Amelogenin is a cell adhesion protein.

Amelogenin, the major protein component of tooth enamel, is shown to be a cell adhesion protein. Since it had been shown that an amelogenin-containing preparation, Emdogain, possessed cell-adhesive activity, we tested the hypothesis that amelogenin was responsible for cell-adhesive activity. Recombinant amelogenin was found to promote adhesion at less than 15 micro g/60-mm plate and requires divalent cations for activity. While we found that amelogenin does not bind to collagen or heparin under physiological conditions, it was demonstrated previously that amelogenin does bind to hydroxyapatite. The cell-adhesive activity of amelogenin may play a role in development and may provide a partial explanation for the therapeutic effects of Emdogain in periodontal regeneration.

Neutrophil collagenase (MMP-8) is expressed during early development in neural crest cells as well as in adult melanoma cells.

Matrix metalloproteinase-8 (MMP-8) is a neutral metalloproteinase of the fibrillar collagenase family that also includes MMP-1 and MMP-13. In contrast to the other collagenases, MMP-8 has a very limited tissue distribution, thought to be restricted to neutrophils and chondrocytes. In a previous study, we observed MMP-8 expression in human melanoma cells. This observation led us to assess in more detail the expression of MMP-8 in normal and malignant melanocytic cells. We found that MMP-8 was expressed by 11 out of 12 human melanoma cell lines tested and all 10 primary melanomas we examined, but was not expressed by four primary neonatal melanocyte strains. Since melanocytes arise from highly motile neural crest cells, we examined the hypothesis that MMP-8 might be expressed by neural crest cells. RT-PCR analysis of post-implantation mouse embryos indicated the presence of MMP-8 transcripts at E9.5. In situ hybridization and immunohistochemistry of mouse embryos between E9.5-E14.5 demonstrated MMP-8 expression in the surface ectoderm, neural crest cells and chondrocytes. MMP-8 was also detected in neural crest cell migration located in the circumference of the neural tube, branchial arches and the notochord. In addition, MMP-8 expression was observed between the somites, in circumscriptive areas of the developing brain, heart, and eye, and in the interdigital zones of the limbs. In summary, we found MMP-8 to be the first fibrillar collagenase expressed during development. In contrast to its restricted tissue expression post-partum, MMP-8 was present in multiple embryonic tissues, including neural crest cells. The production of MMP-8 by migrating neural crest cells may contribute to their ability to degrade fibrillar collagen matrices while in transit.

Overview of expression of matrix metalloproteinases (MMP-17, MMP-18, and MMP-20) in cultured human cells.

We recently described the cell type distribution of several matrix metalloproteinases (MMP-1 through MMP-16). In this report we extend this study by analysis of three recently described MMPs. PCR primers for MMP-17, MMP-18, and MMP-20 were optimized for use in RT-PCR. The results demonstrate one or more cell lines or tissue that express mRNA for each of these newly described MMPs.

Extraction of RNA from single frozen sections.

The sensitivity of the reverse transcriptase-polymerase chain reaction (RT-PCR) makes it ideally suited for the detection of changes in gene expression. Unfortunately, traditional methods for RNA isolation require time-consuming procedures that are not appropriate for small samples, such as individual frozen sections. This report describes a new technique that permits the rapid extraction of RNA from individual frozen histological sections. RNA is extracted by incubating a frozen section in an RT-PCR compatible buffer solution containing RNase inhibitor and dithiothreitol. RNA isolated from frozen sections is stable at room temperature for up to 3 h under the conditions described. Alternatively, extracts can be frozen for later use. When maintained in a dry state at room temperature, RNA in sections remained stable for 2 weeks. Histological, immunohistochemical, or in situ analyses can be carried out with sections that are immediately adjacent to those used for extracting RNA. The simplicity and economy of this procedure may foster the development of prognostic screens that can be performed in parallel with traditional histopathological analysis.

Overview of matrix metalloproteinase expression in cultured human cells.

