EFFECT OF THE MYXOVIRUS RESISTANCE PROTEIN, MX1, ON SECRETION BY A UTERINE CELL LINE.

Open Access
Author:
Boone, Shannon Rae
Graduate Program:
Animal Science
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
June 28, 2011
Committee Members:
  • Troy Ott, Thesis Advisor
Keywords:
  • Reproductive Physiology
  • MX1
  • MYXOVIRUS RESISTANCE PROTEIN
  • ovine
  • secretion
Abstract:
In cattle, substantial embryonic loss occurs during the peri-implantation period resulting in losses approaching half a billion dollars annually. During the peri-implantation period the conceptus must signal to the maternal uterus to maintain pregnancy. In ruminants, this signal is interferon tau (IFNT), which blocks the endometrial luteolytic mechanism, allowing the corpus luteum (CL) to continue producing progesterone and support pregnancy. Interferon-stimulated genes (ISGs) are upregulated by IFNT during this period and are thought to play crucial roles in supporting conceptus growth, differentiation, and embryo implantation. Two of these ISGs encode the myxovirus resistance proteins MX1 and MX2. Recently, MX1 was demonstrated to be a secreted protein that apparently regulated secretion of another protein, interferon stimulated gene-15 (ISG15). Interestingly, both of these proteins are secreted via one of the relatively uncharacterized “unconventional” secretory pathways leading to the hypothesis that MX1 is a regulator of unconventional secretion. This would be critical during early pregnancy because the conceptus is entirely dependent upon endometrial secretions for survival prior to attachment and placentation. Methods for the quantification of the MX1 protein are only semi-quantitative, consisting of protein immunoblotting/ western blotting, which can be quite variable from blot to blot. Therefore, the first objective of these studies was to develop a Meso Scale Discovery (MSD) assay for ovine MX1 (oMX1). This assay was validated by detecting known amounts of recombinant (r) oMX1 in different diluents over the range of 2.4-10,000 ng/mL and in bovine plasma over the range of 2.4-40,000 ng/mL. Once this assay was developed and successfully measuring MX1 protein, we began the main objective of the present studies, which was to determine the effects of MX1 on secretion by uterine epithelial cells using a combination of inhibition and overexpression assays. For the inhibition assay, morpholino antisense oligonucleotides were used to induce transient “knock-down” of the MX1 protein in a uterine epithelial cell line, oGE. The MSD assay was then used to quantify the inhibition of MX1 in oGE cell lysates. The assay quantified the MX1 reduction in lysates to be approximately 52% ± 52 ng of MX1/mL. Potentially regulated proteins included ISG15 and galectin-1 (LGALS1), which were both reduced with the inhibition of MX1. Interestingly, glalectin-15 (LGALS15) increased in secretions with the inhibition of MX1. These three proteins are all unconventionally secreted proteins. The secretion of the conventionally secreted protein, cathepsin L (CSTL), showed increased secretion of the 5-kDa active form/subunit with the inhibition of MX1 as well. For the overexpression assay, an MX1 cDNA was cloned into a pCMV-Myc mammalian expression vector, which was used to overexpress the MX1 protein in oGE cells. Treatment with the overexpression plasmid resulted in robust overexpression of the MX1 protein in both lysates and secretions from these cells, but there was ultimately no difference in the unconventionally or conventionally secreted proteins examined. As a second objective, we examined cellular proteins whose patterns of secretion change when MX1 is altered using 2D SDS PAGE and mass spectrometry. Samples from the overexpression and inhibition assays previously described were utilized in this study. In the MX1 inhibitions gels, 8 proteins were potentially identified that were inhibited when MX1 was inhibited (WD repeat-containing protein 38 (WDR38), serpin peptidase inhibitor (SERPIN), alpha-2-HS-glycoprotein (AHSG), triosephosphate isomerase-1(TPI1), peroxiredoxin-6 (GPX6), malate dehydrogenase 1B (MDH1B), ankyrin repeat and IBR domain-containing 1(ANKIB1), lim domain 7 (LMO7)), and 2 proteins were potentially identified in spots that increased when MX1 was inhibited (cystatin E/M (CST6), toll-like receptor 1(TLR1)). In the overexpression gels, 4 proteins were potentially identified in spots that were increased as well (SERPIN, glial fibrillary acidic protein (GFAP), lactate dehydrogenase B (LDHB), MDH1B). The quality score and the percent coverage (percent of matched peptides between the potential proteins and the peptides recovered from the chosen spot) varied for each of the potentially identified proteins. The experiments in this thesis further define the role of MX1 in secretion and provide evidence that does not support the hypothesis that MX1 is a broad regulator of unconventional secretion. However, because some unconventionally secreted proteins but no conventionally secreted proteins were reduced when MX1 was inhibited, our hypothesis is partially supported and MX1 may regulate a specific pathway in unconventional secretion. Overall, the results presented here point to a more complex role of MX1 with some proteins of each class being affected. The free-floating preattached embryo is completely dependent upon the endometrial secretory milieu for survival prior to attachment and placentation. If MX1 has a role in regulating endometrial secretory processes during early pregnancy, it would therefore have a direct role in embryo survival. From this work, it will be important to; 1) determine if MX1 regulates secretion in any other tissues; 2) determine what specific proteins are regulated by MX1 and; 3) gather more evidence about the complex role MX1 possesses in regulating secretion. Understanding the factors associated with conceptus survival, including how the conceptus regulates endometrial secretion, could lead to techniques to reduce early embryonic mortality. Importantly, MX1 may play a direct role in regulating these functions by regulating endometrial secretory processes, necessary for embryo survival until placentation.