Major internal nuclear matrix proteins are common to different human cell types

Cancer invasion and metastasis are associated with matrix degradation. We describe a novel in vivo model of invasion by squamous epithelial neoplastic cells derived from transgenic mice grown on acellular human dermis. Human dermis was subjected to multiple freeze-thaw cycles to render it acellular, maintaining the basement membrane of the former dermal-epidermal junction. Cells representing discrete stages of a multistep transgenic mouse model of epidermal carcinogenesis (neonatal transgenic keratinocytes, moderately/poorly differentiated squamous cell carcinoma, and lymph node metastasis) were seeded onto the basement membrane surface, grown in culture for 4 days, grafted in a subpannicular pocket of athymic mice, and harvested after 3 weeks. Histological analysis demonstrated that neonatal transgenic keratinocytes did not degrade the basement membrane or invade the underlying dermis. In contrast, malignant cells derived from both a moderately differentiated squamous carcinoma and a lymph node metastasis were highly invasive. Immunohistochemical analysis revealed collagenase only in nests of invading malignant cells in contact with the dermal matrix, but not in the tumor mass remaining above the basement membrane, suggesting that this proteinase may be required for stromal invasion. This novel model recapitulates the events seen in malignant invasion: transgenic keratinocytes are unable to penetrate the dermis while cells from a moderately differentiated carcinoma and from lymph node metastasis consistently invade.     

Common nuclear matrix proteins in rat tissues

Nuclear matrix proteins have been defined as insoluble residual proteins resulting from treatment of isolated nuclei with nucleases, detergents and high ionic strength buffers. They are considered as in part representing the proteins constituting the three-dimensional framework of the interphase nucleus. Though cell-specific nuclear matrix proteins have been differentiated from ubiquitously occurring (common) nuclear matrix proteins, the number and types of common nuclear matrix proteins have not yet been unequivocally established. In the present study nuclear matrix proteins were prepared from isolated nuclei of rat kidney, liver, lung, spleen and testes. The matrix proteins were separated by two-dimensional (2-D) electrophoresis and silver stained. Then the spot patterns were compared by computer-assisted image analysis. Composite images were derived for nuclear matrix proteins of individual tissues. Finding between 396-483 spots per tissue, a total of 964 individual spots were registered. Of these, 102 were common nuclear matrix proteins, as appearing in each of the tissue-characteristic images. The apparent molecular mass and pI data may serve for further identification of these nuclear proteins.     

The major immediate-early proteins IE1 and IE2 of human cytomegalovirus colocalize with and disrupt PML-associated nuclear bodies at very early times in infected permissive cells

The major immediate-early (MIE) gene products of human cytomegalovirus (HCMV) are nuclear phosphoproteins that are thought to play key roles in initiating lytic cycle gene regulation pathways. We have examined the intranuclear localization pattern of both the IE1 and IE2 proteins in virus-infected and DNA-transfected cells. When HCMV-infected human diploid fibroblast (HF) cells were stained with specific monoclonal antibodies, IE1 localized as a mixture of nuclear diffuse and punctate patterns at very early times (2 h) but changed to an exclusively nuclear diffuse pattern at later times. In contrast, IE2 was distributed predominantly in nuclear punctate structures continuously from 2 to at least 12 h after infection. These punctate structures resembled the preexisting PML-associated nuclear bodies (ND10 or PML oncogenic domains [PODs]) that are disrupted and dispersed by the IE110 protein as a very early event in herpes simplex virus (HSV) infection. However, HCMV differed from HSV by leading instead to a change in both the PML and SP100 protein distribution from punctate bodies to uniform diffuse patterns, a process that was complete in 50% of the cells at 2 h and in 90% of the cells by 4 h after infection. Confocal double-label indirect immunofluorescence assay analysis confirmed that both IE1 and IE2 colocalized transiently with PML in punctate bodies at very early times after infection. In transient expression assays, introduction of IE1-encoding plasmid DNA alone into Vero or HF cells produced the typical total redistribution of PML into a uniform nuclear diffuse pattern together with the IE1 protein, whereas introduction of IE2-encoding plasmid DNA alone resulted in stable colocalization of the IE2 protein with PML in the PODs. A truncated mutant form of IE1 gave large nuclear aggregates and failed to redistribute PML, and similarly a deleted mutant form of IE2 failed to colocalize with the punctate PML bodies, confirming the specificity of these effects. Furthermore, both Vero and U373 cell lines constitutively expressing IE1 also showed total PML relocalization together with the IE1 protein into a nuclear diffuse pattern, although a very small percentage of the cells which failed to express IE1 reverted to a punctate PML pattern. Finally, the PML redistribution activity of IE1 and the direct association of IE2 with PML punctate bodies were both confirmed by infection with E1A-negative recombinant adenovirus vectors expressing either IE1 or IE2 alone. These results confirm that transient colocalization with and disruption of PML-associated nuclear bodies by IE1 and continuous targeting to PML-associated nuclear bodies by IE2 are intrinsic properties of these two MIE regulatory proteins, which we suggest may represent critical initial events for efficient lytic cycle infection by HCMV.     

