Objective
Previously we demonstrated anthrax toxin receptor 2 knockout ( Antxr2 −/− ) mice are fertile but fail to deliver their pups at term. This parturition defect is associated with overaccumulation of extracellular matrix proteins and decreased myometrial cell content in the uterus. Myometrial cell loss in Antxr2 −/− uterine tissue prompted us to evaluate if ANTXR2 is essential for human uterine smooth muscle cell viability and function.
Study Design
We subjected human uterine smooth muscle cell to lentiviral-mediated knock down or retroviral-mediated overexpression of ANTXR2. Flow cytometry confirmed lentiviral-mediated knock down or retroviral-mediated overexpression in cell lines vs control. Cell behavior and function in control, lentiviral-mediated knock down and retroviral-mediated overexpression cells were evaluated for apoptosis via TUNEL assay, migration via Boyden chamber assay and with oxytocin-mediated collagen contraction assays. Matrix metalloproteinase activity was evaluated using gelatin zymography. Cell lines and samples were run in duplicate. Student t test was used for statistical analysis.
Results
ANTXR2 is expressed by human uterine smooth muscle cell. Human uterine smooth muscle cell-lentiviral-mediated knock down cells exhibited increased apoptosis ( P < .05) and decreased migration ( P < .05), although human uterine smooth muscle cell-retroviral-mediated overexpression cells exhibited no change in apoptosis ( P = .91) and increased migration ( P = .05) vs control. Human uterine smooth muscle cell-lentiviral-mediated knock down cells contracted significantly less than control, although human uterine smooth muscle cell-retroviral-mediated overexpression cells showed no difference in contractility vs control. Matrix metalloproteinase activity 2 activity appeared slightly decreased in human uterine smooth muscle cell-lentiviral-mediated knock down cells and increased in human uterine smooth muscle cell-retroviral-mediated overexpression cells vs control.
Conclusion
ANTXR2 is expressed by human uterine smooth muscle cell and appears important for normal human uterine smooth muscle cell viability, migration and contractility. Further studies are needed to delineate if ANTXR2 is important for normal and abnormal labor patterns.
The anthrax roxin receptor (ANTXR) proteins, ANTXR1 and ANTXR2 (also known as tumor endothelial marker 8 [TEM8] and capillary morphogenesis gene 2 [CMG2], respectively), are most commonly recognized for their ability to bind anthrax toxin. These transmembrane receptors are also known to be critical for angiogenic processes such as endothelial cell proliferation, migration and network formation in part by binding to and interacting with extracellular matrix (ECM) proteins.
Recently, we demonstrated that ANTXR2 is essential for parturition in mice. Antxr2 knockout ( Antxr2 − /− ) mice are fertile and carry pregnancies to term, but then fail to deliver their fetuses that results in maternal death or fetal reabsorption. Gross analysis of the reproductive tracts revealed enlarged and fibrotic uterine and cervical tissue. Histological analysis of uterine and cervical tissue from the Antxr2 −/− mice demonstrated excessive accumulation of ECM proteins, particularly, collagen. It also showed striking disruption of smooth muscle cell layers in the myometrium. It was unclear if the disruption of the smooth muscle cell layer was due to the direct effect of absent ANTXR2 expression on the myometrial cells or whether it was secondary to the overaccumulation of collagen/ECM proteins. The loss of smooth muscle cells was so severe in aged mice that the myometrial layers could not be delineated. We hypothesized that the parturition defect in Antxr2 −/− mice was due in part to decreased smooth muscle cell content and therefore possible dystocia because of inadequate contractile force.
To investigate the mechanism responsible for the aberrant ECM remodeling in Antxr2 −/− mouse uterine tissue, we evaluated the activity of matrix metalloproteinases (MMPs), enzymes involved in ECM remodeling. MMP2 activity in uterine lysates and conditioned medium from Antxr2 −/− mouse embryonic fibroblasts was reduced in the Antxr2 −/− samples. ANTXR2 was found to form a complex with and enhance the activity of MT1-MMP (a membrane bound MMP responsible for MMP2 activation). These findings suggested that disruption of the Antxr2 gene in mice resulted in decreased MT1-MMP activity and thus decreased MMP2 activation. The lack of MMP2 activity in Antxr2 −/− mouse likely led to decreased ECM remodeling, eventually caused fibrosis in the reproductive tracts of Antxr2 −/− mice. Despite these findings, the role of ANTXR2 in normal murine uterine smooth muscle cell function and viability remained unclear. In particular, it was unclear if the uterine smooth muscle cells in the myometrium of the Antxr2 −/− mouse were being lost because of increased rates of apoptosis and if loss of ANTXR2 disrupted the intrinsic function of smooth muscle cells that includes migration, contraction, and ECM remodeling.
