Abstract
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Regulation of Human Endometrial Stromal Proliferation and Differentiation by C/EBPβ Involves Cyclin E-cdk2 and STAT3
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Abstract
During each menstrual cycle, the human uterus undergoes a unique transformation, known as decidualization, which involves endometrial stromal proliferation and differentiation. During this process, the stromal cells are transformed into decidual cells, which produce factors that prepare the uterus for potential embryo implantation. We previously identified the transcription factor CCAAT/enhancer-binding protein (C/EBP)β as a regulator of endometrial stromal proliferation and differentiation in mice. In this study, we addressed the role of C/EBPβ in human endometrial decidualization. Using small interfering RNA targeted to C/EBPβ mRNA, we demonstrated that C/EBPβ controls the proliferation of primary human endometrial stromal cells (HESCs) by regulating the expression of several key cell cycle-regulatory factors during the G1-S phase transition. Additionally, loss of C/EBPβ expression blocked the differentiation of HESCs in response to estrogen, progesterone, and cyclic AMP. Gene expression profiling of normal and C/EBPβ-deficient HESCs revealed that the receptor for the cytokine IL-11 and its downstream signal transducer signal transducer and activator of transcription 3 (STAT3) are targets of regulation by C/EBPβ. Chromatin immunoprecipitation analysis indicated that C/EBPβ controls the expression of STAT3 gene by directly interacting with a distinct regulatory sequence in its 5′-flanking region. Attenuation of STAT3 mRNA expression in HESCs resulted in markedly reduced differentiation of these cells, indicating an important role for STAT3 in decidualization. Gene expression profiling, using STAT3-deficient HESCs, showed an extensive overlap of pathways downstream of STAT3 and C/EBPβ during stromal cell differentiation. Collectively, these findings revealed a novel functional link between C/EBPβ and STAT3 that is a critical regulator of endometrial differentiation in women.
During each menstrual cycle, the human endometrium undergoes cyclical changes, including proliferation, differentiation, and menstruation, strictly controlled by the ovarian steroids, 17β-estradiol (E) and progesterone (P) (1, 2). Each cycle is initiated by an E-dominated proliferative phase (d 1–14), during which extensive epithelial and stromal regeneration occurs. It then enters a P-dominated secretory phase (d 15–28). The putative window of implantation in women opens for a short period of time in the midsecretory phase between the cycle d 20–24 (3). In preparation for implantation, predecidualization starts in the stromal cells surrounding the spiral arteries during early-midsecretory phase (2). This process involves the transformation of fibroblast-like endometrial stromal cells into large, rounded, and secretory decidual cells. If pregnancy ensues, decidualization spreads throughout the stroma under the continued influence of E and P, as well as yet incompletely characterized embryonic products thought to signal via cyclic AMP (cAMP)-mediated pathways. Impaired decidualization of endometrial stroma is associated with recurrent miscarriage, unexplained infertility, and other clinical disease states, such as endometriosis and endometrial cancer (4–10).
The identities of the steroid-regulated pathways with defined functions during human endometrial decidualization remain largely unknown. However, transgenic mouse models have been highly informative and establish functional roles of several genes in the decidualization process. These include the P receptor (PGR), homeobox genes 10 and 11, cycloxygenase 2, IL-11 receptor (IL-11R)α, CCAAT/enhancer-binding protein (C/EBP)β, bone morphogenetic protein (BMP)2, and steroid receptor coactivator 2 (11–17). Studies employing an in vitro system, in which human endometrial stromal cells (HESCs) isolated from the proliferative phase of the cycle undergo differentiation in the presence of a hormonal cocktail containing E, P, and a cAMP analog, indicate that several genes, such as PGR and forkhead box O1, play important roles during human endometrial decidualization (18, 19).
Our previous studies identified C/EBPβ as a novel mediator of the biological actions of E and P during decidualization in the mouse (16). This transcription factor belongs to a family of basic region/leucine zipper DNA-binding proteins (20), which regulate the transcription of target genes by binding to a conserved sequence, known as CCAAT box, in their promoter regions. Numerous studies have shown that C/EBPβ is a key regulator of proliferation and/or differentiation in multiple tissues, including the ovary, testis, liver, adipose tissue, immune system, skin cells, and the mammary gland (21–30). Using a knockout mouse model, we have previously reported that the C/EBPβ-null uterus fails to undergo decidualization (16). Analyses of these uteri indicated that loss of this transcription factor leads to an arrest in stromal cell cycle progression at the G2-M boundary (31). It was revealed that C/EBPβ controls the expression of multiple cell cycle-regulatory molecules in mouse uteri, including cyclins B1 and B2, the phosphatase cell division cycle 25 homolog C, and the cell cycle inhibitors p21, p27, and p53, all of which control the G2-M phase transition.
In this study, we investigated the role of C/EBPβ in HESC proliferation and differentiation. We found that C/EBPβ controls HESC proliferation by regulating the G1-S phase transition of the cell cycle. It controls the induction of several key factors, such as cyclin E and E2F transcription factor 1 (E2F1), which promote DNA replication. We also noted that, under decidualization conditions, the attenuation of C/EBPβ expression in HESC by small interfering RNA (siRNA) methods led to markedly reduced expression of IL-11Rα and its signal transducer and activator of transcription 3 (STAT3). Our studies further revealed that C/EBPβ interacts directly with the STAT3 promoter to regulate its activity and that pathways downstream of STAT3 are critical for differentiation of HESC. Collectively, these studies showed that C/EBPβ controls proliferation and decidualization in human endometrium by regulating cyclin E-cyclin-dependent kinase (cdk)2 complexes and a unique signaling network involving IL-11Rα and STAT3, respectively.
Materials and Methods
Primary HESC culture, synchronization, and in vitro decidualization
Our studies involving human endometrial biopsies and endometrial cell cultures adhere to the regulations set forth for the protection of human subjects participating in clinical research and are approved by the Institutional Review Boards of Emory University, Wake Forest University (Winston-Salem, North Carolina), and the University of Illinois at Urbana-Champaign. Endometrial samples from early proliferative stage of the menstrual cycle were obtained by Pipelle biopsy at Emory University Medical Center from fertile volunteers providing written informed consent.
