Expression of Mouse LSP1/S37 Isoforms S37 Is Expressed in Embryonic Mesenchymal Cells

Journal of Cell Science 107, 3591-3600 (1994) 3591 Printed in Great Britain © The Company of Biologists Limited 1994 Expression of mouse LSP1/S37 isoforms S37 is expressed in embryonic mesenchymal cells V. L. Misener1, C.-c. Hui2, I. A. Malapitan1, M.-E. Ittel1, A. L. Joyner2,3 and J. Jongstra1,* 1The Arthritis Centre-Research Unit, The Toronto Hospital Research Institute and Department of Immunology, University of Toronto, Toronto, Ontario, Canada 2Division of Molecular and Developmental Biology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada 3Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario, Canada *Author for correspondence at: Toronto Western Hospital, Room 13-419, 399 Bathurst Street, Toronto, Ontario, M5T 2S8 Canada SUMMARY Mouse LSP1 is a 330 amino acid intracellular F-actin 18 bp encoding the 6 amino acids HLIRHQ of the acidic binding protein expressed in lymphocytes and domain. Therefore, the Lsp1 gene encodes four protein macrophages but not in non-hematopoietic tissues. A 328 isoforms: full-length LSP1 and S37 proteins, designated amino acid LSP1-related protein, designated S37, is LSP1-I and S37-I and the same proteins without the expressed in murine bone marrow stromal cells, in fibro- HLIRHQ sequence, designated LSP1-II and S37-II. By in blasts, and in a myocyte cell line. The two proteins differ situ hybridization analysis we show that the S37 isoforms only at their N termini, the first 23 amino acid residues of are expressed in mesenchymal tissue, but not in adjacent LSP1 being replaced by 21 different residues in S37. The epithelial tissue, of several developing organs during mouse presence of different amino termini suggests that the LSP1 embryogenesis. This, together with our finding that S37 is and S37 proteins are encoded by transcripts arising an F-actin binding protein, suggests that S37 is a cytoskele- through alternative exon splicing. Here we report the tal protein of mesenchymal cells, which may play a role in genomic organization of the Lsp1 gene and show that the mesenchyme-induced epithelial differentiation during distinct N termini of LSP1 and S37 are encoded by two organogenesis. alternatively used exons, each containing a translational start codon. We also demonstrate that alternative 3′ acceptor sites are used in the splicing of exon 5. This results Key words: LSP1, S37, gene structure, expression, embryonic in LSP1 and S37 transcripts that either do or do not contain mesenchyme, F-actin binding INTRODUCTION detergent-insoluble residue after lysis of B-lymphoma cells or normal spleen cells with the non-ionic detergent NP-40 Mouse LSP1 (Jongstra et al., 1988a), also designated pp52 (Jongstra-Bilen et al., 1992; M.-E. Ittel, unpublished). Fur- (Gimble et al., 1993), is a 330 amino acid intracellular phos- thermore, we showed that upon treatment of B-lymphoma cell phoprotein expressed in normal B-cells and transformed B-cell lines with anti-immunoglobulin (Ig) antibody to induce lines, in normal T-cells and non-transformed functional T-cell capping of membrane Ig molecules, the intracellular LSP1 lines, but not in transformed T-cell lines (Jongstra et al., 1988a; protein aggregates directly underneath the clusters of Klein et al., 1989). Human LSP1 has a similar expression membrane IgM (Klein et al., 1990). These results suggest that pattern (Jongstra et al., 1988a) and, in addition, has been found LSP1 protein is associated with the cytoskeleton. Our finding to be expressed in human neutrophils (Howard et al., 1994). A that mouse LSP1 protein binds to filamentous actin (F-actin) comparison of the mouse and human LSP1 cDNA sequences with a Kd of 0.2 µM, but does not bind to globular actin (G- predicts that the LSP1 proteins have a two-domain structure actin) suggests that LSP1 protein associates with the cytoskele- with an amino-terminal domain rich in acidic residues and a ton by binding directly to the F-actin-containing microfila- carboxy-terminal domain rich in basic residues. The basic ments. We also showed that both F-actin binding in vitro and domains of the mouse and human LSP1 proteins are 85% cytoskeletal binding in vivo occur through the conserved basic identical, suggesting that the basic domain of LSP1 is of par- domain (Jongstra-Bilen et al., 1992). ticular importance for LSP1 function (Jongstra-Bilen et al., Mouse LSP1 protein is encoded by a 1.6 kb mRNA tran- 1990). script expressed in lymphoid cells, lymphoma cell lines Intracellular fractionation studies showed that a significant (Jongstra et al., 1988a; Klein et al., 1989) and transformed fraction of the intracellular LSP1 protein associates with the macrophage cell lines (this paper). Expression is undetectable 3592 V. L. Misener and others in several other myeloid cell types tested, including peripheral Bilen et al., 1992), except that polymerization reactions were done at blood granulocytes, erythroleukemia cell