Abstract
A novel human keratinocyte-derived autocrine factor (KAF) was purified from conditioned medium by using heparin affinity chromatography as the first step. Purified KAF stimulated the growth of normal human keratinocytes, mouse AKR-2B cells, and a mouse keratinocyte cell line (BALB/MK). Heparin sulfate inhibited KAF mitogenic activity on all cell types tested and inhibited the ability of KAF to compete with epidermal growth factor for cell surface binding. Interestingly, KAF stimulated the growth of BALB/MK cells at high cell density but failed to stimulate these cells at clonal density. Protein microsequencing of the first 20 NH2-terminal amino acid residues of purified KAF revealed identity to the NH2 terminus of human amphiregulin (AR). Northern (RNA) blot analysis with AR-specific cRNA demonstrated that human keratinocytes, as well as mammary epithelial cell cultures, expressed high levels of AR mRNA. In contrast, AR mRNA was not detected in normal human fibroblasts or melanocytes and was present at reduced levels in several mammary tumor cell lines. The mitogenic activity of purified AR was also shown to be inhibited by heparin sulfate, and an AR-specific enzyme-linked immunosorbent assay (ELISA) revealed that KAF and AR are antigenically related. We have previously shown that human keratinocytes can grow in an autocrine manner. Our present study demonstrates that one of the growth factors responsible for this autocrine growth (KAF) is similar or identical to AR and that KAF and AR bioactivity can be negatively regulated by heparin sulfate.
Full text
PDF










Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Barrandon Y., Green H. Cell migration is essential for sustained growth of keratinocyte colonies: the roles of transforming growth factor-alpha and epidermal growth factor. Cell. 1987 Sep 25;50(7):1131–1137. doi: 10.1016/0092-8674(87)90179-6. [DOI] [PubMed] [Google Scholar]
- Burgess W. H., Maciag T. The heparin-binding (fibroblast) growth factor family of proteins. Annu Rev Biochem. 1989;58:575–606. doi: 10.1146/annurev.bi.58.070189.003043. [DOI] [PubMed] [Google Scholar]
- Clowes A. W., Karnowsky M. J. Suppression by heparin of smooth muscle cell proliferation in injured arteries. Nature. 1977 Feb 17;265(5595):625–626. doi: 10.1038/265625a0. [DOI] [PubMed] [Google Scholar]
- Coffey R. J., Jr, Derynck R., Wilcox J. N., Bringman T. S., Goustin A. S., Moses H. L., Pittelkow M. R. Production and auto-induction of transforming growth factor-alpha in human keratinocytes. 1987 Aug 27-Sep 2Nature. 328(6133):817–820. doi: 10.1038/328817a0. [DOI] [PubMed] [Google Scholar]
- Cook P. W., Coffey R. J., Jr, Magun B. E., Pittelkow M. R., Shipley G. D. Expression and regulation of mRNA coding for acidic and basic fibroblast growth factor and transforming growth factor alpha in cells derived from human skin. Mol Endocrinol. 1990 Sep;4(9):1377–1385. doi: 10.1210/mend-4-9-1377. [DOI] [PubMed] [Google Scholar]
- Cook P. W., Edwards C. P., Haraguchi T., Firestone G. L. Partial characterization of a glucocorticoid suppressible mitogenic activity secreted from a rat hepatoma cell line hypersensitive to the antiproliferative effects of glucocorticoids. J Biol Chem. 1989 Aug 25;264(24):14151–14158. [PubMed] [Google Scholar]
- Danielson P. E., Forss-Petter S., Brow M. A., Calavetta L., Douglass J., Milner R. J., Sutcliffe J. G. p1B15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA. 1988 May;7(4):261–267. doi: 10.1089/dna.1988.7.261. [DOI] [PubMed] [Google Scholar]