HESCs were isolated from biopsies taken from the proliferative stage endometrium of regularly cycling women on no hormonal medications. The cells were isolated and cultured in DMEM/F-12 (Invitrogen, Carlsbad, CA) containing 5% (vol/vol) fetal bovine serum (FBS) (HyClone, Logan, UT), 50 μg/ml penicillin, and 50 μg/ml streptomycin (Invitrogen) as described previously (32). Synchronization of HESC in the G1 phase was achieved by two consecutive rounds of thymidine block. Briefly, HESCs were cultured to 60% confluence in DMEM/F12–5% FBS and exposed to 2 mm thymidine for 12 h. Cells were then washed three times with PBS, and the thymidine block was released by culturing the cells in fresh DMEM/F12–5% FBS for 12 h. The cells were subjected to a second round of thymidine block in the presence of DMEM/F12–5% FBS containing 2 mm thymidine for another 12 h. Finally, the block was released by washing the cells with PBS and placing them in fresh DMEM/F12–5% FBS. At this time (indicated as time zero), the cells were synchronized in the G1 phase and poised to progress through the cell cycle in the presence of complete medium.
To induce in vitro decidualization, the cells were treated with medium, containing 10 nm E (Sigma, St. Louis, MO), 1 μm P (Sigma), and 0.5 mm 8-bromo-cAMP (Sigma) (E+P+cAMP). The media were changed every 48 h, and the cultures were maintained for up to 12 d.
Prolactin (PRL) assay
HESCs were cultured in six-well plates, and decidualization was induced as described above. The culture medium was collected every 48 h and replaced with fresh medium. The PRL production was analyzed by an ELISA kit (Calbiotech, Spring Valley, CA). The assay was performed in triplicate, according the manufacturer's instructions. Control samples (unconditioned culture media) were included in each assay. The lower detection limit of the assay was 0.3 ng/ml of PRL. The inter- and intraassay coefficients of variation were 5.8 and 2.3%, respectively.
siRNA interference
HESCs were transfected with siRNA constructs targeted to mRNAs corresponding to C/EBPβ, IL-11Rα, and STAT3. Scrambled siRNA was used as control (Silencer Select Pre-designed; Ambion, Austin, TX). Briefly, SilentFect transfection reagent (Bio-Rad Laboratories, Hercules, CA) was mixed with 40 nm siRNA and added to HESCs at 80% confluency. After 24–48 h, siRNA was removed, and cells were treated with media containing E+P+cAMP to induce decidualization. Cells were harvested at various time points after the hormone treatment. Gene expression was examined by quantitative real-time PCR using gene-specific primers.
Immunocytochemistry
HESCs cultured in chamber slides were fixed in formalin solution (Sigma) at room temperature for 10 min followed by washing with PBS for 10 min. The cells were permeabilized by 0.25% Triton X-100 in PBS for 10 min, and nonspecific binding of antibodies was blocked with 10% donkey serum for 1 h at room temperature. Primary antibodies included anti-C/EBPβ (C-19; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anticyclin D1 (Santa Cruz Biotechnology, Inc.), anti-E2F1 (Abcam, Cambridge, MA), anticyclin E (Abcam), antiphospho-retinoblastoma (Rb) (Ser-780; Santa Cruz Biotechnology, Inc.), antiphospho-cdk2 (T-160, Abcam), anti-STAT3 (C-20; Santa Cruz Biotechnology, Inc.), and antiphospho-STAT3 (Ser-727; Cell Signaling Technology, Beverly, MA). Incubations with these antibodies were performed overnight at 4 C. Fluorescence (cyanine 3 or Delight 488)-conjugated antimouse IgG or antirabbit IgG (The Jackson Laboratory, Bar Harbor, ME) was used as secondary antibody. 4′,6-Diamidino-2-phenylindole (1 μg/ml in PBS) was used as counter staining.
Adenovirus-mediated expression of dominant negative C/EBPβ
An adenoviral vector expressing a dominant negative mutant of C/EBPβ (A-C/EBP) was provided by Charles Vinson (National Cancer Institute/National Institutes of Health, Bethesda, MD). This vector expresses A-C/EBP under the control of cytomegalovirus promoter. The protein is expressed with a hemagglutinin epitope tag. An adenoviral vector lacking the A-C/EBP insert was used as a control. Control or A-C/EBP-carrying adenovirus was added to HESC monolayers (multiplicity of infection, MOI, 10:1) at 80% confluence. After transduction for 24 h, the viral particles were removed, and the cells were treated with E+P+cAMP to induce decidualization.
Chromatin immunoprecipitation (ChIP) of the STAT3 promoter
ChIP analysis was performed using the EZ ChIP (Upstate Biotechnology, Waltham, MA) method, according to the manufacturer's protocol. Briefly, HESCs were treated with hormone cocktail for 3 d to induce the decidualization. Thereafter, cells (8 × 106) were placed in PBS buffer and cross-linked with 1% formaldehyde for 10 min. The cross-linked cells were harvested, lysed using sodium dodecyl sulfate lysis buffer, and sonicated. After preclearing the lysates with salmon sperm DNA-protein A at 4 C for 2 h, the DNA-protein complexes in the supernatant were immunoprecipitated using antibodies against RNA polymerase II, rabbit IgG (Upstate Biotechnology), or C/EBPβ (C-19; Santa Cruz Biotechnology, Inc.). The immune complexes were recovered by adding protein A-agarose. The beads were then washed repeatedly, and the bound complexes were eluted using the elution buffer. The cross-linking was reversed, and then proteins were digested using 0.5 mg/ml proteinase K. Purified DNA served as the template for PCR using various primer sets to amplify specific regions of the STAT3 promoter.
For the experiments using the dominant negative mutant A-C/EBP, HESCs were first transduced with control or A-C/EBP adenovirus as described above. After 24 h of transduction, the viral particles were removed, and the cells were treated with E+P+cAMP to induce decidualization. After 3 d of hormone, cells were harvested to perform the ChIP assay.
Real-time RT-PCR analysis
Total RNA was extracted from cultured HESCs using the TRIzol RNA purification kit (Invitrogen) according the manufacturer's instructions. Reverse transcription was performed using a cDNA synthesis kit (Invitrogen) as recommended. The expression of mRNAs corresponding to PGR, C/EBPβ, Rb, E2F1, cyclin E, cdk2, cdk4, PRL, IGF binding protein (IGFBP)-1, BMP2, wingless-type MMTV integration site family, member 4 (Wnt4), STAT3, IL-11, IL-11Rα, vascular endothelial growth factor (VEGF)-A, and sphingosine kinase 1 (SPHK1) was examined by real-time RT-PCR analysis using SYBR Green Supermix (Bio-Rad Laboratories) and gene-specific primers (Supplemental Table 1, published on The Endocrine Society's Journals Online web site at http://mend.endojournals.org). The gene expression level at any given time point or under a given condition was quantified as fold change (mean ± sd) relative to that at 0 h or control condition after normalization with respect to the internal control 36B4, a constitutive gene encoding a ribosomal protein.
