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J Merregaert

Publications and source records attributed to J Merregaert.

At least 19 recordsLinked to original sources
PubMed2001

Recombinant human extracellular matrix protein 1 inhibits alkaline phosphatase activity and mineralization of mouse embryonic metatarsals in vitro.

Two mRNAs are transcribed from the extracellular matrix protein 1 gene (Ecm1): Ecm1a and an alternatively spliced Ecm1b. We studied Ecm1 mRNA expression and localization during endochondral bone formation and investigated the effect of recombinant human (rh) Ecm1a protein on organ cultures of embryonic mouse metatarsals. Of the two transcripts, Ecm1a mRNA was predominantly expressed in fetal metacarpals from day 16 to 19 after gestation. Ecm1 expression was not found in 16- and 17-day-old metatarsals of which the perichondrium was removed. In situ hybridization and immunohistochemistry demonstrated Ecm1 expression in the connective tissues surrounding the developing bones, but not in the cartilage. Biological effects of rhEcm1a protein on fetal metatarsal cultures were biphasic: at low concentrations, Ecm1a stimulated alkaline phosphatase activity and had no effect on mineralization, whereas at higher concentrations, Ecm1a dose dependently inhibited alkaline phosphatase activity and mineralization. These results suggest that Ecm1a acts as a novel negative regulator of endochondral bone formation.

Alkaline Phosphatase↗
PubMed2001

Extracellular matrix protein 1 (ECM1) has angiogenic properties and is expressed by breast tumor cells.

Tumor growth and metastasis are critically dependent on the formation of new blood vessels. The present study found that extracellular matrix protein 1 (ECM1), a newly described secretory glycoprotein, promotes angiogenesis. This was initially suggested by in situ hybridization studies of mouse embryos indicating that the ECM1 message was associated with blood vessels and its expression pattern was similar to that of flk-1, a recognized marker for endothelium. More direct evidence for the role of ECM1 in angiogenesis was provided by the fact that highly purified recombinant ECM1 stimulated the proliferation of cultured endothelial cells and promoted blood vessel formation in the chorioallantoic membrane of chicken embryos. Immunohistochemical staining with specific antibodies indicated that ECM1 was expressed by the human breast cancer cell lines MDA-435 and LCC15, both of which are highly tumorigenic. In addition, staining of tissue sections from patients with breast cancer revealed that ECM1 was present in a significant proportion of primary and secondary tumors. Collectively, the results of this study suggest that ECM1 possesses angiogenic properties that may promote tumor progression.

Angiogenesis Inducing Agents↗
PubMed2000

Differentiation-dependent alternative splicing and expression of the extracellular matrix protein 1 gene in human keratinocytes.

The human extracellular matrix protein 1 (Ecm1) gene is located at chromosome band 1q21 close to the epidermal differentiation complex and is transcribed in two discrete mRNAs: a full length Ecm1a and a shorter, alternatively spliced, Ecm1b transcript, the expression of which is restricted to tonsils and skin. The chromosomal localization and the Ecm1b expression in skin prompted us to investigate the role of Ecm1 in keratinocyte differentiation. In this study, we provide evidence for the existence of a relationship between keratinocyte differentiation and expression of the Ecm1b transcript. Cultures of subconfluent undifferentiated normal human keratinocytes express only Ecm1a. Upon reaching confluence, the cells start to differentiate, as measured by keratin K10 mRNA expression. Concomitantly Ecm1b mRNA expression is induced, although expression of Ecm1a mRNA remains unchanged. In addition, treatment of undifferentiated normal human keratinocyte cells with 12-O-tetradecanoyl-phorbol-13-acetate strongly induces the expression of Ecm1b mRNA. Expression of Ecm1b can also be induced by coculturing normal human keratinocytes with lethally irradiated feeder cells and by a diffusible factor secreted by stromal cells. In adult human skin, Ecm1a mRNA is expressed throughout the epidermis with the strongest expression in the basal and first suprabasal cell layers, whereas expression of Ecm1b mRNA is predominantly found in spinous and granular cell layers. Immunohistochemically, Ecm1a expression is almost completely restricted to the basal cell layer, whereas Ecm1b is detected in the suprabasal layers. These results are strongly suggestive of a role for Ecm1b in terminal keratinocyte differentiation, which is also supported by the localization of the Ecm1 gene at 1q21. Refinement of its genomic localization, however, placed Ecm1 centromeric of the epidermal differentiation complex.

