Figure 1.
Expression of HA and its receptor CD44 in DSCs from human early pregnancy.
HA and CD44 expression was confirmed by immunohistochemistry, immunofluorescence, ELISA and flow cytometry, respectively. (A) The decidual tissues from normal early pregnancy were stained for HA and CD44. Slides were pretreated with HYAL to determine the specificity of HA. The red and yellow arrows showed HA staining in the cytoplasm and extracellular matrix, respectively (C) Primary DSC cells were seeded at 2×105, 5×105, or 1×106 cells/ml in cell culture plates. After 12, 24, 36, 48, 60 and 72 h of culture, the supernatants were collected and measured by ELISA. Each point represents the mean ± SE of triplicate values from four independent experiments. (D, E) The primary DSCs were stained by CD44 and vimentin, and the percentage of CD44+Vimentin+ DSC cells was analyzed by flow cytometry (FCM). The flow cytometric picture is from three different experiments.
Figure 2.
Pregnancy-associate hormones promote HA secretion from DSCs.
DSCs were seeded at 2×105 cells/ml in cell culture plates and were stimulated with different concentrations of estradiol (A), progesterone (B), hCG (C) for 72 h. Supernatants were harvested and measured for HA by ELISA. Data are the mean±SE of triplicate values from three independent experiments. *P<0.05, **P<0.01, compared to the control.
Figure 3.
Pregnancy-associated hormones enhance HAS mRNA transcription of DSCs.
DSCs were seeded at 5×105 cells/ml in cell culture plates and were stimulated with different concentrations of estradiol (A), progesterone (B), hCG (C) for 24 h. The cells were harvested and subjected to RNA extraction and real-time PCR for detection of mRNA levels of HAS1, HAS2, and HAS3. Data are the mean±SE of triplicate values from three independent experiments. *P<0.05, **P<0.01, compared to the control.
Figure 4.
HMW-HA promotes growth of DSCs via binding to CD44.
The primary DSCs in 96-well plates or 24-well plates were treated with different sizes of HA for 48 h. BrdU assay (A, B) and flow cytometry (C, D) were used to analyze DSC proliferation and apoptosis, respectively. The proliferation index of DSC cells under different conditions was normalized to the unstimulated control. Apoptotic cells were defined as AnnexinV + PI- DSCs. Control, isotype control IgG; HA, 100 µg/ml; α-CD44, anti-CD44 neutralizing antibody 20 µg/ml; Cpep, control HA peptide, 100 µg/ml; Pep-1, HA antagonist peptide, 100 µg/ml. Data represent the mean±SE of four separate experiments. *P<0.05, **P<0.01, compared to the control; # P<0.05, # # P<0.01, compared to HMW-HA treatment.
Figure 5.
PI3K/AKT and MAPK/ERK1/2 pathways are involved in the HA/CD44-regulated DSC proliferation and apoptosis.
The DSCs were treated for 48 h with either 0.1% DMSO (control/vehicle), 100 µg/ml HMW-HA, 50 µM PI3K/AKT inhibitor LY294002, 30 µM MEK1/2 inhibitor U0126, or 100 µg/ml HWW-HA combined with 50µM LY294002 or 30µM U0126. BrdU proliferation assay (A) and flow cytometry (B) were used to detect the proliferation and apoptosis, respectively. In-cell Western (C, D) HMW-HA activated in vitro PI3K/AKT and MAPK/ERK1/2 of DSC cells. The primary-cultured DSCs were starved with FBS-free media for 12 h and then treated with 100 µg/ml HMW-HA for different time. The levels of phosphor- and total- AKT and ERK1/2 were determined by In-cell Western. The pictures were from the typical one of three individual experiments. The red represents phospho-AKT or ERK1/2 and the green represents total- AKT or ERK1/2. Data represent the mean±SE of three experiments performed in triplicate wells. *P≤0.05, **P≤0.01 compared to the control; # P<0.05, compared to HMW-HA treatment.
Figure 6.
Differential expression of HA, HAS2 and CD44 in DSCs from normal pregnancy and unexplained miscarriage.
(A) Primary DSCs from normal pregnancy and unexplained miscarriage were seeded at 1×106 cells/ml in cell culture plates and cultured for 72h. The DSC supernatants were collected and measured by ELISA. Each dot represents results from different sample. (B, C) Molecular size of HA produced by DSCs from normal pregnancy and unexplained miscarriages was displayed through electrophoresis on polyacrylamide gels. Histogram showed the relative molecular mass of HA as determined by using ImageJ software. Data represent the mean ±SE of three independent experiments with a total of 8 samples from unexplained miscarriage and 8 samples from normal early pregnancy. (C) is one representative picture. (D) Primary DSCs from normal pregnancy and unexplained miscarriage were obtained and subjected to real time RT-PCR. HAS2 mRNA level in DSCs was compared between normal pregnancy and unexplained miscarriage. HAS2 mRNA level in DSCs from normal pregnancy was higher than that from unexplained miscarriage. (E) FCM analysis of the expression of CD44 on DSCs from normal and miscarriage. Data are mean ± SE from 4 repeated experiments, respectively. NP is normal pregnancy, and MC is unexplained miscarriage.
Figure 7.
A proposed model for the role of HA in growth of DSCs in human early pregnancy.
DSCs from normal pregnancy express higher HAS2, which synthesize higher molecular size of HA. HMW-HA activates PI3K/Akt and MAPK/ERK1/2 signalings via binding to CD44 in DSC membrane and then promotes cell proliferation while inhibits cell apoptosis, which is beneficial to normal pregnancy maintenance.