Western blotting
HESCs were cultured with or without hormone cocktail treatment for 24 or 48 h. The cells were harvested and lysed in ice-cold lysis buffer containing 25 mm Tris/HCl (pH 7.4), 50 mm NaF, 100 mm NaCl, 1 mm sodium vanadate, 5 mm EGTA, 1 mm EDTA, 1% (vol/vol) Triton X-100, 10 mm sodium pyrophosphate, 1 mm benzamidine, 0.1 mm phenylmethylsulfonylfluoride, 0.27 m sucrose, 2 μm microcystin, and 0.1% (vol/vol) 2-mercaptoethanol. The cell debris was removed by centrifugation, and the protein concentration of the lysate was determined by bicinchoninic acid protein assay, according the manufacturer's instruction (Pierce, Rockford, IL). The cell lysates were analyzed by standard Western blotting, using antibodies against C/EBPβ (1:1000; Santa Cruz Biotechnology, Inc.) and calnexin (1:5000, as loading control; Cell Signaling Technology Corp.).
Microarray analysis
Three independent HESC lines were established from individual biopsies of proliferative stage endometria from three different women. These cultures were transfected with control (scrambled), C/EBPβ-specific, or STAT3-specific siRNA for 24 h and then treated for an additional 24 h in the presence or absence of E+P+cAMP. siRNAs were then removed, and incubation with hormone cocktail was continued for another 48 h. Cells were harvested, and total RNA was prepared by TRIzol reagent (Invitrogen). RNA integrity was verified using an Agilent 2100 bioanalyser (Agilent Technologies, Inc., Santa Clara, CA) at the Biotechnology Center of the University of Illinois at Urbana-Champaign. All samples had RNA integrity numbers greater than nine. Each RNA sample was processed for microarray hybridization using GeneChip Human Genome U133 A Plus 2.0 arrays (Affymetrix, Inc., Santa Clara, CA), following the established protocol. Quality control assessment, data processing, and statistical analysis were done in R, a software module for statistical computing and graphics. Fold differences in gene expression and false discovery rates set at more than 1.5 and more than 0.05, respectively, were considered significant. The significantly regulated genes were further sorted by gene ontology and pathways using PANTHER Classification Software.
Statistical analysis
The RNA and protein samples were prepared from at least three separate primary cultures subjected to the same experimental treatments. The real-time RT-PCR results are expressed as mean ± sd of three separate experiments. Statistical significance was assessed by ANOVA at a significance level of P < 0.05, indicated by an asterisk in the figures.
Results
C/EBPβ is required for HESC proliferation
During the proliferative phase of the menstrual cycle, HESCs undergo repeated cell divisions to rebuild the functionalis layer of the endometrium. This proliferative activity is recapitulated in primary cultures, where undifferentiated HESC isolated from proliferative phase endometria undergo many rounds of cell division when maintained in the presence of serum. Under these basal conditions, HESCs express high levels of C/EBPβ mRNA and protein (Fig. 1, A, none, and B, left panel).
C/EBPβ controls HESC proliferation. Primary cultures of HESC were transfected without (none) or with C/EBPβ siRNA or control siRNA (scrambled) as described in Materials and Methods. A, The mRNA expression of C/EBPβ was measured by real-time RT-PCR using C/EBPβ-specific primers. B, The expression of C/EBPβ protein was monitored using immunocytochemistry 72 h after siRNA transfection. C, Immunostaining showing the BrdU-positive cells in HESC cultures treated with control or C/EBPβ siRNA at different times (4, 7, and 10 h) after BrdU addition. HESCs were synchronized at G1 phase by double thymidine block and then transfected with siRNA (40 nm) targeted against C/EBPβ or control siRNA (scrambled) for 36 h with thymidine present in the medium. Cells were then washed and placed in fresh medium containing 5% FBS and harvested at different times after relief of thymidine block. D, The expression of cyclin E1 was measured by real-time RT-PCR and compared with the level in control siRNA-treated cells at 0 h. E and F, The cells were fixed and subjected to immunocytochemistry using antibodies against cyclin E (E) and phosphorylated (at Thr-160) cdk2 (F). *, Statistically significant change (P < 0.05) when compared with control siRNA treatment.
To investigate the role of C/EBPβ during HESC proliferation, we employed a siRNA strategy to suppress its mRNA expression. As shown in Fig. 1A, transfection of HESCs with C/EBPβ-specific siRNA reduced its mRNA expression approximately 70% within 24 h and approximately 85% within 48 h of treatment. Transfection of control siRNA had no appreciable effect on C/EBPβ mRNA levels (Fig. 1A). Consistent with this observation, immunocytochemical analysis indicated a drastic reduction in the level of C/EBPβ protein upon treatment with C/EBPβ-specific siRNA (Fig. 1B, right panel).
Despite preparation from proliferative phase tissues, once established, HESC cultures consist of populations of stromal cells at various stages of the cell cycle. To synchronize the growth of these cells, we subjected them to two consecutive rounds of thymidine block, a well-established method of cell synchronization (33). When mammalian cells are switched to a thymidine-rich medium, the excess thymidine causes a nucleotide imbalance in the cell, blocking DNA replication and arresting the cell cycle at the late G1-S phase (33). To ascertain complete synchronization, the cells are usually subjected to a second round of thymidine treatment. The cell cycle block is released, and S phase entry is induced upon removal of the extra thymidine from the medium.
To monitor HESC proliferation, we assessed bromodeoxyuridine (BrdU) incorporation at different time points after the cells are released from thymidine block. No incorporation of BrdU was detected when it was added before the release of the thymidine block, confirming that the cell cycle is effectively arrested (data not shown). To monitor the time course of DNA synthesis, BrdU was added to the HESC cultures at the time of release of the block, and its incorporation into DNA was monitored by immunocytochemistry. As shown in Fig. 1C, the HESC exhibited substantial BrdU incorporation at 4, 7, and 10 h after the release of the G1-S arrest. After attenuation of C/EBPβ expression by siRNA in synchronized HESC, we observed that BrdU incorporation is markedly reduced in C/EBPβ-deficient cells relative to the control cells (Fig. 1C and Supplemental Fig. 1A). These results indicated that the down-regulation of C/EBPβ impairs the entry and progression of HESC into the S phase.
