A novel selective progesterone receptor modulator asoprisnil (J867) down-regulates the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured uterine leiomyoma cells

BACKGROUND: This study was conducted to evaluate the effects of a novel selective progesterone receptor modulator (SPRM) asoprisnil on the expression of growth factors and their receptors and on growth factor-induced proliferation of cultured uterine leiomyoma and matching myometrial cells. METHODS: The expression of epidermal growth factor (EGF), insulin-like growth factor-I (IGF-I) and transforming growth factor (TGFbeta3) was assessed by immunocytochemistry and semi-quantitative RT-PCR. The expression of phosphorylated EGF receptor (p-EGFR), IGF-I receptor alpha subunit (IGF-IRalpha) and phosphorylated TGFbeta receptor type II (p-TGFbeta RII) was assessed by Western blot analysis. Cell proliferation was assessed by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay. RESULTS: Treatment with 10(-7) M asoprisnil decreased EGF, IGF-I and TGFbeta3 mRNA and protein expression as well as p-EGFR, IGF-IRalpha and p-TGFbeta RII protein expression in leiomyoma cells cultured for 72 h. EGF (100 ng/ml), IGF-I (100 ng/ml) and TGFbeta3 (10 ng/ml) increased the number of viable leiomyoma cells cultured for 72 h, whereas the concomitant treatment with 10(-7) M asoprisnil antagonized the growth factor-induced increase in leiomyoma cell proliferation. In cultured myometrial cells, however, asoprisnil affected neither the growth factor and their receptor expression nor the cell proliferation. CONCLUSION: Asoprisnil inhibits the expression of EGF, IGF-I, TGFbeta3 and their receptors in cultured leiomyoma cells without affecting their expressions in myometrial cells.     

Progesterone receptor modulator CDB-2914 down-regulates vascular endothelial growth factor, adrenomedullin and their receptors and modulates progesterone receptor content in cultured human uterine leiomyoma cells

BACKGROUND: This study was conducted to evaluate the effects of graded concentrations (10(-8), 10(-7) and 10(-6) M) of progesterone receptor (PR) modulator CDB-2914 on the protein contents of PR, of vascular endothelial growth factor (VEGF), adrenomedullin (ADM) and their receptors in cultured human uterine leiomyoma and matching myometrial cells. METHODS: PR-A, PR-B, VEGF-A, VEGF-B, VEGF receptor (VEGFR)-1, VEGFR-2, ADM and ADM receptor (ADMR) contents were assessed by Western blot analysis. RESULTS: Treatment with 100 ng/ml progesterone increased VEGF-A, VEGF-B and ADM contents in cultured leiomyoma cells and normal myometrial cells. The concomitant treatment with 10(-6) M CDB-2914 significantly decreased the progesterone-induced VEGF-A, VEGF-B and ADM contents in cultured leiomyoma cells but not in normal myometrial cells. CDB-2914 treatment alone decreased VEGFR-1, VEGFR-2 and ADMR contents in cultured leiomyoma cells but not in normal myometrial cells. CDB-2914 treatment increased PR-A and decreased PR-B contents in cultured leiomyoma cells in a dose-dependent manner compared with untreated cultures, whereas no significant changes in PR isoform contents were observed in normal myometrial cells. CONCLUSIONS: These results suggest that CDB-2914 down-regulates VEGF, ADM and their receptor contents and modulates PR isoform contents in cultured leiomyoma cells in a cell-type-specific manner.     

Gene expression and immunolocalization of heparin-binding epidermal growth factor-like growth factor and human epidermal growth factor receptors in human corpus luteum

