Transforming growth factor B1 stimulated DNA synthesis in the granulosa cells of preantral follicles: negative interaction with epidermal growth factor

EGF or TGFB1 alone stimulates but together attenuate granulosa cell DNA synthesis. Intact preantral follicles from hamsters were cultured with TGFB1, EGF, or both to reveal the mechanisms of such unique regulation. Follicular CCND2 (also known as cyclin D2), CDKN1B (also known as p27(kip1)), and the involvement of appropriate signaling intermediaries and kinases were examined. TGFB1, acting via SMAD2 and SMAD3, antagonized the degradation of CCND2 protein by blocking its phosphorylation. In contrast, TGFB1 supported CDKN1B degradation by involving MAPK14 (also known as p38 Map Kinase) and PKC, resulting in CDK4 activation and DNA synthesis. EGF via MAPK3/1 maintained functional levels of CCND2 through CCND2 synthesis as well as degradation. EGF and TGFB1 together inhibited CDK4 activation and DNA synthesis. EGF attenuated TGFB1 stimulated phosphorylation of SMAD3, TGFB1-induced activation of MAPK14 and PKC, and TGFB1 suppression of CCND2 degradation. In contrast, TGFB1 suppressed EGF-induced increase in CCND2 mRNA levels. The final outcome was CCND2 degradation without replenishment and decreased activities of MAPK14 and PKC leading to suppression of CDK4 activation. The results indicate that each growth factor involves a separate mechanism to maintain an effective level of CCND2 in granulosa cells for the activation of CDK4 and induction of DNA synthesis. However, their simultaneous action is inhibitory to follicular DNA synthesis because they counteract each other's activity by interfering at specific sites. Because both EGF and TGFB1 are present in granulosa cells, this mechanism may explain how their effects are temporally modulated for granulosa cell proliferation and folliculogenesis.     

Comprehensive gene expression analysis reveals multiple signal pathways associated with prostate cancer

Prostate cancer (PC) depends on androgenic signaling for growth and survival. To data, the exact molecular mechanism of hormone controlling proliferation and tumorigenesis in the PC remains unclear. Therefore, in this study, we explored the differentially expressed genes (DEGs) and identified featured genes related to hormone stimulus from PC. Two sets of gene expression data, including PC and normal control sample, were downloaded from Gene Expression Omnibus (GEO) database. The t-test was used to identify DEGs between PC and controls. Gene ontology (GO) functional annotation was applied to analyze the function of DEGs and screen hormone-related DEGs. Then these hormone-related DEGs were further analyzed in constructed cancer network and Human Protein Reference Database to screen important signaling pathways they participated in. A total of 912 DEGs were obtained which included 326 up-regulated genes and 586 down-regulated genes. GO functional enrichment analysis identified 50 hormone-related DEGs associated with PC. After pathway and PPI network analysis, we found these hormone-related DEGs participated in several important signaling pathways including TGF-β (TGFB2, TGFB3 and TGFBR2), MAPK (TGFB2, TGFB3 and TGFBR2), insulin (PIK3R3, SHC1 and EIF4EBP1), and p53 signaling pathways (CCND2 and CDKN1A). In addition, a total of five hormone-related DEGs (SHC1, CAV1, RXRA, CDKN1A and SRF) were located in the center of PPI network and 12 hormone-related DEGs formed six protein modules. These important signal pathways and hormone-related DEGs may provide potential therapeutic targets for PC.     

MiR-17-92 cluster regulates cell proliferation and collagen synthesis by targeting TGFB pathway in mouse palatal mesenchymal cells

