Cellular Localization and Function of the Antiviral Protein, Ovine Mx1 (oMx1): II. The oMx1 Protein...

Cellular Localization and Function of the Antiviral Protein, OvineMx1 (oMx1): I. Ovine Mx1 Is Secreted By Endometrial EpithelialCells Via an ‘Unconventional’ Secretory PathwayKoji Toyokawa, Stacy J. Carling*, Troy L. Ott

Department of Animal and Veterinary Science, Center for Reproductive Biology, University of Idaho, Moscow, ID, USA

Introduction

The myxovirus resistance, or Mx, proteins are 70–

80 kD intracellular antiviral proteins induced by vir-

uses and type I interferons (IFNs).1 Mx proteins are

well characterized as antiviral proteins, and form an

important early line of defense against virus infec-

tion.2 However, cytoplasmic rat Mx33 and human

MxB (Mx2 homolog) show no antiviral activity.4

Human MxB is postulated to be involved in normal

cellular functions such as nuclear import and cell

cycle progression.4 In fact, it is becoming increas-

ingly clear that Mx proteins may possess important

roles in normal cellular functions.

Interest in the roles of Mx1 proteins outside the

antiviral response comes from studies showing that

Keywords

Antiviral, Mx1, pregnancy, secretion, uterus

Correspondence

Troy L. Ott, PhD, PAS, Department of Dairy

and Animal Science, Pennsylvania State

University, 324 Henning Bldg., University Park,

PA 16802, USA.

Email: [email protected]

*Department of Human Physiology, Penning-

ton Biomedical Research Center, Baton Rouge,

LA, USA.

Submitted May 1, 2006;

revised August 16, 2006;

accepted October 13, 2006.

Citation

Toyokawa K, Carling SJ, Ott TL. Cellular

localization and function of the antiviral

protein, ovine Mx1 (oMx1): I ovine Mx1 is

secreted by endometrial epithelial cells via an

‘unconventional’ secretory pathway. Am J

Reprod Immunol 2007; 57:13–22

doi:10.1111/j.1600-0897.2006.00444.x

Problem

Embryonic loss is a major contributor to infertility. Understanding fac-

tors contributing to embryonic loss will aid in development of technol-

ogies to improve/regulate fertility in animals and humans.

Method of study

We tested the hypothesis that the antiviral protein, ovine Mx1 (oMx1),

is secreted by uterine epithelial cells. Uterine flushes were obtained from

cyclic and early pregnant ewes and examined for levels of oMx1 protein.

The pathway for ovine Mx1 secretion in ovine glandular epithelial

(oGE) cells was determined using brefeldin A (BFA), an inhibitor of the

conventional secretory pathway. Effects of BFA were determined using

b2-microglobulin (b2MG) as a marker for the conventional secretory

pathway, and interferon stimulated gene 15 (ISG15) and Galectin-1

(Gal-1) as markers for the unconventional secretory pathways.

Results

Ovine Mx1 protein levels were low in uterine flushes from cyclic ewes

and levels increased in pregnant ewes after D 15. Ovine GE cells secre-

ted oMx1 in response to interferon and secretion was not reduced by

BFA, suggesting oMx1 was secreted via an unconventional secretory

pathway. b2MG secretion was reduced by BFA, whereas ISG15 and

Gal-1 were not.

Conclusion

This is the first report that the antiviral protein, oMx1, is secreted and

provides evidence that secretion occurs via unconventional secretory

pathway(s).