Adult↗
PubMed2000

Collagenase-3 (MMP-13) and integral membrane protein 2a (Itm2a) are marker genes of chondrogenic/osteoblastic cells in bone formation: sequential temporal, and spatial expression of Itm2a, alkaline phosphatase, MMP-13, and osteocalcin in the mouse.

Endochondral bone formation requires the action of cells of the chondrocytic and osteoblastic lineage, which undergo continuous differentiation during this process. To identify subpopulations of resting, proliferating, and hypertrophic chondrocytes and osteoblasts involved in bone formation, we have identified here two novel marker genes present in endochondral and intramembranous ossification. Using Northern blot analysis and in situ hybridization on parallel sections of murine embryos and bones of newborn mice we compared the expression pattern of the recently cloned Itm2a and MMP-13 (collagenase-3) genes with that of established marker genes for bone formation, such as alkaline phosphatase (ALP), osteocalcin (OC), and collagen type X, during endochondral and intramembranous ossification. During embryonic development expression of Itm2a and ALP was detectable at midgestation (11.5 days postcoitum [dpc]) and increased up to 16.5 dpc. MMP-13 and OC expression started at 14.5 dpc and 16.5 dpc, respectively. This temporal expression was reflected in the spatial distribution of these markers in the growth plate of long bones. In areas undergoing endochondral ossification Itm2a expression was found in chondrocytes of the resting and the proliferating zones. Expression of ALP and MMP-13 are mutually exclusive: ALP transcripts were found only in collagen type X positive hypertrophic chondrocytes of the upper zone. MMP-13 expression was restricted to chondrocytes of the lower zone of hypertrophic cartilage also expressing collagen type X. In osteoblasts involved in endochondral and intramembranous ossification Itm2a was not present. ALP, MMP-13, and OC were mutually exclusively expressed in these cells suggesting a differentiation-dependent sequential expression of ALP, MMP-13, and OC. The identification of the continuum of sequential expression of Itm2a, ALP, MMP-13, and OC will now allow us to establish a series of marker genes that are highly suitable to characterize bone cells during chondrocytic and osteoblastic differentiation in vivo.

Alkaline Phosphatase↗
PubMed2000

The monoclonal antibodies 18d7/91f2 recognize a receptor regulatory protein on mouse bone marrow stromal cells.

Two monoclonal antibodies 18D7 and 91F2 were developed by immunizing rats with the mouse bone marrow-derived osteogenic cell line MN7. Hybridomas secreting rat antibodies against MN7 cell surface markers were selected by flow cytometry analysis. Both the monoclonal antibody 18D7 and the monoclonal antibody 91F2 are directed against the same cell surface antigen present on MN7 cells. Here, we report on the immunopurification of the 18D7/91F2 antigen and its identification as the prostaglandin F2 alpha receptor regulatory protein (FPRP). FPRP is expressed as a single messenger RNA (mRNA) of approximately 6 kilobases (kb) in MN7 cells and is differentially expressed in developing osteogenic cultures of bone marrow cells of the mouse. However, addition of the monoclonal antibodies 18D7 and 91F2 to these cultures did not inhibit bone formation in vitro. Both monoclonal antibodies reacted with mouse stromal cell lines established from bone marrow, thymus, spleen, and mandibular condyles. Immunohistochemical analysis of mature tibia of mice using the monoclonal antibody 18D7 revealed the presence of a distinct population of bone marrow cells close to trabecular and endosteal bone surfaces. In the central bone marrow, hardly any positive cells were found. In 17-day-old fetal mouse radius 18D7 immunoreactivity was restricted to cells in the periosteum in close vicinity to the bone collar. Mature osteoblasts, osteoclasts, osteocytes, growth plate chondrocytes, and mature macrophages were all negative. Taken together, these results suggest that FPRP plays a role in the osteogenic differentiation process.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterization of the mouse Ecm1 gene and its 5' regulatory sequences.