To date, no studies have evaluated ANTXR2 expression on human uterine smooth muscle cells (HUMSC) nor have studies addressed the role of ANTXR2 during normal HUSMC function. Studies have documented that individuals harboring a mutation in the ANTXR2 gene have a rare, autosomal recessive disease called systemic hyalinosis, which is further characterized as 2 syndromes, infantile systemic hyalinosis (ISH) and juvenile hyaline fibromatosis (JHF). Patients with ISH and JHF have abnormal collagen and glycosaminoglycan deposition in various tissues resulting in gingival hypertrophy, progressive joint contractures, osteolysis, osteoporosis, recurrent subcutaneous fibromas, and hyaline depositions. However, studies are lacking on the role of ANTXR2 in human reproductive tissues and during parturition. We sought to evaluate ANTXR2 expression in HUSMC and delineate the role of ANTXR2 in HUSMC function and ECM homeostasis.
Materials and Methods
DNA constructs
For ANTXR2 knockdown, lentiviral pLKO.1 vectors encoding scrambled control shRNA and shRNA targeting ANTXR2 transcripts were purchased from Sigma Aldrich (St. Louis, MO). The pLKO.1 vector carries puromycin resistance allowing for selection of shRNA expressing cells. The ANTXR2 siRNA target sense sequence was 5′-CCTGCACCTATCCTGAATAAA-3′. For ANTXR2 overexpression, we used a retroviral vector engineered to express recombinant receptor-EGFP fusion protein as previously described. HUSMCs (Promocell, Heidelberg, Germany) were cultured in smooth muscle cell growth medium 2 (Promocell) according to manufacturer’s instructions. Cells were cultured under standard conditions in a humidified incubator at 37°C, 5% CO 2 .
ANTXR2/CMG2 gene silencing and overexpression
Lentivirus was produced in transfected 293T cells and retrovirus was produced in GP2-293 cells and used to transduce HUSMCs as previously described. Briefly, lentiviral-containing supernatants and retroviral-containing supernatants were collected 24 hours after transfection, filtered, diluted 1:1 with HUSMC growth medium 2 and added to low passage HUSMC. This process was repeated at 24 hours, 36 hours, and 48 hours. At 48 hours after the initial infection, lentiviral cell lines were selected for 5 days with puromycin (Invitrogen, Carlsbad, CA) at 1 μg/mL and then maintained at 0.5 μg/mL. Retroviral cell lines were selected for 5 days with 300 μg/mL hygromycinB (Invitrogen). ANTXR2 gene silencing and overexpression were confirmed via flow cytometry as described previously described.
Evaluation of cell viability–apoptosis assay
HUSMC cell lines were seeded at equivalent numbers on type I collagen-coated coverslips. Samples were run in duplicate. After allowing the cells to adhere to the plates, the medium was changed to basal medium. After 48 hours, TUNEL (TdT mediated dUTP Nick End Labeling) staining of cells was carried out using the ApopTag Red In Situ Apoptosis Detection Kit (Millipore, Billerica, MA) according to the manufacturer’s instructions. DAPI (Vector Laboratories, Burlingame, CA) was used to counterstain nuclei. Cells were visualized on a Nikon ECLIPSE E 800 microscope (Nikon Inc, Melville, NY). Five pictures were taken at 20× magnification (top, middle, bottom, left, and right) for each coverslip and the number of TUNEL-positive cells was counted per high power field. An average of the number of TUNEL positive cells in each of the 5 pictures per coverslip was obtained for each sample and used for analysis. Apoptotic cells were also detected using the Annexin V-FITC Apoptosis Detection Kit (BioVision, Mountain View, CA) according to the manufacturer’s instructions. Quantification of apoptotic cells was performed using flow cytometry. Samples were run in duplicate.
Evaluation of HUSMC migration–Boyden chamber assay
We performed cell migration assays using a specialized Boyden migration chamber that included a 24-well tissue culture plate with cell culture inserts (BD Falcon, San Jose, CA). The inserts contained an 8-μm pore size polycarbonate membrane that was precoated with matrigel. Briefly, we seeded equal numbers of the HUSMC cell lines on the membrane of the culture inserts. Basal HUSMC medium supplemented with 10 ng/mL of epidermal growth factor (EGF) was added on top of the cells in each insert. As a control in this migration test, the same medium (basal HUSMC medium supplemented with 10 ng/mL of EGF) was also added to the lower compartment of each of the cell lines. To test the migration of HUMSC-R2KD and HUSMC-R2OE cell lines, basal HUSMC medium supplemented with 10 ng/mL of EGF and 20 ng/mL of the chemoattractant, platelet-derived growth factor subunit B ( PDGF-B), was added to the lower compartments in another set of each of the cell lines. After 48 hours of incubation at 37°C, the migrated cells were fixed in 4% PFA. The HUSMC from the upper surface of the membrane were removed using a moist cotton-tipped swab. We stained the migrating cells on the lower surface of the membrane, which had migrated through the polycarbonate membrane, with crystal violet staining solution for 20 minutes and rinsed them 3 times with distilled water. Three pictures were obtained at 10× (top, middle, bottom) of each membrane and the number of migrating cells per image was obtained. The average of the number of migrated cells was then calculated from each of the 3 images. This was repeated for each membrane in all cell lines. Samples were run in duplicate and the experiment was repeated to confirm results.
