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1.
Oncogene ; 28(40): 3526-36, 2009 Oct 08.
Article in English | MEDLINE | ID: mdl-19617899

ABSTRACT

Growing evidence indicates that microRNAs have a significant role in tumor development and may constitute robust biomarkers for cancer diagnosis and prognosis. In this study, we evaluated the clinical and functional relevance of microRNA-122 (miR-122) expression in human hepatocellular carcinoma (HCC). We report that miR-122 is specifically repressed in a subset of primary tumors that are characterized by poor prognosis. We further show that the loss of miR-122 expression in tumor cells segregates with specific gene expression profiles linked to cancer progression, namely the suppression of hepatic phenotype and the acquisition of invasive properties. We identify liver-enriched transcription factors as central regulatory molecules in the gene networks associated with loss of miR-122, and provide evidence suggesting that miR-122 is under the transcriptional control of HNF1A, HNF3A and HNF3B. We further show that loss of miR-122 results in an increase of cell migration and invasion and that restoration of miR-122 reverses this phenotype. In conclusion, miR-122 is a marker of hepatocyte-specific differentiation and an important determinant in the control of cell migration and invasion. From a clinical point of view, our study emphasizes miR-122 as a diagnostic and prognostic marker for HCC progression.


Subject(s)
Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , MicroRNAs/physiology , Carcinoma, Hepatocellular/mortality , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Gene Expression Profiling , Humans , Liver Neoplasms/mortality , Liver Neoplasms/pathology , MicroRNAs/analysis , Neoplasm Invasiveness , Neoplasm Metastasis , Phenotype , Prognosis
2.
Oncogene ; 26(31): 4580-9, 2007 Jul 05.
Article in English | MEDLINE | ID: mdl-17297465

ABSTRACT

Two related Rho GTPase-activating proteins, DLC-1 (deleted in liver cancer 1) and DLC-2, are emerging as bona fide tumor suppressor genes that inhibit cancer cell growth. In this report, we characterized a gene on chromosome Xq13 that encodes DLC-3 (also known as KIAA0189 and STARD8), a third member of the DLC family. The DLC-3 gene has transcripts with alternative 5' ends, one of which, DLC-3alpha, encodes an 1103-amino acid polypeptide highly similar to DLC-1 and DLC-2. A second isoform (DLC-3beta) would yield a protein lacking the N-terminal sterile alpha motif domain. The DLC-3 gene is widely expressed in normal tissues, but DLC-3 mRNA levels were low or absent in a significant number of breast, ovarian, liver and prostate cancer cell lines. Using a cancer profiling array to compare matched tumor and normal human tissues, downregulation of DLC-3 mRNA was observed in kidney, lung, ovarian, uterine and breast cancer samples. By quantitative reverse transcriptase-polymerase chain reaction, DLC-3 expression was reduced in primary prostate carcinomas relative to normal prostate tissue. Transfection of human breast and prostate cancer cells with a DLC-3alpha expression vector inhibited cell proliferation, colony formation and growth in soft agar. These results indicate that deregulation of DLC-3 may contribute to breast and prostate tumorigenesis.


Subject(s)
GTPase-Activating Proteins/metabolism , Neoplasms/metabolism , Tumor Suppressor Proteins/genetics , Amino Acid Sequence , Breast/metabolism , Breast Neoplasms/metabolism , Cell Proliferation , Down-Regulation , Female , Genes, Tumor Suppressor , Humans , Male , Molecular Sequence Data , Prostate/metabolism , Prostatic Neoplasms/metabolism , Reverse Transcriptase Polymerase Chain Reaction
3.
Br J Cancer ; 91(4): 753-9, 2004 Aug 16.
Article in English | MEDLINE | ID: mdl-15266310

