DNA methylation is not likely to be responsible for aggrecan down regulation in aged or osteoarthritic cartilage.

BACKGROUND: Expression of aggrecan is reduced during aging and osteoarthritic cartilage degeneration. CpG methylation may have a role in the down regulation of aggrecan transcriptions. OBJECTIVE: To investigate whether a correlation between gene methylation and expression of aggrecan in chondrocytes exists. METHODS: The human aggrecan promoter region was analysed computationally for CpG-rich regions. These were investigated for the methylation of C residues in normal (aged) and osteoarthritic chondrocytes by the bisulphite method for modifying DNA as well as sequence analysis using DNA directly extracted from normal and osteoarthritic cartilage tissue. Additionally, chondrocytic cell lines were investigated for methylation within the aggrecan promoter region. RESULTS: The CpG-rich promoter region of the human aggrecan gene contains a 0.6 kb region that meets the criteria of a CpG island as defined by prediction programmes. A significant correlation of aggrecan mRNA expression levels and methylation status in normal (aged) and osteoarthritic chondrocytes as well as in different chondrocytic cell lines was not found. CONCLUSIONS: Expression of aggrecan in normal cartilage and diseased states is not modulated by gross changes of CpG methylation of its promoter region. CpG methylation does not have a central role in the switch off of aggrecan promoter activity in human adult articular chondrocytes.

Fibroblast-mediated delivery of growth factor complementary DNA into mouse joints induces chondrogenesis but avoids the disadvantages of direct viral gene transfer.

OBJECTIVE: To assess the advantages and disadvantages of a direct adenoviral and a cell-mediated approach to the induction of cartilage formation in joints by transfer of growth factor genes. METHODS: Adenoviral vectors carrying insulin-like growth factor 1 (IGF-1) or bone morphogenetic protein 2 (BMP-2) complementary DNA were constructed and applied to primary human and murine chondrocytes or fibroblasts. Transgene expression was quantified by enzyme-linked immunosorbent assay. Direct injection of these vectors or AdLacZ, a reporter gene vector, into mouse knee joints was compared with the transplantation of syngeneic fibroblasts (infected ex vivo with the same vectors) with respect to virus spread, immune response, and cartilage formation by use of histologic, immunohistochemical, and molecular analyses. RESULTS: AdIGF-1 and AdBMP-2 efficiently infected all cell types tested. Human cells secreted biologically relevant levels of protein over a period of at least 28 days. Direct transfer of AdLacZ into mouse knee joints resulted in positively stained synovial tissues, whereas AdLacZ-infected fibroblasts settled on the surface of the synovial membranes. Inadvertent spread of vector DNA into the liver, lung, and spleen was identified by nested polymerase chain reaction in all mice that had received the vector directly; this rarely occurred following fibroblast-mediated gene transfer. Direct injection of AdBMP-2 induced the synthesis of new cartilage in periarticular mesenchyme, accompanied by extensive osteophyte formation. When AdBMP-2 was administered by injecting ex vivo-infected fibroblasts, cartilage formation was observed only in regions near the injected cells. AdIGF-1 treatment did not lead to morphologic changes. Importantly, fibroblast-mediated gene transfer avoided the strong immune response to adenovirus that was elicited following direct application of the vector. CONCLUSION: Our results indicate that cell-mediated gene transfer provides sufficient BMP-2 levels in the joint to induce cartilage formation while avoiding inadvertent vector spread and immune reactions.

The integrin alpha 7 cytoplasmic domain regulates cell migration, lamellipodia formation, and p130CAS/Crk coupling.

The integrin alpha(7)beta(1) is the major laminin-binding integrin in skeletal, heart, and smooth muscle and is a receptor for laminin-1 and -2. It mediates myoblast migration on laminin-1 and -2 and thus might be involved in muscle development and repair. Previously we have shown that alpha(7)B as well as the alpha(7)A and -C splice variants induce cell motility on laminin when transfected into nonmotile HEK293 cells. In this study we have investigated the role of the cytoplasmic domain of alpha(7) in the laminin-induced signal transduction of alpha(7)beta(1) integrin regulating cell adhesion and migration. Deletion of the cytoplasmic domain did not affect assembly of the mutated alpha(7)Deltacyt/beta(1) heterodimer on the cell surface or adhesion of alpha(7)Deltacyt-transfected cells to laminin. The motility of these cells on the laminin-1/E8 fragment, however, was significantly reduced to the level of mock-transfected cells; lamellipodia formation and polarization of the cells were also impaired. Adhesion to the laminin-1/E8 fragment induced tyrosine phosphorylation of the focal adhesion kinase, paxillin, and p130(CAS) as well as the formation of a p130(CAS)-Crk complex in wild-type alpha(7)B-transfected cells. In alpha(7)BDeltacyt cells, however, the extent of p130(CAS) tyrosine formation was reduced and formation of the p130(CAS)-Crk complex was impaired, with unaltered levels of p130(CAS) and Crk protein levels. These findings indicate adhesion-dependent regulation of p130(CAS)/Crk complex formation by the cytoplasmic domain of alpha(7)B integrin after cell adhesion to laminin-1/E8 and imply alpha(7)B-controlled lamellipodia formation and cell migration through the p130(CAS)/Crk protein complex.

