Journal
MOLECULAR AND CELLULAR ENDOCRINOLOGY
Volume 332, Issue 1-2, Pages 106-115Publisher
ELSEVIER IRELAND LTD
DOI: 10.1016/j.mce.2010.10.002
Keywords
BMP15; Bone morphogenetic protein; Granulosa cell; Human
Categories
Funding
- Academy of Finland
- Finnish Funding Agency for Technology and Innovation
- Juselius Foundation
- Jalmari and Rauha Ahokas Foundation
- Novo Nordisk Foundation
- Orion-Farmos Research Foundation
- Helsinki University
- Paulo Foundation
- Jenny and Antti Wihuri Foundation
- New Zealand Foundation for Research Science and Technology
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Oocyte-derived bone morphogenetic protein-15 (BMP15) is critical for the regulation of mammalian fertility. Previously we have found that a C-terminal His(6)-tag destroys the bioactivity of growth differentiation-9 (GDF9, a homolog of BMP15). In this study we found that recombinant human BMP15 is produced by HEK-293T cells in an active form, but the bioactivity is lost by C-terminal modification, specifically, fusion to a Flag tag. After purification the mature BMP15wt is active in transcriptional reporter assays specific for Smad1/5/8 in human granulosa-luteal (hGL) and COV434 granulosa tumor cells, whereas BMP15 with a carboxy-terminal Flag tag remains inactive. Using these same cell models we found that treatment with purified mature BMP15wt causes a rapid phosphorylation of Smad1. The purified BMP15wt is a potent stimulator of rat granulosa cell DNA synthesis, which could be antagonized by the BMPRII ectodomain-Fc fusion molecule, whereas the BMP15C-Flag was completely inactive. Further, the BMP15wt form is a potent stimulator of inhibin B production in hGL cells. We found that the purified BMP15wt consists of P16 and -17, both of which are post-translationally modified forms. This is the first characterization of a purified untagged human BMP15 mature region, which is stable and highly bioactive in human and rodent granulosa cells and as such is of importance for studies on human fertility. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
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