To further examine the role of C/EBPβ in HESC proliferation, we examined the mitotic activity of these cells, using an antibody that recognizes the unique mitotic phase marker phosphorylated Ser 10 of histone 3 (P-His3) (34). We observed that control HESC displayed specific staining of P-His3 by 13 h after the release of thymidine block (Supplemental Fig. 1, B and C). The number of P-His-positive cells increased further at 16.5 h, and the positive staining was sustained until 18 h. In contrast, P-His3 staining was drastically reduced in C/EBPβ-deficient HESC. These results indicate that the majority of the C/EBPβ-deficient HESCs did not undergo the G2-M transition, consistent with their arrest at the G1-S boundary.
C/EBPβ regulates the expression of cyclin E and activation of cyclin E-dependent kinase in proliferating HESC
We next investigated whether C/EBPβ controls the G1-S transition of proliferating HESCs by regulating the expression and activation of key cyclins and cdks that specifically control this checkpoint. We monitored the expression of mRNAs and proteins corresponding to cyclins D, E, and A, and cdks 2 and 4, which are well-known regulators of G1 and S phases of the cell cycle, in HESC in the presence or absence of C/EBPβ. No significant alteration was noted in the expression of cyclin D1, cyclin A, cdk2, and cdk4 (Supplemental Fig. 2). However, we noted that the expression of cyclin E mRNA, which reaches a peak at 4 h after the release of the thymidine block at 0 h, was significantly reduced in cells treated with C/EBPβ siRNA (Fig. 1D). Consistent with this observation, the overall levels and particularly the nuclear localization of the cyclin E protein were reduced in C/EBPβ-deficient HESC (Fig. 1E). Because cyclin E is required to phosphorylate cdk2 at Thr-160 and activate the enzyme (35), we next examined the functional status of cdk2 in these cells. We found that activated phosho-cdk2 was indeed absent in the C/EBPβ-deficient HESCs compared with the control HESCs (Fig. 1F). Therefore, the lack of functional cyclin E-cdk2 complexes is the likely cause of the G1-S arrest of the C/EBPβ-deficient cells.
C/EBPβ-regulated pathways control the expression of E2F1 and phosphorylation of Rb in HESC
We also examined whether the expression of the E2F family of transcription factors, a major regulator of cell cycle progression, is controlled by C/EBPβ in HESC. The mRNA corresponding to E2F1 is prominently expressed in HESC during the cell cycle, whereas those of E2F2 and E2F3 are maintained at relatively low levels (data not shown). As shown in Fig. 2A, the level of E2F1 mRNA rose rapidly upon the release of the thymidine block as HESCs entered the S phase and cell proliferation resumes. This induction of E2F1 mRNA expression was impaired in C/EBPβ-deficient cells. The consequent reduction in the levels of E2F1 protein was confirmed by immunocytochemistry (Fig. 2B).
Reduced E2F1 expression and Rb phosphorylation in C/EBPβ-deficient HESC. HESCs were synchronized at G1 phase and then transfected with siRNA (40 nm) targeted against C/EBPβ or control siRNA (scrambled) for 36 h with thymidine present in the medium and then harvested at different times. A and C, The expression of mRNAs corresponding to E2F1 (A) and Rb (C) was monitored by real-time RT-PCR and compared with the level of control siRNA-treated cells at 0 h. The values represent mean ± sd of three separate experiments. *, Statistically significant change (P < 0.05) when compared with control siRNA treatments. B and D, The cells were fixed and subjected to immunocytochemistry using antibodies against E2F1 (B) and p-Rb (S780) (D). p, Phospho.
The Rb protein is another important regulator of cell cycle progression (36). In its hypophosphorylated form, Rb binds to the E2F family factors, suppressing their transcriptional activity. In the G1 phase, cyclin D-cdk4 and cyclin E-cdk2 complexes sequentially phosphorylate Rb at T798 and T780, respectively (37, 38). Hyperphosphorylated Rb disassociates from the E2F factors, allowing the latter to regulate downstream genes required for entry into the S phase (39). To assess whether the function of Rb is affected during G1-S arrest in C/EBPβ-deficient HESCs, we examined its expression level and phosphorylation status. We found that the level of Rb mRNA was not significantly altered between control and C/EBPβ siRNA-treated HESCs (Fig. 2C). However, the phosphorylation of Rb at T780 is markedly reduced in C/EBPβ-deficient HESCs (Fig. 2D), consistent with the observed lack of cyclin E/cdk2 activity in these cells. This finding suggests that hypophosphorylated Rb remains bound to the low levels of the E2Fs present in the C/EBPβ-deficient HESCs and inhibits their transcriptional activation, further preventing the G1-S transition of these cells.
Analysis of the role of C/EBPβ in HESC differentiation
Our laboratory and others have previously described an in vitro decidualization system using primary cultures of HESC, isolated from proliferative phase human endometrium (32, 40–43). Addition of E+P+cAMP to these cultures initiates differentiation of these cells as indicated by the induction of expression of several well-characterized decidual markers, such as PRL, IGFBP-1, and IL-11 (Supplemental Fig. 3). We also noted that the HESCs, which continued to divide in complete medium without hormones, had a doubling time of approximately 0.8 d. However, after addition of the E+P+cAMP cocktail, the synchronized cells failed to proliferate (doubling time, ~4.0 d) (Fig. 3A). Therefore, the initiation of the stromal differentiation process is accompanied by a cessation of their proliferative activity.
In vitro decidualization of HESCs arrests proliferation and induces differentiation. Primary cultures of HESCs were grown in DMEM/F-12 (1:1) medium containing 5% FBS and treated with or without a hormone cocktail containing 10 nm E, 1 mm P, and 0.5 mm 8-bromo-cAMP. The culture was continued for the indicated number of days. A, The cell numbers were determined using a hemocytometer from three independent experiments and reported as mean ± sd. B, HESCs were synchronized via double thymidine block and then treated with the hormone cocktail for 3 d with or without removal of the block. After 3 d, the expression of decidualization markers PRL and IGFBP-1 was monitored by real-time RT-PCR and compared with untreated controls. C, The expression of C/EBPβ was measured by real-time RT-PCR. Graphs represent means ± sems from at least three separate experiments. *, Significantly different (P ≤ 0.05) from untreated controls. D, HESCs were collected at 0, 24, and 48 h of hormone cocktail treatment, whole-cell extracts were prepared and subjected to Western blotting using antibodies against C/EBPβ. Two C/EBPβ-related bands, at 35 and 38 kDa, were observed. Immunostaining of calnexin served as a loading control. E, Immunocytochemical staining of C/EBPβ in HESC at 48 h in the presence or absence of hormone cocktail.