ORIGINAL ARTICLE

American Journal of Reproductive Immunology 57 (2007) 13–22 ª 2007 The Authors

Journal compilation ª 2007 Blackwell Munksgaard 13

oMx1 protein is upregulated in the endometrium in

response to conceptus derived-IFNs during early

pregnancy in domestic ruminants (cattle and sheep)

and swine.5–8 In addition, ovine Mx1 (oMx1) was

recently shown to be upregulated in peripheral

blood leukocytes of pregnant ewes9and cows.10 All

three of these species have conceptuses (embryo and

associate extraembryonic membranes) that secrete a

type I IFN during early pregnancy.11

In addition to Mx proteins, conceptus-derived

IFNs induces a number of interferon stimulated

genes (ISGs) in the uteri of cattle and sheep. These

include signal transducers and activators of transcrip-

tion 1 (STAT1) and STAT2,12 interferon regulatory

factor1 (IRF-1) and IRF-2,13b2-microglobulin

(b2MG),14 interferon stimulated genes 15 (ISG15),15

granulocyte chemotactic protein-2 (GCP-2),16 and

2¢,5¢-oligoadenylate synthetase (OAS).17 In fact, it is

now clear that a large number of ISGs are regulated

in the endometrium during early pregnancy in rumi-

nants.18 A definitive role for any of these genes in

establishment and maintenance of pregnancy has

yet to be determined, but is an area of active investi-

gation.18

Among known IFNs-induced genes in the uterus,

b2MG and ISG15 were detected in uterine secretions

(uterine flushings).19,20 In addition, b2MG was

shown to be secreted in cell culture by conventional

secretory pathways involving the endoplasmic reti-

culum-golgi-trans-golgi network,21 whereas ISG15

was secreted from both non-immune and immune

cells utilizing the poorly characterized unconven-

tional secretory pathways.20

The majority of extracellular proteins are secreted

via the ER/Golgi-dependent secretory pathway; also

termed the classical secretory pathway.22 Proteins

secreted via this pathway typically contain a 13–30

amino acid hydrophobic signal peptide at their N-

terminus.23 These signal peptides are cleaved before

completion of protein transport across the membrane

of the endoplasmic reticulum.24 However, a number

of secreted proteins do not contain these signal pep-

tides including fibroblast growth factor-2 (FGF-2),25

galectin-1 (Gal-1),26 and ISG15.15,20 These secretory

proteins are exported from cells in the absence of an

intact ER/Golgi transport system27 via ‘unconven-

tional’ secretory pathways.28 Proteins that are secre-

ted via unconventional secretory pathways have

some common features; including translation on free

ribosomes, protein localization throughout the cyto-

plasm,28 the absence of hydrophobic signal

sequence,23 and insensitivity to drugs that block

classical secretion such as brefeldin A (BFA)29 and

monensin.30 Although many secreted proteins are

now known to be secreted via unconventional secre-

tory pathways, the precise mechanisms involved in

the unconventional secretory pathways remain

poorly described.

Recent results utilizing uterine gland knockout

(UGKO) ewes have highlighted the key role that

uterine gland secretions play in support of pre-

attachment conceptus development.31 Mx proteins

are large GTPases that belong to the superfamily of

mechanochemical enzymes of which dynamin is the

prototype.32 Members of this family are molecular

motors that perform cellular work. For example,

dynamin is involved in the final pinching off process

of endocytosis33 and cytokinesis.34 In addition,

human MxA was recently shown to be able to bind

and tubulate lipids in a GTP-dependent fashion.35

On the basis of these results and structural similarit-

ies with dynamins, we postulate that oMx1 is

involved in intracellular protein trafficking or secre-

tion in the uterine secretory epithelium. However, a

role for oMx1 outside the antiviral response has not

been established. Likewise, it is not known whether

oMx1 protein is itself secreted.

In studies presented here our objective was to

determine if oMx1 protein was secreted into the

uterine lumen and to address the pathway by

which it was secreted using a uterine epithelial-

derived cell line. We hypothesized that oMx1

expression would be increased during early preg-

nancy in ovine uterine flushings. On the basis of

its apparent lack of a secretion signal sequence, we

postulated that oMx1 secretion would be mediated

via the ER/Golgi-independent, or ‘unconventional’

secretory pathways.