BACKGROUND: The objective of this study was to elucidate gene expression and immunolocalization of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and human epidermal growth factor receptor (HER) family in the human ovary during luteal growth and regression. METHODS: Ovaries obtained from pre-menopausal women were used for immunohistochemistry and semiquantitative RT-PCR analysis. RESULTS: Immunoreactive HB-EGF was not detected in follicles or oocyte, while HB-EGF became apparent in granulosa luteal cells in the early luteal phase, and most abundant in the mid-luteal phase, but less abundant in the late luteal phase. Immunostaining for HER1 was very weak in granulosa luteal cells in the early and mid-luteal phases, and was not detected in the late luteal phase. Immunoreactive HER4 was abundant in the early luteal phase and became less abundant in the mid-luteal phase, whereas it was negative in the late luteal phase. Semiquantitative RT-PCR analysis revealed that HB-EGF and HER1 mRNA levels were high in the mid-luteal phase, whereas HER4 mRNA expression was high in the early luteal phase. CONCLUSIONS: HB-EGF may play a vital role in regulating luteal growth in a juxtacrine manner and through activating HER4 signalling.     

Progesterone down-regulates insulin-like growth factor-I expression in cultured human uterine leiomyoma cells

BACKGROUND: Insulin-like growth factor-I (IGF-I) plays crucial roles in uterine leiomyoma cell growth through stimulating proliferation and inhibiting apoptosis. The present study was conducted to elucidate whether progesterone affects IGF-I and its receptor expression in cultured leiomyoma cells. METHODS: Isolated leiomyoma cells were subcultured in Phenol Red-free Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum for 120 h and then stepped down to serum-free conditions for an additional 48 h in the presence or absence of 17beta-estradiol (E(2)) (10 ng/ml) or progesterone (100 ng/ml). IGF-I and its receptor mRNA and immunoreactive IGF-I in the cultured cells were assessed by quantitative RT-PCR with Southern blot analysis and by radioimmunoassay with Seppak C18 chromatography, respectively. The presence of estrogen receptor (ER) and progesterone receptor (PR) in cultured leiomyoma cells was immunocytochemically examined. RESULTS: Both treatment with progesterone alone and treatment with E(2) and progesterone combined significantly decreased IGF-I mRNA and protein expression in cultured leiomyoma cells compared with that in untreated cultures, but treatment with E(2) alone did not. IGF-I receptor mRNA expression in those cells was not affected by treatment with either E(2) or progesterone. Immunocytochemical analysis revealed that PR protein expression in cultured leiomyoma cells maintained in a serum-free condition for 48 h whereas ER protein expression in the cells remarkably decreased after 24 h culture under the serum-free condition. CONCLUSIONS: The present study provided evidence for the first time that progesterone down-regulates IGF-I mRNA and protein expression in cultured leiomyoma cells without affecting IGF-I receptor mRNA expression.     

Selective progesterone receptor modulator asoprisnil down-regulates collagen synthesis in cultured human uterine leiomyoma cells through up-regulating extracellular matrix metalloproteinase inducer

BACKGROUND: A recent clinical trial demonstrated that selective progesterone receptor modulator asoprisnil is effective in reducing uterine leiomyoma volume. We investigated the effects of asoprisnil in vitro on the expression of the extracellular matrix (ECM)-remodeling enzymes and collagens in cultured leiomyoma and matching normal myometrial cells. METHODS: The expression of extracellular matrix metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagens were assessed by western blot analysis. RESULTS: Untreated cultured leiomyoma cells had significantly lower EMMPRIN (P < 0.05), MMP-1 (P < 0.05) and membrane type 1-MMP (MT1-MMP) (P < 0.01) protein contents, but significantly higher TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.05) and type III (P < 0.01) collagen protein contents compared with untreated cultured myometrial cells. Treatment with asoprisnil at concentrations > or =10(-7) M for 48 h significantly (P < 0.05) increased EMMPRIN, MMP-1 and MT1-MMP protein contents, and decreased TIMP-1 (P < 0.05), TIMP-2 (P < 0.01), type I (P < 0.01) and type III (P < 0.05 at 10(-7) M; P < 0.01 at 10(-6) M) collagen protein contents in cultured leiomyoma cells compared with control cultures. However, asoprisnil treatment did not affect the protein contents of ECM-remodeling enzymes and collagens in cultured myometrial cells. CONCLUSIONS: These results suggest that asoprisnil may reduce collagen deposit in the ECM of cultured leiomyoma cells through decreasing collagen synthesis and enhancing the expression of EMMPRIN, MMPs and TIMPs without comparable effects on cultured myometrial cells.     