ABSTRACT

The WWOX (WW-domain containing oxidoreductase) is a candidate tumour suppressor gene spanning the same chromosome region, 16q23, as the second most common fragile site (FS), FRA16D. Deletions detected by comparative genomic hybridisation (CGH) and loss of heterozygosity at microsatellite markers on chromosome 16q are common in many human cancers including hepatocellular carcinoma (HCC). The development of human HCC is closely associated with exposure to oncogenic viruses and chemical carcinogens, agents known to frequently target common FS. We examined the status of WWOX genomic DNA, RNA and protein in 18 cell lines derived from human HCC and found recurrent alterations of the gene. Loss of DNA copy-number confined to band 16q23 was detected by CGH in several cell lines. Although homozygous deletions of the WWOX gene were not detected, WWOX mRNA expression was absent or lower in 60% of cell lines. The occurrence of aberrant WWOX reverse transcription-PCR products with deletion of exons 6-8 correlated significantly with altered WWOX expression. All of the cell lines showing mRNA downregulation had a decreased or undetectable level of WWOX protein as demonstrated by Western blotting with antibody to WWOX. Furthermore, 13 out of the 18 cell lines expressed decreased levels or no WWOX protein when compared with normal liver. These results show that WWOX gene is frequently altered in HCC and raise the possibility that this gene is implicated in hepatocarcinogenesis.


Subject(s)
Carcinoma, Hepatocellular/genetics , Chromosomes, Human, Pair 16/genetics , Gene Deletion , Gene Expression Regulation, Neoplastic , Liver Neoplasms/genetics , Oxidoreductases/biosynthesis , Apoptosis , Carcinoma, Hepatocellular/pathology , Chromosome Fragile Sites , Down-Regulation , Humans , Liver Neoplasms/pathology , Loss of Heterozygosity , Microsatellite Repeats , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, Cultured , Tumor Suppressor Proteins , WW Domain-Containing Oxidoreductase
4.
Mol Cell Biol ; 21(22): 7817-25, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11604516

ABSTRACT

Tetranectin is a plasminogen-binding, homotrimeric protein belonging to the C-type lectin family of proteins. Tetranectin has been suggested to play a role in tissue remodeling, due to its ability to stimulate plasminogen activation and its expression in developing tissues such as developing bone and muscle. To test the functional role of tetranectin directly, we have generated mice with a targeted disruption of the gene. We report that the tetranectin-deficient mice exhibit kyphosis, a type of spinal deformity characterized by an increased curvature of the thoracic spine. The kyphotic angles were measured on radiographs. In 6-month-old normal mice (n = 27), the thoracic angle was 73 degrees +/- 2 degrees, while in tetranectin-deficient 6-month-old mice (n = 35), it was 93 degrees +/- 2 degrees (P < 0.0001). In approximately one-third of the mutant mice, X-ray analysis revealed structural changes in the morphology of the vertebrae. Histological analysis of the spines of these mice revealed an apparently asymmetric development of the growth plate and of the intervertebral disks of the vertebrae. In the most advanced cases, the growth plates appeared disorganized and irregular, with the disk material protruding through the growth plate. Tetranectin-null mice had a normal peak bone mass density and were not more susceptible to ovariectomy-induced osteoporosis than were their littermates as determined by dual-emission X-ray absorptiometry scanning. These results demonstrate that tetranectin plays a role in tissue growth and remodeling. The tetranectin-deficient mouse is the first mouse model that resembles common human kyphotic disorders, which affect up to 8% of the population.


Subject(s)
Blood Proteins/physiology , Kyphosis/etiology , Lectins, C-Type , Lectins/physiology , Animals , Blood Proteins/genetics , Bone Density , Disease Susceptibility , Female , Gene Deletion , Gene Targeting/methods , Kyphosis/genetics , Kyphosis/pathology , Lectins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Osteoporosis/etiology , Ovariectomy , Thoracic Vertebrae/abnormalities , Thoracic Vertebrae/pathology
5.
Eur J Biochem ; 268(13): 3797-806, 2001 Jul.
Article in English | MEDLINE | ID: mdl-11432748

ABSTRACT

The laminin beta2 chain is a basement membrane component expressed in a tissue- and developmental stage-specific manner. In this report we have examined the transcriptional and post-transcriptional regulation of the human laminin beta2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma and clone A colon carcinoma cells express the laminin beta2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the beta2 chain. Segments of the beta2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested by transient transfection. Sequences downstream of the transcription start site between nucleotides +91 and +120 were found to be essential for luciferase activity in the two cell lines. Additional positive regulatory regions were present further upstream, between nucleotides -164 to -667 and between nucleotides -667 to -1724. Genomic DNA at the 3' end of the gene also appeared to have enhancer activity, as a 1.1-kb fragment located downstream of the last exon stimulated the luciferase activity of the nucleotides -667/+297 promoter segment approximately threefold. Alternative splicing of the first intron of the human laminin beta2 chain gene generates two isoforms of the 5' untranslated region of the beta2 chain mRNA. The translational efficiencies of the two laminin beta2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell beta2 chain mRNA, transient transfection of chimeric beta2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates.