Characterization of human type X procollagen and its NC-1 domain expressed as recombinant proteins in HEK293 cells.

Type X collagen is a short-chain, network-forming collagen found in hypertrophic cartilage in the growth zones of long bones, vertebrae, and ribs. To obtain information about the structure and assembly of mammalian type X collagen, we generated recombinant human type collagen X by stable expression of full-length human alpha1(X) cDNA in the human embryonal kidney cell line HEK293 and the fibrosarcoma cell line HT1080. Stable clones were obtained secreting recombinant human type X collagen (hrColX) in amounts of 50 microg/ml with alpha1(X)-chains of apparent molecular mass of 75 kDa. Pepsin digestion converted the native protein to a molecule migrating as one band at 65 kDa, while bands of 55 and 43 kDa were generated by trypsin digestion. Polyclonal antibodies prepared against purified hrColX reacted specifically with type X collagen in sections of human fetal growth cartilage. Circular dichroism spectra and trypsin/chymotrypsin digestion experiments of hrColX at increasing temperatures indicated triple helical molecules with a reduced melting temperature of 31 degrees C as a result of partial underhydroxylation. Ultrastructural analysis of hrColX by rotary shadowing demonstrated rodlike molecules with a length of 130 nm, assembling into aggregates via the globular noncollagenous (NC)-1 domains as reported for chick type X collagen. NC-1 domains generated by collagenase digestion of hrColX migrated as multimers of apparent mass of 40 kDa on SDS-polyacrylamide gel electrophoresis, even after reduction and heat denaturation, and gave rise to monomers of 18-20 kDa after treatment with trichloroacetic acid. The NC-1 domains prepared by collagenase digestion comigrated with NC-1 domains prepared as recombinant protein in HEK293 cells, both in the multimeric and monomeric form. These studies demonstrate the potential of the pCMVsis expression system to produce recombinant triple helical type X collagens in amounts sufficient for further studies on its structural and functional domains.

Localization of silencer and enhancer elements in the human type X collagen gene.

Collagen type X is a short, network-forming collagen expressed temporally and spatially tightly controlled in hypertrophic chondrocytes during endochondral ossification. Studies on chicken chondrocytes indicate that the regulation of type X collagen gene expression is regulated at the transcriptional level. In this study, we have analyzed the regulatory elements of the human type X collagen (Col10a1) by reporter gene constructs and transient transfections in chondrogenic and nonchondrogenic cells. Four different promoter fragments covering up to 2,864 bp of 5'-flanking sequences, either including or lacking the first intron, were linked to luciferase reporter gene and transfected into 3T3 fibroblasts, HT1080 fibrosarcoma cells, prehypertrophic chondrocytes from the resting zone, hypertrophic chondrocytes, and chondrogenic cell lines. The results indicated the presence of three regulatory elements in the human Col10a1 gene besides the proximal promoter. First, a negative regulatory element located between 2.4 and 2.8 kb upstream of the transcription initiation site was active in all nonchondrogenic cells and in prehypertrophic chondrocytes. Second, a positive, but also non-tissue-specific positive regulatory element was present in the first intron. Third, a cell-type-specific enhancer element active only in hypertrophic chondrocytes was located between -2.4 and -0.9 kb confirming a previous report by Thomas et al. [(1995): Gene 160:291-296]. The enhancing effect, however, was observed only when calcium phosphate was either used for transfection or included in the culture medium after lipofection. These findings demonstrate that the rigid control of human Col10a1 gene expression is achieved by both positive and negative regulatory elements in the gene and provide the basis for the identification of factors binding to those elements.

Cooperative and competitive interactions of regulatory elements are involved in the control of divergent transcription of human Col4A1 and Col4A2 genes.

The genes COL4A1 and COL4A2, coding for the two subunit chains alpha1(IV) and alpha2(IV) of collagen IV [alpha1(IV)2alpha2(IV)] are found closely linked on the human chromosome 13 in a unique head-to-head arrangement resulting in opposite strand transcription starting from a shared promoter region. Transient transfection experiments defined a shared promoter and two symmetrically arranged, downstream located and gene-specific activating elements in each gene. The shared promoter does not exhibit any transcriptional activity and efficient transcription depends on the cooperative effect of downstream elements. Mutual inhibitory effects between the two activating elements indicate competitive interactions with the shared promoter. Symmetry, cooperativity and competitivity of cis-elements are also reflected by the binding of transacting factors to the promoter and activating elements. From these data we propose a model for the coordination of divergent transcription of COL4 genes based on the cooperative and competitive interactions of the shared promoter and gene-specific regulating elements.