Expression of the epidermal growth factor system in human endometrium during the menstrual cycle

The epidermal growth factor (EGF) system is ubiquitous in humans and plays fundamental roles in embryogenesis, development, proliferation and differentiation. As the endometrium of fertile women is characterized by proliferation and differentiation, we hypothesize a role for the EGF system. Fourteen premenopausal women had endometrial samples removed on day 6 +/- 1 and day 6 +/- 1 and 12 +/- 1 after ovulation during one menstrual cycle. RNA was extracted and analysed by real-time PCR, and immunohistochemistry was performed to localize the components of the EGF system. Human EGF Receptor 1 (HER1) showed highest expression during the proliferative phase, HER2 and HER4 during the early and HER3 during the late secretory phase. Amphiregulin (AR) and transforming growth factor alpha (TGFalpha) expression is highest in proliferative phase. Heparin binding (HB)-EGF and betacellulin (BCL) show no variation. Epiregulin (EP) is detectable in some samples. EGF is undetectable. HER1, HER2, HER3 and HER4 were localized to the epithelium and glands HER3 and HER4 solely in the secretory phase. Amphiregulin was seen in leucocytes and stromal cells, TGFalpha and betacellulin in the epithelial lining, epiregulin in stromal cells whereas HB-EGF and EGF are undetectable. In conclusions, we observed cyclical expression of the four EGF receptors and two ligands and localized all four receptors and four ligands in endometrial biopsies. This suggests a role for the EGF system in growth of the endometrium.     

Progesterone receptor modulator CDB-2914 induces extracellular matrix metalloproteinase inducer in cultured human uterine leiomyoma cells

Effects of progesterone receptor modulator CDB-2914 on the expression of the extracellular matrix (ECM) components were examined in cultured human uterine leiomyoma and myometrial cells. ECM metalloproteinase inducer (EMMPRIN), matrix metalloproteinases (MMPs), tissue inhibitors of MMP (TIMPs) and collagen levels were assessed by Western blot analysis, MMP activity assay and real-time RT-PCR. RNA interference (RNAi) of EMMPRIN was performed using small interfering mRNA. In cultured leiomyoma cells, CDB-2914 treatment at concentrations greater than or equal to 10(-8) M significantly increased EMMPRIN, MMP-1 and MMP-8 protein contents and MMP-1, MMP-2, MMP-3 and MMP-9 mRNA levels, and activity of MMP-1, MMP-2, MMP-3 and MMP-9 in the medium. TIMP-1 and TIMP-2 were significantly decreased at mRNA and protein levels by CDB-2914 treatment at concentrations > or =10(-7) M in these cells. CDB-2914 treatment decreased types I and III collagen protein contents. However, CDB-2914 treatment did not affect the ECM component expression in cultured myometrial cells. RNAi of EMMPRIN abrogated CDB-2914-mediated both induction of MMPs and reduction of TIMPs and collagens in cultured leiomyoma cells. These results suggest that CDB-2914 modulates the expression of EMMPRIN, MMPs, TIMPs and collagens in cultured leiomyoma cells without comparable effects on myometrial cells.     

Insulin-like growth factor binding protein-1 inhibits the DNA amplification induced by insulin-like growth factor I in human granulosa-luteal cells.

In order to study the effects of insulin-like growth factor (IGF-I) and insulin-like growth factor binding protein (IGFBP-1) on human granulosa cell proliferation after in vitro fertilization, cells were obtained after oocyte retrieval and cultured in the presence or absence of graded amounts of recombinant IGF-I, purified IGFBP-1 and [3H]thymidine. Physiological concentrations of IGF-I (2-200 ng/ml) were found to stimulate [3H]thymidine incorporation into the cells in a concentration-dependent manner. Half-maximal stimulation of [3H]thymidine incorporation was obtained with 10 ng/ml exogenous IGF-I, which was chosen for suppression experiments with graded amounts of purified IGFBP-1. Suppression of IGF-stimulated thymidine incorporation was observed when 200 ng/ml or more of IGFBP-1 was added to the culture medium. The same concentration of IGFBP-1 also markedly inhibited binding of [125I]iodotyrosyl IGF-I to the cells. It is concluded that: (i) after a refractory period, granulosa cells from hyperstimulated follicles retained their mitogenic activity; (ii) IGF-I is capable of stimulating DNA amplification in granulosa cells; and (iii) IGFBP-1 inhibits the IGF-I stimulated proliferation in these cells. In view of our previous studies showing that IGFBP-1 is synthesized by the granulosa cells as they luteinize, the present results suggest that IGFBP-1 is one of the endogenous factors locally regulating the growth and differentiation of granulosa cells.     

