2-Methoxyestradiol causes functional repression of transforming growth factor ß3 signaling by ameliorating Smad and non-Smad signaling pathways in immortalized uterine fibroid cells.

OBJECTIVE: To investigate the effects and the mechanism of action of 2-methoxyestradiol (2ME(2)) on transforming growth factor (TGF) ß3-induced profibrotic response in immortalized human uterine fibroid smooth muscle (huLM) cells. DESIGN: Laboratory study. SETTING: University research laboratory. PATIENTS(S): Not applicable. INTERVENTIONS(S): Not applicable. MAIN OUTCOME MEASURE(S): huLM cells were treated with TGF-ß3 (5 ηg/mL) in the presence or absence of specific Smad3 inhibitor SIS3 (1 µmol/L), inhibitor of the PI3K/Akt (LY294002, 10 µmol/L), or 2ME(2) (0.5 µmol/L), and the expression of collagen (Col) type I(αI), Col III(αI), plasminogen activator inhibitor (PAI) 1, connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA) were determined by real-time reverse-transcription polymerase chain reaction and immunoblotting. The effect of 2ME(2) on Smad-microtubule binding was evaluated by coimmunoprecipitation. RESULT(S): Our data revealed that TGF-ß3-induced fibrogenic response in huLM is mediated by both Smad-dependent and Smad-independent PI3K/Akt/mTOR signaling pathways. 2ME(2) abrogates TGF-ß3-induced expression of Col I(αI), Col III(αI), PAI-1, CTGF, and α-SMA. Molecularly, 2ME(2) ameliorates TGF-ß3-induced Smad2/3 phosphorylation and nuclear translocation. In addition, 2ME(2) inhibits TGF-ß3-induced activation of the PI3K/Akt/mTOR pathway. CONCLUSION(S): TGF-ß3-induced profibrotic response in fibroid cells is mediated by Smad-dependent and Smad-independent PI3K/Akt/mTOR pathways. 2ME(2) inhibits TGF-ß3 profibrotic effects in huLM cells by ameliorating both Smad-dependent and Smad-independent signaling pathways.

Folic acid rescue of ATRA-induced cleft palate by restoring the TGF-ß signal and inhibiting apoptosis.

BACKGROUND: Cleft palate is a frequent congenital malformation with a heterogeneous etiology, for which folic acid (FA) supplementation has a protective effect. To gain more insight into the molecular pathways affected by FA, TGF-ß signaling and apoptosis in mouse embryonic palatal mesenchymal (MEPM) cells of all-trans retinoic acid (ATRA)-induced cleft palate in organ culture were tested. METHODS: C57BL/6J mice embryonic palates were explanted on embryonic day 14 and cultured in DMEM/F12 medium with or without ATRA or FA for 72 h. The palatal fusion was examined by light microscopy. Immunohistochemistry was used to detect TGFß3/TGF receptor II and caspase 9 in MEPM cells. TUNEL was used to detect apoptosis. RESULTS: Similar to development in vivo, palatal development and fusion were normal in control medium. ATRA inhibited palatal development and induced cleft palate, which can be rescued by FA. A higher apoptosis rate and caspase-9 in MEPM cells were detected in the ATRA group than in the control or the ATRA+FA group. Compared with the control or the ATRA+FA group, ATRA had little effect on TGF-ß3 in MEPM cells but significantly inhibited TGF-ß receptor II. CONCLUSIONS: Folic acid can rescue the cultured palates to continue developing and fusing that were inhibited and resulted in cleft palate by ATRA. Apoptosis and TGFß signaling in MEPM cells were involved in folic acid rescued ATRA-induced cleft palate.

Transforming growth factor beta3 increases chondrocyte proliferation and decreases apoptosis in murine cricoid cartilage in vitro.

OBJECTIVE: To determine if the luminal epithelium and/or exogenous transforming growth factor beta (TGFbeta) affects growth of the cricoid. DESIGN: Subglottises from 20 neonatal mice were subdivided into four groups: A, five subglottises with luminal epithelium grown in basic medium; B, five epithelium-free subglottises in basic medium; C, five epithelium-free subglottises in basic medium with supplemental TGFbeta1, and D, five epithelium-free subglottises in basic medium with supplemental TGFbeta3. RESULTS: Groups A and D demonstrated the greatest luminal area expansion. Group A rings demonstrated statistically higher chondrocyte proliferation than Groups B and C and lesser amounts of luminal apoptosis. Groups B and C rings demonstrated the least amount of cell proliferation, and greater luminal apoptosis relative to Group A. Groups A and D rings had similar apoptotic and proliferative results. CONCLUSIONS: Luminal epithelium exerts influence over the cricoid by increasing chondrocyte proliferation and decreasing the relative proportion of luminal chondrocytes that undergo apoptosis. Exogenous TGFbeta3, not TGFbeta1, also increases chondrocyte proliferation within the cricoid and appears to influence apoptosis as well.