The matrix metalloproteinases (MMP) have been implicated in tumor invasion and metastasis both by immunohistochemical studies and from the observation that specific metalloproteinase inhibitors block tumor invasion and metastasis. Oligonucleotide primers for thirteen MMPs (MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-16) were optimized for use in RT-PCR. A semi-quantitative RT-PCR assay was used to determine the pattern of MMP mRNA expression in 84 normal and transformed or carcinogen transformed human cell lines and strains derived from different tissues. The results demonstrate one or more cell lines which express thirteen members of the MMP family. In addition, various oncogene transfected human fibroblast cell strains were analyzed for MMP expression. We confirm that over-expression of the H-ras oncoprotein correlates with up-regulation of MMP-9 and demonstrate that over-expression of v-sis also up-regulates MMP-9. A cell line immortalized following myc expression was found to up-regulate MMP-7, MMP-11 and MMP-13. Inappropriate expression of several MMP mRNAs was detected in breast, prostate, bone, colon and oral tumor derived cell lines. Identification of at least one cell line expressing each of thirteen MMPs and the observation of oncogene induced expression of several MMPs should facilitate analysis of the transcriptional mechanisms controlling each MMP.

Affinity chromatography of RNase inhibitor.

Of the reagents used in reverse transcription-PCR (RT-PCR), RNase Inhibitor is the most costly on a per assay basis. A simple method for purification of RNase Inhibitor from bovine liver is described. Approximately 40,000 units of RNase Inhibitor can be purified to homogeneity from 400 g of bovine liver within two days using inexpensive reagents. The method described employs (a) facile procedures to rapidly obtain a clarified liver extract, (b) affinity chromatography of RNase Inhibitor on RNase-A-Sepharose, and (c) batchwise concentration of the purified protein with a molecular sieve resin.

Use of oxygen-permeable silicone rubber pouches for growing mass cultures of bacteria.

Growth of most bacteria often involves the use of expensive incubated shaker systems. In this report, oxygen-permeable silicone rubber pouches, with oxygen permeability over 100 times higher than other polymers, were employed for growing bacterial cultures. With little, if any, agitation oxygen-permeable silicone rubber pouches produced bacterial growth rates equivalent to growth rates obtained in shaker flasks. The silicone rubber pouch described has a glass cuvette integrated into its design that permits readings of bacterial density without opening the pouch. One can sterilize and store powdered bacterial culture medium in silicone rubber pouches; therefore, bacterial cultures can be initiated by simply adding water and bacteria.

RT-PCR without RNA isolation.

Reverse transcription-PCR (RT-PCR) has traditionally required time-consuming RNA extraction and purification. This report demonstrates that one can completely avoid the RNA extraction step in RT-PCR by basing the comparison of samples on cell number rather than micrograms of total RNA. A new method for lysing cells while preserving RNA is described. RT-PCR is carried out (i) by rapidly freezing cells in the presence of ribonuclease inhibitor (RNase inhibitor) plus dithiothreitol and (ii) by using extracts of 250 or fewer cells directly in the RT-PCR assay. Aldolase mRNA, extracted by freeze-thawing cells in the presence of RNase inhibitor, was found to be stable at 42 degrees C for over three hours. Since the RT step can be completed within 1 h, there is minimal degradation of mRNA. This simple procedure avoids the use of harsh reagents, which may inhibit enzymes involved in RT-PCR, and produces results virtually identical to methods that employ guanidinium thiocyanate and phenol for RNA extraction. Optimized conditions for each parameter of the procedure are described that permit amplification of mRNA from as few as four cells.

Chondrocyte cultures express matrix metalloproteinase mRNA and immunoreactive protein; stromelysin-1 and 72 kDa gelatinase are localized in extracellular matrix vesicles.

Previous studies have shown that costochondral cartilage cell cultures produce extracellular matrix vesicles which contain metalloproteinase activity. In the present study, we examined whether two matrix metalloproteinases (MMPs) known to be present in cartilage, stromelysin-1 and 72 kDa gelatinase, are expressed by fourth passage resting zone and growth zone costochondral chondrocytes and whether they are specifically incorporated into matrix vesicles produced by the cells. We also examined whether the cells synthesize tissue inhibitor of metalloproteinase-1 and -2 (TIMP-1 and TIMP-2). Oligonucleotide primers for stromelysin-1, 72 kDa gelatinase, tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2), and GAPDH were synthesized and optimized for use in the reverse transcription-polymerase chain reaction (RT-PCR). It was found that both resting zone and growth zone chondrocytes produced mRNA for both MMPs and the two TIMPs. Further, immunostaining of cell layers with antibodies to 72 kDa gelatinase and stromelysin-1 showed that both cell types produced these MMPs in culture. Substrate gel electrophoresis and Western analysis were used to characterize MMP activity in matrix vesicles, media vesicles, or plasma membranes as well as in conditioned media produced by the chondrocyte cultures. It was found that matrix vesicles but not plasma membranes or media vesicles were selectively enriched in stromelysin-1. Also, 72 kDa gelatinase was found in matrix vesicles, but to a lesser extent than seen in media vesicles. The relative activity of each enzyme detected was cell maturation-dependent. No MMP activity was detected in conditioned media produced by either cell type. The results of this study show that MMPs are expressed by resting zone and growth zone chondrocytes in culture and differentially distributed among three different membrane compartments. This suggests that, in addition to the well-known activators and inhibitors of MMP activity in the matrix, differential membrane distribution may enable more precise control over the site, rate, and extent of matrix degradation by the cell.