lines and the masto- room temperature for 1 hour and contained 4 µM G-actin (from rabbit cytoma cell line P815. In addition, expression of mouse LSP1 skeletal muscle, a gift from Dr P. A. Janmey, Brigham and Women’s RNA is not detected in a variety of non-hematopoietic tissues Hospital, Boston) and 2 µM recombinant protein. such as adult liver, brain and kidney (Jongstra et al., 1988a). Oligonucleotides Recently, an LSP1-related cDNA designated S37, was isolated from the murine bone marrow stromal cell line BMS2. These Sequences of oligonucleotides used in this study are given below. Nucleotides represented in upper case letters correspond to either cells express a 2 kb RNA transcript that hybridizes with the sense or anti-sense Lsp1 sequences. Those in lower case letters are LSP1 cDNA and which is also detected in the fibroblast cell nonhomologous sequences, incorporating restriction sites designed to lines NIH3T3 and L-cells, and in the myocyte cell line G7 facilitate the subcloning of amplified fragments: (Gimble et al., 1993). The nucleotide sequence of the stromal A1 (sense): 5′-ATGGCGGAGGCTGCCATCGATCCCAGA-3′; cell-derived S37 clone predicts that it encodes a 328 amino A2 (anti-sense): 5′-aggtcgaCAGCTCAACGGTGTCTTCTA-3′; acid protein that is identical to the LSP1 protein expressed in A17 (sense): 5′-CCTGCAGCATGAATGGCCCCGCACTCC-3′; lymphocytes and macrophages, except that the 23 amino- JJ24 (sense): 5′-aggtcgacAACAGAGGTGCTGGGCCAGA-3′; terminal residues of LSP1 are replaced by a different amino JJ25 (anti-sense): 5′-aggtcgaCTCAGCAGCTTCTCCAGGC-3′; terminus of 21 amino acid residues in S37. JJ53 (sense): 5′-agggatccAGAGAAACACCAGGAGCC-3′; The F-actin binding property and the cytoskeletal localiza- JJ54 (anti-sense): 5′-aggtcgacCCTTCGCTGTCTTCTGCA-3′. tion of LSP1 protein led us to suggest that this protein is involved in the proper functioning of the immune system, in RNA isolation and northern blot analysis which it is uniquely expressed, through its involvement in Isolation of poly(A)+ RNA from mouse embryos and isolation of total cytoskeleton-dependent aspects of biological processes such as cytoplasmic RNA from cell lines, were carried out as described signal transduction, cellular adhesion or cell motility. As a (Jongstra et al, 1988b; Chomczynski and Sacchi, 1987; Kingston, 1987). Samples of poly(A)+ RNA (10 µg) or of total cytoplasmic prelude to generating an LSP1-deficient mouse strain by RNA (10 µg) were subjected to agarose/formaldehyde gel elec- targeted disruption of the Lsp1 gene in embryonic stem cells, trophoresis, transferred to nitrocellulose and hybridized with 32P- we determined the genomic organization of the Lsp1 gene and labelled DNA probes (Jongstra et al., 1988b). investigated its expression in the developing mouse embryo. The genomic organization of the Lsp1 gene demonstrates that In situ hybridization analysis the different N termini of the LSP1 and S37 proteins are In situ hybridization analysis of fixed and acetylated mouse embryo encoded by separate exons. Further isoform diversity is created sections with the full-length LSP1 probe was carried out essentially by alternative splicing of exon 5, which encodes amino acid as described (Hui and Joyner, 1993). In experiments using the S37- residues in the acidic domain. In addition, we extend the specific Emb 5′ probe the hybridization and wash conditions were findings of previous expression studies (Jongstra et al., 1988a; modified as described below. The Emb 5′ probe contains 106 bp of ′ Klein et al., 1989), and show that transcripts encoding LSP1 5 untranslated sequence and 62 bp of N-terminal coding sequence of are expressed in macrophage cell lines and that transcripts S37 RNA. Sense and anti-sense RNA probes were prepared by in vitro transcription of DNA templates in the cloning vector pBluescript II encoding S37 are expressed in certain mesenchymal tissues KS(+), using either T3 or T7 RNA polymerase. The Emb 5′ probe during mouse embryonic development. was hybridized overnight at 50°C. Modified post-hybridization washes were as follows: slides were immersed at 65°C for 10 minutes in washing buffer (50% formamide, 2× SSC, 0.1% β-mercap- MATERIALS AND METHODS toethanol), rinsed three times (10 minutes each) at 37°C in NTE (0.5 M NaCl, 10 mM Tris-HCl, 5 mM EDTA, pH 7.5), treated for 30 Cell lines minutes at 37°C with 20 µg/ml RNase A in NTE, and immersed for The macrophage cell lines J774A.1 and IC-21 were purchased form a further 15 minutes in NTE. Slides were then washed sequentially in the ATCC and cultured according to the instructions provided. B- washing buffer at 65°C for 5 minutes, 2× SSC at 37°C for 10 minutes lymphoma cell lines BAL17 and WEHI-231 were obtained from M. and 0.1× SSC at 37°C for 10 minutes, followed by dehydration. Slides M. Davis, Stanford University. The T-lymphoma cell line BW5147 were coated with Kodak NTB 2 emulsion and exposed at 4°C for 4- was obtained from N.

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