To determine whether the proliferation of HESCs is a necessary prerequisite for their differentiation, we arrested HESC proliferation at the G1 phase by double thymidine block and then added E+P+cAMP to these cells with or without releasing the cell cycle block (Fig. 3B). When the block was released, treatment with hormone cocktail induced differentiation as evidenced by the enhanced expression of PRL and IGFBP-1. Interestingly, HESCs that are still arrested at the G1 phase of the cell cycle by thymidine block also exhibited a similar robust induction of the decidualization markers (Fig. 3B), indicating that the differentiation of HESCs can proceed even in the absence of cell proliferation.
To assess the functional role of C/EBPβ in HESC differentiation, we first monitored its expression during in vitro decidualization. We observed that the level of C/EBPβ mRNA increased significantly within 12 h of addition of E+P+cAMP cocktail (Fig. 3C). This elevated expression of C/EBPβ mRNA continued until 48 h. It declined by 72 h but was maintained at a significant plateau for up to 9 d of culture. Western blotting data indicated that the C/EBPβ protein levels were up regulated at 24 and 48 h after hormone cocktail treatment (Fig. 3D). Two bands, specific for C/EBPβ, were observed at 35 and 38 kDa. The lower band (35 kDa) corresponds to full-length C/EBPβ, whereas the upper band (38 kDa) presumably represents the phosphorylated form of the protein. Immunocytochemical analysis further confirmed the enhanced nuclear expression of C/EBPβ protein by 48 h after treatment with E+P+cAMP (Fig. 3E).
Next, we suppressed the expression of C/EBPβ, using siRNA, and then examined its effect on decidualization by monitoring the expression of PRL and IGFBP-1. As shown in Fig. 4A, siRNA-mediated down-regulation of C/EBPβ in the HESC led to a significant reduction in expression of PRL and IGFBP-1. In contrast, the level of progesterone receptor remained unaltered in cells treated with either C/EBPβ or control siRNA. These results revealed an essential role of C/EBPβ in the stromal differentiation process.
C/EBPβ regulates HESC differentiation. HESCs were transfected with siRNA (40 nm) targeted against C/EBPβ or control (scrambled) siRNA as described in Materials and Methods. A and B, Twenty-four hours after transfection, cells were treated with or without E+P+cAMP and harvested 72 h after treatment. The expressions of PGR, C/EBPβ, PRL, IGFBP-1, BMP2, Wnt4, IL-11Rα, STAT3, and VEGF-A were measured and compared with no hormone cocktail controls. *, Statistically significant change (P < 0.05) when compared with control cells subjected to mock transfection. C, The expression of C/EBPβ and STAT3 proteins was monitored by immunocytochemistry after 72 h of hormone cocktail treatment, using antibodies against C/EBPβ and STAT3, respectively.
Identification of C/EBPβ-regulated gene networks that control human endometrial decidualization
Because C/EBPβ critically controls the differentiation of HESCs, we sought to explore the downstream pathways of this transcription factor. One approach involved attenuation of C/EBPβ expression by C/EBPβ-specific siRNA followed by gene expression profiling, using Affymetrix human GeneChip arrays, to identify its target genes. Three independent primary HESC cultures were established from biopsies derived from different individuals and subjected to in vitro decidualization after treatment with control or C/EBPβ-specific siRNA. Total RNA isolated from these cells was subjected to gene expression profiling. The expression of 665 genes was found to be up-regulated and that of 674 genes was down-regulated in all three expression profiles upon treatment with C/EBPβ-specific siRNA (>1.5-fold, P < 0.05) (results submitted to the Gene Expression Omnibus database repository, GEO accession no. GSE41473). The candidate C/EBPβ-regulated genes were classified by the Panther Classification System according to their known biological functions. These pathways involve a variety of biological processes, such as extracellular matrix and integrin signaling, inflammatory responses to cytokines and chemokines, angiogenesis, and signaling by the TGFβ, epidermal growth factor receptor, and Wnt families. Given their complexity, redundancy, and Darwinian importance, all of these pathways are likely to play roles in decidualization and implantation.
We confirmed the results of our microarray analysis by performing quantitative real-time PCR analysis for selected genes, using RNA isolated from the HESCs treated with C/EBPβ-specific or control siRNA. We observed that siRNA targeted to C/EBPβ mRNA significantly inhibited the expression of mRNAs encoding BMP2, WNT4, IL-11Rα, STAT3, and VEGF-A (Fig. 4B). Down-regulation of total STAT3 protein, which showed both cytoplasmic and nuclear localization, was further confirmed by immunocytochemistry (Fig. 4C). Previous studies indicated that BMP2, a morphogen belonging to the TGFβ superfamily, is an essential regulator of decidualization in both mouse and human (32, 44). WNT4, a downstream target of BMP2, also controls endometrial stromal decidualization in mouse and human (32, 70). It was also reported that IL-11Rα and the signal transducer STAT3, components of the IL-11 cytokine pathway, are important regulators of decidualization in the mouse (15, 45–47). VEGF-A, a well-known angiogenesis promoter, is activated during decidualization (48, 49). The finding that BMP2, WNT4, IL-11Rα, STAT3, and VEGF-A, well-established regulators of stromal differentiation and angiogenesis, are also downstream targets of regulation by C/EBPβ during HESC differentiation indicated that the function of this transcription factor during decidualization is conserved among species.
Activation of IL-11 signaling pathway during human endometrial decidualization
Identification of IL-11Rα and its signaling transducer STAT3 as downstream targets of C/EBPβ prompted us to examine the roles of these factors during decidualization of HESCs. We observed that IL-11Rα and STAT3 displayed similar expression patterns during in vitro decidualization (Fig. 5A). The levels of their transcripts were elevated early in the decidualization process, reached a peak by d 2–3, and then gradually declined to the basal level by d 6. STAT3 is a major mediator of signaling by the IL-6 family of cytokines, which include IL-6, IL-11, and leukemia inhibitory factor (LIF) (50). Our study indicated that primary HESCs robustly produce IL-11 during decidualization (Supplemental Fig. 3), whereas they secrete lower levels of IL-6 (49) and LIF (data not shown). This raised the possibility that IL-11 signals via IL-11Rα to activate STAT3 during the stromal differentiation process.
