Abstract
BeWo is a choriocarcinoma cell line that generates an extracellular matrix (ECM) rich in laminin and is a useful model for human trophoblast. Immunofluorescence with monoclonal antibodies demonstrates that BeWo ECM contains laminin subunits beta1 and gamma1. Immunoprecipitation from conditioned medium shows that the cells secrete two distinct laminin trimers both containing beta1 and gamma1 but with alpha subunits of approx. 400 and 450 kDa. The culture medium also contains a species thought to be beta1 gamma1 dimer. Immunoprecipitation with monoclonal antibody 4C7, previously thought to recognize the alpha1 subunit, isolates complexes containing only the smaller alpha subunit. A second complex containing the larger alpha subunit along with beta1, gamma1 and a 150 kDa polypeptide is precipitated from 4C7-depleted medium with an anti-(laminin 1) polyclonal antibody. Peptide sequencing demonstrates that the 4C7-reactive species is alpha5, which is present as two similarly sized polypeptides. mRNA species encoding laminin subunits alpha1, alpha5, beta1, beta2 and gamma1 are all present in the cells. These results demonstrate the secretion of a novel laminin isoform, laminin 10, the subunit composition of which is alpha5 beta1 gamma1. Laminin 1 is also produced. No evidence for the secretion of beta2-containing laminin isoforms could be derived despite the presence of beta2 mRNA. Analysis with reverse transcriptase-mediated PCR also showed the presence of laminin alpha5 in first-trimester placenta and decidua.
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- Aplin J. D. Expression of integrin alpha 6 beta 4 in human trophoblast and its loss from extravillous cells. Placenta. 1993 Mar-Apr;14(2):203–215. doi: 10.1016/s0143-4004(05)80261-9. [DOI] [PubMed] [Google Scholar]
- Aplin J. D., Sattar A., Mould A. P. Variant choriocarcinoma (BeWo) cells that differ in adhesion and migration on fibronectin display conserved patterns of integrin expression. J Cell Sci. 1992 Oct;103(Pt 2):435–444. doi: 10.1242/jcs.103.2.435. [DOI] [PubMed] [Google Scholar]
- Burgeson R. E., Chiquet M., Deutzmann R., Ekblom P., Engel J., Kleinman H., Martin G. R., Meneguzzi G., Paulsson M., Sanes J. A new nomenclature for the laminins. Matrix Biol. 1994 Apr;14(3):209–211. doi: 10.1016/0945-053x(94)90184-8. [DOI] [PubMed] [Google Scholar]
- Church H. J., Vićovac L. M., Williams J. D., Hey N. A., Aplin J. D. Laminins 2 and 4 are expressed by human decidual cells. Lab Invest. 1996 Jan;74(1):21–32. [PubMed] [Google Scholar]
- Durkin M. E., Loechel F., Mattei M. G., Gilpin B. J., Albrechtsen R., Wewer U. M. 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. FEBS Lett. 1997 Jul 14;411(2-3):296–300. doi: 10.1016/s0014-5793(97)00686-8. [DOI] [PubMed] [Google Scholar]
- Ecay T. W., Valentich J. D. Basal lamina formation by epithelial cell lines correlates with laminin A chain synthesis and secretion. Exp Cell Res. 1992 Nov;203(1):32–38. doi: 10.1016/0014-4827(92)90036-8. [DOI] [PubMed] [Google Scholar]
- Ekblom P. Receptors for laminins during epithelial morphogenesis. Curr Opin Cell Biol. 1996 Oct;8(5):700–706. doi: 10.1016/s0955-0674(96)80112-8. [DOI] [PubMed] [Google Scholar]
- Engvall E., Davis G. E., Dickerson K., Ruoslahti E., Varon S., Manthorpe M. Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site. J Cell Biol. 1986 Dec;103(6 Pt 1):2457–2465. doi: 10.1083/jcb.103.6.2457. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Engvall E., Earwicker D., Haaparanta T., Ruoslahti E., Sanes J. R. Distribution and isolation of four laminin variants; tissue restricted distribution of heterotrimers assembled from five different subunits. Cell Regul. 1990 Sep;1(10):731–740. doi: 10.1091/mbc.1.10.731. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Haaparanta T., Uitto J., Ruoslahti E., Engvall E. Molecular cloning of the cDNA encoding human laminin A chain. Matrix. 1991 Jun;11(3):151–160. doi: 10.1016/s0934-8832(11)80153-8. [DOI] [PubMed] [Google Scholar]
- Hey N. A., Graham R. A., Seif M. W., Aplin J. D. The polymorphic epithelial mucin MUC1 in human endometrium is regulated with maximal expression in the implantation phase. J Clin Endocrinol Metab. 1994 Feb;78(2):337–342. doi: 10.1210/jcem.78.2.8106621. [DOI] [PubMed] [Google Scholar]
- Iivanainen A., Sainio K., Sariola H., Tryggvason K. Primary structure and expression of a novel human laminin alpha 4 chain. FEBS Lett. 1995 May 29;365(2-3):183–188. doi: 10.1016/0014-5793(95)00462-i. [DOI] [PubMed] [Google Scholar]
- Kallunki T., Ikonen J., Chow L. T., Kallunki P., Tryggvason K. Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene. J Biol Chem. 1991 Jan 5;266(1):221–228. [PubMed] [Google Scholar]