Heparin-binding epidermal growth factor-like growth factor suppresses experimental liver fibrosis in mice

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cytoprotective agent in several organ systems but its roles in liver fibrosis are unclear. We studied the roles of HB-EGF in experimental liver fibrosis in mice and during hepatic stellate cell (HSC) activation. Thioacetamide (TAA; 100 mg/kg) was administered by intraperitoneal injection three times a week for 4 weeks to wild-type HB-EGF(+/+) or HB-EGF-null (HB-EGF(-/-)) male mice. Livers were examined for histology and expression of key fibrotic markers. Primary cultured HSCs isolated from untreated HB-EGF(+/+) or HB-EGF(-/-) mice were examined for fibrotic markers and/or cell migration either during culture-induced activation or after exogenous HB-EGF (100 ng/ml) treatment. TAA induced liver fibrosis in both HB-EGF(+/+) and HB-EGF(-/-) mice. Hepatic HB-EGF expression was decreased in TAA-treated HB-EGF(+/+) mice by 37.6% (P<0.05) as compared with animals receiving saline alone. HB-EGF(-/-) mice treated with TAA showed increased hepatic α-smooth muscle actin-positive cells and collagen deposition, and, as compared with HB-EGF(+/+) mice, TAA-stimulated hepatic mRNA levels in HB-EGF(-/-) mice were, respectively, 2.1-, 1.7-, 1.8-, 2.2-, 1.2- or 3.3-fold greater for α-smooth muscle actin, α1 chain of collagen I or III (COL1A1 or COL3A1), transforming growth factor-β1, connective tissue growth factor or tissue inhibitor of metalloproteinase-1 (P<0.05). HB-EGF expression was detectable in primary cultured HSCs from HB-EGF(+/+) mice. Both endogenous and exogenous HB-EGF inhibited HSC activation in primary culture, and HB-EGF enhanced HSC migration. These findings suggest that HB-EGF gene knockout in mice increases susceptibility to chronic TAA-induced hepatic fibrosis and that HB-EGF expression or action is associated with suppression of fibrogenic pathways in HSCs.     

The expression of Abl interactor 2 in leiomyoma and myometrium and regulation by GnRH analogue and transforming growth factor-beta

BACKGROUND: Abelson (Abl) interactor 2 (Abi-2) has been considered as a key regulator of cell/tissue structural organization and is differentially expressed in leiomyomas. The objective of this study was to evaluate the expression of Abi-2 in leiomyoma/myometrium during the menstrual cycle and following GnRH analogue (GnRHa) therapy, as well as regulation by transforming growth factor (TGF)-beta1 in leiomyoma and myometrial smooth muscle cells (LSMC and MSMC). METHODS: We used real-time PCR, Western blotting and immunohistochemistry to determine the expression of Abi-2 in paired leiomyoma and myometrium (n = 27) from proliferative (n = 8) and secretory (n = 12) phases of the menstrual cycle and from patients who received GnRHa therapy (n = 7). Time-dependent action of TGF-beta1 (2.5 ng/ml) and GnRHa (0.1 microM) on Abi-2 expression was determined in LSMC and MSMC. RESULTS: Leiomyomas express elevated levels of Abi-2 as compared with myometrium from the proliferative but not the secretory phase of the menstrual cycle, with a significant reduction following GnRHa therapy (P < 0.05). Western blotting showed a similar trend in Abi-2 protein expression in leiomyoma/myometrial tissue extracts, which was immunolocalized in LSMC and MSMC, connective tissue fibroblasts and arterial walls. The expression of Abi-2 in LSMC and MSMC was increased by TGF-beta1 (2.5 ng/ml) and was inhibited by GnRHa (0.1 microM) in a time- and cell-dependent manner, and pretreatment with Smad3 SiRNA and U0126, an MEK-1/2 inhibitor, respectively, reversed their actions. CONCLUSION: Based on the menstrual cycle-dependent expression, the influence of GnRHa therapy, and regulation by TGF-beta in LSMC/MSMC, we conclude that Abi-2 may have a key regulatory function in leiomyomas cellular/tissue structural organization during growth and regression.     

