 might modulate those aspects of cell migration, positioning, and development that are guided by adhesion to extracellular matrices.

The molecular forms of fibronectin (FN) in gingival crevicular fluid of five subjects with at least two sites exhibiting clinical signs of inflammation and pockets of at least 4 mm (test group) and five subjects with clinically healthy periodontium (control group) were investigated. Samples were collected with standard filter paper strips. In the test group samples from both diseased and healthy sites were collected. After collection the test group received one episode of periodontal treatment (scaling and root planning). The sampling and clinical recording were repeated for the diseased sites after about 2 wk. The crevicular fluid FN was analyzed using sodium dodecyl sulphate gel electrophoresis followed by western blotting with polyclonal antibodies against FN. Both intact FN and FN fragments were found in all samples. A larger proportion of FN was in degraded form in the diseased sites than in the healthy or the treated sites. FN was also degraded into smaller peptide fragments in the diseased than in the treated sites. These results suggest that crevicular fluid FN is partially degraded both in periodontal health and disease and that the degree of degradation of FN increases with periodontal inflammation and decreases with periodontal treatment.

The integrins form a large family of cell adhesion receptors. All multicellular animals express integrins, indicating that the family evolved relatively early in the history of metazoans, and homologous sequences of the component domains of integrin alpha and beta subunits are seen in prokaryotes. Some integrins, however, seem to be much younger. For example, the alphaI domain containing integrins, including collagen receptors and leukocyte integrins, have been found in chordates only. Here, we will discuss what conclusions can be drawn about integrin function by studying the evolutionary conservation of integrins. We will also look at how studying integrins in organisms such as the fruit fly and mouse has helped our understanding of integrin evolution-function relationships. As an illustration of this, we will summarize the current understanding of integrin involvement in skeletal muscle formation.

Collagenase-3 (MMP-13) is a matrix metalloproteinase capable of cleaving a multitude of extracellular matrix proteins in addition to fibrillar collagens. Human MMP-13 is expressed by fibroblasts in chronic cutaneous ulcers, but not in normally healing adult skin wounds. However, MMP-13 is produced by fibroblasts in adult gingival and in fetal skin wounds characterized by rapid collagen remodeling and scarless healing. Here, we have examined the role of human MMP-13 in remodeling of three-dimensional (3D) collagenous matrix by primary adult human skin fibroblasts. The high level of human MMP-13 expression by fibroblasts achieved by adenoviral gene delivery resulted in potent enhancement of remodeling and contraction of 3D collagen. Fibroblasts expressing MMP-13 in 3D collagen possessed altered filamentous actin morphology with patch-like actin distribution in cell extensions. The expression of MMP-13 promotes survival and proliferation of fibroblasts in floating collagen gel, and results in activation of Akt and extracellular signal-regulated kinase-1/2 by these cells. The results provide evidence for a novel role for human MMP-13 in regulating dermal fibroblast survival, proliferation, and interaction in 3D collagen, which may be an important survival mechanism for fibroblasts in chronic skin ulcers and contribute to scarless healing of adult gingival and fetal skin wounds.

In the present work we demonstrate that non-activated, cultured rat peritoneal macrophages deplete arginine from their culture medium and that the use of this medium in fibroblast cultures may lead to decreased synthesis of collagen by fibroblasts.

Dermatan sulphate proteoglycans (DSPGs) synthesized in the presence of 35SO4 were characterized in culture media of fibroblast lines obtained from skin, synovium, and gingiva. The molecular mass of DSPG varied from 95-130 kDa as estimated by SDS/polyacrylamide-gel electrophoresis. Gingival fibroblasts constantly produced larger DSPGs than skin fibroblasts. This was due to the larger dermatan sulphate (DS) chains, which also showed tissue-related heterogeneity in the distribution of 4- and 6-sulphated disaccharide units. The N-glycosylated cores (44 and 47 kDa) obtained following chondroitinase ABC treatment were of identical size in all tissues. The cores from the different tissues were also of the same size (38 kDa) when addition of the N-linked oligosaccharides was inhibited by tunicamycin or when they were removed by N-glycanase treatment. No evidence for low-molecular-mass sulphated oligosaccharides was found. All tissues contained two mRNA species (1.6 and 1.9 kb) for the DSPG core protein. These data suggest that the pattern of transferase activities involved in the construction of DS chains differs from one tissue to another. This variation may modulate the functions of DSPG in the extracellular matrix.

Human skin fibroblasts were exposed to various concentrations (from 0.01 to 5.0 units/ml) of human recombinant interleukin-1 alpha and interleukin-1 beta (IL-1 alpha and IL-1 beta). Both IL-1 alpha and IL-1 beta were found to increase dermatan-sulphate-proteoglycan (DSPG) core-protein mRNA levels. Maximal increase (3.0-fold) was seen at 48 h after addition of 1 unit of IL-1 beta/ml. In spite of the elevated DSPG-core-protein mRNA only a slight increase (from 10 to 18%) could be seen in the production of DSPG to cell-culture medium. No changes in the molecular mass of DSPG could be detected.

