A potential role for tetranectin in mineralization during osteogenesis.

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.

Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor.

Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'-untranslated region of 2.2 kb. The open reading frame encodes a 1,245-residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asn-linked glycosylation sites, and two potential Ca2+-binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells.

Deleted in liver cancer 3 (DLC-3), a novel Rho GTPase-activating protein, is downregulated in cancer and inhibits tumor cell growth.

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.

Mice with a targeted deletion of the tetranectin gene exhibit a spinal deformity.

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.

Characterization of the 5' end of the mouse Ent gene encoding the basement membrane protein, entactin.

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.

Tissue-specific expression of the human laminin alpha5-chain, and mapping of the gene to human chromosome 20q13.2-13.3 and to distal mouse chromosome 2 near the locus for the ragged (Ra) mutation.

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.

Laminin beta 2 chain and adhalin deficiency in the skeletal muscle of Walker-Warburg syndrome (cerebro-ocular dysplasia-muscular dystrophy).

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.

Loss of miR-122 expression in liver cancer correlates with suppression of the hepatic phenotype and gain of metastatic properties.

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.

Entactin: structure and function.

Entactin is an integral and ubiquitous component of the basement membrane. The amino acid sequences of the mouse and human molecules have been determined and exhibit 85% sequence identity. The molecule is organized into three structural domains, an N-terminal globule (I) is linked to a smaller C-terminal globule (III) by a rigid stalk (II) largely consisting of cysteine-rich EGF-like homology repeats and a cysteine-rich thyroglobulin homology repeat. The molecule binds calcium ions and supports cell adhesion. However, its major function may be the assembly of the basement membrane. The carboxyl globule binds tightly to one of the short arms of laminin at the inner rodlike segment. This same region is also believed to be responsible for the attachment of entactin to type IV collagen at approximately 80 nm from its carboxyl noncollagenous end. Entactin therefore could serve as a bridge between the two most abundant molecules in the basement membrane. Supporting evidence for this role has been obtained from transfection of human choriocarcinoma, JAR, cells with the entactin gene. JAR cells synthesize laminin and type IV collagen but not entactin. Transfection of entactin into the cells stimulated incorporation of laminin and type IV collagen along with entactin into the extracellular matrix and into structures resembling focal contacts. The calcium-binding activity of entactin may play a role in the matrix assembly process. The protease sensitivity of entactin suggests that it may be a target for proteolytic activity during tissue remodeling, metastasis, and other events requiring the turnover of the basement membrane.

Exon organization of the mouse entactin gene corresponds to the structural domains of the polypeptide and has regional homology to the low-density lipoprotein receptor gene.

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.

Control of laminin synthesis during differentiation of F9 embryonal carcinoma cells. A study using cDNA clones complementary to the mRNA species for the A, B1 and B2 subunits.

Molecular clones complementary to the mRNA species for the A, B1 and B2 chains of murine laminin were identified by hybrid-selection and in vitro translation. Northern blot analysis demonstrated that the three clones, p59 (A), p2 (B1) and p16 (B2) hybridized to mRNA species 9.8, 6.0, and 8.0 kb in length, respectively. The three clones were used as probes to monitor the steady-state levels of laminin mRNA species during differentiation of F9 embryonal carcinoma cells induced by treatment with retinoic acid and dibutyryl cyclic AMP. The steady-state levels of the three mRNA species appeared to increase in a coordinate manner. Undetectable levels at the beginning of induction were followed by a dramatic increase in the levels of the three mRNA species between 48 and 72 h. The kinetics parallel the increase in laminin synthesis and the striking morphological changes previously reported.

Laminin B1 expression is required for laminin deposition into the extracellular matrix of PC12 cells.

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.

Biological functions of entactin.

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.

Integration of a c-myc transgene results in disruption of the mouse Gtf2ird1 gene, the homologue of the human GTF2IRD1 gene hemizygously deleted in Williams-Beuren syndrome.

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.

Assignment of the gene for human tetranectin (TNA) to chromosome 3p22-->p21.3 by somatic cell hybrid mapping.

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.

Synthesis of laminin and entactin by F9 cells induced with retinoic acid and dibutyryl cyclic AMP.

Mouse embryonal carcinoma F9 cells were exposed to retinoic acid and dibutyryl cyclic AMP. The treated cells synthesized and secreted into the culture medium the basal lamina components, laminin (GP-1 and GP-2) and entactin. The time course of secretion of the basal lamina components was examined by electron microscopic and immunochemical procedures. The induction of the cells resulted in major morphological changes and the deposition of both laminin and entactin at the cell surface and cell junctions. Intracellular deposits of laminin could be localized to the endoplasmic reticulum and membrane-bound intracytoplasmic vacuoles. Concomitant with the appearance of laminin and entactin, there was a loss of fibronectin synthesis and a marked decrease in a 190,000-Da sulfated glycoprotein that appeared to be related to entactin. In the induced cells, laminin and entactin were associated in a complex that could be dissociated with low concentrations of sodium dodecyl sulfate. The induction of laminin and entactin seem to be independent. The enhanced synthesis of laminin appeared to be under transcriptional regulation since it was found that induced F9 cells contained translatable mRNA for GP-2 when tested in a rabbit reticulocyte lysate system. The uninduced cells did not contain detectable quantities of translatable GP-2 mRNA.

Evaluation of Lama5 as a candidate for the mouse ragged (Ra) mutation.

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.

Regulation of laminin beta2 chain gene expression in human cancer cell lines.

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.

Risk factors for injuries due to the 1990 earthquake in Luzon, Philippines.

On 16 July 1990, an earthquake measuring 7.7 on the Richter scale struck the island of Luzon, Philippines. A case-control study was carried out to identify the risk factors for earthquake-related injuries and at the same time observations were made on the rescue efforts. Being hit by falling objects was the leading cause of injury (34%). Those injured during the tremor were more likely to have been inside buildings constructed of concrete or mixed materials (odds ratio, 2.6; 95% confidence interval (CI), 1.7-4.1) and to have been on the middle floors of multistorey buildings (odds ratio, 3.4; 95% CI, 2.2-5.5). Leaving a building during the earthquake was a protective behaviour (odds ratio, 0.3; 95% CI, 0.2-0.8). Of the 235 survivors who were trapped and rescued alive from the rubble, 99% were rescued within 48 hours of the impact of the tremor. These findings should prove useful in developing seismic safety codes. People should be taught proper evasive actions to take during earthquakes, and training in basic first aid and methods of rescue should be an integral part of community preparedness programmes.

Frequent downregulation and loss of WWOX gene expression in human hepatocellular carcinoma.

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.