Materials and methods

Animals and Experimental Design

Mature ewes of primarily Rambouillet breeding were

observed daily for estrus behavior in the presence of

vasectomized rams. Following two estrous cycles of

normal length (16–18 days), ewes were randomly

assigned to either cyclic or pregnant status. Ewes

assigned to pregnant status were bred to intact rams

at estrus, and 12 and 24 hr later. At estrus ewes

were randomly assigned to treatment and under-

went ovariectomy–hysterectomy (n ¼ 3 ewes/day)

TOYOKAWA ET AL.


14 Journal compilation ª 2007 Blackwell Munksgaard

on day 15 of the estrous cycle (day 0 ¼ estrus) and

days 15 or 17 of pregnancy (day 0 ¼ mating). At

surgery, 20 mL of sterile saline was used to lavage

the uterine lumen of each ewe. Uterine flushings

were then recovered and frozen at )80�C until

assayed. Pregnancy was confirmed by the presence

of apparently normal conceptuses in the uterine flu-

shes. All procedures using animals were review and

approved by the Animal Care and Use Committee

(Animal Use Protocol 7–286).

Western Blot Analysis

Total protein (8 lg) samples were dissolved in 1X

sample buffer [4X: 7.5 mL dH2O/760 mg Tris base/

2 g sodium dodecyl sulfate (SDS)/10 mL glycerol/pH

to 6.8/5 mL 2-bmercaptoethanol/300 lL bromphenol

blue]. Proteins were separated on a 15% SDS–poly-

acrylamide gel electrophoresis (PAGE) gel with 8%

stacking gel in 1X electrode buffer (10X: 30.3 g Tris

base/144.2 g glycine/10 g SDS/pH to 8.3/add dH2O to

1.0 L) at a constant current 70 mA for approximately

2 hr. The proteins in the gel were transferred to

nitrocellulose membranes (Protran BA83; pore

size ¼ 0.2 lm, Schleicher & Schuell, Keene, NH,

USA) in a Mini-Protean II Cell apparatus (Bio-Rad

Laboratories, Hercules, CA, USA) at a constant 70 V

for 90 min with an ice pack. Non-fat milk was used

as a blocker during detection of oMx1 and beta-actin,

and bovine serum albumin (BSA; Fisher Scientific,

Pittsburgh, PA, USA) was used as a blocker during

detection of b2MG, ISG15 and Gal-1. After blocking

non-specific binding with 5% non-fat milk or BSA

for 2 hr, immunoblotting was performed with tris-

buffered saline tween-20 (TBST) (20 mL 1 m Tris pH

7.5/137 mL 1 m NaCl/500 lL Tween 20/dH2O to

1.0 L). Immunoblotting was performed with 1:1000

dilution of rabbit polyclonal human b2MG antiserum

(Abcam, Cambridge, MA, USA), 1:1000 dilution of

an amino terminal rabbit polyclonal ovine Mx1 anti-

serum36 (90618 bleed no. 2, prepared by Multiple

Peptide Systems, San Diego, CA, USA) or 1:2000

dilution of rabbit polyclonal bovine ISG15 antiserum

(Provided by Dr Thomas R. Hansen, Colorado State

University) or 1:500 dilution of goat anti-human

Gal-1 (R&D, MN) in TBST including 2% BSA at 4�Covernight. Following washing with TBST for 5 min

four times on a rocker, the membranes were then

incubated with TBST including 2% non-fat milk or

BSA and 1:100,000 dilution of goat anti-rabbit

immunoglobulin G (IgG)-horseradish peroxidase

conjugate (Pierce, Rockford, IL, USA) or rabbit anti-

goat IgG-horseradish peroxidase conjugate on a

rocker at room temperature for 1 hr. Following

washing with TBST four times for 10 min on a

rocker, the membranes was incubated with Super

Signal� West Femto Maximum Sensitivity Substrate

chemiluminescence kit (Pierce) to detect immuno-

reactive proteins for 10 min at room temperature.

Detection of the chemiluminescence signal was per-

formed and quantified using the Bio-Rad Fluor-S

Multiimager system (40-mm lens, F-stop ¼ 2.7,

exposure ¼ 360 s) and Quantity One software (Bio-

Rad Laboratories).