- Elder J. T., Fisher G. J., Lindquist P. B., Bennett G. L., Pittelkow M. R., Coffey R. J., Jr, Ellingsworth L., Derynck R., Voorhees J. J. Overexpression of transforming growth factor alpha in psoriatic epidermis. Science. 1989 Feb 10;243(4892):811–814. doi: 10.1126/science.2916128. [DOI] [PubMed] [Google Scholar]
- Hammond S. L., Ham R. G., Stampfer M. R. Serum-free growth of human mammary epithelial cells: rapid clonal growth in defined medium and extended serial passage with pituitary extract. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5435–5439. doi: 10.1073/pnas.81.17.5435. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hök M., Kjellén L., Johansson S. Cell-surface glycosaminoglycans. Annu Rev Biochem. 1984;53:847–869. doi: 10.1146/annurev.bi.53.070184.004215. [DOI] [PubMed] [Google Scholar]
- Imamura T., Mitsui Y. Heparan sulfate and heparin as a potentiator or a suppressor of growth of normal and transformed vascular endothelial cells. Exp Cell Res. 1987 Sep;172(1):92–100. doi: 10.1016/0014-4827(87)90096-6. [DOI] [PubMed] [Google Scholar]
- Kraus M. H., Issing W., Miki T., Popescu N. C., Aaronson S. A. Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9193–9197. doi: 10.1073/pnas.86.23.9193. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Madtes D. K., Raines E. W., Sakariassen K. S., Assoian R. K., Sporn M. B., Bell G. I., Ross R. Induction of transforming growth factor-alpha in activated human alveolar macrophages. Cell. 1988 Apr 22;53(2):285–293. doi: 10.1016/0092-8674(88)90390-x. [DOI] [PubMed] [Google Scholar]
- Majesky M. W., Schwartz S. M., Clowes M. M., Clowes A. W. Heparin regulates smooth muscle S phase entry in the injured rat carotid artery. Circ Res. 1987 Aug;61(2):296–300. doi: 10.1161/01.res.61.2.296. [DOI] [PubMed] [Google Scholar]
- McKeehan W. L., Ham R. G. Methods for reducing the serum requirement for growth in vitro of nontransformed diploid fibroblasts. Dev Biol Stand. 1976 Dec 13;37:97–98. [PubMed] [Google Scholar]
- O'Keefe E. J., Chiu M. L., Payne R. E., Jr Stimulation of growth of keratinocytes by basic fibroblast growth factor. J Invest Dermatol. 1988 May;90(5):767–769. doi: 10.1111/1523-1747.ep12560956. [DOI] [PubMed] [Google Scholar]
- Pittelkow M. R., Lindquist P. B., Abraham R. T., Graves-Deal R., Derynck R., Coffey R. J., Jr Induction of transforming growth factor-alpha expression in human keratinocytes by phorbol esters. J Biol Chem. 1989 Mar 25;264(9):5164–5171. [PubMed] [Google Scholar]
- Platt E. J., Karlsen K., Lopez-Valdivieso A., Cook P. W., Firestone G. L. Highly sensitive immunoadsorption procedure for detection of low-abundance proteins. Anal Biochem. 1986 Jul;156(1):126–135. doi: 10.1016/0003-2697(86)90163-6. [DOI] [PubMed] [Google Scholar]
- Plowman G. D., Green J. M., McDonald V. L., Neubauer M. G., Disteche C. M., Todaro G. J., Shoyab M. The amphiregulin gene encodes a novel epidermal growth factor-related protein with tumor-inhibitory activity. Mol Cell Biol. 1990 May;10(5):1969–1981. doi: 10.1128/mcb.10.5.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Plowman G. D., Whitney G. S., Neubauer M. G., Green J. M., McDonald V. L., Todaro G. J., Shoyab M. Molecular cloning and expression of an additional epidermal growth factor receptor-related gene. Proc Natl Acad Sci U S A. 1990 Jul;87(13):4905–4909. doi: 10.1073/pnas.87.13.4905. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rahemtulla F., Moorer C. M., Wille J. J., Jr Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum-free medium. J Cell Physiol. 1989 Jul;140(1):98–106. doi: 10.1002/jcp.1041400113. [DOI] [PubMed] [Google Scholar]
