Ming Ting Lee

Interplay of PI3K and cAMP/PKA signaling, and rapamycin-hypersensitivity in TGFbeta1 enhancement of FSH-stimulated steroidogenesis in rat ovarian granulosa cells.

Transforming growth factor (TGF) beta1 facilitates FSH-induced differentiation of rat ovarian granulosa cells. The signaling crosstalk between follicle stimulating hormone (FSH) and TGFbeta receptors remains unclear. This study was to investigate the interplay of cAMP/protein kinase A (PKA) and phosphatidylinositol-3-kinase (PI3K) signaling including mammalian target of rapamycin (mTOR)C1 dependence in FSH- and TGFbeta1-stimulated steroidogenesis in rat granulosa cells. To achieve this aim, inhibitors of PKA (PKAI), PI3K (wortmannin), and mTORC1 (rapamycin) were employed. PKAI and wortmannin suppressions of the FSH-increased progesterone production were partly attributed to decreased level of 3beta-HSD, and their suppression of the FSH plus TGFbeta1 effect was attributed to the reduction of all the three key players, steroidogenic acute regulatory (StAR) protein, P450scc, and 3beta-HSD. Further, FSH activated the PI3K pathway including increased integrin-linked kinase (ILK) activity and phosphorylation of Akt(S473), mTOR(S2481), S6K(T389), and transcription factors particularly FoxO1(S256) and FoxO3a(S253), which were reduced by wortmannin treatment but not by PKAI. Interestingly, PKAI suppression of FSH-induced phosphorylation of cAMP regulatory element-binding protein (CREB(S133)) disappeared in the presence of wortmannin, suggesting that wortmannin may affect intracellular compartmentalization of signaling molecule(s). In addition, TGFbeta1 had no effect on FSH-activated CREB and PI3K signaling mediators. We further found that rapamycin reduced the TGFbeta1-enhancing effect of FSH-stimulated steroidogenesis, yet it exhibited no effect on FSH action. Surprisingly, rapamycin displayed a suppressive effect at concentrations that had no effect on mTORC1 activity. Together, this study demonstrates a delicate interplay between cAMP/PKA and PI3K signaling in FSH and TGFbeta1 regulation of steroidogenesis in rat granulosa cells. Furthermore, we demonstrate for the first time that TGFbeta1 acts in a rapamycin-hypersensitive and mTORC1-independent manner in augmenting FSH-stimulated steroidogenesis in rat granulosa cells.

Inhibitory effects of a luteinizing hormone-releasing hormone agonist on basal and epidermal growth factor-induced cell proliferation and metastasis-associated properties in human epidermoid carcinoma a431 cells

The purpose of this study was to investigate the effects of a potent LHRH agonist, [D‐Trp6]LHRH on the basal and EGF‐induced cell proliferation and the metastasis‐associated properties in A431 human epidermoid carcinoma. [D‐Trp6]LHRH time‐dependently inhibited the basal and EGF‐stimulated growth of A431 cancer cells. It is assumed that phosphorylation/dephosphorylation of cellular proteins is highly related to cell growth. This study demonstrates that [D‐Trp6]LHRH decreased the basal and EGF‐induced total cellular kinase activity, particularly the tyrosine phosphorylation of several cellular proteins including the EGFR. In contrast, [D‐Trp6]LHRH did not cause detectable changes in basal and EGF‐stimulated serine/threonine phosphorylation of A431 cellular proteins. The inhibitory effect of [D‐Trp6]LHRH on A431 cell proliferation was associated with apoptosis as evidenced by the cell morphology and DNA integrity (ladder pattern), the expression of interleukin 1β‐converting enzyme (ICE) and activation of caspase. Furthermore, EGF could rescue the remaining attached A431 cells following [D‐Trp6]LHRH treatment for 48 hr, which suggests that limited exposure to [D‐Trp6]LHRH did not channel all cells to irreversible apoptotic process. We also determined the effects of [D‐Trp6]LHRH on metastasis‐associated properties in A431 cells. [D‐Trp6]LHRH reduced both basal and EGF‐stimulated secretion of MMP‐9 and MMP‐2. In addition, [D‐Trp6]LHRH suppressed the basal and EGF‐induced invasive activity of A431 cells based on an in vitro invasion assay. In conclusion, this study indicates that [D‐Trp6]LHRH may act partly through activating tyrosine phosphatase activity to inhibit cell proliferation and the metastasis‐associated properties of A431 cancer cells. Our work suggests that [D‐Trp6]LHRH may be therapeutically useful in limiting the tumor growth and metastasis of some neoplasms.