Immunofluorescence

Immortalized ovine glandular epithelial (oGE)

cells37(300,000 cells/well) were cultured in 6-well

plates (Coster� 3516; Corning Inc., Corning, NY,

USA) for 24 hr in Dulbecco’s Modified Eagle’s Med-

ium (15.63 g/L DMEM; Sigma, St Louis, MO, USA)

with 10% FBS under 5% CO2 at 38.5�C. Cells were

plated on three cover slips per well in 6-well plates.

Cells on the cover slips were collected at 0, 1, 2, 3,

4, 8, and 72 hr after IFNs treatment (10,000 U/mL;

provided by Fuller W. Bazer, Texas A&M Univer-

sity). Cells on the cover slips were rinsed with 1 mL

of 1X phosphate-buffered saline (PBS) (10X: 80 g

NaCl/2 g KCl/14.4 g Na2HPO4/2.4 g KH2PO4/dH2O

to 1.0 L/adjust pH to 7.2). Following washing, cells

were fixed with 1 mL of 3% formaldehyde (Poly-

sciences Inc., Warrington, PA, USA) for 10 min on a

rocker and were washed with 1 mL of 1X PBS for

5 min twice on a rocker. Non-specific binding was

blocked with 1 mL of Base (1X PBS/0.05% Triton X-

100/2% BSA) overnight at 4�C. Following overnight

blocking with Base, cells were incubated in 1 mL of

1X PBS-2% BSA including 1:1000 dilution of an

amino terminal rabbit polyclonal ovine Mx1 anti-

serum and 1:200 dilution of mouse monoclonal to

58 K Golgi protein (Abcam) for approximately 6 hr

at room temperature. For oMx1 staining, pre-

immune rabbit serum (Multiple Peptide Systems) at

a dilution of 1:1000 was used as a negative control.

Cells were also incubated in 1X PBS–2% BSA with-

out immune serum as a negative control (no pri-

mary Ab control). After incubation with primary

antibodies, cells were washed with Base three times

for 5 min on a rocker at RT. Following washing,

cells were incubated in 1 mL of 1X PBS–2% BSA

including 1:2000 dilution of goat anti-rabbit Alexa

UTERINE SECRETION OF THE MYXOVIRUS RESISTANCE PROTEIN MX1



488 (Molecular Probe Inc., Eugene, OR, USA) and

goat anti-mouse Alexa 555 (Molecular Probe Inc.)

for 1 hr at room temperature. Nuclei of cells were

stained by Hoechst reagent (Sigma) for 5 min on a

rocker. After washing cells with 1X PBS, each cover

slip was whole-mounted in a mounting medium

(Vector, Burlingame, CA, USA) on a glass slide

sealed by nail polish. Immunofluorescence signals

were detected using an Eclipse E 1000 fluorescence

photomicroscope (Nikon, Tokyo, Japan) and Meta-

Morph version 6.1 (Universal Imaging Corpora-

tionTM, Downingtown, PA, USA).

Amino Acid Sequences

Prediction of the signal peptide cleavage sites in

amino acid sequences was analyzed using SignalP

3.0.38 Amino acid sequences of ovine b2MG, oMx1,

ISG15 and Gal-1 were obtained from NCBI (acces-

sion numbers AY549962, X66093, AF152103 and

AY694970, respectively).

Cell culture and brefeldin A treatment

Immortalized oGE cells (1,200,000 cells/dish)37 were

cultured in Ten-twenty-nineTM Petri dishes (Falcon,

100 X 15 mm style, Becton Dickinson Labware,

Franklin Lakes, NJ, USA) for 24 hr in DMEM with

10% FBS under 5% CO2 at 38.5�C. Following 24 hr

culture, cells (approximately 80% confluent) were

further cultured for another 24 hr in DMEM with

10% FBS in the presence of IFNs (10,000 U/mL).