Fibrillin-1: organization in microfibrils and structural properties.

To investigate the microfibrillar organization and structural properties of fibrillin-1, we produced overlapping recombinant peptides in human cells which altogether span the fibrillin-1 molecule. The peptides were purified under non-denaturing conditions and extensive characterization indicated correct folding. The purified proteins were used to map monoclonal antibodies 26, 69 and 201. The binding sites are located at the N-terminal end between amino acid residues 45 and 450 (mAb 26), 451 and 909 (mAb 201) and at the C-terminal end between residues 2093 and 2871 (mAb 69). Immunolocalization of these antibodies to extended beaded structures (microfibrils) demonstrated that the N- and C-terminal ends of fibrillin-1 are located in proximity and on opposite sides of the beads, and more central parts of the molecule are located between the beads. Each epitope is present once between each bead. These data allow two possible models for the organization of fibrillin in microfibrils. However, comparison of distances between antibody binding sites on the recombinant peptides and labeling events in tissue suggests that fibrillin molecules are compacted within their tissue form as microfibrils. Additional analysis of the recombinant peptides provide new information regarding the eight-cysteine motif, a novel domain present in fibrillins and TGF beta binding proteins, and suggest that fibrillins are processed at their N-and C-terminal ends.

Structural and functional analysis of the globular domain IVa of the laminin alpha 1 chain and its impact on an adjacent RGD site.

The globular domain IVa (about 250 residues) of the laminin alpha1 chain was obtained in recombinant form from mammalian cell clones. It was prepared either with (alpha1IVa-R) or without (alpha1IVa) an adjacent cell-adhesive RGD site which seems to be masked in laminin-1. The recombinant products could be visualized as globular structures by rotary shadowing, were resistant to trypsin and shared immunological epitopes with laminin-1, indicating folding into a native structure. Sequence analysis of pepsin fragments demonstrated the insertion of the globular domain into an epidermal growth factor-like scaffold which is characteristic of the extracellular laminin domain IV (L4) module. Only little immunological cross-reaction was found, however, with other L4 modules from perlecan and different laminin isoforms. Fragment alpha1IVa-R, but not fragment alpha1IVa, bound to alphaVbeta3 integrin, although to a distinctly lower level than a laminin fragment where the RGD site is fully exposed. The fragments also had no or only little cell attachment activity. This confirmed previous predictions that the globular domain alpha 1IVa masks the RDG site in laminin-1. Domain alpha 1IVa showed, in addition, a weak binding activity for the basement-membrane protein fibulin-1.

Specific induction of cell motility on laminin by alpha 7 integrin.

Laminin, the major glycoprotein of basement membranes, actively supports cell migration in development, tissue repair, tumor growth, metastasis, and other pathological processes. Previously we have shown that the locomotion of murine skeletal myoblasts is specifically and significantly enhanced on laminin but not on other matrix proteins. One of the major laminin receptors of myoblasts is the alpha 7 beta 1 integrin, which was first described in human MeWo melanoma cells and Rugli glioblastoma cells. In order to investigate and directly test the role of the alpha 7 integrin in cell migration on laminin, we expressed the murine alpha 7B splice variant in human 293 kidney cells and 530 melanoma cells which cannot migrate on laminin and are devoid of endogenous alpha 7. Northern blotting of the transfected cells showed that the alpha 7 mRNA was expressed efficiently, and the protein was detected on the cell surface by immunofluorescence and fluorescence-activated cell sorter analysis. Cell motility measurements by computer-assisted time-lapse videomicroscopy of the alpha 7-transfected cells revealed an 8-10-fold increase in motility on laminin-1 and its E8 fragment, but not on fibronectin. Mock-transfected cells did not migrate significantly of alpha 7-transfected 293 cells through laminin-coated filters in a Boyden chamber assay was significantly enhanced in comparison to mock transfected cells. These findings prove that alpha 7 integrin expression confers a gain of function-motile phenotype to immobile cells and may be responsible for transduction of the laminin-induced cell motility.

Identification and characterization of a novel transcriptional silencer in the human collagen type IV gene COL4A2.