In the present study we show that highly purified human interleukin-1 increases collagen production nearly 2-fold and mRNA levels of type I and III collagen over 2.5-fold in cultured normal human dermal fibroblasts. To minimize the effects of transient prostaglanding E2 production in fibroblasts treated with interleukin-1, the cell cultures were preincubated for 24 h before these measurements were made. The effects of interleukin-1 were also tested on scleroderma fibroblasts exhibiting increased collagen production. Although collagen synthesis was stimulated by interleukin-1 to some degree, the cells grown from both affected and unaffected skin areas were found to be relatively unresponsive to the effects of interleukin-1, suggesting a role for this monokine in the earlier stages of the disease process. The results also suggest that interleukin-1 has a role in stimulation of collagen synthesis under certain normal and pathological conditions in addition to stimulating fibroblast proliferation.

In in vivo studies 0.5 U human interleukin 1 (IL-1) was incoculated daily into a subcutaneously implanted viscose cellulose sponge. IL-1 significantly decreased the dry weight (7.8%) and the hydroxyproline content (24.2%) of granulomas. When the cultured rat granulation tissue cells were exposed to IL-1 (0.5–2.0 U/ml) their collagen production decreased to 80% of that in controls. No effect on cell proliferation was detected.

Rheumatoid synovia were enzymatically digested and the in vitro morphology of different types of plastic adherent cells was observed. Four main types of cells were found after 24 hours in culture: stellate cells which had nuclei resembling those of classical cultured fibroblasts, but which stained positively with I2 antibody (anti-HLA-DR antibody); fibroblastic cells; cells which resembled morphologically in vitro macrophages and which were I2 and OKM-1 positive; round monocytes. The stellate cells did not stain with anti-S-100 or OKT-6 antibodies, which are used to detect classical antigen presenting dendritic cells. Furthermore, in the presence of indomethacin the stellate shaped cells were replaced by new I2 positive cells with a typical fibroblast shape. These results support the view that the stellate cells in synovial cell cultures represent HLA-DR positive fibroblasts, probably B cells of synovial lining.

Subcutaneously implanted cylindrical hollow viscose cellulose sponges were used to study the effect of locally applied epidermal growth factor (EGF) on methylprednisolone-induced inhibition of granulation tissue formation in rats. In in vivo studies the sponges were treated immediately after implantation with a single injection of 2 mg (approximately 1.7 x 10(-3) M) of depot methylprednisolone or with its carrier solution only. Thereafter the implants were injected daily with 5 micrograms of EGF or with its carrier solution 0.1% albumin for 7 days. Methylprednisolone treatment decreased the accumulation of nucleic acids, collagen, and glycosaminoglycans in the developing granulation tissue. After daily injections of EGF the concentrations of these tissue components returned close to the control values. In cultures of rat granulation tissue fibroblasts, 10(-4) M and 10(-3)M methylprednisolone decreased collagen synthesis by 41 and 81% from the control level, respectively. In the presence of methylprednisolone EGF treatment could not increase collagen synthesis of fibroblasts. Methylprednisolone treatment resulted in a dose-dependent reduction in pro alpha 1(I) collagen mRNA levels, which was partially inhibited by low EGF concentrations (1 and 10 ng/ml). In the presence of methylprednisolone all concentrations of EGF increased fibronectin mRNA levels in a dose-dependent manner. It is concluded that EGF treatment can prevent the inhibitory effect of methylprednisolone on wound healing by stimulating fibroblast proliferation but does not stimulate collagen synthesis per cell.

Long-term synovial fibroblast cultures were exposed to interleukin 1 (IL-1) or prostaglandin E2 (PGE2). The normally spindle-shaped fibroblasts changed to stellate-shaped cells, resembling the HLA-DR-positive, collagenase-producing cells which are normally seen only in primary cultures from enzyme-digested rheumatoid synovial tissue. However, the IL-1- or PGE2-induced fibroblasts were not HLA-DR-positive. This suggests that these cell populations represent originally different cell lines or that the expression of HLA-DR antigens is not induced by the agents used. For further characterization of these stellate cells, the location of fibronectin and type I collagen was studied by specific antibodies and the pericellular coat around fibroblasts was visualized by the erythrocyte exclusion method. Both IL-1 and PGE2 treatments destroyed the intercellular fibronectin network. Type I collagen was detected as intracellular granules. The stellate fibroblasts were usually full of these granules in contrast to intact fibroblasts in which the number of collagen fluorescence granules varied greatly. The pericellular coat known to be formed mainly by hyaluronic acid was similar around spindle and stellate-shaped fibroblasts. Rheumatoid arthritis-derived fibroblasts did not differ from their non-rheumatoid counterparts in any of the experiments. The effect of IL-1 and PGE2 on fibroblasts simulates the interaction between mononuclear cells and fibroblasts in synovial stroma and also potentially the interactions between different cell types in synovial lining.