Elongation and elevation of palatal shelves, mainly caused by proliferation and extra-cellular matrix synthesis of palatal mesenchymal cells (PMCs), are essential for normal palatal development. Transforming growth factor beta (TGFB) pathway could induce proliferation inhibition and collagen synthesis in PMCs. Recent studies found that miRNA-17-92 (miR-17-92) cluster, including miR-17, miR-18a, miR-19a, miR-20a, miR-19b, and miR-92a, expressed in the 1st bronchial arch of mouse embryos during the period of palatal shelf elongation and elevation, and directly targeted TGFB pathway in cancer cell lines. Whether miR-17-92 cluster expresses and targets TGFB pathway in PMCs has not yet been studied. Using quantitative real-time RT-PCR, we found that miR-17-92 expressed in PMCs and decreased from embryonic day (E) 12 to E14 in palatal shelves. MTT assay and Western blot showed that miR-17-92 inhibited TGFB1 induced proliferation inhibition and collagen synthesis in PMCs by decreasing TGFBR2, SMAD2, and SMAD4 protein level. Further luciferase assay showed that miR-17 and miR-20a directly targeted 3′UTR of TGFBR2, and that miR-18a directly targeted 3′UTR of SMAD2 and SMAD4. We thus conclude that miR-17-92 cluster could inhibit TGFB pathway induced proliferation inhibition and collagen synthesis in PMCs by directly targeting TGFBR2, SMAD2, and SMAD4.     

TGF-β-SMAD3 signaling mediates hepatic bile acid and phospholipid metabolism following lithocholic acid-induced liver injury

Transforming growth factor-β (TGFβ) is activated as a result of liver injury, such as cholestasis. However, its influence on endogenous metabolism is not known. This study demonstrated that TGFβ regulates hepatic phospholipid and bile acid homeostasis through MAD homolog 3 (SMAD3) activation as revealed by lithocholic acid-induced experimental intrahepatic cholestasis. Lithocholic acid (LCA) induced expression of TGFB1 and the receptors TGFBR1 and TGFBR2 in the liver. In addition, immunohistochemistry revealed higher TGFβ expression around the portal vein after LCA exposure and diminished SMAD3 phosphorylation in hepatocytes from Smad3-null mice. Serum metabolomics indicated increased bile acids and decreased lysophosphatidylcholine (LPC) after LCA exposure. Interestingly, in Smad3-null mice, the metabolic alteration was attenuated. LCA-induced lysophosphatidylcholine acyltransferase 4 (LPCAT4) and organic solute transporter β (OSTβ) expression were markedly decreased in Smad3-null mice, whereas TGFβ induced LPCAT4 and OSTβ expression in primary mouse hepatocytes. In addition, introduction of SMAD3 enhanced the TGFβ-induced LPCAT4 and OSTβ expression in the human hepatocellular carcinoma cell line HepG2. In conclusion, considering that Smad3-null mice showed attenuated serum ALP activity, a diagnostic indicator of cholangiocyte injury, these results strongly support the view that TGFβ-SMAD3 signaling mediates an alteration in phospholipid and bile acid metabolism following hepatic inflammation with the biliary injury.     

Wild-type TP53 inhibits G(2)-phase checkpoint abrogation and radiosensitization induced by PD0166285, a WEE1 kinase inhibitor

The WEE1 protein kinase carries out the inhibitory phosphorylation of CDC2 on tyrosine 15 (Tyr15), which is required for activation of the G(2)-phase checkpoint in response to DNA damage. PD0166285 is a newly identified WEE1 inhibitor and is a potential selective G(2)-phase checkpoint abrogator. To determine the role of TP53 in PD0166285-induced G(2)-phase checkpoint abrogation, human H1299 lung carcinoma cells expressing a temperature-sensitive TP53 were used. Upon exposure to gamma radiation, cells cultured under nonpermissive conditions (TP53 mutant conformation) underwent G(2)-phase arrest. However, under permissive conditions (TP53 wild-type conformation), PD0166285 greatly inhibited the accumulation of cells in G(2) phase. This abrogation was accompanied by a nearly complete blockage of Tyr15 phosphorylation of CDC2, an increased activity of CDC2 kinase, and an enhanced sensitivity to radiation. However, under permissive conditions (TP53 wild-type conformation), PD0166285 neither disrupted the G(2)-phase arrest nor increased cell death. The compound inhibited Tyr15 phosphorylation only partially and did not activate CDC2 kinase activity. To understand the potential mechanism(s) by which TP53 inhibits PD0166285-induced G(2)-phase checkpoint abrogation, two TP53 target proteins, 14-3-3rho and CDKN1A (also known as p21), that are known to be involved in G(2)-phase checkpoint control in other cell models were examined. It was found that 14-3-3rho was not expressed in H1299 cells, and that although CDKN1A did associate with CDC2 to form a complex, the level of CDKN1A associated with CDC2 was not increased in response to radiation or to PD0166285. The level of cyclin B1, required for CDC2 activity, was decreased in the presence of functional TP53. Thus inhibition of PD0166285-induced G(2)-phase checkpoint abrogation by TP53 was achieved at least in part through partial blockage of CDC2 dephosphorylation of Tyr15 and inhibition of cyclin B1 expression.     