Subject(s)
Gene Expression Regulation, Neoplastic , Laminin/genetics , Transcription, Genetic , Alternative Splicing , Amino Acid Sequence , Base Sequence , Choriocarcinoma , Colonic Neoplasms , Enhancer Elements, Genetic , Fluorescent Antibody Technique, Indirect , Humans , Laminin/analysis , Molecular Sequence Data , Promoter Regions, Genetic , Protein Biosynthesis , RNA Splicing , RNA, Messenger/genetics , Recombinant Proteins/analysis , Recombinant Proteins/biosynthesis , Regulatory Sequences, Nucleic Acid , Rhabdomyosarcoma , Transfection , Tumor Cells, Cultured
6.
Genomics ; 73(1): 20-7, 2001 Apr 01.
Article in English | MEDLINE | ID: mdl-11352562

ABSTRACT

Transgenic mice expressing c-myc under the control of the albumin promoter and enhancer develop liver tumors and have served as a useful model for studying the progression of hepatocarcinogenesis. The chromosomes of one line of c-myc transgenic mice carry the reciprocal translocation t(5;6)(G1;F2) adjacent to the transgene insertion site on the 5G1-ter segment translocated to chromosome 6. To characterize the genomic alterations in the c-myc transgenic animals, we have cloned the mouse DNA flanking the transgene array. By linkage mapping, the transgene integration site was localized to the region of distal chromosome 5 syntenic to the region on human chromosome 7q11.23 that is hemizgygously deleted in Williams-Beuren syndrome, a multisystemic developmental disorder. Comparison of the genomic DNA structure in wildtype and transgenic mice revealed that the transgene integration had induced an approximately 40-kb deletion, starting downstream of the Cyln2 gene and including the first exon of the Gtf2ird1 gene. Gtf2ird1 encodes a polypeptide related to general transcription factor TFII-I, and it is the mouse orthologue of GTF2IRD1 (WBSCR11), one of the genes commonly deleted in Williams-Beuren syndrome patients. Loss of the 5' end of the Gtf2ird1 gene resulted in greatly reduced expression of Gtf2ird1 mRNA in mice homozygous for the transgene.


Subject(s)
Genes, myc , Transcription Factors/genetics , Williams Syndrome/genetics , Animals , Base Sequence , Chromosome Mapping/methods , Chromosomes, Human, Pair 7 , DNA , Exons , Gene Deletion , Gene Expression , Genetic Linkage , Helix-Loop-Helix Motifs , Humans , Mice , Mice, Transgenic , Models, Animal , Molecular Sequence Data , RNA, Messenger/genetics , Transgenes , Translocation, Genetic
7.
Cytogenet Cell Genet ; 84(3-4): 173-8, 1999.
Article in English | MEDLINE | ID: mdl-10393422

ABSTRACT

The laminin beta2 chain is an important constituent of certain kidney and muscle basement membranes. We have generated a detailed physical map of a 110-kb genomic DNA segment surrounding the human laminin beta2 chain gene (LAMB2) on chromosome 3p21.3-->p21.2, a region paralogous with the chromosome 7q22-->q31 region that contains the laminin beta1 chain gene locus (LAMB1). Several CpG islands and a novel polymorphic microsatellite marker (D3S4594) were identified. The 3' end of LAMB2 lies 16 kb from the 5' end of the glutaminyl tRNA synthetase gene (QARS). About 20 kb upstream of LAMB2 we found a gene encoding a transcribed, non-processed LAMB2-like pseudogene (LAMB2L). The sequence of 1.75 kb of genomic DNA at the 3' end of LAMB2L was similar to exons 8-12 of the laminin beta2 chain gene. The LAMB2L-LAMB2-QARS cluster lies telomeric to the gene encoding the laminin-binding protein dystroglycan (DAG1).


Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Genetic Linkage , Laminin/genetics , Physical Chromosome Mapping , Pseudogenes/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosomes, Artificial, Yeast/genetics , Chromosomes, Human, Pair 3/genetics , Chromosomes, Human, Pair 7/genetics , Cloning, Molecular , CpG Islands/genetics , Cytoskeletal Proteins/genetics , Dystroglycans , Evolution, Molecular , Exons/genetics , Genetic Markers/genetics , Genome, Human , Humans , Introns/genetics , Membrane Glycoproteins/genetics , Mice , Microsatellite Repeats/genetics , Molecular Sequence Data
8.
Biochem Biophys Res Commun ; 250(1): 125-30, 1998 Sep 08.
Article in English | MEDLINE | ID: mdl-9735344

ABSTRACT

The laminin alpha5 chain is a component of the basement membranes of many developing and adult tissues. The mouse laminin alpha5 chain gene (Lama5) has been mapped close to the locus of the semidominant ragged (Ra) mutation on distal chromosome 2. The cause of the Ra mutation, which is usually lethal in the homozygous state, has not been determined. We have investigated whether a defect in Lama5 is responsible for the ragged mutation, using the RaJ strain. No differences in the level of the laminin alpha5 chain transcript were found in placental RNA from homozygous RaJ mutant embryos compared to normal littermates. Antiserum raised against a recombinant laminin alpha5 chain polypeptide stained the basement membranes of both normal and homozygous mutant embryos to a similar extent. More precise mapping of Lama5 on an interspecific Ra backcross indicated that Lama5 is proximal to the Ra locus. These results exclude Lama5 as a candidate gene for the Ra mutation.


Subject(s)
Laminin/genetics , Mutation , Animals , Base Sequence , Chromosome Mapping , DNA , Female , Heterozygote , Homozygote , Male , Mice , Mice, Inbred C3H , Mice, Mutant Strains , Molecular Sequence Data
9.
Dev Biol ; 200(2): 247-59, 1998 Aug 15.
Article in English | MEDLINE | ID: mdl-9705231

ABSTRACT

Tetranectin, a plasminogen-binding protein with a C-type lectin domain, is found in both serum and the extracellular matrix. In the present study we report that tetranectin is closely associated with myogenesis during embryonic development, skeletal muscle regeneration, and muscle cell differentiation in vitro. We find that tetranectin expression coincides with muscle differentiation and maturation in the second half of gestation and further that tetranectin is enriched at the myotendinous and myofascial junctions. The tetranectin immunostaining declines after birth and no immunostaining is observed in normal adult muscle. However, during skeletal muscle regeneration induced by the intramuscular injection of the myotoxic anesthetic Marcaine, myoblasts, myotubes, and the stumps of damaged myofibers exhibit intense tetranectin immunostaining. Tetranectin is also present in regenerating muscle cells in dystrophic mdx mice. Murine C2C12 myogenic cells and pluripotent embryonic stem cells can undergo muscle cell differentiation in vitro. Tetranectin is not expressed in the undifferentiated myogenic cells, but during the progression of muscle differentiation, tetranectin mRNA is induced, and both cytoplasmic and cell surface tetranectin immunostaining become apparent. Finally, we demonstrate that while tetranectin mRNA is translated to a similar degree in developing limbs and lung, the protein does not seem to be tissue associated in the lung as it is in the limbs. This indicates that in some tissues, such as the limbs, tetranectin may function locally, whereas in other tissues, such as the lung, tetranectin production may be destined for body fluids. In summary, these results suggest that tetranectin is a matricellular protein and plays a role in myogenesis.


Subject(s)
Biomarkers, Tumor/metabolism , Blood Proteins/metabolism , Cell Differentiation/physiology , Lectins, C-Type , Muscle Development , Muscle, Skeletal/growth & development , Animals , Bupivacaine/metabolism , Cell Line , Embryonic and Fetal Development , Gene Expression Regulation, Developmental/genetics , Immunohistochemistry , Mice , Mice, Inbred Strains , Mice, Inbred mdx , Polyribosomes/metabolism , Protein Biosynthesis/genetics , RNA, Messenger/metabolism , Regeneration
10.
Am J Pathol ; 151(2): 621-31, 1997 Aug.
Article in English | MEDLINE | ID: mdl-9273822