Crucial Role of Estrogen Receptor-α Interaction with Transcription Coregulators in Follicle-Stimulating Hormone and Transforming Growth Factor β1 Up-Regulation of Steroidogenesis in Rat Ovarian Granulosa Cells

This study was to explore estrogen receptor (ER) involvement in FSH and TGFbeta1-stimulated steroidogenesis in rat ovarian granulosa cells. We first determined the specific involvement of ERalpha and ERbeta in the process, and then investigated the molecular interaction of ERalpha and transcription coregulators in FSH and TGFbeta1 up-regulation of steroidogenic gene expression. Primary culture of ovarian granulosa cells from antral follicles of gonadotropin-primed immature rats was used. Interestingly, a selective ERalpha antagonist methyl-piperidino-pyrazole (MPP) [like ER antagonist ICI-182,780 (ICI)] decreased FSH +/- TGFbeta1-stimulated progesterone production, whereas an androgen receptor antagonist hydroxyflutamide and particularly a selective ERbeta antagonist 4-[2-Phenyl-5,7-bis(trifluoromethyl) pyrazolo [1,5-a] pyrimidin-3-yl] phenol had no significant effect. Consistent with this, a selective ERbeta agonist diarylpropionitrile (unlike 17beta-estradiol) also had no effect on FSH +/- TGFbeta1-stimulated progesterone production. Furthermore, a selective ERalpha agonist 4,4,4-(4-Propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol (like 17beta-estradiol) enhanced FSH-stimulated progesterone production, and this was abolished by pretreatment with MPP. Immunoblotting and chromatin immunoprecipitation analyses indicate that MPP/ICI suppression of FSH +/- TGFbeta1 action is partly attributed to the reduced ERalpha-mediated expression of Hsd3b and Cyp11a1 genes, but not steroidogenic acute regulatory protein. Furthermore, FSH +/- TGFbeta1 increased ERalpha association with histone acetylases (CBP and SRC-1) and coactivator of peroxisome proliferator-activated receptor gamma (PGC-1alpha), and MPP/ICI dramatically reduced these interactions. In addition, FSH +/- TGFbeta1 increased CBP, SRC-1, and PGC-1alpha binding to Hsd3b and Cyp11a1 genes. Together, we demonstrate for the first time that ERalpha interaction with transcription coregulators, histone acetylases (CBP/SRC-1), and PGC-1alpha is crucial to FSH and TGFbeta1-up-regulated expression of Hsd3b and Cyp11a1, and, thus, progesterone production in rat ovarian granulosa cells.

Role of tissue transglutaminase 2 in the acquisition of a mesenchymal-like phenotype in highly invasive A431 tumor cells

BackgroundCancer progression is closely linked to the epithelial-mesenchymal transition (EMT) process. Studies have shown that there is increased expression of tissue tranglutaminase (TG2) in advanced invasive cancer cells. TG2 catalyzes the covalent cross-linking of proteins, exhibits G protein activity, and has been implicated in the modulation of cell adhesion, migration, invasion and cancer metastasis. This study explores the molecular mechanisms associated with TG2s involvement in the acquisition of the mesenchymal phenotype using the highly invasive A431-III subline and its parental A431-P cells.ResultsThe A431-III tumor subline displays increased expression of TG2. This is accompanied by enhanced expression of the mesenchymal phenotype, and this expression is reversed by knockdown of endogenous TG2. Consistent with this, overexpression of TG2 in A431-P cells advanced the EMT process. Furthermore, TG2 induced the PI3K/Akt activation and GSK3β inactivation in A431 tumor cells and this increased Snail and MMP-9 expression resulting in higher cell motility. TG2 also upregulated NF-κB activity, which also enhanced Snail and MMP-9 expression resulting in greater cell motility; interestingly, this was associated with the formation of a TG2/NF-κB complex. TG2 facilitated acquisition of a mesenchymal phenotype, which was reversed by inhibitors of PI3K, GSK3 and NF-κB.ConclusionsThis study reveals that TG2 acts, at least in part, through activation of the PI3K/Akt and NF-κB signaling systems, which then induce the key mediators Snail and MMP-9 that facilitate the attainment of a mesenchymal phenotype. These findings support the possibility that TG2 is a promising target for cancer therapy.