Effects of peripheral blood mononuclear cells on cultured synovial fibroblasts were studied. When mononuclear cells from normal or rheumatoid blood were incubated on synovial fibroblast cultures, a part of the cells adhered to the fibroblasts. They were mainly T lymphocytes but also some B lymphocytes and monocytes. After a 10-hour incubation, adhered mononuclear cells induced morphologic alterations to synovial fibroblasts: appearance of stellate cells and thinning and branching of fibroblasts. No changes were seen when the cells were incubated in the presence of indomethacin. Cytotoxicity of peripheral blood mononuclear cells from 8 rheumatoid patients was also tested against three rheumatoid and three normal synovial fibroblast strains. Only 2 out of 48 combinations were cytotoxic. The potentially cytotoxic mononuclear cells were bound equally well to rheumatoid and control synovial fibroblast cultures.

The role of indomethacin in the regulation of extracellular matrix synthesis was studied in dermal fibroblast cultures. Indomethacin (10 μM) blocked totally the prostaglandin secretion and markedly increased the synthesis of collagen. In parallel, measurement of fibronectin, type I and type III procollagen mRNA levels showed a substantial increase under the action of indomethacin. On the other hand, indomethacin did not modify the mRNA levels of dermatan sulfate proteoglycan core protein. Measurement of collagen production estimated as the amount of collagenase digestible protein and by specific radioimmunoassay indicated a good correlation with the corresponding mRNA levels. These results suggest that indomethacin can regulate the extracellular matrix deposition at a transcriptional level.

The synthesis of collagen and its deposition into the extracellular matrix were studied in scleroderma and normal skin fibroblast cultures using immunocytochemistry, electron microscopy and electrophoretic analysis. Indirect immunofluorescence localized type I collagen to intracellular granules of normal skin fibroblasts but not to the extracellular matrix. A 48-h ascorbic acid-treatment of scleroderma and normal skin fibroblasts induced the appearance of fibrillar, collagen-containing deposits in the extracellular space as indicated by indirect immunofluorescence and electron microscopy. Interestingly, similar type I collagen-containing fibrils were observed in cultures of scleroderma skin fibroblasts even without preceding ascorbic acid treatment. Electrophoretic analysis revealed that ascorbic acid increases the conversion of procollagen to collagen to same extent both in scleroderma and control cell layers. The accumulation of collagen to the cell layer of scleroderma fibroblasts was increased when compared to normal skin fibroblasts; whereas the relative conversion of procollagen to collagen was identical in scleroderma and normal fibroblasts. The results show that the conversion of procollagen to collagen in the cell layers of both scleroderma and normal skin fibroblasts is dependent on ascorbic acid, due to improved secretion of procollagen molecules in the presence of ascorbic acid.

Cell-matrix interactions and intergrin-type cell adhesion receptors are involved in the regulation of tumor cell invasion and metastasis. We have analyzed the expression of matrix proteins and their cellular receptors in human osteosarcoma cells (HOS) and in their virally (KHOS-NP) and chemically (HOS-MNNG) transformed tumorigenic subclones. Transformation decreased dramatically the cellular mRNA levels of alpha 1(I) collagen. Concomitantly with down-regulation of collagen mRNA levels the synthesis of the collagen receptor, alpha 2 beta 1 integrin, was induced. No alpha 2 integrin mRNA was found in HOS cells, suggesting that its expression was regulated most probably at the transcriptional level. 5-Azacytidine alone or combined with alpha 2 integrin-stimulating cytokines, transforming growth factor-beta 1, and interleukin-1 beta, did not turn on the alpha 2 integrin gene. In chemically transformed cells, however, alpha 2 integrin expression could be regulated by cytokines. Thus, we suggest that HOS cells have a strong element, probably other than cell culture-generated de novo promoter methylation, suppressing alpha 2 integrin expression and that this factor is lost in both chemical and viral transformation. Furthermore, the mechanism used by cytokines and malignant transformation to increase alpha 2 integrin expression seems not to be identical. Other transformation-related changes in beta 1 integrins were (i) reduction of the intracellular pool of precursor beta 1 (in HOS-MNNG cells), leading to faster maturation rate of beta 1 subunit and slower maturation rate of alpha subunits, and (ii) decreased electrophoretic mobility of both alpha and beta 1 subunits. At the cellular level both chemical and viral transformation increased cell adhesion to type I collagen.