The medium was replaced at 24 hr after IFNs treat-

ment with DMEM in the absence of FBS or phenol

red (Sigma). Cells were randomly assigned to con-

trols or treatments. Controls were non-treatment

(NT) without IFNs or BFA, IFN with IFNs plus BFA

at 0.01, 0.025 or 0.05 lg/mL (BFA 0.01, BFA 0.025,

and BFA 0.05). Conditioned media were collected

12 hr later. At harvest, cells were approximately

95% confluent and showed no visual evidence of

toxicity. Each experiment was replicated three inde-

pendent times with three dishes per treatment

within each experiment. Conditioned media were

centrifuged at 10,000 · g for 10 min to remove cell

debris and concentrated using Amicon� Ultra-4 cen-

trifugal Filter Devices (Millipore, Billerica, MA, USA)

according to manufacturer’s directions. Cell lysates

from the same dish were also collected using Mam-

malian Protein Extraction ReagentTM (MPERTM;

Pierce) to detect intracellular levels of b2MG, oMx1,

ISG15, and Gal-1. Protein concentrations in condi-

tioned media and cell lysates were determined by

BCA protein assay (Pierce). Total protein (40 lg)

from concentrated conditioned media or cell lysates

was used for Western blot assay to determine levels

of b2MG, oMx1, ISG15 and Gal-1 in conditioned

media or cell lysates.

Data Analysis

For effects of BFA on b2MG in conditioned media,

data were analyzed using the GLM procedures of the

Statistical Analysis System (SAS, version 8.0; SAS

Institute, Cary, NC, USA). The model tested the

main effects of treatment (NT, IFN, IFN + BFA),

experiment (three independent replicates) and their

interaction on levels of b2MG in conditioned media.

Experiment was not a significant source of variation

and was dropped from the model for all subsequent

analyses. Following a significant F-test (P < 0.05),

the following set of orthogonal contrasts were tested;

NT versus IFN and NT versus IFN + BFA. Because of

problems associated with heterogenous variances

that could not be alleviated with data transforma-

tion, effects of BFA on oMx1, ISG15 and Gal-1 in

condition media were determined using the Bonfer-

roni procedure (SAS, version 8.0). This approach is

considered conservative and holds the experiment-

wise error rate and alpha. Protein levels are reported

as least squares means ± pooled standard error of

the mean (pSEM).

Results

Ovine Mx1 Expression in Uterine Flushes from

Cyclic and Pregnant Ewes

Uterine flushes from cyclic and pregnant ewes were

examined for presence of oMx1 protein (Fig. 1).

Ewe 1 2 3 1 2 3 1 2 3

D15PD15C D17P PC

oMx1

Fig. 1 Western blot analysis of oMx1 protein in uterine flushes from

cyclic and pregnant ewes. D, day; C, cyclic; P, pregnant; PC, positive

control of consisting of proteins from D15P ovine endometrium; n,

three animals for each status and day. Ovine Mx1 was detected

approximately at 80 kDa in ovine uterine flushes.

TOYOKAWA ET AL.



Ovine Mx1 was detected at approximately � 80 kDa

in ovine uterine flushes from all three day-17 preg-

nant ewes, whereas oMx1 expression in uterine

flushes from day 15 cyclic ewes was relatively low,

but detectable. Ovine Mx1 levels in flushes from day

15 pregnant ewes were intermediate but variable.

Time Course Induction of oMx1 in oGE Cells

To examine the induction and localization of intra-

cellular oMx1, we conducted time course experi-

ment in oGE cells treated with IFNs using

immunofluorescence microscopy (Fig. 2). At 0 hr,

expression of oMx1 was very low to undetectable.

Through 1 and 2 hr after induction, expression of

oMx1 increased around the nucleus. Ovine Mx1

protein localization started to expand towards the

periphery of the cells at 3–4 hr after induction.

There was no apparent co-localization of oMx1 with

the Golgi apparatus (stained red in Fig. 2). Strong

expression of oMx1 was observed in the cytoplasm

at 8 hr after induction and this staining pattern per-

sisted through 72 hr after induction. Negative con-

trols included use of pre-immune serum and no

primary antibody in cells collected at 8 hr after treat-

ment with IFNs.