Effect of heparin-binding EGF-like growth factor and amphiregulin on the MAP kinase-induced production of vascular endothelial growth factor by human granulosa cells

The function of granulosa cells is regulated by various hormones and growth factors. Our aim is to clarify the regulation of vascular endothelial growth factor (VEGF) production induced by heparin-binding EGF-like growth factor (HB-EGF) and amphiregulin (AR) in a human granulosa cell line, KGN. KGN cells were cultured and incubated for 24 h with HB-EGF and AR. The levels of VEGF in the culture media were measured by an enzyme-linked immunosorbent assay. The activation of MAP kinase in KGN cells was detected by Western blot analysis. VEGF production was significantly increased by HB-EGF or AR alone in a dose-dependent manner, whereas it was decreased by AG1478 or U0126. The MAP kinase activity was increased by treatment with HB-EGF or AR. The results suggested that VEGF is induced by HB-EGF and AR through mechanisms involving MAP kinase. The increase in VEGF may contribute to neovascularization, which in turn would promote various ovulation phenomena as well as follicular growth.     

Human albumin enhances expression of vascular endothelial growth factor in cultured human luteinizing granulosa cells: importance in ovarian hyperstimulation syndrome.

Ovarian hyperstimulation syndrome (OHSS) is a severe complication of ovarian stimulation for assisted reproductive techniques. Clinical manifestations are massive extravascular fluid accumulation and haemoconcentration. Vascular endothelial growth factor (VEGF) has been demonstrated to mediate the development of OHSS. Intravenous albumin at the time of oocyte aspiration has been suggested as an effective prophylactic treatment against the occurrence of severe OHSS. Here it is reported that in cultured human luteinizing granulosa cells, VEGF mRNA expression was enhanced by human albumin and maximum expression was observed in cultured granulosa cells obtained from patients with serum oestradiol concentrations >2000 pg/ml on the day of human chorionic gonadotrophin injection (P < 0. 05).     

Hepatocyte growth factor concentrations are elevated in peritoneal fluid of women with endometriosis.

The concentrations of hepatocyte growth factor (HGF) in peritoneal fluid (PF) from women with endometriosis (n = 36) and without endometriosis (n = 40) were measured. All of the PF samples examined contained detectable concentrations of HGF. The HGF concentrations in PF from women with stage III/IV endometriosis (0.906 ng/ml, 0. 561-1.185; median, interquartile range) were significantly higher (P < 0.0001) than those from women without endometriosis (0.315 ng/ml, 0.251-0.472). The HGF concentrations from women with stage I/II endometriosis (0.417 ng/ml, 0.310-1.023) appeared to be intermediate. There were no apparent variations detected among the HGF concentrations in women in the follicular or luteal phases regardless of the presence of endometriosis. Interestingly, HGF concentrations in PF from women on gonadotrophin releasing hormone analogues, independent of the presence of endometriosis, were comparable with those from untreated women. Given the known mitogenic property of HGF in human endometrial cells, these results suggest that HGF might play a role in the progression of endometriosis.     