Different human tenascin-C variants in the extracellular matrix of cultured human fibroblasts

Using an immunoadsorbent prepared with a monoclonal antibody specific for the high molecular mass isoform of human tenascin-C, we purified tenascin-C molecules containing at least one large subunit from the extracellular matrix of cultured normal human fibroblasts. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis and immunoblotting analyses have shown that both high and low molecular mass subunits are present in these tenascin-C preparations. Because the monoclonal antibody used is able to bind only the high molecular mass isoform, the present data show that part of the tenascin-C present in the fibroblast extracellular matrix is made up of heterohexameric molecules.     

Localization of proteins to the Golgi apparatus

A case-control study was performed on 9,175 Italian adult outpatients in 5 hospitals in Rome. The study was carried out to clarify the role of some less investigated risk factors (RF) in the spread of hepatitis C virus (HCV) infection. All subjects were contacted by interviewers, who completed a questionnaire. Their sera were stored and subsequently tested for both HCV and hepatitis B virus core (HBc) antibodies. 365 subjects, positive for anti-HCV and anti-HBc-negative, and who had denied intravenous drug use (IDU) (cases) were compared with an equal number of suitable random controls negative for anti-HCV and anti-HBc. Gender, age and region of birth and residence were matched. The prevalence of 13 RFs were statistically compared by univariate and multivariate analysis. A positive anti-HCV test was significantly associated, by multivariate analysis with intravenous treatments and minor surgical procedures (both before 1975) (p < 0.001), blood transfusions (before 1991) (p < 0.01), diabetes (p < 0.01), and deliveries in hospital (p < 0.05) (both before 1975). After 1975 (1991 for transfusions), all associations lost their significance. Intra-familial (sexual and non sexual), occupational RFs and dental care were not significantly associated with the presence of anti-HCV. We suggest that non-disposable syringes, commonly used until 1975 in Italy for i.v. treatments, have been the major route for HCV transmission in Italy among non-IDU subjects.     

Comparative analysis of the virion structural proteins of 3 nuclear polyhedrosis viruses

The composition of structural proteins of virions of nuclear polyhedrosis viruses (NPV) of Barathra brassicae L., Lymantria dispor L. and Aporia crataegi L. was studied by polyacrylamide gel-SDS electrophoresis (PAGE-SDS). The object of the study included fractions of virion bundles in the envelope. All the baculoviruses under study were shown to have in virion structural proteins 23-24 polypeptides with molecular weights ranging from 12500 to 95000 daltons. Alongside with polypeptides of similar molecular weight, each baculovirus was shown to have a specific set of structural proteins allowing them to be readily identified. Specific distribution of major polypeptides of the virions was found. All the protein fractions were readily reproducible in repeated analyses with the exception of two minor polypeptides of NPV of Barathra brassicae L. The study showed that the composition of structural proteins of virions may be an important criterion for identification of baculoviruses under identical electrophoresis conditions.     