Prediction of the Signal Peptide Cleavage Sites in

Amino Acid Sequences from b2MG, oMx1, ISG15

and Gal-1

Detection of a signal peptide cleavage site(s) in

amino acid sequences from ovine b2MG, oMx1,

ISG15 and Gal-1 was accomplished using SignalP

3.0.38b2MG contained a conventional secretion sig-

nal peptide and was predicted (probability 1.000) to

be a classically secreted protein (Fig. 3a). The predic-

ted cleavage site was between amino acids 20 and

21 in the ovine b2MG sequence. In contrast, oMx1,

ISG15 and Gal-1 did not contain a signal peptide

and are predicted to be secreted via non-classical

secretory pathways (Fig. 3b–d).

Effects of BFA on b2MG, oMx1, ISG15, and Gal-1

Levels in Secretion

Ovine glandular epithelial cells were treated with dif-

ferent concentrations of BFA to examine effects on

oMx1 secretion; a protein that was previously not

known to be secreted (Fig. 4). b2MG is an IFNs-

stimulated protein known to be secreted via the

classical pathway that is a component of uterine

secretions. ISG15 is an IFNs-stimulated protein in

uterine secretions that is secreted via an unconven-

tional secretory pathway. Gal-1 is an unconvention-

ally secreted protein that is not known to be induced

by IFNs. Western blot analysis detected b2MG in

medium conditioned by oGE cells treated with IFNs.

As expected, secretion of the 11.8 kDa b2MG was

blocked by BFA in a dose-dependent manner with-

out affecting intracellular levels of b2MG. Ovine

Mx1, ISG15, and Gal-1 were detected at approxi-

mately � 80 kDa, � 17 kDa and � 15 kDa, respect-

ively, and their levels in oGE-conditioned medium

were not reduced by BFA. As expected, levels of

oMx1 and ISG15 in cell lysates were increased in

0 hr

4 hr

No 1A

8 hr 72 hr Preimmune

1 hr 2 hr 3 hr

Fig. 2 Immunofluorescence analysis of the

time course for induction of oMx1 in inter-

feron-s (IFNs)-treated oGE cells. Green, red,

and blue show expression of oMx1, Golgi, and

nuclei of oGE cells, respectively. Pre-immune

serum and no primary antibody were used as

negative controls. Magnification used was

100·.




response to IFNs, whereas Gal-1 was constitutively

expressed and not affected by IFNs treatment. Levels

of oMx1, ISG15 and Gal-1 in secretions were

increased by BFA in a dose-dependent manner with

no changes in intracellular levels of these proteins.

Levels of b2MG, oMx1, and ISG15 in conditioned

media were assayed in triplicate in three independ-

ent experiments for b2MG, oMx1, ISG15, and Gal-1

in two independent experiments (Fig. 5). IFNsinduced a modest increase in amount of b2MG in

oGE-conditioned culture media compared to NT

(1.5-fold increase; P ¼ 0.03). Levels of b2MG in oGE

cells treated with 0.05 lg/mL BFA were reduced

compared to NT alone (50% reduction; P < 0.01).

IFN alone did not increase oMx1 (P ¼ 0.18), ISG15

(P ¼ 0.6) or Gal-1 (P ¼ 0.86) in oGE-conditioned

media compared with NT at 12 hr. In contrast, levels

of oMx1, ISG15, and Gal-1 in conditioned media

were dramatically enhanced by BFA treatment com-

pared with NT (6.4-fold, 14-fold, and 16-fold

increase P < 0.01, respectively).

Discussion

Results presented here provide the first evidence

that the myxovirus resistance protein, oMx1, is a

secreted protein. We showed that oMx1 was pre-

sent in uterine flushes from pregnant ewes and

Fig. 3 Prediction of the signal peptide clea-

vage sites in amino acid sequences from

b2MG, (a) oMx1 (b), ISG15 (c) and Gal-1 (d)

using SignalP 3.0. C (red), S (green) and Y

(blue)-scores indicate cleavage site, ‘signal

peptideness’ and combined cleavage site

predictions, respectively.