Activation of IL-11 signaling pathway during decidualization. HESCs were subjected to decidualization in vitro upon addition of E+P+cAMP as described above. A, The expression of IL-11Ra and STAT3 mRNAs were measured at different time points and compared with 0 h untreated controls. *, Statistically significant change (P < 0.05) when compared with untreated control. B, The level of phospho-STAT3 (Ser 727) was monitored at different days for up to 6 d of in vitro decidualization by immunocytochemistry using an antibody that specifically recognizes active phosphorylation at Ser 727 of STAT3. C, HESCs were transfected with IL-11Rα siRNA or control siRNA; 24 h after transfection, HESCs were treated with hormone cocktail. Cells were harvested at 72 h after hormone treatment, and the expression of IL-11Rα, PGR, PRL, IGFBP-1, and STAT3 mRNAs was measured. The expression of these genes in IL-11Rα siRNA-treated cells (gray bars) is normalized to that in control siRNA-treated cells (black bars, set as 1.0). *, Statistically significant change (P < 0.05) when compared with control siRNA treatment. D, STAT3 activation was monitored after 72 h of hormone cocktail treatment by immunocytochemistry using antibodies that recognize phospho-STAT3.
Previous reports indicated that downstream of cytokine signaling, STAT3 is phosphorylated at Ser-727, which activates its transcriptional function and allows it to modulate gene expression (50). As shown in Fig. 5B, coincident with increasing IL-11 production in differentiating stromal cells, phosphorylated STAT3 is formed and translocated to the nucleus. The phosphorylated state of STAT3 was maintained for several days, although its intensity was diminished slightly by d 6. These results pointed to a potential role of STAT3 activation downstream of IL-11 signaling during decidualization.
To establish that signaling via IL-11Rα actives STAT3 during HESC differentiation, we employed a siRNA strategy to down-regulate IL-11Rα mRNA levels in these cells. We then examined the effect of this intervention on STAT3 activation and decidualization. As shown in Fig. 5C, although siRNA-mediated attenuation of IL-11Rα mRNA did not significantly affect the levels of STAT3 mRNA, it markedly reduced the activating phosphorylation of STAT3 and suppressed the expression of differentiation markers PRL and IGFBP-1 (Fig. 5D). Thus, IL-11 signaling via its receptor is critical for STAT3 activation and HESC differentiation.
STAT3 is a direct target of regulation by C/EBPβ during HESC differentiation
To investigate the mechanism by which C/EBPβ regulates STAT3 expression, we analyzed its interaction with the promoter of STAT3 gene. In silico analysis of the STAT3 promoter revealed that it contains four candidate C/EBPβ binding sites within the 2-kb 5′-flanking region of the gene at nucleotides −61, −521, −931, and −1470, relative to the transcription start site (Fig. 6A). To test whether C/EBPβ actually binds to one or more of these sites, we employed ChIP using an antibody specific for C/EBPβ. As shown in Fig. 6A, minimal binding of C/EBPβ was seen at the promoter sites −61, −521, and −1470, whereas relatively strong binding of this transcription factor was observed at the −931 region of the STAT3 promoter.
C/EBPβ controls the expression of STAT3 in differentiating stromal cells. A, upper panel, The nucleotide positions of the candidate C/EBPβ binding sites within the four promoter regions analyzed by ChIP; lower panel, ChIP assays were performed using anti-C/EBPβ, anti-RNA polymerase II (RPII), and antirabbit IgG antibodies and then using PCR to amplify specific regions of the STAT3 promoter. The representative data are shown here. B, HESCs were transduced with adenovirus expressing dominant negative mutant A-C/EBP or control virus (MOI, 10:1); 24 h after viral transduction, HESCs were treated with hormone cocktail for 72 h, and ChIP assays were performed to determine promoter occupancy by C/EBPβ at the −931 site of the STAT3 promoter. For ChIP experiments, RPII IP served as a positive control and IgG IP served as a negative control. C, HESCs were transduced with adenovirus expressing A-C/EBP or empty vector (MOI, 10:1). After 24 h of viral transduction, HESCs were treated with hormone cocktail for 72 h. RNA was isolated, and the expression of mRNAs corresponding to C/EBPβ, PGR, PRL, IGFBP-1, and STAT3 was analysis by real-time PCR using gene-specific primers. The mRNA levels were normalized with respect to untreated (no virus) control. *, Statistically significant change (P < 0.05) when compared with no adenovirus treatment.
To confirm that the CCAAT motif at −931 is indeed the site where endogenous C/EBPβ regulates STAT3 expression, we employed a dominant negative mutant A-C/EBP protein that forms inactive complexes with wild-type C/EBPβ and inhibits DNA binding. As shown in Fig. 6B, adenovirus-mediated expression of A-C/EBP blocked the occupancy of endogenous C/EBPβ at the −931 region of the STAT3 promoter. A-C/EBP also strongly reduced the expression of PRL, IGFBP-1, and STAT3 mRNAs (Fig. 6C). These data suggested that the −931 region of the STAT3 promoter represents a bona fide C/EBPβ binding site and mediates the direct transcriptional regulation of this cytokine signal transducer by C/EBPβ.
STAT3 controls human endometrial decidualization
To examine whether STAT3 plays a regulatory role in the stromal differentiation process, we employed siRNA strategy to reduce its expression in HESCs. Attenuation of STAT3 mRNA expression (Fig. 7A) and consequent down-regulation of STAT3 protein (Fig. 7B) led to a marked reduction in the expression of mRNAs corresponding to PRL and IGFBP-1 but did not significantly affect the mRNA levels of C/EBPβ and PGR (Fig. 7A). The reduction of the PRL protein secretion was further confirmed by ELISA analysis (Fig. 7C). To further assess whether the STAT3 signaling pathway is a critical regulator of HESC differentiation, we tested whether blockade of STAT3 activation upon administration of a potent inhibitor of Janus kinases (JAK)/STAT3 signaling pathway, JSI-124, affects the HESC decidualization process. As shown in Supplemental Fig. 4, treatment with JSI-124 blocks the morphological transformation characteristic of stromal differentiation (Supplemental Fig. 4A). The fibroblastic stromal cells, which are normally transformed into cuboidal and epithelioid decidual cells in response to E+P+cAMP, failed to undergo such transformation in the presence of JSI-124. Furthermore, treatment with JSI-124 suppressed the expression of decidualization markers, PRL and IGFBP-1, but not STAT3 (Supplemental Fig. 4B). Collectively, these results established that STAT3 plays an essential role in human endometrial stromal decidualization.