Collagen type IV [alpha 1(IV)2 alpha 2(IV)] is the basic structural component of all basement membranes. The two subunit genes COL4A1 and COL4A2 are found closely linked in the human and murine genomes and are transcribed divergently from a common promoter. Previously, activating elements had been detected within both genes which are indispensable for efficient transcription. An additional negative regulatory element has now been identified within the third intron of the COL4A2 gene which is able to inhibit transcription of both COL4 genes from their shared promoter, as well as the nonrelated herpes simplex virus thymidine kinase promoter. The element exerts its inhibitory effect largely independently from its relative orientation and distance from the initiation site of transcription. Therefore, the element represents a silencer which is named the "COL4 silencer." The minimal functional silencer could be narrowed down by deletion mapping to a sequence element located within intron 3 of the COL4A2 gene. This motif is specifically recognized by a nuclear protein, named "SILBF," and the binding site of which was determined by footprinting assays. Mutation studies and deletion analysis proved that the presence of this sequence element and its interaction with SILBF is not only essential but also sufficient for the silencing function. We assume that the COL4 silencer plays an important role in the control of overall expression and the balance of divergent transcription of both COL4 genes.

Recombinant expression and properties of the Kunitz-type protease-inhibitor module from human type VI collagen alpha 3(VI) chain.

The Kunitz-type inhibitor motif (domain C5) present at the C-terminus of the human collagen alpha 3(VI) chain was prepared in a recombinant form from the culture medium of stably transfected kidney cell clones. The 76-residue protein was disulfide bonded and showed a high stability against protease treatment. The recombinant protein lacked, however, any inhibitory activity for trypsin, thrombin, kallikrein and several other proteases, which could be due to a few unusual substitutions in the region crucial for inhibitor binding. A sensitive radioimmunoassay detected low concentrations of C5 epitopes in normal human serum and fibroblast culture medium and showed a lack of cross-reaction with aprotinin. Antibodies against C5 immunoprecipitated collagen VI obtained from fibroblast medium. The C5 epitopes could not be detected on intact collagen VI purified from guanidine extracts of human placenta. Collagen VI was shown to possess several alpha 3(VI) chain bands (approximately 200 kDa) and reacted strongly with antibodies to an N-terminal recombinant fragment. Immunofluorescence with anti-C5 antibodies failed to stain several human tissues but produced a distinct intracellular staining of cultured fibroblasts. The data indicate the rapid loss of the C5 domain after biosynthesis of collagen VI.

Expression of human collagen type IV genes is regulated by transcriptional and post-transcriptional mechanisms.

The molecules of the basement membrane specific collagen type IV are heterotrimers consisting of two alpha 1(IV) and one alpha 2(IV) polypeptide chains. Comparison of the ratios of transcription by nuclear run-on analysis and mRNAs by RNAse protection assay indicates the involvement of transcriptional as well as post-transcriptional events in the control of overall collagen type IV expression. The relative ratios of transcription of the respective genes COL4A1 and COL4A2 remained near 2:1 in most cells, whereas the ratio of mRNA steady-state levels alpha 1(IV)/alpha 2(IV) varied from 0.3:1 to 1:1 and did not parallel the subunit structure of the protein. Nevertheless, secreted protein shows a 2:1 ratio of the subunit polypeptides. This indicates that post-translational processes during chain selection, aggregation and secretion finally determine the amount of secreted protein.

Regulation of divergent transcription of the genes coding for basement membrane type IV collagen.

The genes coding for the two polypeptide chains, alpha 1(IV) and alpha 2(IV), which form together the molecule of the basement membrane type IV collagen, were found to have a special and unusual genomic arrangement. The two genes are very closely linked, they are transcribed in opposite directions, and they apparently use a common and bidirectional promoter with a length of 127 bp. This region is characterized by a symmetrical arrangement of typical elements and by the palindromic structure of the sequence. In accordance with the symmetry of the promoter itself, a symmetrical organization of sequence motifs (SP1, CCAAT) was also observed in flanking regions. For the promoter and the flanking regions we could detect specific binding of nuclear factors that indicates their involvement in transcriptional activation. This suggests that the intrinsic symmetry of the type IV collagen promoter and its flanking regions may be a structural prerequisite for its bidirectional function. In transient gene expression systems no significant activity of the type IV collagen promoter was observed in either direction. This implies that additional enhancing elements are essential for the efficient and tissue-specific transcription of both type IV collagen genes. The screening for such controlling elements within the alpha 1(IV) and the alpha 2(IV) gene led to the observation that the transcription in direction of the alpha 2(IV) gene is activated by an element located in the first intron of the alpha 2 gene. Its enhancing effect is strictly dependent on the intact genomic structure of this region. Alternation of orientation and distance to the promoter destroys its activity completely. This element, located about 100-600 bp downstream from the start site of alpha 2(IV) transcription, seems to form a synergistically acting unit with the common promoter, essential for transcriptional activity in alpha 2 direction. We have not found additional enhancing elements in other regions of both genes. Explanations for the discrepancy with previous data, which define an enhancing element within the first intron of the alpha 1(IV) gene of mouse, are only speculative at present.