Regulation of matrix metalloproteinases following cellular transformation.

During progression towards malignancy, many tumor cells display changes in their repertoire of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs). The recent finding that many members of the MMPs are regulated by protooncogenes may explain the frequent observation of changes in MMP gene expression during progression of many tumor types. While studies involving enzymatic assays of MMPs are usually confined to one or a few MMPs, reverse transcription-PCR (RT-PCR) permitted the analysis of seven members of the MMP family and two members of the TIMP family in several normal and transformed cell lines. RT-PCR permitted us to confirm the observation that MMP-9 is activated following transformation and also to observe the previously unreported activation of MMP-7 in SV40-transformed cells. It has previously been found that MMP-1, -2, -3, -8, and -9 are upregulated by phorbol esters; we have found that MMP-10 is also upregulated by phorbol esters. The phorbol ester upregulation of MMP-1, -3, and -10 was found to be abolished in cells transformed by SV40 virus. Several studies have shown that MMP-1 is upregulated by an integrin-mediated signal transduction pathway. This study demonstrates that MMP-3 and MMP-10 are also regulated by integrin-mediated signal transduction and that upregulation by this pathway is abolished following SV40 transformation. In summary, the more global view of MMP expression afforded by RT-PCR indicates that MMP-1, -3, and -10 are regulated by both integrin-mediated signal transduction and phorbol esters. While fibroblasts and transformed bone cells express several members of the MMP gene family, several other cell types do not express MMPs.

Cyclic-AMP deficient MDCK cells form tubules.

It has been known for many years that MDCK cells form blister-like structures, termed domes. During an examination of the morphology of a large number of MDCK clones, we found that two stable morphotypes exist in an MDCK cell population-namely, dome-forming and tubule-forming clones. When maintained at high cell density, tubule-forming clones displayed large numbers of anastomosing tubules which contained lumens. The frequency of observation of the tubule-forming clones in an MDCK population was 0.7%. Tubule-forming MDCK clones should be useful in studying tubule morphogenesis. While agents that affect protein kinase A activity increased dome formation, the same agents abolished the formation of tubules in all tubule-forming clones. In contrast, drugs that stimulate protein kinase C activity (phorbol esters and staurosporine) decreased dome formation an increased tubule morphogenesis in all MDCK morphotypes. Tubule-forming clones were found to have lower resting levels of cyclic-AMP and to respond to forskolin stimulation of adenylate cyclase less readily. Hence, signals transmitted by the protein kinase C pathway appear to lead to tubule formation in MDCK cells, while signals transmitted through the protein kinase A pathway lead to dome formation.

Growth factor effects on the expression of collagenase and TIMP-1 in periodontal ligament cells.

The fibroblast is a prominent cellular component of the periodontal ligament. It is believed to play an important role in collagen metabolism in health and disease. The turnover of collagen in the periodontal ligament is believed to be controlled by the balance between collagen synthesis and degradation. The family of matrix metalloproteinases and their inhibitors is one of the mechanisms which regulates this balance. The factors that regulate the synthesis of collagenase and its inhibitor, TIMP-1, by the periodontal ligament cell are poorly understood. The present study was undertaken to assess the effect of interleukin-1 beta (IL-1 beta), platelet-derived growth factor (PDGF), and transforming growth factor-beta 1 (TGF-beta) on the expression of collagenase (MMP-1) and TIMP-1 mRNA in periodontal derived fibroblasts using reverse transcription polymerase chain reaction (RT-PCR). Early passage periodontal ligament derived fibroblasts were treated with IL-1 beta (10 and 100 pg/ml), two isoforms of PDGF, -AA and -BB (4 and 20 ng/ml) and TGF-beta (1 and 10 ng/ml). Treatment with growth factors from 2 to 24 hours revealed that the largest effects on MMP-1 mRNA occurred after 24 hours. IL-1 beta induced a 5 to 9 fold increase in MMP-1 mRNA. The two isoforms of PDGF had less of an effect (3 to 5 fold) on MMP-1 mRNA whereas TGF-beta induced a 25 to 50% decrease in the expression of this message. None of the growth factors had an effect on TIMP-1 mRNA expression.(ABSTRACT TRUNCATED AT 250 WORDS)

A heat-shock-like response with cytoskeletal disruption occurs following hydrostatic pressure in MG-63 osteosarcoma cells.