- Robertson P. L., Goldstein G. W. Heparin inhibits the growth of astrocytes in vitro. Brain Res. 1988 May 3;447(2):341–345. doi: 10.1016/0006-8993(88)91137-7. [DOI] [PubMed] [Google Scholar]
- Schwab M., Alitalo K., Varmus H. E., Bishop J. M., George D. A cellular oncogene (c-Ki-ras) is amplified, overexpressed, and located within karyotypic abnormalities in mouse adrenocortical tumour cells. Nature. 1983 Jun 9;303(5917):497–501. doi: 10.1038/303497a0. [DOI] [PubMed] [Google Scholar]
- Shipley G. D., Childs C. B., Volkenant M. E., Moses H. L. Differential effects of epidermal growth factor, transforming growth factor, and insulin on DNA and protein synthesis and morphology in serum-free cultures of AKR-2B cells. Cancer Res. 1984 Feb;44(2):710–716. [PubMed] [Google Scholar]
- Shipley G. D., Ham R. G. Improved medium and culture conditions for clonal growth with minimal serum protein and for enhanced serum-free survival of Swiss 3T3 cells. In Vitro. 1981 Aug;17(8):656–670. doi: 10.1007/BF02628401. [DOI] [PubMed] [Google Scholar]
- Shipley G. D., Keeble W. W., Hendrickson J. E., Coffey R. J., Jr, Pittelkow M. R. Growth of normal human keratinocytes and fibroblasts in serum-free medium is stimulated by acidic and basic fibroblast growth factor. J Cell Physiol. 1989 Mar;138(3):511–518. doi: 10.1002/jcp.1041380310. [DOI] [PubMed] [Google Scholar]
- Shipley G. D., Pittelkow M. R. Control of growth and differentiation in vitro of human keratinocytes cultured in serum-free medium. Arch Dermatol. 1987 Nov;123(11):1541a–1544a. [PubMed] [Google Scholar]
- Shoyab M., McDonald V. L., Bradley J. G., Todaro G. J. Amphiregulin: a bifunctional growth-modulating glycoprotein produced by the phorbol 12-myristate 13-acetate-treated human breast adenocarcinoma cell line MCF-7. Proc Natl Acad Sci U S A. 1988 Sep;85(17):6528–6532. doi: 10.1073/pnas.85.17.6528. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shoyab M., Plowman G. D., McDonald V. L., Bradley J. G., Todaro G. J. Structure and function of human amphiregulin: a member of the epidermal growth factor family. Science. 1989 Feb 24;243(4894 Pt 1):1074–1076. doi: 10.1126/science.2466334. [DOI] [PubMed] [Google Scholar]
- Sternfeld M. D., Hendrickson J. E., Keeble W. W., Rosenbaum J. T., Robertson J. E., Pittelkow M. R., Shipley G. D. Differential expression of mRNA coding for heparin-binding growth factor type 2 in human cells. J Cell Physiol. 1988 Aug;136(2):297–304. doi: 10.1002/jcp.1041360212. [DOI] [PubMed] [Google Scholar]
- Weissman B. E., Aaronson S. A. BALB and Kirsten murine sarcoma viruses alter growth and differentiation of EGF-dependent balb/c mouse epidermal keratinocyte lines. Cell. 1983 Feb;32(2):599–606. doi: 10.1016/0092-8674(83)90479-8. [DOI] [PubMed] [Google Scholar]
- Wille J. J., Jr, Pittelkow M. R., Shipley G. D., Scott R. E. Integrated control of growth and differentiation of normal human prokeratinocytes cultured in serum-free medium: clonal analyses, growth kinetics, and cell cycle studies. J Cell Physiol. 1984 Oct;121(1):31–44. doi: 10.1002/jcp.1041210106. [DOI] [PubMed] [Google Scholar]
- Wright T. C., Jr, Castellot J. J., Jr, Petitou M., Lormeau J. C., Choay J., Karnovsky M. J. Structural determinants of heparin's growth inhibitory activity. Interdependence of oligosaccharide size and charge. J Biol Chem. 1989 Jan 25;264(3):1534–1542. [PubMed] [Google Scholar]
- Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]