Abstract
The protease inhibitor leupeptin inhibits the degradation process of 125I-labeled epidermal growth factor (125I-EGF) by cultured bovine granulosa cells. At 80 μg/ml, leupeptin inhibited the appearance of degradation products of 125I-EGF in the medium by 95% during 1 hr of incubation and by 90% during 24 hr of incubation when the cells were exposed to 5 ng of 125I-EGF per ml. In contrast, cultures exposed to either saturating (10 ng/ml) or nonsaturating (0.1 ng/ml) concentrations of EGF in the presence of leupeptin (80 μg/ml) exhibited an increase in DNA synthesis that was 70-80% that of cultures exposed to EGF alone. Cultures responded to either EGF or fibroblast growth factor with a logarithmic increase in cell number and, over a period of 8 days, the number of cells increased 10- to 18-fold. Addition of leupeptin did not diminish the growth rate of the cultures. In the presence of leupeptin, 125I-EGF accumulated within the granulosa cells and was in a form that was precipitable with antiserum against EGF and that comigrated on isoelectric focusing with native 125I-EGF. That a full mitogenic response can be obtained despite a 90-95% inhibition of EGF degradation at either saturating or nonsaturating concentrations of the mitogen suggests that a proteolytic degradation of a given mitogen may not be involved in the induction of a proliferative response.
Keywords: mitogen degradation, leupeptin, granulosa cells, cell proliferation, DNA synthesis
Full text
PDF
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Brown M. S., Goldstein J. L. Receptor-mediated endocytosis: insights from the lipoprotein receptor system. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3330–3337. doi: 10.1073/pnas.76.7.3330. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Byyny R. L., Orth D. N., Cohen S. Radioimmunoassay of epidermal growth factor. Endocrinology. 1972 May;90(5):1261–1266. doi: 10.1210/endo-90-5-1261. [DOI] [PubMed] [Google Scholar]
- Carpenter G., Cohen S. 125I-labeled human epidermal growth factor. Binding, internalization, and degradation in human fibroblasts. J Cell Biol. 1976 Oct;71(1):159–171. doi: 10.1083/jcb.71.1.159. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chen L. B., Buchanan J. M. Mitogenic activity of blood components. I. Thrombin and prothrombin. Proc Natl Acad Sci U S A. 1975 Jan;72(1):131–135. doi: 10.1073/pnas.72.1.131. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Das M., Fox C. F. Molecular mechanism of mitogen action: processing of receptor induced by epidermal growth factor. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2644–2648. doi: 10.1073/pnas.75.6.2644. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gospodarowicz D., Bialecki H., Greenburg G. Purification of the fibroblast growth factor activity from bovine brain. J Biol Chem. 1978 May 25;253(10):3736–3743. [PubMed] [Google Scholar]
- Gospodarowicz D., Bialecki H. The effects of the epidermal and fibroblast growth factors on the replicative lifespan of cultured bovine granulosa cells. Endocrinology. 1978 Sep;103(3):854–865. doi: 10.1210/endo-103-3-854. [DOI] [PubMed] [Google Scholar]
- Gospodarowicz D., Ill C. R., Birdwell C. R. Effects of fibroblast and epidermal growth factors on ovarian cell proliferation in vitro. I. Characterization of the response of granulosa cells to FGF and EGF. Endocrinology. 1977 Apr;100(4):1108–1120. doi: 10.1210/endo-100-4-1108. [DOI] [PubMed] [Google Scholar]
- HUNTER W. M., GREENWOOD F. C. Preparation of iodine-131 labelled human growth hormone of high specific activity. Nature. 1962 May 5;194:495–496. doi: 10.1038/194495a0. [DOI] [PubMed] [Google Scholar]
- Haigler H. T., McKanna J. A., Cohen S. Direct visualization of the binding and internalization of a ferritin conjugate of epidermal growth factor in human carcinoma cells A-431. J Cell Biol. 1979 May;81(2):382–395. doi: 10.1083/jcb.81.2.382. [DOI] [PMC free article] [PubMed] [Google Scholar]
- O'Farrell P. Z., Goodman H. M., O'Farrell P. H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins. Cell. 1977 Dec;12(4):1133–1141. doi: 10.1016/0092-8674(77)90176-3. [DOI] [PubMed] [Google Scholar]
- Savage C. R., Jr, Cohen S. Epidermal growth factor and a new derivative. Rapid isolation procedures and biological and chemical characterization. J Biol Chem. 1972 Dec 10;247(23):7609–7611. [PubMed] [Google Scholar]
- Schechter Y., Hernaez L., Schlessinger J., Cuatrecasas P. Local aggregation of hormone-receptor complexes is required for activation by epidermal growth factor. Nature. 1979 Apr 26;278(5707):835–838. doi: 10.1038/278835a0. [DOI] [PubMed] [Google Scholar]
- Schlessinger J., Shechter Y., Willingham M. C., Pastan I. Direct visualization of binding, aggregation, and internalization of insulin and epidermal growth factor on living fibroblastic cells. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2659–2663. doi: 10.1073/pnas.75.6.2659. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shechter Y., Hernaez L., Cuatrecasas P. Epidermal growth factor: biological activity requires persistent occupation of high-affinity cell surface receptors. Proc Natl Acad Sci U S A. 1978 Dec;75(12):5788–5791. doi: 10.1073/pnas.75.12.5788. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Umezawa H. Structures and activities of protease inhibitors of microbial origin. Methods Enzymol. 1976;45:678–695. doi: 10.1016/s0076-6879(76)45058-9. [DOI] [PubMed] [Google Scholar]
- Vlodavsky I., Brown K. D., Gospodarowicz D. A comparison of the binding of epidermal growth factor to cultured granulosa and luteal cells. J Biol Chem. 1978 May 25;253(10):3744–3750. [PubMed] [Google Scholar]