Abstract
Background
The human maspin gene encodes a protein in the serine proteinase inhibitor (serpin) family with tumor-suppressing functions in cell culture and in nude mice. In order to examine the role of maspin in an intact mammal, we cloned and sequenced the cDNA of mouse maspin. The recombinant protein was produced and its activity in cell culture was assessed.
Materials and Methods
Mouse maspin (mMaspin) was cloned by screening a mouse mammary gland cDNA library with the human maspin cDNA probe. Northern blot analysis was used to examine the expression patterns in mouse tissues, mammary epithelial cells, and carcinomas. Recombinant mMaspin protein was produced in E. coli. Invasion and motility assays were used to assess the biological function of mMaspin.
Results
mMaspin is 89% homologous with human maspin at the amino acid level. Like its human homolog, mMaspin is expressed in normal mouse mammary epithelial cells and down-regulated in mouse breast tumor cell lines. The expression is altered at different developmental stages in mammary gland. Addition of the recombinant mMaspin protein to mouse tumor cells was shown to inhibit invasion in a dose-dependent manner. As with the human protein, recombinant mMaspin protein also inhibited mouse mammary tumor motility. Deletion in the putative mMaspin reactive site loop (RSL) region resulted in the loss of its inhibitory functions.
Conclusions
mMaspin is the mouse homolog of a human tumor suppressor gene. The expression of mMaspin is down-regulated in tumor cells and is altered at different developmental stages of mammary gland. mMaspin has inhibitory properties similar to those of human maspin in cell culture, suggesting that the homologous proteins play similar physiological roles in vivo.
Similar content being viewed by others
References
Zou Z, Anisowicz A, Hendrix MJC, Thor A, Neveu M, Sheng S, Rafidi K, Seftor E, Sager R. (1994) Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells. Science 263: 526–529.
Sager R, Sheng S, Pemberton P, Hendrix MJC. (1995) Maspin, a tumor suppressing serpin. Curr. Top. Microbiol. Immunol. 1: 51–64.
Sheng S, Carey J, Seftor AE, Dias L, Hendrix JCM, Sager R. (1996) Maspin acts at the cell membrance to inhibit invasion and motility of mammary and prostate cancer cells. Proc. Natl. Acad. Sci. U.S.A. 93: 11669–11674.
Hopkins P, Whisstock J, Sager R. (1994) Function of maspin. Science 265: 1893–1894.
Pemberton P, Wong D, Gibson H, Kiefer M, Fitzpatrick P, Sager R, Barr P. (1995) The tumor suppressor maspin does not undergo the stressed to relaxed transition or inhibit trypsin-like serine proteases. J. Biol. Chem. 270: 15832–15837.
Swisshelm K, Ryan K, Lee X, Tsou HC, Peacocke M, Sager R. (1994) Down-regulation of retinoic acid receptor β in mammary carcinoma cell lines and its up-regulation in senescing normal mammary epithelial cells. Cell Growth Differ. 5: 133–141.
Laborda J. (1991) 36B4 cDNA used as an estradiol-independent mRNA control is the cDNA for human acidic ribosomal phosphoprotein PO. Nucl. Acids Res. 19: 3998.
Sambrook J, Fritsch EF, Maniatis T (ed). (1989) Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Harbor Laborotory, Cold Spring Harbor, NY.
Smith DB, Johnson KS. (1988) Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67: 31–40.
Hendrix MJC, Seftor EA, Seftor REB, Fidler IJ. (1987) A simple quantitative assay for studying the invasive potential of high and low human metastatic variants. Cancer Lett. 38: 137–147.
Chu YW, Runyan RB, Oshima RG, Hendrix MJC. (1993) Expression of complete keratin filaments in mouse L cells augments cell migration and invasion. Proc. Natl. Acad. Sci. U.S.A. 90: 4261–4265.
Travis J, Guzdek A, Potempa J, Watorek W. (1990) Serpins: Structure and mechanism of action. Biol. Chem. Hoppe Seyler 371: 3–11.
Huber R, Carrell RW. (1989) Implications of the three dimensional structure of a1-antitrypsin for structure and function of serpins. Biochemistry 28: 8951.
Coleman S, Silberstein GB, Daniel CW. (1988) Ductal morphogenesis in the mouse mammary gland: Evidence supporting a role for epidermal growth factor. Dev. Biol. 127: 304–315.
Robinson SD, Silberstin A, Roberts K, Flanders K, Daniel CW. (1991) Regulated expression and growth inhibitory effects of transforming factor. Development 113: 867–878.
Tulchinsky E, Ford HL, Kramerov D, Reshetnyak E, Grigorian M, Zain S, Lukanidin E. (1992) Transcriptional analysis of the mtsl gene with specific reference to 5′ flanking sequences. Proc. Natl. Acad. Sci. U.S.A. 89: 9146–9150.
Tulchinsky E, Kramerov D, Ford HL, Reshetnyak E, Lukanidin E, Zain S. (1993) Characterization of a positive regulatory element in the mts1 gene. Oncogene 8: 79–86.
Sakakura T. (1991) New aspects of stromal-parenchyma relations in mammary gland differentiation. Int. Rev. Cytol. 125: 165–202.
Imagawa W, Bandyopadhyay GK, Nandi S. (1990) Regulation of mammary epithelial cell growth in mice and rats. Endocr. Rev. 11: 494–523.
Snedeker S, Brown C, DiAugustine R. (1991) Expression and functional properties of transforming factor alpha and epidermal growth factor during mouse mammary gland ductal morphogenesis. Proc. Natl. Acad. Sci. U.S.A. 88: 276–280.
Lefebvre O, Wolf C, Limacher J, Hutin P, Wendling C, Lemeur M, Basset P, Rio M. (1992) The breast cancer-associated stromelysis-3 gene is expressed during mouse mammary gland apoptosis. J. Cell Biol. 119: 997–1002.
Ossowski L, Biegel D, Reich E. (1979) Correlation with involution, hormonal modulation and comparison between normal and neoplastic tissues. Cell 16: 867–878.
Talhouk RS, Chin JR, Unemori EN, Werb Z, Bissell MJ. (1991) Proteinases of the mammary gland: developmental regulation in vivo and vectorial secretion in culture. Development 112: 439–449.
Talhouk RS, Bissell MJ, Werb Z. (1992) Coordinated expression of extracellular matrix-degrading proteinase and their inhibitors regulate mammary epithelial function during involution. J. Cell Biol. 118: 1271–1282.
Sheng S, Pemberton PA, Sager R. (1994) Production, purification, and characterization of recombinant maspin proteins. J. Biol. Chem. 269: 30988–30993.
Acknowledgments
The authors thank Dr. Phil Pemberton (LXR, Inc.) for critical reading of the manuscript. This work was supported in part by an NIH grant (CA61253) to R.S. and an NIH fellowship (5-T32-CA 09361-14) to M.Z., and by LXR Biotechnology.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Zhang, M., Sheng, S., Maass, N. et al. mMaspin: The Mouse Homolog of a Human Tumor Suppressor Gene Inhibits Mammary Tumor Invasion and Motility. Mol Med 3, 49–59 (1997). https://doi.org/10.1007/BF03401667
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03401667