Concordant repression and aberrant methylation of transforming growth factor-beta signaling pathway genes occurs early in gastric cardia adenocarcinoma

The loss of transforming growth factor-β (TGF-β) response due to the dysregulation of TGF-β receptor type I (TGFBR1), type II (TGFBR2) and Smad4 is well known for its contribution to oncogenesis, although the role of the genes of TGF-β/Smad signalling pathway in gastric cardia adenocarcinoma (GCA) is poorly understood. In the present study, the methylation status and expression of TGF-β receptor type I (TGFBR1), type II (TGFBR2), and Smad4 was investigated in GCA and dysplasia. MSP approach was used to detect the methylation status of TGFBR1, TGFBR2, and Smad4. Immunohistochemistry and quantitative RT-PCR methods were used respectively to examine the protein and mRNA expression of them in tissues. The methylation frequency of TGFBR1 and TGFBR2 in the tissues of high grade dysplasia and GCA was significantly higher than that in corresponding normal tissues (p?相似文献   

CD109-mediated degradation of TGF-β receptors and inhibition of TGF-β responses involve regulation of SMAD7 and Smurf2 localization and function

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that regulates a wide variety of cellular processes including proliferation, differentiation, and extracellular matrix deposition. Dysregulation of TGF-β signaling is associated with several diseases such as cancer and tissue fibrosis. TGF-β signals through two transmembrane proteins known as the type I (TGFBR1) and type II (TGFBR2) receptors. The levels of these receptors at the cell surface are tightly regulated by several mechanisms, including degradation following recruitment of the E3 ubiquitin ligase SMAD ubiquitination regulatory factor (Smurf) 2 by SMAD7. In addition, TGF-β co-receptors can modulate TGF-β signaling receptor activity in a cell-specific manner. We have previously identified a novel TGF-β co-receptor, CD109, a glycosyl phosphatidylinositol (GPI)-anchored protein that negatively regulates TGF-β signaling. Despite CD109's potential relevance as a regulator of TGF-β action in vivo, the mechanisms by which CD109 regulates TGF-β signaling are still incompletely understood. Previously, we have shown that CD109 downregulates TGF-β signaling by promoting TGF-β receptor localization into the lipid raft/caveolae compartment and by enhancing TGF-β receptor degradation. Here, we demonstrate that CD109 enhances SMAD7/Smurf2-mediated degradation of TGFBR1 in a ligand-dependent manner. Moreover, we show that CD109 regulates the localization and the association of SMAD7/Smurf2 with TGFBR1. Finally, we demonstrate that CD109's inhibitory effect on TGF-β signaling and responses require SMAD7 expression and Smurf2 ubiquitin ligase activity. Taken together, these results suggest that CD109 is an important regulator of SMAD7/Smurf2-mediated degradation of TGFBR1.     