The mouse Ecm1 (extracellular matrix protein 1) gene codes for an extracellular protein of 85kDa. We have determined the primary structure of this gene and analysed 1665 bases of its 5' upstream regulatory region. The gene is approximately 5kb long and contains 11 exons. The exons range in size from 45 to 375bp, whereas the intron sizes ranges from 95 to 1115bp. All splice donor/acceptor sites conform to the GT/AG rule. The 5' upstream sequences contain a TATA-box, a CCAAT-box and an inverted CCAAT-box. We have analysed the Ecm1 regulatory elements by reporter gene constructs and transient transfections in the stromal osteogenic cell line MN7. Progressive deletion of the Ecm1 promoter revealed the presence of a region with a repressive activity between -110 and -317 and showed that a 110-bp fragment, containing potential binding sites for AP1, Sp1, GATA and Ets family of transcription factors, is sufficient for CAT expression in MN7 cells. Except for the GATA binding site, these regulatory sequences are conserved in the human promoter. Point mutation analysis revealed that the AP1, Sp1 and Ets binding sites are absolutely necessary for Ecm1 expression in MN7.

Animals↗
PubMed1999

Schizosaccharomyces pombe stt3+ is a functional homologue of Saccharomyces cerevisiae STT3 which regulates oligosaccharyltransferase activity.

The Saccharomyces cerevisiae STT3 (ScSTT3) gene encodes a protein which is involved in protein glycosylation via the regulation of oligosaccharyltransferase activity. We have cloned and isolated the Schizosaccharomyces pombe STT3 homologous gene (Spstt3+). The Spstt3+ gene encodes a protein consisting of 749 amino acid residues which has significant homology with ScStt3p and the mouse Stt3p-homologue Itm1p. Disruption of the Spstt3+ gene shows that this gene is essential for growth. Like Itm1, Spstt3+ partially suppressed the temperature sensitivity of the stt3-1 mutation of S. cerevisiae, indicating that Spstt3+ is a functional and structural homologue of the ScSTT3 gene.

Amino Acid Sequence↗
PubMed1999

Genomic organization and chromosomal localization of the Itm2a gene.

Itm2A is a novel type II integral membrane protein that is involved in osteo- and chondrogenic differentiation. Itm2a cDNA was originally isolated from a cDNA library of organ cultures from prenatal mouse mandibular condyles, by subtractive hybridization and differential screening. The Itm2a gene was isolated from a BALB/c liver genomic library. In total, 9.4 kb of the gene were sequenced, of which 2649 bp are 5' flanking sequences. The Itm2a gene contains six exons and five introns. The splice sites conform to the GT/AG rule. The 5' flanking region, which contains the presumed promoter sequence, lacks the common TATAA and CCAAT sequences, but contains consensus binding sites for various transcription factors. Several of these transcription factors are known to play a role in transcriptional regulation of cartilage- or bone-specific genes (e.g. Cbfa1, Cart-1, MHox, HES-1, and CIIS1). Itm2a was mapped to mouse chromosome position XA2-XA3 by fluorescent in situ hybridization (FISH) analysis. The human homolog, ITM2A, was mapped to chromosome position Xq13.3-Xq21.2.

Animals↗
PubMed1998

cDNA sequence analysis, chromosomal assignment and expression pattern of the gene coding for integral membrane protein 2B.

The complete cDNA of the mouse integral membrane protein 2B gene (Itm2b) was determined by sequence analysis of expressed sequence tag (EST) clone L26775 and a clone isolated from a cDNA library of the osteogenic stromal cell line MN7 (Mathieu et al., 1992. Calcif. Tissue Int. 50, 362-371) and by 5' rapid amplification of cDNA ends (RACE). Alignment of different mouse ESTs confirmed the entire sequence. Northern blot analysis of different neonatal and adult mouse tissues showed that Itm2b is ubiquitously expressed. There are three mRNAs with different lengths in neonatal as well as in adult tissues, originating from alternative polyadenylation by usage of one consensus and two additional variant polyadenylation signals. The cDNA sequence of the human Itm2b homolog (ITM2B) was assembled using data from available human ESTs. Both the mouse and the human gene code for a protein of 266 amino acids (aa) that is homologous to a previously described integral membrane protein, Itm2A, of which the expression is restricted to osteo- and chondrogenic tissues. Itm2A and Itm2B belong to a family of type II integral membrane proteins, which contains a third member, Itm2C (Deleersnijder et al., 1996. J. Biol. Chem. 271, 19475-19482). The human ITM2B and mouse Itm2b genes were previously mapped as unknown ESTs to conserved syntenic regions Homo sapiens 13q12-13 and Mus musculus 14.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Identification and molecular characterization of TM7SF2 in the FAUNA gene cluster on human chromosome 11q13.