Human osteosarcoma cells, MG-63, were exposed to a hydrostatic pressure shock of 4.0 MPa for 20 min. Changes in subcellular distribution of the cytoskeletal elements and heat shock protein 70 (hsp70) were followed by indirect immunofluorescence and by avidin-biotin-peroxidase protocols. During recovery, total cellular RNA was determined and actin and aldolase mRNA content was followed using reverse transcription-polymerase chain reaction techniques. Hydrostatic pressure caused cell rounding (but not cell death), disruption of microtubules, collapse of intermediate filaments to a perinuclear location, collapse of actin stress fibers into globular aggregates in the cytoplasm, and the formation of several large elongated intranuclear actin inclusions. During recovery, the cells flattened, reorganized microtubules, and redistributed intermediate filaments prior to the reappearance of actin stress fibers. At 20 and 60 min following the initiation of hydrostatic pressure, there was increased anti-hsp 70 staining at the nuclear membrane and concentration of hsp70 in four to six granules in the nucleus. At 120 min following the hydrostatic pressure, hsp70 showed intense staining in the cytoplasm and hsp70-containing granules in the nucleus disappeared. Cellular RNA decreased during the first 120-min posthydrostatic pressure shock and then recovered to near prehydrostatic pressure treatment levels by 240 min. Actin mRNA abundance, in relation to aldolase mRNA abundance, showed the same temporal pattern of initial decrease, followed by increase as did total RNA. Review of the literature indicated that eukaryotic cells respond to heat shock and to hydrostatic pressure by disruption of the cytoskeletal elements and by similar modifications in genetic expression. In this study, the observed responses of MG-63 cells to a 4-MPa hydrostatic pressure shock was like the reported response of mammalian cells to a 43 degrees C heat shock.

Regulation of integrin gene expression by substrate adherence.

Under substrate adherent conditions, integrin gene expression can be regulated by transforming growth factor-beta, interleukin-1 beta, and prostaglandins. This report demonstrates a new mechanism that can differentially control the expression of several integrins. When MG-63 osteosarcoma cells are maintained in suspension, up-regulation of several integrin alpha-subunits takes place. Within as little as 4 h, the mRNA levels for both the alpha 2- and alpha 4-subunits are increased 4- and 6-fold, respectively. It was found that mRNA levels for the alpha 2-, alpha 4-, and alpha v-subunits were markedly increased in several differentiated cell lines under nonadherent conditions; however, cells that did not express a given integrin under substrate adherent conditions also did not express this integrin when maintained in suspension. The alpha 5-subunit did not upregulate during suspension growth. By immunocytochemistry, changes in integrin mRNA levels were confirmed at the protein level. Both cytochalasin B and a phorbol ester were found to induce the expression of the alpha 2-subunit, but not the alpha 4- and alpha 5-subunits, in a dose-dependent fashion. Many investigators have documented changes in gene expression that result from changes in "cell shape." These phenomena may result from up-regulation of integrin gene expression induced by the lack of substrate adherence.

Alpha 5 integrin subunit expression changes during myogenesis.

Fibronectin and its cellular receptor, the alpha 5 beta 1 integrin, are involved in the transmembrane signalling events that control muscle cell differentiation. In this study, the expression of the alpha 5 integrin subunit was followed by reverse transcription-polymerase chain reaction (RT-PCR) to determine alterations during myogenesis. In studies of murine muscle, we found a 90% reduction in the level of the alpha 5 integrin subunit mRNA during early postnatal development. Concurrently, the fibronectin alternative splicing pattern changed markedly in the EIIIB and V exons. In-vitro analyses of these molecules during myoblast differentiation revealed changes that followed trends similar to those observed in vivo, although of lesser magnitude. These observations imply an important role of fibronectin and the alpha 5 integrin subunit in muscle development.