Suppression of STAT3 expression blocks HESC decidualization. HESCs were transfected with STAT3 siRNA or control siRNA for 24 h and then treated with a hormonal cocktail containing E+P+cAMP for 72 h. A, Cells were harvested at 72 h after hormone treatment, and total RNA was isolated. The expression of mRNAs corresponding to STAT3, C/EBPβ, PGR, PRL, and IGFBP-1 was monitored by real-time RT-PCR as described above. STAT3 siRNA-treated cells (gray bars) is shown relative to that in control siRNA-treated cells (black bars, set as 1.0). *, Statistically significant change (P < 0.05) when compared with control siRNA treatment. B, The level of STAT3 protein was monitored after 72 h of hormone cocktail treatment by immunocytochemistry using an antibody that recognizes total STAT3. C, Culture medium was collected at different time points after hormone cocktail treatment, and the level of secreted PRL protein was measured by ELISA. D, Venn diagrams indicate the number of genes that are up or down regulated by C/EBPβ and STAT3.
Pathways downstream of STAT3 overlap with those regulated by C/EBPβ
To identify the downstream gene networks regulated by STAT3 during HESC differentiation, we compared gene expression profiles of HESC treated with control and STAT3 siRNA. This microarray analysis identified 759 genes whose expression is down-regulated and 1046 genes whose expression is up-regulated (>1.5-fold) upon treatment with STAT3 siRNA (Fig. 7D, results submitted to the Gene Expression Omnibus database repository). A comparison of the pathways downstream of C/EBPβ and STAT3 revealed a substantial overlap of the gene networks regulated by these transcription factors (Fig. 7D and Supplemental Table 2). We found that 428 genes are up-regulated and 474 genes are down-regulated by both C/EBPβ and STAT3 siRNA treatment (Fig. 7D). These common pathways, which include genes involved in inflammation mediated by chemokine/cytokine (e.g. IL-11Rα), Wnt signaling (e.g. WNT4), angiogenesis (e.g. VEGF-A, SPHK1), and TGFβ signaling pathway (e.g. BMP2), support our findings that C/EBPβ and STAT3 participate in a linear pathway that controls a variety of biological events during HESC decidualization.
Discussion
In human endometrium, the stromal cells undergo mitotic expansion during the proliferative phase of the menstrual cycle and initiate differentiation, known as predecidualization, during the secretory phase of the cycle (51). The postovulatory increase of P, and putative factors from the implanting blastocyst, elevates intercellular cAMP, thereby activating the protein kinase A signaling pathway and full stromal differentiation (19). Several factors are known to play important roles in controlling human endometrium proliferation and differentiation (18, 32, 52–54). Our recent studies described C/EBPβ expression in both epithelial and stromal compartments of human endometrium during proliferative and secretory phases of the menstrual cycle (55). An intense nuclear expression of C/EBPβ was observed in glandular epithelium and differentiating stroma during the midsecretory phase of the cycle (55). These findings raised the possibility that C/EBPβ-regulated pathways control HESC function during various stages of the menstrual cycle. In the present study, we employed primary cultures of endometrial stromal cells to demonstrate that C/EBPβ alternatively controls HESC proliferation as well as differentiation via its downstream signaling pathways.
Several laboratories have shown that primary HESCs can be maintained in a proliferating state for many generations but subsequently can be induced to undergo differentiation in response to a hormonal cocktail containing E+P+cAMP (40, 51, 56–58). We observed prominent expression of C/EBPβ in proliferating HESCs, and its expression was further elevated during the initial phase of differentiation. The primary HESC culture system, therefore, presents a unique opportunity to explore the role of C/EBPβ in stromal proliferation and differentiation. A loss-of-function approach using siRNAs showed that C/EBPβ is a key regulator of both of these processes.
The cyclical changes in the expression and localization of various cyclins and the sequential activation of specific cyclin/cdk complexes drive the mammalian cell cycle through distinct stages: G1, S, G2, and M (59). Our study showed that down-regulation of C/EBPβ expression in synchronized HESCs inhibits the expression of cyclin E and impairs its nuclear accumulation. This, in turn, prevented cyclin E-dependent phosphorylation and activation of cdk2. The formation and activation of the cyclin E-cdk2 complex is critical for G1-S transition (59–62). When knocked down by siRNA, C/EBPβ failed to activate cdk2 and prevented phosphorylation of Rb and its dissociation from the E2Fs (36, 37, 39, 63, 64). Additionally, E2F1 mRNA and protein expression were diminished in C/EBPβ-deficient HESCs. The combination of decreased production of E2F1 and a block in its transcriptional activation due to a failure to dissociate from Rb contributed to the G1-S arrest in the C/EBPβ-deficient cells.
Using a C/EBPβ-null mouse model, our laboratory previously reported that, in response to a decidual stimulation, mouse uterine stromal cells lacking C/EBPβ were able to enter the S phase of the cell cycle but were arrested at the G2-M checkpoint (31). Interestingly, in the mouse, loss of C/EBPβ did not affect the expression or activity of D-, A-, or E-type cyclins or their target kinases cdk2 and cdk4, which control G1-S transition of uterine stromal cells. Our analysis revealed that C/EBPβ regulated the expression of several key molecules that control G2-M transition in proliferating murine uterine stromal cells. These included cyclins B1 and B2, the phosphatase cell division cycle 25 homolog C, and the cell cycle inhibitors p21, p27, and p53. Although the G2-M transition, mitotic activity, and cyclin B1/B2 expression are also blocked in C/EBPβ-deficient HESCs, our current findings reveal that the primary cell cycle step controlled by this transcription factor during HESC proliferation is entry into S phase and progression of DNA replication. In this regard, the mechanisms underlying C/EBPβ regulation of endometrial stromal proliferation in the human are distinct from that in the mouse.
Previous reports described that C/EBPβ regulates the expression of decidual prolactin, a biomarker of decidualization of human stromal cells (65, 66). In stromal primary cultures in which decidualization can be efficiently induced upon addition of a hormonal cocktail containing E, P, and a cAMP analog, enhanced expression of C/EBPβ was observed early during the decidual program, consistent with the hypothesis that it regulates downstream pathways critical for this process. Indeed, when we suppressed C/EBPβ with siRNA targeting its mRNA, the differentiation process was significantly inhibited, providing strong evidence that C/EBPβ plays an essential role in human stromal cell decidualization.