BFA µg/mLIFN

β2MG

oMx1

Secretions

Cell lysates

Secretions

Cell lysates

Secretions

Cell lysates

Secretions

Cell lysates

ISG15

Galectin-1

0– + + + +

0 0.01 0.025 0.05

Fig. 4 Western blot analysis of the effects of brefeldin A (BFA) on

b2MG, oMx1, ISG15 and Gal-1 levels in secretions from oGE cells at

12 hr after IFNs treatment. Concentrations of BFA used in this study

were 0, 0.01, 0.025, and 0.05 lg/mL. Secretions and cell lysates within

each treatment were derived from the same dish. Each lane shows

representative secretions and cell lysates from the same sample.

TOYOKAWA ET AL.



confirmed that observation by demonstrating that

oMx1 is secreted by an ovine uterine epithelial-

derived cell line in vitro.39 In the present study, we

used a well-characterized inhibitor of the classical

(ER–Golgi–TGN) secretory pathway, BFA, to deter-

mine if oMx1 was secreted via the classical secretory

pathway. Results clearly showed that secretion of

oMx1, ISG15, and Gal-1 were not inhibited by BFA.

This is consistent with their lack of a classical secre-

tion signal sequence at their amino termini and their

cellular localization. These results strongly suggest

that all three are secreted via unconventional secre-

tory pathways.

The fact that levels of oMx1 protein in uterine flu-

shes increased between days 15 and 17 of early

pregnancy suggests that it may be a bona fide secre-

tory protein. This is further supported by the fact

that oGE cells secreted oMx1 in vitro and that levels

increased in response to IFNs. However, it remains a

possibility that levels of oMx1 increased in uterine

flushes as a result of lysis of epithelial cells during

the process of forming the synepitheliochorial pla-

centa that is characteristic of domestic ruminants.40

It is also possible that immune cells could be present

in the uterine lumen and have contributed to

increased levels of oMx1 in uterine flushes; how-

ever, there are no reports on the presence of free

immune cells in uterine flushes during this period of

early pregnancy. Furthermore, we have not been

able to detect oMx1 mRNA in conceptus tissue (S.J.

Carling, unpublished observations). Therefore, the

simplest explanation of the present results is that the

uterine epithelium in vivo, like we showed in vitro, is

secreting oMx1 into the uterine lumen. The pres-

ence of oMx1 protein in endometrial secretions begs

the question of whether it affects conceptus growth

and development during the peri-implantation per-

iod.

We further wanted to address the mechanism of

oMx1 secretion by determining whether oMx1 was

secreted via ‘classical’ or ‘unconventional’ pathways.

Proteins secreted via unconventional pathways have

a number of features in common. First of all, uncon-

ventionally secreted proteins tend to be localized

throughout the cytoplasm.28 Our immunofluores-

cence analysis demonstrated that upon IFNs treat-

ment oMx1 was distributed diffusely throughout the

cytoplasm. Among other Mx proteins, human

MxA,41,42 rat Mx2 and Mx343 and canine Mx1 and

Mx244 are also localized in the cytoplasm. Secondly,

unconventionally secreted proteins lack a hydropho-

bic signal peptide.23 Based on predictions (SignalP

3.0), oMx1, ISG15 and Gal-1 do not contain a classi-

cal secretion signal peptide, whereas b2MG does (see

Fig. 3). In agreement with this analysis, secretion of

b2MG is known to occur in an ER/Golgi/TGN-

dependent fashion,22 whereas ISG15 is secreted via

an unconventional secretory pathway.28 Finally,

inhibitors of the ER/Golgi/TGN-dependent secretion

such as BFA do not block unconventional secretory

processes.28 We observed in the present study that

BFA treatment resulted in higher secretion levels of

two IFNs-stimulated proteins, oMx1 and ISG15

without affecting intracellular levels of these

700

(a) (b)

(c) (d)

700

500

300

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400

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0

0NT IFN NT IFNIFN+BFA0.05 IFN+BFA0.05

NT IFN IFN+BFA0.05 NT IFN IFN+BFA0.05

Fig. 5 Quantitation of effects of brefeldin A

(BFA) on b2MG, (A) oMx1 (B), ISG15 (C) and

Gal-1 (D) levels in secretions from oGE cells.