Influence of TGFBR2, TGFB3, DNMT1, and DNMT3A Knockdowns on CTGF,TGFBR2, and DNMT3A in Neonatal and Adult Human Dermal Fibroblasts Cell Lines

Dermal fibroblasts are responsible for the production of the extracellular matrix that undergoes significant changes during the skin aging process. These changes are partially controlled by the TGF-β signaling, which regulates tissue homeostasis dependently on several genes, including CTGF and DNA methyltransferases. To investigate the potential differences in the regulation of the TGF-β signaling and related molecular pathways at distinct developmental stages, we silenced the expression of TGFB1, TGFB3, TGFBR2, CTGF, DNMT1, and DNMT3A in the neonatal (HDF-N) and adult (HDF-A) human dermal fibroblasts using the RNAi method. Through Western blot, we analyzed the effects of the knockdowns of these genes on the level of the CTGF, TGFBR2, and DNMT3A proteins in both cell lines. In the in vitro assays, we observed that CTGF level was decreased after knockdown of DNMT1 in HDF-N but not in HDF-A. Similarly, the level of DNMT3A was decreased only in HDF-N after silencing of TGFBR2, TGFB3, or DNMT1. TGFBR2 level was lower in HDF-N after knockdown of TGFB3, DNMT1, or DNMT3A, but it was higher in HDF-A after TGFB1 silencing. The reduction of TGFBR2 after silencing of DNMT3A and vice versa in neonatal cells only suggests the developmental stage-specific interactions between these two genes. However, additional studies are needed to explain the dependencies between analyzed proteins.     

Disruption of postnatal folliculogenesis and development of ovarian tumor in a mouse model with aberrant transforming growth factor beta signaling

Background

Transforming growth factor beta (TGFB) superfamily signaling is implicated in the development of sex cord-stromal tumors, a category of poorly defined gonadal tumors. The aim of this study was to determine potential effects of dysregulated TGFB signaling in the ovary using Cre recombinase driven by growth differentiation factor 9 (Gdf9) promoter known to be expressed in oocytes.

Methods

A mouse model containing constitutively active TGFBR1 (TGFBR1^CA) using Gdf9-iCre (termed TGFBR1-CA^G9Cre) was generated. Hematoxylin and eosin (H & E) staining, follicle counting, and immunohistochemistry and immunofluorescence analyses using antibodies directed to Ki67, forkhead box L2 (FOXL2), forkhead box O1 (FOXO1), inhibin alpha (INHA), and SRY (sex determining region Y)-box 9 were performed to determine the characteristics of the TGFBR1-CA^G9Cre ovary. Terminal deoxynucleotidyl transferase (TdT) labeling of 3’-OH ends of DNA fragments, real-time PCR, and western blotting were used to examine apoptosis, select gene expression, and TGFBR1 activation. RNAscope in situ hybridization was used to localize the expression of GLI-Kruppel family member GLI1 (Gli1) in ovarian tumor tissues.

Results

TGFBR1-CA^G9Cre females were sterile. Sustained activation of TGFBR1 led to altered granulosa cell proliferation evidenced by high expression of Ki67. At an early age, these mice demonstrated follicular defects and development of ovarian granulosa cell tumors, which were immunoreactive for granulosa cell markers including FOXL2, FOXO1, and INHA. Further histochemical and molecular analyses provided evidence of overactivation of TGFBR1 in the granulosa cell compartment during ovarian pathogenesis in TGFBR1-CA^G9Cre mice, along with upregulation of Gli1 and Gli2 and downregulation of Tgfbr3 in ovarian tumor tissues.

Conclusions

These results reinforce the role of constitutively active TGFBR1 in promoting ovarian tumorigenesis in mice. The mouse model created in this study may be further exploited to define the cellular and molecular mechanisms of TGFB/activin downstream signaling in granulosa cell tumor development. Future studies are needed to test whether activation of TGFB/activin signaling contributes to the development of human granulosa cell tumors.

     