Effects of dietary flavonoids, luteolin, and quercetin on the reversal of epithelial–mesenchymal transition in A431 epidermal cancer cells

Highly invasive A431‐III cells, which are derived from parental A431‐P cells, were originally isolated by three successive passages through a Boyden chamber using a Matrigel‐coated membrane support. The greater invasion potential shown by A431‐III cells was due to their increased ability to spread/migrate, which was associated with enhanced MMP activity. The tumor progression events evoked by A431‐P cells compared to A431‐III cells may help identify useful strategies for evaluating the epithelial–mesenchymal transition (EMT) and these cell lines could be a reliable model for evaluating tumor metastasis events. Using this approach, we evaluated the effects of luteolin and quercetin using the A431‐P/A431‐III EMT model. These flavonoids reversed cadherin switching, downregulated EMT markers, and nullified the invasion ability of A431‐III cells. Overexpression of MMP‐9 resulted in induction of the EMT in A431‐P cells and this could be reversed by treating with luteolin or quercetin. Cotreatment of A431‐P and A431‐III cells with epidermal growth factor (EGF) plus luteolin or quercetin resulted in a more epithelial‐like morphology, led to reduced levels of EGF‐induced markers of EMT, and caused the restoration of cell–cell junctions. E‐cadherin was decreased by EGF, but increased by luteolin and quercetin. Our results suggest that luteolin and quercetin are potentially beneficial agents that target and prevent the occurrence of EMT in epidermal carcinoma cells. These chemicals also have the ability to attenuate tumor progression in A431‐III cells. Luteolin and quercetin show inherent potential as chemopreventive/antineoplastic agents and do this by abating tumor progression through a reversal of EMT. (Cancer Sci 2011; 102: 1829–1839)

The Modulatory Role of Transforming Growth Factor β1 and Androstenedione on Follicle-Stimulating Hormone-Induced Gelatinase Secretion and Steroidogenesis in Rat Granulosa Cells

Abstract To investigate the potential roles of matrix metalloproteinases (MMPs) in ovarian granulosa cell differentiation, we studied the interactive effects of FSH and local ovarian factors, transforming growth factor β1 (TGFβ1) and androstenedione, on gelatinase secretion and progesterone production in rat ovarian granulosa cells. Granulosa cells of eCG-primed immature rats were treated once with various doses of FSH and TGFβ1 and androstenedione alone or in combinations for 2 days. Conditioned media were analyzed for gelatinase activity using gelatin-zymography/densitometry and progesterone levels using enzyme immunoassay. Cell lysates were analyzed for steroidogenic acute regulatory (StAR) and cholesterol side-chain-cleavage (P450scc) enzyme protein levels. This study demonstrates for the first time that FSH dose-dependently increased the secretion of a major 63-kDa gelatinase and minor 92- and 67-kDa gelatinases. TGFβ1 also dose-dependently increased the secretion of 63-kDa gelatinase, while androstenedione alone had no effect. The 92-kDa gelatinase was identified as the pro-MMP9 that could be cleaved by aminophenylmercuric acetate into the 83-kDa active form. Importantly, we show that TGFβ1 and androgen act in an additive manner to enhance FSH stimulatory effects both on the secretion of gelatinases and the production of progesterone. We further show by immunoblotting that the enhancing effect of TGFβ1 and androstenedione on FSH-stimulated steroidogenesis is partly mediated through the increased level of StAR protein and/or P450scc enzyme. In conclusion, this study indicates that, during antral follicle development, TGFβ1 and androgen act to enhance FSH promotion of granulosa cell differentiation and that the process may involve the interplay of modulating cell- to-matrix/cell-to-cell interaction and steroidogenic activity.