Evaluation of HUSMC contractility–collagen contraction assay
To measure myometrial cell contraction in response to oxytocin (OT) we adapted an assay based upon the technique described by Dallot et al. A collagen gel was made using 1 part RPMI (Sigma Aldrich), 1 part H20, 7 parts type 1A collagen (Cellmatrix, Newport, VA), 1 part neutralization buffer (Cellmatrix). HUSMC cell lines were mixed into the collagen gel at equal numbers and 300 μL of HUSMC/collagen gel mixture was added to individual wells of a 24-well plate and left at 37°C for 30 minutes to allow gelling of the collagen mixture. Then, 500 μL of HUSMC growth medium 2 was then added to each well. At 48 hours, each collagen lattice was detached from the bottom of the well with a sterile pipette tip. The 100 nM of oxytocin (Sigma Aldrich) diluted in HUSCM growth medium 2 or HUSMC without oxytocin was added to each well and left overnight at 37°C. Cell contraction was stopped by fixing the lattices in PBS-4% paraformaldehyde. The lattices were photographed with a digital camera and the surface area of each lattice was calculated. For each lattice, collagen contraction was determined in triplicate and expressed as percentage contraction. Percentage contraction was taken as the percentage of lattice size diminution relative to the size area of the well. The experiment was repeated to confirm results.
Evaluation of MMP activity-gelatin zymography
Gelatin zymography analysis was performed as previously described. After 16-24 hours, the conditioned medium was harvested from CTL, R2KD, and R2OE cells, which were seeded at equal densities. The conditioned medium was cleared by centrifugation at 12,000 rpm for 10 minutes and subjected to analysis by SDS-substrate gel electrophoresis (zymography) under nondenaturing conditions in 8.0% SDS-polyacrylamide gels impregnated with 1 mg/mL gelatin as previously described. The gels were incubated at 37°C overnight in 50 mM Tris (pH 7.5), 5 mM CaCl 2 , 1 mM ZnCl 2, and stained with Coomassie Brilliant Blue R25 (Bio-Rad, Hercules, CA). Destained gel images were captured by Kodak EL Logic 100 Imaging System (Kodak, Rochester, NY). Samples were tested in duplicate. All of the experiments were repeated twice. ImageJ 1.45 s (National Institutes of Health, Washington, DC) was used to quantify zymography band intensities.
Statistical analysis
Statistical significance was evaluated using the unpaired Student’s t test with a P value less than .05 considered statistically significant.
Results
To delineate the function of ANTXR2 in HUSMC, we evaluated cell viability, migration and contractility on ANTXR2 knockdown or overexpression. Flow cytometry confirmed the presence of ANTXR2 protein on the surface of HUSMC and we were able to achieve approximately 40% lentiviral-mediated knock down and 30% retroviral-mediated overexpression relative to control (CTL). Figure 1 , A, represents results from 1 of 2 flow cytometry assessments that were performed.
We first asked whether HUSMC viability was affected when ANTXR2 expression was reduced or increased. HUSMC-R2KD cells exhibited increased apoptosis via TUNEL assay ( P < .05) as compared with CTL cells ( Figure 1 , B). The HUSMC-R2OE cells exhibited no significant change in apoptosis ( Figure 1 , B; P = .9), relative to CTL. These findings were replicated using the ANNEXIN-V assay (data not shown), where R2KD displayed increased apoptosis. Higher rates of R2KD resulted in cell death and inability to sustain HUSMC-R2KD cell lines (data not shown).
In pregnancy, it is well established that the uterus undergoes myometrial hyperplasia. Cell migration is required during this process for proper formation and function of the myometrial layers. As such, we assessed HUSMC migration towards a migratory signal using a Boyden chamber assay. HUSMC cell lines were allowed to migrate across a filter toward media supplemented with platelet derived growth factor-B, a factor known to promote migration of smooth muscle cells. We found that HUSMC-R2KD cells migrated to PDGF-B to a lesser extent than CTL cells ( P < .05) indicating that ANTXR2 expression is necessary for chemokine-directed migration ( Figure 2 ). HUSMC-R2OE cells exhibited increased migration relative to CTL ( P = .05) ( Figure 2 ).