碱性成纤维细胞生长因子及胰岛素样生长因子1对精原干细胞增殖凋亡影响的机制

文题释义：碱性成纤维细胞生长因子：是细胞生长和分化的重要调节因子,具有促血管生成、细胞增殖、细胞趋化、细胞迁移等活性,在细胞分化和机体发育过程中发挥重要作用。碱性成纤维细胞生长因子通过与细胞膜表面的特异性配体结合,进而引发细胞内的一系列级联反应,从而产生各种生物学效应。 胰岛素样生长因子1：是多功能细胞增殖调控因子,主要分布在肝脏中,其与胰岛素样生长因子2、胰岛素及其受体一起构成了胰岛素样生长因子家族,其对细胞生长和代谢具有多效作用。 背景：生长因子作为体外细胞培养和体内细胞生长及增殖必需的调节因子,一直被广泛的关注。 目的：探讨碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)与胰岛素样生长因子1(insulin-like growth facter,IGF-1)联合作用对小鼠精原干细胞增殖、凋亡的影响。 方法：从6-8 d龄昆明雄性小鼠睾丸内分离培养精原干细胞并进行鉴定。将精原干细胞接种于经丝裂霉素C处理过的胚胎成纤维细胞饲养层上,分组干预：对照组加入正常DMEM培养基进行培养;bFGF、IGF-1组分别加入含20 μg/L bFGF、20 μg/L IGF-1的DMEM培养基进行培养;bFGF+IGF-1组同时加入含20 μg/L bFGF及20 μg/L IGF-1的DMEM培养基进行培养。采用CCK-8、EDU染色法分别检测精原干细胞增殖活性,流式细胞仪检测精原干细胞生长周期和细胞凋亡情况,Western blot检测增殖和凋亡相关蛋白PCNA、Bax、Bcl-2的表达。 结果与结论：①与对照组比较,bFGF组、IGF-1组、bFGF+IGF-1组吸光度值显著升高,与bFGF组、IGF-1组比较,bFGF+IGF-1组吸光度值进一步升高(P < 0.05),EDU染色得到与CCK-8实验一致的结论;②bFGF+IGF-1组S+G₂/M期细胞比例明显高于其他3组(P < 0.05),IGF-1组、bFGF组S+G₂/M期细胞比例高于对照组(P < 0.05);③与对照组比较,bFGF组、IGF-1组、bFGF+IGF-1组凋亡细胞降低;与bFGF组、IGF-1组比较,bFGF+IGF-1组凋亡细胞进一步降低;④与对照组比较,bFGF组、IGF-1组、bFGF+IGF-1组细胞中Bax蛋白相对表达水平显著下降(P < 0.01),Bcl-2和PCNA蛋白相对表达水平均显著升高(P < 0.05)。与bFGF组、IGF-1组比较,bFGF+IGF-1组细胞中Bax蛋白相对表达水平进一步下降(P < 0.01),Bcl-2和PCNA蛋白相对表达水平进一步升高(P < 0.05);⑤结果表明,bFGF、IGF-1通过上调PCNA和Bcl-2蛋白的表达,下调Bax蛋白的表达,促进细胞增殖,抑制细胞凋亡,二者联合作用效果最佳。 ORCID: 0000-0001-5693-3713(李宏) 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

The influence of epidermal growth factor on progesterone production by human granulosa--luteal cells in culture

Epidermal growth factor (EGF) stimulates progesterone productionby human granulosa—luteal cells in culture. The presentstudy investigated some of the parameters that affect the magnitudeof human granulosa—luteal cells' response to EGF. Cellsfrom pre-ovulatory follicles obtained 36 h post-human chorionicgonadotrophin (HCG) were cultured for 12 days with or withoutEGF (20 ng/ml). Medium was changed every 48 h and assayed forprogesterone by radio-immunoassay. DNA content of the culturedcells was determined fluorometrically. EGF was added every otherday to the culture medium, starting on either day 4, 6 or 8of culture, up to day 10, and compared with controls. When EGFwas initiated on day 4, the medium had significantly higherprogesterone concentration than control samples on days 6, 8,10 and 12 of culture (P < 0.01). When EGF was withheld untilday 6 or 8, progesterone concentrations were not significantlyhigher than control values. When EGF was added on day 4 anddiscontinued on day 8 or 10, progesterone concentrations werereduced significantly (P < 0.001) compared with the groupwhere EGF was added continuously from day 4 to 10. These datasuggest that: (i) human granulosa—luteal cells requirethe early exposure and continuous presence of EGF for the stimulatoryeffect on progesterone secretion, (ii) cells not exposed initiallyto EGF do not respond in a similar way, (iii) EGF is capableof maintaining progesterone production for a period > 12days. Therefore, normal luteal function may require the earlyand continuous presence of EGF.     