In this report, the identification and molecular characterization of a novel gene, designated TM7SF2, is reported. This gene was found in the FAU neighboring area (FAUNA) to which other genes have been mapped previously. The FAUNA gene cluster is located at chromosome 11q13 between landmarks H4B and D11S2196E. The TM7SF2 gene contains eight coding exons, and their splice site consensus sequences are consistent with AG/GT rule. Northern blot analysis with a cDNA probe corresponding to TM7SF2 revealed varying expression levels of a 1.7-kb transcript in adult human heart, brain, pancreas, lung, liver, skeletal muscle, kidney, ovary, prostate, and testis, but no detectable expression in placenta, spleen, thymus, small intestine, colon (mucosal lining), or peripheral blood leukocytes. The open reading frame in the cDNA sequence codes for a protein of 590 amino acids that is rich in glycine (23%) and arginine (17%) residues in its amino-terminal half and contains seven transmembrane domains in its carboxy-terminal half. The transmembrane region of the putative TM7SF2 protein shows amino acid sequence similarity to those of the lamin B receptor and the C14/C24 sterol reductase.

Adult↗
PubMed1997

The human extracellular matrix gene 1 (ECM1): genomic structure, cDNA cloning, expression pattern, and chromosomal localization.

The Ecm1 gene encodes an 85-kDa protein of unknown function that was originally identified as a secretory protein of the murine osteogenic stromal cell line MN7. This paper describes the isolation of a genomic clone containing the human ECM1 gene from a chromosome 1 cosmid library and the characterization of its exon-intron structure. The protein coding region comprises 10 exons. The ECM1 gene maps at chromosome 1q21 outside the epidermal differentiation complex region. The ECM1 gene appears to be expressed as a 1.8-kb transcript predominantly in placenta and heart, while an additional 1.4-kb alternatively spliced message was detected in tonsils. The full-length human ECM1 transcript contains 1838 bp and has an overall homology of 79.6% with the mouse Ecm1 cDNA. This transcript codes for a protein of 540 amino acids that is 69.4% identical and 81.3% similar to the corresponding mouse protein. The alternatively spliced variant, 1450 bp long, encodes a protein of 415 amino acids.

Alternative Splicing↗
PubMed1997

Construction of a 1.2-Mb sequence-ready contig of chromosome 11q13 encompassing the multiple endocrine neoplasia type 1 (MEN1) gene. The European Consortium on MEN1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant familial cancer syndrome characterized by parathyroid, pancreatic, and anterior pituitary tumors. The MEN1 locus has been previously localized to chromosome 11q13, and a 2-Mb gene-rich region flanked by D11S1883 and D11S449 has been defined. We have pursued studies to facilitate identification of the MEN1 gene by narrowing this critical region to a 900-kb interval between the VRF and D11S1783 loci through melotic mapping. This was achieved by investigating 17 cosmids for microsatellite polymorphisms, which defined two novel polymorphisms at the VRF and A0138 loci, and utilizing these to characterize recombinants in MEN1 families. In addition, we have established a 1200-kb sequence-ready contig consisting of 26 cosmids, eight BACs, and eight PACs that encompass this region. The precise locations for 19 genes and three ESTs within this contig have been determined, and three gene clusters consisting of a centromeric group (VRF, FKBP2, PNG, and PLCB3), a middle group (PYGM, ZFM1, SCG1, SCG2 (which proved to be the MEN1 gene), and PPP2R5B), and a telomeric group (H4B, ANG3, ANG2, ANG1, FON, FAU, NOF, NON, and D11S2196E) were observed. These results represent a valuable transcriptional map of chromosome 11q13 that will help in the search for disease genes in this region.

Chromosome Mapping↗
PubMed1996

Isolation of markers for chondro-osteogenic differentiation using cDNA library subtraction. Molecular cloning and characterization of a gene belonging to a novel multigene family of integral membrane proteins.