Although C/EBPβ is a major regulator of uterine functions in the mouse and the human, little is known about its target genes in this tissue. Using gene expression profiling during in vitro decidualization, we identified as many as 1329 known genes as potential targets of up- and down-regulation by C/EBPβ during the stromal differentiation process. These genes encoded factors with a wide range of biological functions, such as cellular signaling, regulation of cytoskeleton structure, inflammation and immune regulation, enzyme metabolism, transport, and ion channels. The identification of many different molecular pathways downstream of C/EBPβ is not unexpected, given the complexity of the decidualization process, which involves biological phenomena as varied as differentiation, vascular remodeling, and maternal immune suppression. These genes could be directly regulated by C/EBPβ or be downstream of primary target genes. Our results indicate that a key pathway downstream of C/EBPβ is the BMP2-WNT4 pathway, which is intimately related to the formation and function of the decidual tissue (32, 44). Previous studies established that mice deficient in uterine BMP2 are infertile and exhibit a severe defect in stromal differentiation (44). We identified Wnt4 as a downstream target of BMP2 regulation in stromal cells undergoing decidualization (32). Attenuation of Wnt4 expression by siRNA greatly reduced stromal differentiation in vitro, suggesting that it is a candidate mediator of BMP2-induced decidualization (Bagchi, I. C., and M. K. Bagchi, unpublished data).
A major finding of this study is that C/EBPβ regulates IL-11 signaling during decidualization. IL-11 activates a cell surface receptor complex comprising IL-11Rα and the common signaling protein glycoprotein 130, which act through the Janus kinase/STAT pathway (67). A number of studies have shown that IL-11 signaling is important in decidualization. Female mice bearing a null mutation in the IL-11Rα are infertile due to defective decidualization and improper trophoblast invasion (15, 45). In human endometrium, IL-11 and IL-11Rα both are expressed in decidualizing stromal cells of mid-late secretory phase, suggesting a possible role in implantation (68). In primary HESCs, addition of IL-11 stimulated P-induced decidualization (69), and conditions associated with low IL-11 in the endometrium have compromised decidualization and infertility (6, 8). Our microarray analysis revealed that IL-11Rα mRNA expression is regulated by C/EBPβ, establishing an important functional link between this transcription factor and a cytokine signal pathway invoked in the successful establishment of pregnancy.
Although locally produced cytokines are thought to facilitate decidualization, little is known about the underlying mechanisms by which these factors regulate differentiation (53, 69). Previous studies identified STAT3 as a major mediator of signaling by the IL-6 family of cytokines, which include IL-6, IL-11, and LIF (50), and it was reported that inhibition of STAT3 activation impairs implantation in the mouse (46). Our study confirmed that primary HESCs produce ample amounts of IL-11 during decidualization (Supplemental Fig. 3), and the temporal production of IL-11 by human stromal cells overlapped with the activation profile of STAT3 (Fig. 5 and Supplemental Fig. 3). Using siRNA-mediated silencing of C/EBPβ, we showed that it regulates the expression of STAT3. This conclusion was further strengthened via ChIP experiments, which revealed that C/EBPβ directly binds to the −931 region of the STAT3 promoter that contains a CCAAT motif. Expression of the dominant negative mutant A-C/EBP, which blocked DNA binding by endogenous C/EBPβ, also suppressed the STAT3 promoter occupancy by this transcription factor and as a consequence markedly reduced STAT3 mRNA production. Furthermore, using a siRNA strategy, we demonstrated that the loss of STAT3 expression inhibits human stromal decidualization. DNA microarray-based gene expression analysis revealed extensive overlap between pathways regulated downstream of C/EBPβ and STAT3 (Fig. 7D and Supplemental Table 2). Taken together, these results provided convincing evidence that STAT3 is an essential mediator of C/EBPβ-driven human endometrial stromal differentiation. It is also of interest that STAT3, acting downstream of C/EBPβ, regulates the stromal expression of the angiogenic factors, VEGF-A and SPHK1. Although VEGF-A is a key promoter of angiogenesis, SPHK1 is required for proper uterine decidualization and blood vessel stability during mouse pregnancy (71).
In summary, the present study demonstrated that C/EBPβ is a critical regulator of HESC proliferation and differentiation. This work also indicated that key regulators of stromal differentiation, such as BMP2 and WNT4, appear to operate in a linear pathway downstream of C/EBPβ during decidualization. Our study also uncovered a unique signaling network, involving C/EBPβ, IL-11Rα, and STAT3, which plays an important role in regulating stromal differentiation, angiogenesis, and inflammatory responses during decidualization.
Acknowledgments
This work was supported by National Institutes of Health (NIH) Grant U54 HD055787 as part of the Eunice Kennedy Shriver National Institute of Child Health and Human Development/NIH Centers Program in Reproduction and Infertility Research.
Disclosure Summary: The authors have nothing to disclose.
Footnotes
Abbreviations:
- BMP
- Bone morphogenetic protein
- BrdU
- bromodeoxyuridine
- cAMP
- cyclic AMP
- cdk
- cyclin-dependent kinase
- C/EBP
- CCAAT/enhancer-binding protein
- ChIP
- chromatin immunoprecipitation
- E
- 17β-estradiol
- E+P+cAMP
- 10 nm E, 1 μm P, and 0.5 mm 8-bromo-cAMP
- E2F1
- E2F transcription factor 1
- FBS
- fetal bovine serum
- HESC
- human endometrial stromal cell
- IGFBP
- IGF binding protein
- IL-11R
- IL-11 receptor
- JSI-124
- Janus kinases/STAT3 inhibitor-124
- LIF
- leukemia inhibitory factor
- MOI
- multiplicity of infection
- P
- progesterone
- PGR
- P receptor
- P-His3
- phosphorylated Ser 10 of histone 3
- PRL
- prolactin
- Rb
- retinoblastoma
- siRNA
- small interfering RNA
- SPHK1
- sphingosine kinase 1
- STAT3
- signal transducer and activator of transcription 3
- VEGF
- vascular endothelial growth factor
- Wnt4
- wingless-type MMTV integration site family, member 4.
References
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Funding
Funders who supported this work.
NCATS NIH HHS (1)
Grant ID: UL1 TR000454
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Grant ID: UL1 RR025008
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Grant ID: U54 HD055787