ABSTRACT

We have investigated the distribution of the laminin beta 2 chain (previously s-laminin) in human fetal and adult skeletal muscle and compared it to the distribution of laminin beta 1. Immunoblotting and transfection assays were used to characterize a panel of monoclonal and polyclonal antibodies to the laminin beta 2 chain. We found that laminin beta 1 chain was detected at all times during development from 10 weeks of gestation. Laminin beta 2 chain was first detected in 15 to 22-week-old fetal skeletal muscle as distinct focal immunoreactivity in the sarcolemmal basement membrane area of some myofibers. In the adult skeletal muscle, laminin beta 2 chain immunoreactivity was found along the entire perimeter of each of the individual myofibers in a large series of different muscles studied. Laminin beta 2 chain was similarly found in the skeletal muscle basement membranes in patients with Duchenne and Becker muscular dystrophy. Immunoaffinity chromatography of muscle extracts with a monoclonal antibody to the laminin alpha 2 chain followed by immunoblotting with various antibodies to the beta 2 chain demonstrated the presence of the laminin-4 (alpha 2-beta 2-gamma 1) isoform. Together the present results demonstrate a prominent extrasynaptic localization of laminin beta 2 in the human muscle, suggesting that it may have an important function in the sarcolemmal basement membrane.


Subject(s)
Laminin/analysis , Muscle, Skeletal/metabolism , Sarcolemma/metabolism , Adult , Antibodies, Monoclonal , Female , Humans , Immunohistochemistry , Laminin/immunology , Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Muscle, Skeletal/innervation , Muscular Dystrophies/metabolism , Muscular Dystrophies/pathology , Neuromuscular Junction/metabolism , Pregnancy , Transfection
11.
FEBS Lett ; 411(2-3): 296-300, 1997 Jul 14.
Article in English | MEDLINE | ID: mdl-9271224

ABSTRACT

To investigate the function of the laminin alpha5-chain, previously identified in mice, cDNA clones encoding the 953-amino-acid carboxy terminal G-domain of the human laminin alpha5-chain were characterized. Northern blot analysis showed that the laminin alpha5-chain is expressed in human placenta, heart, lung, skeletal muscle, kidney, and pancreas. The human laminin alpha5-chain gene (LAMA5) was assigned to chromosome 20q13.2-q13.3 by in situ hybridization, and the mouse gene (Lama5) was mapped by linkage analysis to a syntonic region of distal chromosome 2, close to the locus for the ragged (Ra) mutation.


Subject(s)
Chromosome Mapping , Chromosomes, Human, Pair 20/genetics , Laminin/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Gene Expression Regulation , Genetic Linkage , Humans , In Situ Hybridization , Laminin/chemistry , Mice , Molecular Sequence Data , Mutation , RNA, Messenger/analysis , Sequence Analysis, DNA , Sequence Homology, Amino Acid
12.
Cytogenet Cell Genet ; 76(1-2): 39-40, 1997.
Article in English | MEDLINE | ID: mdl-9154122

ABSTRACT

Tetranectin is a plasminogen-binding protein that is induced during the mineralization phase of osteogenesis. By screening a human chromosome 3 somatic cell hybrid mapping panel, we have localized the human tetranectin gene (TNA) to 3p22-->p21.3, which is distinct from the loci of two human connective tissue disorders that map to the short arm of chromosome 3, MFS2 and LRS1.


Subject(s)
Biomarkers, Tumor/genetics , Blood Proteins/genetics , Chromosomes, Human, Pair 3 , Lectins, C-Type , Base Sequence , Chromosome Mapping , DNA Primers , Humans , Hybrid Cells , Molecular Sequence Data
13.
J Biol Chem ; 271(23): 13407-16, 1996 Jun 07.
Article in English | MEDLINE | ID: mdl-8662701

ABSTRACT

We have determined the structural organization of the human and mouse genes that encode the laminin beta2 chain (s-laminin), an essential component of the basement membranes of the neuromuscular synapse and the kidney glomerulus. The human and mouse genes have a nearly identical exon-intron organization and are the smallest laminin chain genes characterized to date, due to the unusually small size of their introns. The laminin beta2 chain genes of both species consist of 33 exons that span