A rapid and sensitive radioimmunoassay for the detection of human cytomegalovirus binding and infection of human fibroblasts

A rapid and sensitive radioimmunoassay for the quantitation of HCMV binding and infection of human fibroblasts (HFF) was developed. The protocol involves the use of a monoclonal antibody (27-156) reactive with HCMV gB (alpha-gB), followed by an 125I-labeled second antibody to mouse IgG. Antibody to gB bound specifically to HFF inoculated with HCMV when compared to sham inoculated cells or cells inoculated with HSV (strain KOS). Antibody to gB also bound to HFF infected with HCMV 48 h prior to assay. The binding of antibody to HFF inoculated with HCMV was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Moreover, antibody binding was directly dependent on the concentration of the virus inoculum, using either conventional viral preparations or gradient purified HCMV. The binding of antibody to HFF inoculated with HCMV at 4 degrees C was found to be dependent on antibody concentration and to demonstrate saturable kinetics. Displacement of HCMV binding to HFF with the proteoglycan heparin sulfate could be detected, thus allowing for competitive binding studies. This binding assay allows for the relative quantitation of HCMV binding to cells and will be useful for examining the early events of cell-viral interactions.     

Ceramides are bound to structural proteins of the human foreskin epidermal cornified cell envelope

An important component of barrier function in human epidermis is contributed by ceramides that are bound by ester linkages to undefined proteins of the cornified cell envelope (CE). In this paper, we have examined the protein targets for the ceramide attachment. By partial saponification of isolated foreskin epidermal CEs followed by limited proteolysis, we have recovered several lipopeptides. Biochemical and mass spectroscopic characterization revealed that all contained near stoichiometric amounts of ceramides of masses ranging from about 690 to 890 atomic mass units, of which six quantitatively major species were common. The array of ceramides was similar to that obtained from pig skin, the composition of which is known, thereby providing strong indirect data for their fatty acid and sphingosine compositions. The recovered peptides accounted for about 20% of the total foreskin CE ceramides. By amino acid sequencing, about 35% of the peptides were derived from ancestral glutamine-glutamate-rich regions of involucrin, an important CE structural protein. Another 18% derived from rod domain sequences of periplakin and envoplakin, which are also known or suspected CE proteins. Other peptides were too short for unequivocal identification. Together, these data indicate that involucrin, envoplakin, periplakin, and possibly other structural proteins serve as substrates for the attachment of ceramides by ester linkages to the CE for barrier function in human epidermis.     

hnRNP proteins and B23 are the major proteins of the internal nuclear matrix of HeLa S3 cells

The nuclear matrix is the structure that persists after removal of chromatin and loosely bound components from the nucleus. It consists of a peripheral lamina-pore complex and an intricate internal fibrogranular structure. Little is known about the molecular structure of this proteinaceous internal network. Our aim is to identify the major proteins of the internal nuclear matrix of HeLa 53 cells. To this end, a cell fraction containing the internal fibrogranular structure was compared with one from which this structure had been selectively dissociated. Protein compositions were quantitatively analyzed after high-resolution two-dimensional gel electrophoresis. We have identified the 21 most abundant polypeptides that are present exclusively in the internal nuclear matrix. Sixteen of these proteins are heterogeneous nuclear ribonucleoprotein (hnRNP) proteins. B23 (numatrin) is another abundant protein of the internal nuclear matrix. Our results show that most of the quantitatively major polypeptides of the internal nuclear matrix are proteins involved in RNA metabolism, including packaging and transport of RNA.     

