Search

Abstract Background Uterine serpins are members of the serine proteinase inhibitor superfamily. Like some other serpins, these proteins do not appear to be functional proteinase inhibitors. The most studied member of the group, ovine uterine serpin (OvUS), inhibits proliferation of several cell types including activated lymphocytes, bovine preimplantation embryos, and cell lines for lymphoma, canine primary osteosarcoma and human prostate cancer (PC-3) cells. The goal for the present study was to evaluate the mechanism by which OvUS inhibits cell proliferation. In particular, it was tested whether inhibition of DNA synthesis in PC-3 cells involves cytotoxic actions of OvUS or the induction of apoptosis. The effect of OvUS in the production of the autocrine and angiogenic cytokine interleukin (IL)-8 by PC-3 cells was also determined. Finally, it was tested whether OvUS blocks specific steps in the cell cycle using both PC-3 cells and lymphocytes. Results Recombinant OvUS blocked proliferation of PC-3 cells at concentrations as low as 8 μg/ml as determined by measurements of [3H]thymidine incorporation or ATP content per well. Treatment of PC-3 cells with OvUS did not cause cytotoxicity or apoptosis or alter interleukin-8 secretion into medium. Results from flow cytometry experiments showed that OvUS blocked the entry of PC-3 cells into S phase and the exit from G2/M phase. In addition, OvUS blocked entry of lymphocytes into S phase following activation of proliferation with phytohemagglutinin. Conclusion Results indicate that OvUS acts to block cell proliferation through disruption of the cell cycle dynamics rather than induction of cytotoxicity or apoptosis. The finding that OvUS can regulate cell proliferation makes this one of only a few serpins that function to inhibit cell growth.

Uterine serpins (USs), designated as SERPINA14, are expressed in the endometrium in response to progesterone. All species identified as having USs exhibit epitheliochorial placentation and are in the Ruminantia and Suidae orders of the Laurasiatheria superorder. The objective was to identify US genes in species within and outside Laurasiatheria and evaluate whether evolution of the US gene was associated with development of the epitheliochorial placenta. Through queries of nucleotide and genomic databases, known US genes were identified (caprine, bovine, porcine, water buffalo), and new US coding sequences were found in dolphins, horses, dogs, and cats. The cat sequence contained several stop codons. No sequence was found in completed genomic sequences for primates, rodents, rabbits, opossums, or duck-billed platypuses. Reverse transcription-polymerase chain reaction confirmed expression of the US gene in the uterus of pregnant horses and dogs. The ratio of nonsynonymous/synonymous substitutions suggests that the US gene evolved under positive selection. In conclusion, the US gene evolved within the Laurasiatheria super-order to play a role in pregnancy for species with epitheliochorial placentation and some but not all Laurasiatheria species that have a different form of placentation. The positive selection taking place in die gene suggests development of species-specific functions. [ABSTRACT FROM AUTHOR]

The uterine serpins have been described in sheep, cattle, and pigs as a highly diverged group of the large superfamily of serpin proteins that typically function as serine proteinase inhibitors. Here, the range of species that possess and express a uterine serpin gene is extended to the goat. Sequencing of cDNA amplified from total RNA from a pregnant goat at day 25 of pregnancy resulted in a 1,292 bp full-length consensus cDNA sequence for caprine uterine serpin (CaUS). The predicted amino acid sequence of the caprine precursor showed 96%, 82%, 55%, and 56% identity to OvUS, BoUS, PoUS1, and PoUS2, respectively. The signal peptide extends from amino acids 1 to 25, resulting in a secreted protein of 404 amino acids and 46,227 Mr (excluding carbohydrate). Both the goat and sheep uterine serpins have a nine amino acid insert in the Helix I region that is not found in bovine or porcine uterine serpins. A total of 13 amino acids in CaUS are different than those for the nearest homologue, ovine uterine serpin. One of these is in the site of cleavage of the signal sequence, where a single nucleotide substitution (G --> C) changed the cysteine for the sheep, bovine, and porcine genes to a serine. In addition, the amino acid at the putative P1-P1' site (the scissile bond for antiproteinase activity) is a valine for CaUS, BoUS, PoUS1, and PoUS2 versus an alanine for OvUS. The hinge region of all five of the uterine serpins (P17-P9) is distinct from the consensus pattern for inhibitory sequences and it is unlikely, therefore, that the uterine serpins possess prototypical proteinase inhibitory activity. The goat uterine serpin was immunolocalized to the glandular epithelium of the endometrium from a pregnant nanny at day 25 of pregnancy. There was also immunoreactive product in scattered luminal epithelial cells. No immunoreaction product was detected in endometrium from a nanny at day 5 of the estrous cycle. Western blotting of uterine fluid collected from the pregnant uterine horn of a unilaterally-pregnant goat revealed the presence of a protein band at Mr approximately 56,000 that reacted with monoclonal antibody to OvUS. In conclusion, the range of species in which uterine serpins are present and expressed in the uterus includes the goat in addition to the previously described sheep, cow, and pig. In all of these species, the uterine serpin is derived primarily from glandular epithelium, is secreted into the uterine lumen, and contains sequence characteristics suggesting it is not an inhibitory serpin., (Copyright 2005 Wiley-Liss, Inc.)