Subject(s)
Laminin/genetics , Alternative Splicing , Amino Acid Sequence , Animals , Base Sequence , Basement Membrane/chemistry , Chromosome Mapping , Conserved Sequence , DNA Primers/genetics , Exons , Gene Expression Regulation , Humans , Introns , Laminin/chemistry , Mice , Molecular Sequence Data , Promoter Regions, Genetic , Sequence Homology, Amino Acid , Species Specificity , Transcription, Genetic
14.
Neurology ; 45(11): 2099-101, 1995 Nov.
Article in English | MEDLINE | ID: mdl-7501167

ABSTRACT

Muscular dystrophy may be caused by disturbances in a number of muscle proteins that appear to be part of a chain of interacting molecules that includes cytoskeletal, cell membrane, and basement membrane components. We found that the skeletal muscle cells in two cases of Walker-Warburg syndrome were severely deficient in the laminin beta 2 chain and in adhalin. The findings indicate that these two proteins are key molecules in the interactive protein complex conferring muscle stability and cell survival.


Subject(s)
Abnormalities, Multiple/metabolism , Cytoskeletal Proteins/analysis , Eye Abnormalities/metabolism , Laminin/analysis , Membrane Glycoproteins/analysis , Muscles/metabolism , Muscular Dystrophies/metabolism , Basement Membrane/metabolism , Child, Preschool , Humans , Immunohistochemistry , Male , Sarcoglycans , Syndrome
15.
Genomics ; 26(2): 219-28, 1995 Mar 20.
Article in English | MEDLINE | ID: mdl-7601446

ABSTRACT

Entactin is a widespread basement membrane protein of 150 kDa that binds to type IV collagen and laminin. The complete exon-intron structure of the mouse entactin gene has been determined from lambda genomic DNA clones. The gene spans at least 65 kb and contains 20 exons. The exon organization of the mouse entactin gene closely corresponds to the organization of the polypeptide into distinct structural and functional domains. The two amino-terminal globular domains are encoded by three exons each. Single exons encode the two protease-sensitive, O-glycosylated linking regions. The six EGF-like repeats and the single thyroglobulin-type repeat are each encoded by separate exons. The carboxyl-terminal half of entactin displays sequence homology to the growth factor-like region of the low-density lipoprotein receptor, and in both genes this region is encoded by eight exons. The positions of four introns are also conserved in the homologous region of the two genes. These observations suggest that the entactin gene has evolved via exon shuffling. Finally, several sequence polymorphisms useful for gene linkage analysis were found in the 3' noncoding region of the last exon.


Subject(s)
Exons/genetics , Membrane Glycoproteins/genetics , Mice/genetics , Receptors, LDL/genetics , Amino Acid Sequence , Animals , Consensus Sequence , Genes , Membrane Glycoproteins/chemistry , Mice, Inbred AKR , Mice, Inbred C57BL , Mice, Inbred CBA , Molecular Sequence Data , Protein Structure, Tertiary , Sequence Homology, Amino Acid
16.
J Cell Biol ; 127(6 Pt 1): 1767-75, 1994 Dec.
Article in English | MEDLINE | ID: mdl-7798325

ABSTRACT

Tetranectin is a protein shared by the blood and the extracellular matrix. Tetranectin is composed of four identical, noncovalently bound polypeptides each with a molecular mass of approximately 21 kD. There is some evidence that tetranectin may be involved in fibrinolysis and proteolysis during tissue remodeling, but its precise biological function is not known. Tetranectin is enriched in the cartilage of the shark, but the gene expression pattern in the mammalian skeletal system has not been determined. In the present study we have examined the expression pattern and putative function of tetranectin during osteogenesis. In the newborn mouse, strong tetranectin immunoreactivity was found in the newly formed woven bone around the cartilage anlage in the future bone marrow and along the periosteum forming the cortex. No tetranectin immunoreactivity was found in the proliferating and hypertrophic cartilage or in the surrounding skeletal muscle. Using an in vitro mineralizing system, we examined osteoblastic cells at different times during their growth and differentiation. Tetranectin mRNA appeared in the cultured osteoblastic cells in parallel with mineralization, in a pattern similar to that of bone sialoprotein, which is regarded as one of the late bone differentiation markers. To explore the putative biological role of tetranectin in osteogenesis we established stably transfected cell lines (PC12-tet) overexpressing recombinant tetranectin as evidenced by Northern and Western blot analysis and immunoprecipitation. Both control PC12 cells and PC12-tet cells injected into nude mice produced tumors containing bone material, as evidenced by von Kossa staining for calcium and immunostaining with bone sialoprotein and alkaline phosphatase antiserum. Nude mice tumors established from PC12-tet cells contained approximately fivefold more bone material than those produced by the untransfected PC12 cell line or by the PC12 cells transfected with the expression vector with no insert (Mann Whitney rank sum test, p < 0.01), supporting the notion that tetranectin may play an important direct and/or indirect role during osteogenesis. In conclusion, we have established a potential role for tetranectin as a bone matrix protein expressed in time and space coincident with mineralization in vivo and in vitro.