To identify novel marker molecules associated with chondro-osteogenic differentiation, we have set up a differential screening system based on a cDNA library subtraction in organ cultures of prenatal mouse mandibular condyles. Differential screening of a cDNA library constructed from in vitro cultured condyles allowed the isolation of a novel gene, named E25. Full-length E25 cDNA is predicted to encode a type II integral membrane protein of 263 amino acid residues. In situ hybridization experiments show that E25 is expressed in the outer perichondrial rim of the postnatal mandibular condyle, which contains the proliferating progenitor cells, but not in the deeper layers of the condyle containing the more differentiated chondroblasts and chondrocytes. Other cartilagenous tissues and their perichondrium were negative. Strong in situ hybridization signals were also detected on bone trabeculae of mature bone in tooth germs and in hair follicles. Northern blot analysis showed strong expression in osteogenic tissues, such as neonatal mouse calvaria, paws, tail, and in skin. This expression profile suggests that E25 could be a useful marker for chondro-osteogenic differentiation. Homology searches of DNA databanks showed that E25 belongs to a novel multigene family, containing three members both in man and mouse. The mouse E25 gene locus (Itm2) was mapped to the X chromosome.

Amino Acid Sequence↗
PubMed1996

Isolation, cDNA, and genomic structure of a conserved gene (NOF) at chromosome 11q13 next to FAU and oriented in the opposite transcriptional orientation.

In our effort to characterize a gene at chromosome 11q13 involved in a t(11;17)(q13;q21) translocation in B-non-Hodgkin lymphoma, we have identified a novel human gene, NOF (Neighbour of FAU). It maps right next to FAU in a head to head configuration separated by a maximum of 146 nucleotides. cDNA clones representing NOF hybridized to a 2. 2-kb mRNA present in all tissues tested. The largest open reading frame appeared to contain 166 amino acids and is proline rich, and the sequence shows no homology with any known gene in the public databases. The NOF gene consists of 4 exons and 3 introns spanning approximately 5 kb, and the boundaries between exons and introns follow the GT/AG rule. The NOF locus is conserved during evolution, with the predicted protein having over 80% identity to three translated mouse and rat ESTs of unknown function. Moreover, the mouse ESTs map in the same organization, closely linked to the FAU gene, in the mouse genome. NOF, however, is not affected by the t(11;17)(q13;q21) chromosomal translocation.

Amino Acid Sequence↗
PubMed1996

Molecular cloning of a highly conserved mouse and human integral membrane protein (Itm1) and genetic mapping to mouse chromosome 9.

We have isolated and characterized a novel cDNA coding for a highly hydrophobic protein (B5) from a fetal mouse mandibular condyle cDNA library. The full-length mouse B5 cDNA is 3095 nucleotides long and contains a potential open reading frame coding for a protein of 705 amino acids with a calculated molecular weight of 80.5 kDa. The B5 mRNA is differentially polyadenylated, with the most abundant transcript having a length of 2.7 kb. The human homolog of B5 was isolated from a cDNA testis library. The predicted amino acid sequence of the human B5 is 98.5% identical to that of mouse. The most striking feature of the B5 protein is the presence of numerous (10-14) potential transmembrane domains, characteristic of an integral membrane protein. Similarity searches in public databanks reveal that B5 is 58% similar to the T12A2.2 gene of Caenorhabditis elegans and 60% similar to the STT3 gene of Saccharomyces cerevisiae. Furthermore, the report of an EST sequence (Accession No. Z13858) related to the human B5, but identical to the STT3 gene, indicates that B5 belongs to a larger gene family coding for novel putative transmembrane proteins. This family exhibits a remarkable degree of conservation in different species. The gene for B5, designated Itm1 (Integral membrane protein 1), is located on mouse chromosome 9.

Animals↗
PubMed1995

Characterization of a processed pseudogene of human FAU1 on chromosome 18.

A member of the human FAU (Finkel-Biskis-Reilly murine sarcoma virus-associated ubiquitously expressed) gene subfamily, encoding the ribosomal protein S30 fused in frame to an ubiquitin-like protein, was cloned, sequenced and analysed. This clone, FAU1P, is a processed pseudogene with a completely intact, although transcriptionally silent, open reading frame of 137 codons. FAU1P exhibits an amplification of the (AAG) triplet repeat present in the S30 coding part of FAU. FAU1P is integrated in an antisense orientation within a sequence homologous to the promoter of the islet amyloid polypeptide (IAPP or amylin)-encoding gene. By means of PCR hybrid panel mapping, FAU1P was assigned to chromosome 18.

Amyloid↗