MARCH5 RNA promotes autophagy,migration, and invasion of ovarian cancer cells

MARCH5 is a crucial regulator of mitochondrial fission. However, the expression and function of MARCH5 in ovarian cancer have not been determined. This study investigated the expression and function of MARCH5 in ovarian cancer with respect to its potential role in the tumorigenesis of the disease as well as its usefulness as an early diagnostic marker. We found that the expression of MARCH5 was substantially upregulated in ovarian cancer tissue in comparison with the normal control. Silencing MARCH5 in SKOV3 cells decreased TGFB1-induced cell macroautophagy/autophagy, migration, and invasion in vitro and in vivo, whereas the ectopic expression of MARCH5 in A2780 cells had the opposite effect. Mechanistic investigations revealed that MARCH5 RNA may function as a competing endogenous RNA (ceRNA) to regulate the expression of SMAD2 and ATG5 by competing for MIR30A. Knocking down SMAD2 or ATG5 can block the effect of MARCH5 in A2780 cells. Also, silencing the expression of MARCH5 in SKOV3 cells can inhibit the TGFB1-SMAD2/3 pathway. In contrast, the ectopic expression of MARCH5 in A2780 cells can activate the TGFB1-SMAD2/3 pathway. In turn, the TGFB1-SMAD2/3 pathway can regulate MARCH5 and ATG5 through MIR30A. Overall, the results of this study identified MARCH5 as a candidate oncogene in ovarian cancer and a potential target for ovarian cancer therapy.     

水貂睾丸退化期间TGFB家族生长因子及其受体基因表达量的变化

为了探究水貂季节性睾丸退化的分子机制,本研究利用HE染色技术对不同退化时期的水貂睾丸组织进行了组织学研究,同时利用荧光定量PCR技术对不同退化时期的水貂睾丸组织中TGFB家族生长因子及其受体基因表达量变化进行了研究。结果发现在水貂睾丸退化期间,其直径逐渐缩短到原来的1/2,重量逐渐降低到原来的1/8。HE染色结果发现,4~7月水貂睾丸经历了严重退化,而且曲细精管的管腔逐渐消失,同时曲细精管的直径减小到其最大尺寸的1/3。荧光定量PCR结果发现TGFb3、INHBA、TGFBR1、ACVR2A、ACVR2B、SMAD2、SMAD4基因相对表达量在水貂睾丸退化期间都呈下降趋势。本研究表明TGFB信号通路可能在水貂睾丸退化期间发挥了重要功能。     

Complexity of the mechanisms of initiation and maintenance of DNA damage-induced G2-phase arrest and subsequent G1-phase arrest: TP53-dependent and TP53-independent roles

Through a detailed study of cell cycle progression, protein expression, and kinase activity in gamma-irradiated synchronized cultures of human skin fibroblasts, distinct mechanisms of initiation and maintenance of G2-phase and subsequent G1-phase arrests have been elucidated. Normal and E6-expressing fibroblasts were used to examine the role of TP53 in these processes. While G2 arrest is correlated with decreased cyclin B1/CDC2 kinase activity, the mechanisms associated with initiation and maintenance of the arrest are quite different. Initiation of the transient arrest is TP53-independent and is due to inhibitory phosphorylation of CDC2 at Tyr15. Maintenance of the G2 arrest is dependent on TP53 and is due to decreased levels of cyclin B1 mRNA and a corresponding decline in cyclin B1 protein level. After transiently arresting in G2 phase, normal cells chronically arrest in the subsequent G1 phase while E6-expressing cells continue to cycle. The initiation of this TP53-dependent G1-phase arrest occurs despite the presence of substantial levels of cyclin D1/CDK4 and cyclin E/CDK2 kinase activities, hyperphosphoryated RB, and active E2F1. CDKN1A (also known as p21(WAF1/CIP1)) levels remain elevated during this period. Furthermore, CDKN1A-dependent inhibition of PCNA activity does not appear to be the mechanism for this early G1 arrest. Thus the inhibition of entry of irradiated cells into S phase does not appear to be related to DNA-bound PCNA complexed to CDKN1A. The mechanism of chronic G1 arrest involves the down-regulation of specific proteins with a resultant loss of cyclin E/CDK2 kinase activity.     