Distribution of cytoskeletal proteins in neomycin-induced protrusions of human fibroblasts

The organization of actin, tubulin, and vimentin was studied in protruding lamellae of human fibroblasts induced by the aminoglycoside antibiotic neomycin, an inhibitor of the phosphatidylinositol cycle. Neomycin stimulates the simultaneous protrusion of lamellae in all treated cells, and the lamellae remain extended for about 15-20 min, before gradually withdrawing. The pattern and distribution of actin, tubulin, and vimentin during neomycin stimulation were analyzed by fluorescence and electron microscopy. F-actin in the newly formed lamellae is localized in a marginal band at the leading edge. Tubulin is colocalized with F-actin in the marginal band, but the newly formed lamellae are initially devoid of microtubules. Over a period of 10 to 20 min after the addition of neomycin, microtubules grow into the lamellae from the adjacent cytoplasm, while the intensity of tubulin staining of the marginal band decreases. Distribution of vimentin remains unchanged in neomycin-treated cells and vimentin filaments do not enter the new protrusions. Treatment of cells with colchicine and Taxol do not inhibit neomycin-induced protrusion but protrusions are no longer localized at the ends of cell processes and occur all around the cell periphery. We conclude that actin filaments are the major component of the cytoskeleton involved in generating protrusions. Microtubules and, possibly, intermediate filaments control the pattern of protrusions by their interaction with actin filaments.     

Immunocytochemical evaluation of protein kinase C translocation to the inner nuclear matrix in 3T3 mouse fibroblasts after IGF-I treatment

The complex pathway which links the agonist-cell membrane receptor binding to the response at the genome level involves, among other elements, protein kinase C (PKC). Agonists acting at the cell membrane can affect an autonomous nuclear polyphosphoinositide signaling system inducing an activation of nuclear phosphoinositidase activity and a subsequent translocation of PKC to the nuclear region. The fine localization of PKC has been investigated by means of electron microscopy quantitative immunogold labeling in 3T3 mouse fibroblasts, mitogenically stimulated by IGF-I. The enzyme, which in untreated cells is present in the cytoplasm, except for the organelles, and in the nucleoplasm, after IGF-I treatment is reduced in the cytoplasm and almost doubled in the nucleus. The PKC isoform translocated to the nucleus is the alpha isozyme, which is found not only associated with the nuclear envelope but mainly with the interchromatin domains. By using in situ matrix preparations, PKC appears to be retained at the nuclear matrix level, both at the nuclear lamina and at the inner nuclear matrix, suggesting a direct involvement in the phosphorylation of nuclear proteins which are responsible for the regulation of DNA replication.     

Boron modulates extracellular matrix and TNF alpha synthesis in human fibroblasts

Boric acid was not mitogenic for human fibroblasts and it did not change cell viability until 0.5% (w/v). Boric acid treatment affected the metabolism of human dermal fibroblasts in culture, decreasing the synthesis of extracellular matrix macromolecules such as proteoglycans, collagen, and total proteins. It also increased the release of these molecules into the culture medium. The principal proteins secreted into the medium after boric acid treatment had molecular masses of 90, 70, 58, 49, and 43 kDa and faint bands were detected by electrophoresis between 14 and 30 kDa. hsp 70 and TNF alpha were detected among the secreted proteins by immunoblotting, and the amount of TNF alpha released was quantified by radioimmunoassay. Total mRNA levels were higher after boric acid treatment and peaked after 6 h of treatment. TNF alpha mRNA was undetectable in unstimulated fibroblasts and two TNF alpha mRNA bands were detected after stimulation: immature mRNA (4.8 kb) and mature TNF alpha mRNA (1.9 kb). Thus, the effects of boric acid observed in wound repair in vivo may be due to TNF alpha synthesis and secretion.     