Each experiment was replicated three inde-

pendent times with three replicate wells per

treatment within each experiment (n ¼ 9 for

b2MG, oMx1, ISG15, n ¼ 6 for Gal-1). NT, no

treatment; IFN, interferon tau alone

(10,000 AVU/mL); and IFN + BFA0.05, BFA at

0.05 lg/mL. Levels of b2MG in conditioned

culture media treated with IFN + 0.05 lg/ml

BFA were reduced compared with NT (� 50%

reduction; P < 0.01). In contrast, levels of

oMx1, ISG15, and Gal-1 in conditioned media

were increased (6.4-fold, 14-fold and 16-fold

increase, respectively; P < 0.01) in IFN + BFA

compared with NT, but were not different

between NT and IFN.




proteins. This effect of BFA to decrease secretion via

classical secretory pathways while increasing secre-

tion of unconventionally secreted proteins has been

reported previously.45 The fact that b2MG levels in

secretions were reduced argues against this resulting

from toxic effects of IFN s or BFA. In fact, we

(unpublished data) and others37 have used 10-fold

higher concentrations of IFNs in this cell line with-

out any evidence of toxicity. In addition, our tran-

sient oMx1 knockout model also demonstrated that

levels of b actin in cell lysates were similar in the

absence and presence of IFNs at the same concentra-

tion utilized in these experiments.39

Galectin-1 is not thought to be stimulated by IFNsand it also increased in secretions in response to

BFA treatment. This is particularly interesting in

light of preliminary results from Yeast 2-hybrid

screens in our laboratory that identified Gal-1 as a

binding partner for oMx1 (J. Welker and T.L. Ott,

unpublished data). Thus, we speculate that oMx1

and Gal-1 may interact in vivo in the uterus although

this interaction remains to be confirmed in vivo.

In the experiments examining the effects of BFA

on secretion, IFNs did not increase levels of oMx1,

and ISG15 in secretions at 12 hr after treatment

(Fig. 4). We chose 12 hr to minimize any potential

toxic effects of BFA on the oGE cells. While levels of

these proteins in the secretions were apparently

increasing they did not achieve significance in the

short 12 hr incubation period. Whereas, it is clear

from the Western blot (Fig. 4) that oMx1 and ISG15

were beginning to accumulate in conditioned media

in response to IFNs, and oMx1 levels were

approaching significance, longer incubation times

are need to detect these effects. In contrast, all three

unconventionally secreted proteins increased at

12 hr in conditioned culture media in response to

IFN + BFA treatment. This indicates that BFA stimu-

lated or enhanced unconventional secretory path-

way(s). Other reports showed a similar phenomenon

for IL-b secretion in the NIH3T3 cell line.45 The

mechanism(s) whereby BFA increases secretion of

unconventionally secreted proteins is not known.

Conclusion

Results from this study provide support for the

hypothesis that oMx1 is a secretory protein in

the ovine uterus and a component of endometrial

secretions in ewes. Furthermore, our results sup-

port the hypothesis that oMx1 is secreted via an

unconventional secretory pathway. Results from

transient oMx1 knockdown using siRNA suggest that

oMx1 may regulate secretion of other unconven-

tionally secreted proteins.39

Acknowledgements

This study was supported in part by USDA NRICGP

grant 2002-02398 and NIH NCRR grant P20-

RR15587-01 to T.L.O.

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Cellular Localization and Function of the Antiviral Protein, Ovine Mx1 (oMx1): II. The oMx1 Protein...

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