Subject(s)
Blood Proteins/physiology , Calcification, Physiologic/physiology , Lectins, C-Type , Osteogenesis/physiology , Animals , Base Sequence , Biomarkers , Blood Proteins/genetics , Bone and Bones/cytology , Cattle , Cell Differentiation , Cells, Cultured , Gene Expression , Immunohistochemistry , Mice , Mice, Nude , Molecular Sequence Data , Neoplasms, Experimental , PC12 Cells , RNA, Messenger/analysis , Rats , Recombinant Proteins/metabolism , Transfection
17.
Genomics ; 24(2): 243-52, 1994 Nov 15.
Article in English | MEDLINE | ID: mdl-7698745

ABSTRACT

Overlapping cDNA clones that encode the full-length human laminin beta 2 chain, formerly called the S chain, were isolated. The cDNA of 5680 nt contains a 5391-nt open reading frame encoding 1797 amino acids. At the amino terminus is a 32-amino-acid signal peptide that is followed by the mature beta 2 chain polypeptide of 1765 amino acids with a calculated molecular mass of 192,389 Da. The human beta 2 chain is predicted to have all of the seven structural domains typical of the beta chains of laminin, including the short cysteine-rich alpha region. The amino acid sequence of human beta 2 chain showed 86.1% sequence identity to the rat beta 2 chain, 50.0% to the human beta 1 chain, and 36.3% to the human beta 3 chain. The greatest sequence identity was in domains VI, V, and III. The sequence of a 24-amino-acid peptide fragment isolated from the beta 2 chain of laminin purified from human amniotic basement membrane matched the sequence predicted from the cDNA, confirming that the cDNA encodes human beta 2 laminin. The cDNA was used to assign the gene (LAMB2) to human chromosome 3p21 by in situ hybridization. It is not linked to genes for human laminin chains alpha 1, beta 1, and gamma 1 or other known laminin genes. Immunostaining showed that the beta 2 chain is localized to the smooth muscle basement membranes of the arteries, while the homologous beta 1 chain is confined to the subendothelial basement membranes. The beta 2 chain was found in the basement membranes of ovarian carcinomas but not colon carcinomas. These results indicate that the expression of the beta 2 chain gene is tightly regulated in normal human tissues and in disease.


Subject(s)
Chromosomes, Human, Pair 3 , Laminin/genetics , Neoplasms/genetics , Amino Acid Sequence , Animals , Base Sequence , Chromosome Mapping , Cloning, Molecular , DNA, Complementary , Female , Gene Expression Regulation, Neoplastic , Humans , Laminin/chemistry , Molecular Sequence Data , Rats , Tumor Cells, Cultured
18.
Gene ; 132(2): 261-6, 1993 Oct 15.
Article in English | MEDLINE | ID: mdl-8224873

ABSTRACT

Entactin (Ent) is a 150-kDa basement membrane glycoprotein that forms a complex with laminin and also binds to type-IV collagen. For the initial characterization of the 5' end of the mouse Ent gene, we isolated genomic clones that encompass the first three exons of the gene. A 3.5-kb EcoRI fragment at the 5' end of the gene was sequenced and shown to contain 1226 bp of the 5'-flanking DNA, the 260-bp first exon, and 2060 bp of the first intron. The second exon is separated from the first exon by an intron that spans greater than 18 kb. The major transcription start point (tsp) was mapped to 35 nucleotides upstream from the translation start codon. The 5'-flanking DNA contains a putative TATA box, two CAAT boxes and two GC boxes. Nine potential AP-2- and two potential AP-1-binding sites were found in the upstream region and the first intron. The first 248 bp of the promoter region are G+C rich, and they are 65% identical to the promoter of human Ent. Fragments of the 5'-flanking DNA linked to a reporter gene were shown to have promoter activity in transient transfection assays.