Fibromodulin is expressed in leiomyoma and myometrium and regulated by gonadotropin-releasing hormone analogue therapy and TGF-beta through Smad and MAPK-mediated signalling

Microarray gene expression profiling revealed fibromodulin (FMOD) is among differentially expressed genes in leiomyoma (L) and myometrium. Using realtime PCR, western blotting and immunohistochemistry, we validated the expression of FMOD in paired leiomyoma and myometrium (N = 20) during the menstrual cycle, from women who received gonadotropin-releasing hormone analogue (GnRHa) therapy (N = 7) and in leiomyoma and myometrial (M) smooth muscle cells (SMC) due to transforming growth factor (TGF)-beta and GnRHa treatment. The results indicated that FMOD is expressed at significantly higher levels in leiomyoma as compared to myometrium from proliferative phase (two- to three-folds; P < 0.05), but not the secretory phase of the menstrual cycle, whereas GnRHa therapy reduced FMOD expression to levels detected in myometrium from proliferative phase (P = 0.05). By using western blotting and immunohistochemistry immunoreactive FMOD was detected in leiomyoma and myometrial tissue-extract and in LSMC and MSMC, connective tissue fibroblasts and arterial walls. In a time- and cell-dependent manner, TGF-beta1 (2.5 ng/ml) increased the expression of FMOD in MSMC, whereas GnRHa (0.1 microM) inhibited that in MSMC and LSMC (P < 0.05). The effect of TGF-beta and GnRHa on FMOD expression was reversed following pretreatment of LSMC and MSMC with Smad3 SiRNA and U0126 (MEK1/2 inhibitor), respectively. In summary, menstrual cycle-dependent expression of FMOD and suppression following GnRHa therapy in leiomyoma and myometrium, as well as differential regulation by TGF-beta and GnRHa in vitro suggests that FMOD, a key regulator of tissue organization, plays a critical role in leiomyoma fibrotic characteristics.     

The effects of progesterone on apoptosis in the human trophoblast-derived HTR-8/SV neo cells

Progesterone (P4) is frequently used in the treatment of threatened abortion, prevention of recurrent miscarriage and threatened preterm labor. However, little is known about the molecular mechanism of P4 in the regulation of extravillous trophoblasts' (EVTs) function. This study was designed to examine the presence of progesterone receptor (PR) in the human trophoblast-derived HTR-8/SV neo cell line, which is a possible model of EVTs, and the effects of P4 on apoptosis in those cells. The HTR-8/SV neo cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 microg/ml streptomycin. When the cell the population reached 50% confluency, the cells were stepped down to serum-free conditions in the presence or absence of graded concentrations of P4 (1, 10 and 100 ng/ml) for 48 h. The cultured cells were used for RT-PCR, terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) assay, immunocytochemistry and western blot analyses. Immunocytochemistry and western blot analyses revealed that PR was evident in HTR-8/SV neo cells. Compared with untreated cultures, treatment with P4 (10 and 100 ng/ml) resulted in significant decreases in the TUNEL-positive rate, Fas, Fas ligand (Fas-L), caspase-8, caspase-3 and poly (ADP-ribose) polymerase (PARP) expression in HTR-8/SV neo cells, and a significant increase in Bcl-2 expression in those cells. Consistently, Fas mRNA expression in those cells was significantly inhibited by the treatment with 10 ng/ml P4 compared with untreated cultures. This study suggests that PR exists in HTR-8/SV neo cells and that P4 inhibits apoptosis by down-regulating Fas, Fas-L, caspase-8, caspase-3 and PARP expression as well as up-regulating Bcl-2 expression in HTR-8/SV neo cells.