Selected nuclear matrix proteins are targets for poly(ADP-ribose)-binding

Recent evidence suggests that poly(ADP-ribose) may take part in DNA strand break signalling due to its ability to interact with and affect the function of specific target proteins. Using a poly(ADP-ribose) blot assay, we have found that several nuclear matrix proteins from human and murine cells bind ADP-ribose polymers with high affinity. The binding was observed regardless of the procedure used to isolate nuclear matrices, and it proved resistant to high salt concentrations. In murine lymphoma LY-cell cultures, the spontaneous appearance of radiosensitive LY-S sublines was associated with a loss of poly(ADP-ribose)-binding of several nuclear matrix proteins. Because of the importance of the nuclear matrix in DNA processing reactions, the targeting of matrix proteins could be an important aspect of DNA damage signalling via the poly ADP-ribosylation system.     

Mechanical forces alter extracellular matrix synthesis by human periodontal ligament fibroblasts

Periodontal ligament fibroblasts (PDLFs) are a heterogeneous population of cells that are involved in the normal maintenance, repair and regeneration of both the ligament and adjacent hard tissues. Additionally, the ability of these cells to respond to mechanical stimulation suggests that they have a central role in mediating the osseous remodeling that underlies physiological and orthodontic tooth movement. To characterize their role further in this process, the current study evaluated the effect of tensional stress on the biosynthesis of extracellular matrix (ECM) proteins by human PDLFs. Cell strains were established from extracted human premolars and third molars. Cells exposed to 5% biaxial deformation (strain) at a frequency of 30 times/min for 24 hr exhibited statistically significant increases in type I collagen and fibronectin synthesis, and a statistically significant decrease in tropoelastin production relative to unstretched controls. Cells exposed to 10% strain exhibited similar responses for fibronectin and tropoelastin while the amount of type I collagen synthesized by stretched cells did not differ from control levels. These results indicate that mechanical stimulation of PDLFs alters type I collagen, tropoelastin and fibronectin production and that these cells are differentially responsive to varying levels of mechanical stress. The ability of these cells to alter ECM protein synthesis in response to specific magnitudes of tensional stress may in part explain how PDLFs regulate ligament and hard tissue remodeling.     

Interactions of rat repetitive sequence MspI8 with nuclear matrix proteins during spermatogenesis

Using the Southwestern blot analysis we have studied the interactions between rat repetitive sequence MspI8 and the nuclear matrix proteins of rat testis cells. Starting from 2 weeks the young to adult animals showed differences in type of testis nuclear matrix proteins recognizing the MspI8 sequence. The same sets of nuclear matrix proteins were detected in some fractions enriched in spermatocytes and spermatides and obtained after fractionation of testis cells of adult animals by the velocity sedimentation technique.     

Studies in vitro on the role of alpha v and beta 1 integrins in the adhesion of human dermal fibroblasts to provisional matrix proteins fibronectin, vitronectin, and fibrinogen

Fibroblasts that migrate into a wound during the early stages of repair use cell surface integrins to interact with extracellular molecules as they move away from the interstitial matrix of normal tissue and into the provisional matrix of the wound. Therefore, to understand a critical phase of wound healing, it is necessary to understand the details of integrin involvement. Normal adult human dermal fibroblasts in culture express many receptors for the provisional matrix proteins fibronectin, vitronectin, and fibrinogen, including the integrins alpha 3 beta 1, alpha 4 beta 1, alpha 5 beta 1, alpha v beta 1, alpha v beta 3, and alpha v beta 5. We used quantitative flow cytometry to estimate the relative numbers of these receptors and immunoprecipitation to confirm the expression of alpha v beta 1. Adult human dermal fibroblasts primarily use beta 1 integrins, alpha 4 beta 1, alpha 5 beta 1, and possibly alpha v beta 1, for attachment to fibronectin. alpha v beta 3 and perhaps other integrins containing the alpha v subunit serve fibroblasts as secondary or auxiliary receptors for fibronectin. In contrast, these cells use alpha v integrins but probably not beta 1 integrins for attachment to vitronectin. alpha v beta 3 and alpha v beta 5 apparently act in concert to mediate attachment to vitronectin, and these two integrins may perform different functions during wound repair. Fibroblast adhesion to certain preparations of fibrinogen occurs, at least partially, through the small amount of fibronectin present in the preparations. Fibroblast attachment to fibrinogen purified free of fibronectin also occurs, and that was demonstrated with a sensitive new assay called electrical cell-substrate impedance sensing. Fibroblast attachment to pure fibrinogen can be inhibited by RGD peptide, suggesting that integrins are involved.     