- Korhonen M., Virtanen I. The distribution of laminins and fibronectins is modulated during extravillous trophoblastic cell differentiation and decidual cell response to invasion in the human placenta. J Histochem Cytochem. 1997 Apr;45(4):569–581. doi: 10.1177/002215549704500409. [DOI] [PubMed] [Google Scholar]
- Lissitzky J. C., Cantau P., Martin P. M. Heterotrimeric configuration is essential to the adhesive function of laminin. J Cell Biochem. 1992 Feb;48(2):141–149. doi: 10.1002/jcb.240480206. [DOI] [PubMed] [Google Scholar]
- Mercurio A. M. Laminin receptors: achieving specificity through cooperation. Trends Cell Biol. 1995 Nov;5(11):419–423. doi: 10.1016/s0962-8924(00)89100-x. [DOI] [PubMed] [Google Scholar]
- Miner J. H., Lewis R. M., Sanes J. R. Molecular cloning of a novel laminin chain, alpha 5, and widespread expression in adult mouse tissues. J Biol Chem. 1995 Dec 1;270(48):28523–28526. doi: 10.1074/jbc.270.48.28523. [DOI] [PubMed] [Google Scholar]
- Miner J. H., Patton B. L., Lentz S. I., Gilbert D. J., Snider W. D., Jenkins N. A., Copeland N. G., Sanes J. R. The laminin alpha chains: expression, developmental transitions, and chromosomal locations of alpha1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel alpha3 isoform. J Cell Biol. 1997 May 5;137(3):685–701. doi: 10.1083/jcb.137.3.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nissinen M., Vuolteenaho R., Boot-Handford R., Kallunki P., Tryggvason K. Primary structure of the human laminin A chain. Limited expression in human tissues. Biochem J. 1991 Jun 1;276(Pt 2):369–379. doi: 10.1042/bj2760369. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peters B. P., Hartle R. J., Krzesicki R. F., Kroll T. G., Perini F., Balun J. E., Goldstein I. J., Ruddon R. W. The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells. J Biol Chem. 1985 Nov 25;260(27):14732–14742. [PubMed] [Google Scholar]
- Pikkarainen T., Eddy R., Fukushima Y., Byers M., Shows T., Pihlajaniemi T., Saraste M., Tryggvason K. Human laminin B1 chain. A multidomain protein with gene (LAMB1) locus in the q22 region of chromosome 7. J Biol Chem. 1987 Aug 5;262(22):10454–10462. [PubMed] [Google Scholar]
- Reing J., Durkin M. E., Chung A. E. Laminin B1 expression is required for laminin deposition into the extracellular matrix of PC12 cells. J Biol Chem. 1992 Nov 15;267(32):23143–23150. [PubMed] [Google Scholar]
- Schuger L., Skubitz A. P., Zhang J., Sorokin L., He L. Laminin alpha1 chain synthesis in the mouse developing lung: requirement for epithelial-mesenchymal contact and possible role in bronchial smooth muscle development. J Cell Biol. 1997 Oct 20;139(2):553–562. doi: 10.1083/jcb.139.2.553. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shevchenko A., Wilm M., Vorm O., Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem. 1996 Mar 1;68(5):850–858. doi: 10.1021/ac950914h. [DOI] [PubMed] [Google Scholar]
- Sorokin L. M., Pausch F., Frieser M., Kröger S., Ohage E., Deutzmann R. Developmental regulation of the laminin alpha5 chain suggests a role in epithelial and endothelial cell maturation. Dev Biol. 1997 Sep 15;189(2):285–300. doi: 10.1006/dbio.1997.8668. [DOI] [PubMed] [Google Scholar]
- Tiger C. F., Champliaud M. F., Pedrosa-Domellof F., Thornell L. E., Ekblom P., Gullberg D. Presence of laminin alpha5 chain and lack of laminin alpha1 chain during human muscle development and in muscular dystrophies. J Biol Chem. 1997 Nov 7;272(45):28590–28595. doi: 10.1074/jbc.272.45.28590. [DOI] [PubMed] [Google Scholar]
- Timpl R., Brown J. C. The laminins. Matrix Biol. 1994 Aug;14(4):275–281. doi: 10.1016/0945-053x(94)90192-9. [DOI] [PubMed] [Google Scholar]
- Timpl R. Macromolecular organization of basement membranes. Curr Opin Cell Biol. 1996 Oct;8(5):618–624. doi: 10.1016/s0955-0674(96)80102-5. [DOI] [PubMed] [Google Scholar]
- Vuolteenaho R., Nissinen M., Sainio K., Byers M., Eddy R., Hirvonen H., Shows T. B., Sariola H., Engvall E., Tryggvason K. Human laminin M chain (merosin): complete primary structure, chromosomal assignment, and expression of the M and A chain in human fetal tissues. J Cell Biol. 1994 Feb;124(3):381–394. doi: 10.1083/jcb.124.3.381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wewer U. M., Gerecke D. R., Durkin M. E., Kurtz K. S., Mattei M. G., Champliaud M. F., Burgeson R. E., Albrechtsen R. Human beta 2 chain of laminin (formerly S chain): cDNA cloning, chromosomal localization, and expression in carcinomas. Genomics. 1994 Nov 15;24(2):243–252. doi: 10.1006/geno.1994.1612. [DOI] [PubMed] [Google Scholar]
- Wewer U. M., Wayner E. A., Hoffstrom B. G., Lan F., Meyer-Nielsen B., Engvall E., Albrechtsen R. Selective assembly of laminin variants by human carcinoma cells. Lab Invest. 1994 Nov;71(5):719–730. [PubMed] [Google Scholar]