Identification of dynamic signatures associated with smoking‐related squamous cell lung cancer and chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) is a risk factor for the development of lung cancer. The aim of this study was to identify early diagnosis biomarkers for lung squamous cell carcinoma (SQCC) in COPD patients and to determine the potential pathogenetic mechanisms. The GSE12472 data set was downloaded from the Gene Expression Omnibus database. Differentially co‐expressed links (DLs) and differentially expressed genes (DEGs) in both COPD and normal tissues, or in both SQCC + COPD and COPD samples were used to construct a dynamic network associated with high‐risk genes for the SQCC pathogenetic process. Enrichment analysis was performed based on Gene Ontology annotations and Kyoto Encyclopedia of Genes and Genomes pathway analysis. We used the gene expression data and the clinical information to identify the co‐expression modules based on weighted gene co‐expression network analysis (WGCNA). In total, 205 dynamic DEGs, 5034 DLs and one pathway including CDKN1A, TP53, RB1 and MYC were found to have correlations with the pathogenetic progress. The pathogenetic mechanisms shared by both SQCC and COPD are closely related to oxidative stress, the immune response and infection. WGCNA identified 11 co‐expression modules, where magenta and black were correlated with the “time to distant metastasis.” And the “surgery due to” was closely related to the brown and blue modules. In conclusion, a pathway that includes TP53, CDKN1A, RB1 and MYC may play a vital role in driving COPD towards SQCC. Inflammatory processes and the immune response participate in COPD‐related carcinogenesis.     

Interactions between NF-κB and SP3 Connect Inflammatory Signaling with Reduced FGF-10 Expression

Inflammation inhibits normal lung morphogenesis in preterm infants. Soluble inflammatory mediators present in the lungs of patients developing bronchopulmonary dysplasia disrupt expression of multiple genes critical for development. However, the mechanisms linking innate immune signaling and developmental programs are not clear. NF-κB activation inhibits expression of the critical morphogen FGF-10. Here, we show that interactions between the RELA subunit of NF-κB and SP3 suppress SP1-mediated FGF-10 expression. SP3 co-expression reduced SP1-mediated Fgf-10 promoter activity, suggesting antagonistic interactions between SP1 and SP3. Chromatin immunoprecipitation of LPS-treated primary mouse fetal lung mesenchymal cells detected increased interactions between SP3, RELA, and the Fgf-10 promoter. Expression of a constitutively active IκB kinase β mutant not only decreased Fgf-10 promoter activity but also increased RELA-SP3 nuclear interactions. Expression of a dominant-negative IκB, which blocks NF-κB nuclear translocation, prevented inhibition of FGF-10 by SP3. The inhibitory functions of SP3 required sequences located in the N-terminal region of the protein. These data suggested that inhibition of FGF-10 by inflammatory signaling involves the NF-κB-dependent interactions between RELA, SP3, and the Fgf-10 promoter. NF-κB activation may therefore lead to reduced gene expression by recruiting inhibitory factors to specific gene promoters following exposure to inflammatory stimuli.     

YWK-II/APLP2 inhibits TGF-β signaling by interfering with the TGFBR2–Hsp90 interaction

Transforming growth factor-β (TGF-β) regulates multiple cellular biological processes by activating TGF-β type I receptors (TGFBR1) and type II receptors (TGFBR2), and Hsp90 stabilizes these receptors through specific interactions. In many malignancies, one of the most deregulated signaling pathways is the TGF-β signaling pathway, which is often inactivated by mutations or deregulation of TGF-β type II receptors (TGFBR2). However, the molecular mechanisms are not well understood. In this study, we show that YWK-II/APLP2, an immediately early response gene for TGF-β signaling, inhibits TGF-β signaling by promoting the degradation of the TGFBR2 protein. Knockdown of YWK-II/APLP2 increases the TGFBR2 protein level and sensitizes cells to TGF-β stimulation, while YWK-II/APLP2 overexpression destabilizes TGFBR2 and desensitizes cells to TGF-β. Mechanistically, YWK-II/APLP2 is associated with TGFBR2 in a TGF-β activity-dependent manner, binds to Hsp90 to interfere with the interaction between TGFBR2 and Hsp90, and leads to enhanced ubiquitination and degradation of TGFBR2. Taken together, YWK-II/APLP2 is involved in negatively regulating the duration and intensity of TGF-β/Smad signaling and suggests that aberrantly high expression of YWK-II/APLP2 in malignancies may antagonize the growth inhibition mediated by TGF-β signaling and play a role in carcinogenesis.     

Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion

Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.