Bacteria inhibit biosynthesis of bone matrix proteins in human osteoblasts

The effect of extracts from Staphylococcus aureus and Staphylococcus epidermidis on bone matrix production were assessed by analyzing the biosynthesis of osteocalcin and Type I collagen in a human osteoblastic osteosarcoma cell line (MG-63). In MG-63 cells, extracts from Staphylococcus aureus and Staphylococcus epidermidis decreased 1,25(OH)2-vitamin D3 stimulated osteocalcin biosynthesis, and insulin-like growth factor I induced production of Type I collagen in a concentration dependent manner. The basal rate of osteocalcin and Type I collagen formation was unaffected by the bacterial extracts. The inhibitory effect of the bacteria on osteocalcin biosynthesis was seen after 24 hours of treatment and was maintained for at least 96 hours. The extracts of Staphylococcus aureus and Staphylococcus epidermidis enhanced prostaglandin E2 formation in the MG-63 cells. Abolition of the prostaglandin E2 response by treatment with indomethacin and flurbiprofen did not affect bacteria induced inhibition of osteocalcin production. Stimulation of osteocalcin biosynthesis by 1,25(OH)2-vitamin D3 was associated with a decreased rate of cell proliferation. The inhibitory action of the bacterial extracts was not linked to any inhibition of [3H]-thymidine incorporation into deoxyribonucleic acid. These data show that extracts of Staphylococcus aureus and Staphylococcus epidermidis have the ability to inhibit the biosynthesis of bone matrix proteins by a nonprostaglandin and noncytotoxic dependent mechanism and suggest that bone loss in inflammatory processes containing Staphylococcus aureus or Staphylococcus epidermidis may not be caused only by enhanced bone resorption but also by decreased bone formation.     

Calmodulin antagonists increase the expression of membrane-type-1 matrix metalloproteinase in human uterine cervical fibroblasts

The treatment of human uterine cervical fibroblasts with concanavalin A (ConA), or a specific calmodulin antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) or trifluoperazine resulted in accumulation of an active form of matrix metalloproteinase 2 (MMP-2, gelatinase A). In contrast, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), a weaker antagonist of calmodulin, did not modulate the activation of proMMP-2. The activation of proMMP-2 was confirmed by the enhanced activity on gelatin and the conversion of proMMP-2 to a 62-kDa form by zymography and western blotting. The plasma membrane, but not the conditioned medium, of the W-7- or trifluoperazine-treated cells activated proMMP-2; this activation was blocked by membrane-type-1 MMP (MT1-MMP) antibody and EDTA. The plasma membrane from trifluoperazine- or ConA-treated cells contained MT1-MMP and tissue inhibitor of metalloproteinases 2. Both trifluoperazine treatment and ConA treatment increased the steady-state levels of MT1-MMP mRNA and proMMP-2 mRNA. These results, together with our previous observations on the production of proMMP-1 (interstitial procollagenase) and proMMP-3 (prostromelysin 1) [Ito, A., Sato, T., Ojima, Y., Chen, L.-C., Nagase, H. & Mori, Y. (1991) J. Biol. Chem. 266, 13598-13601], suggest that calmodulin negatively regulates the matrix turnover by suppressing the production of a number of proMMPs including proMMP-1, proMMP-3 and MT1-MMP, and the activation of proMMP-2 in human uterine cervical fibroblasts.