Subject(s)
Basement Membrane/metabolism , Membrane Glycoproteins/genetics , Amino Acid Sequence , Animals , Base Sequence , DNA , Exons , Humans , Introns , Mice , Molecular Sequence Data , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Restriction Mapping , Sequence Homology, Nucleic Acid , Transfection
19.
Kidney Int ; 43(1): 13-9, 1993 Jan.
Article in English | MEDLINE | ID: mdl-8433553

ABSTRACT

Entactin is a sulfated multidomain glycoprotein component of basement membranes. The molecule consists of 1217 amino acids which are organized into two terminal globular domains linked by a rod-like structure largely composed of four EGF- and one thyroglobulin-like cysteine-rich homology repeats. Entactin binds to laminin, collagen IV, fibrinogen, and fibronectin. In the parietal endoderm M1536-B3 cell line, the laminin-entactin complex is formed intracellularly and transported in membrane enclosed vesicles to the extracellular compartment. Transfection of human choriocarcinoma JAR cells, which do not synthesize entactin, with entactin cDNA results in the synthesis and insertion of entactin into the extracellular matrix where it becomes associated with laminin and collagen IV. Indirect immunofluorescent staining also reveals that entactin co-localizes with fibronectin in the extracellular matrix of the embryonal carcinoma-derived 4CQ cell line. These observations suggest that entactin plays an important role in the assembly and properties of diverse extracellular matrices. In addition, entactin binds to immobilized fibrinogen, and more specifically, to the A alpha and B beta chains. The binding of radiolabeled entactin to immobilized fibrinogen is not dependent on metal ions, and is inhibited by antibodies against either fibrinogen or entactin, soluble fibrinogen, and unlabeled entactin. This interaction combined with the chemotactic and phagocytic promoting activities of entactin may be important in hemostasis and would healing.


Subject(s)
Membrane Glycoproteins/physiology , Animals , Basement Membrane/physiology , Cell Line , Collagen/metabolism , Extracellular Matrix/metabolism , Fibrinogen/metabolism , Hemostasis/physiology , Humans , Laminin/metabolism , Membrane Glycoproteins/genetics
20.
J Biol Chem ; 267(32): 23143-50, 1992 Nov 15.
Article in English | MEDLINE | ID: mdl-1385414

ABSTRACT

The extracellular matrix of rat pheochromocytoma PC12 cells was shown by indirect immunofluorescence to consist of a network of fibronectin. The matrix did not contain laminin. The cells synthesized messenger RNA for fibronectin, laminin B2, and s-laminin but not for entactin or the B1 and A chains of laminin. Laminin B2 but not laminin B1 was detectable in the culture medium and in cell lysates. A full-length cDNA clone for the B1 chain of laminin was constructed in the plasmid p-444, which contains the neomycin-resistance marker and human beta-actin promoter. PC12 cells were transfected with this recombinant plasmid, and stable neomycin-resistant clones were isolated and characterized. Clones that synthesized laminin B1 messenger RNA were found to deposit a laminin-containing matrix. In many of these clones the deposition of the fibronectin matrix was greatly diminished. The laminin matrix was predominantly localized in the intercellular spaces forming a honeycomb pattern. The morphology of the laminin-synthesizing transfected cells was markedly different from the parental cells. The cells grew in tight clusters that were resistant to dissociating agents. It is concluded that the B1 chain of laminin contains information that is required for the formation of a stable laminin-containing extracellular matrix network either by interaction with cell surface receptors or other extracellular matrix components. Furthermore, expression of the laminin B1 gene may be a central regulatory point in determining extracellular matrix composition during embryogenesis.


Subject(s)
Extracellular Matrix/metabolism , Laminin/genetics , Laminin/metabolism , Animals , Blotting, Northern , Blotting, Western , Clone Cells , DNA Probes , Extracellular Matrix/ultrastructure , Fluorescent Antibody Technique , Laminin/isolation & purification , Macromolecular Substances , Mice , PC12 Cells , Poly A/isolation & purification , Poly A/metabolism , RNA/isolation & purification , RNA/metabolism , RNA, Messenger , Transfection
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