CREB coactivator CRTC2/TORC2 and its regulator calcineurin crucially mediate follicle‐stimulating hormone and transforming growth factor β1 upregulation of steroidogenesis

In vitro and in vivo studies implicate that follicle‐stimulating hormone (FSH) and transforming growth factor β1 (TGFβ1) play crucial physiological roles in regulating ovarian granulosa cell function essential to fertility control in females. FSH induces cAMP and calcium signaling, thereby activating transcription factor CREB to upregulate steroidogenic gene expression, and TGFβ1 greatly enhances FSH‐stimulated steroidogenesis. A CREB coactivator CRTC2/TORC2 was identified to function as a cAMP and calcium‐sensitive coincidence sensor. This led us to explore the role of CRTC2 and its regulator calcineurin in FSH and TGFβ1‐stimulated steroidogenesis. Primary culture of granulosa cells from gonadotropin‐primed immature rats was used. Immunoblotting analysis shows that FSH rapidly and transiently induced dephosphorylation/activation of CRTC2, and FSHu2009+u2009TGFβ1 additionally induced late‐phase CRTC2 dephosphorylation. Immunofluorescence analysis further confirms FSHu2009±u2009TGFβ1 promoted CRTC2 nuclear translocation. Using selective inhibitors, we demonstrate that FSH activated CRTC2 in a PKA‐ and calcineurin‐dependent manner, and TGFβ1 acting through its type I receptor (TGFβRI)‐modulated FSH action in a calcineurin‐mediated and PKA‐independent fashion. Next, we investigated the involvement of calcineurin and CRTC2 in FSH and TGFβ1‐stimulated steroidogenesis. Calcineurin and TGFβRI inhibitor dramatically reduced the FSHu2009±u2009TGFβ1‐increased progesterone synthesis and protein levels of StAR, P450scc, and 3β‐HSD enzyme. Furthermore, chromatin‐immunoprecipitation and immunoprecipitation analyses demonstrate that FSHu2009±u2009TGFβ1 differentially increased CRTC2, CREB, and CBP binding to these steroidogenic genes, and CREB nuclear association with CRTC2 and CBP. In all, this study reveals for the first time that CRTC2 and calcineurin are critical signaling mediators in FSH and TGFβ1‐stimulated steroidogenesis in ovarian granulosa cells. J. Cell. Physiol. 227: 2430–2440, 2012.

Phosphorylation of the Zebrafish M6Ab at Serine 263 Contributes to Filopodium Formation in PC12 Cells and Neurite Outgrowth in Zebrafish Embryos

Background Mammalian M6A, a member of the proteolipid protein (PLP/DM20) family expressed in neurons, was first isolated by expression cloning with a monoclonal antibody. Overexpression of M6A was shown to induce filopodium formation in neuronal cells; however, the underlying mechanism of is largely unknown. Possibly due to gene duplication, there are two M6A paralogs, M6Aa and M6Ab, in the zebrafish genome. In the present study, we used the zebrafish as a model system to investigate the role of zebrafish M6Ab in filopodium formation in PC12 cells and neurite outgrowth in zebrafish embryos. Methodology/Principal Findings We demonstrated that zebrafish M6Ab promoted extensive filopodium formation in NGF-treated PC12 cells, which is similar to the function of mammalian M6A. Phosphorylation at serine 263 of zebrafish M6Ab contributed to this induction. Transfection of the S263A mutant protein greatly reduced filopodium formation in PC12 cells. In zebrafish embryos, only S263D could induce neurite outgrowth. Conclusions/Significance Our results reveal that the phosphorylation status of zebrafish M6Ab at serine 263 is critical for its role in regulating filopodium formation and neurite outgrowth.

Impact of Flavonoids on Matrix Metalloproteinase Secretion and Invadopodia Formation in Highly Invasive A431-III Cancer Cells

Metastasis is a major cause of mortality in cancer patients. Invadopodia are considered to be crucial structures that allow cancer cells to penetrate across the extracellular matrix (ECM) by using matrix metalloproteinases (MMPs). Previously, we isolated a highly invasive A431-III subline from parental A431 cells by Boyden chamber assay. The A431-III cells possess higher invasive and migratory abilities, elevated levels of MMP-9 and an enhanced epithelial-mesenchymal transition (EMT) phenotype. In this study, we discovered that A431-III cells had an increased potential to form invadopodia and an improved capacity to degrade ECM compared with the original A431 cells. We also observed enhanced phosphorylation levels of cortactin and Src in A431-III cells; these phosphorylated proteins have been reported to be the main regulators of invadopodia formation. Flavonoids, almost ubiquitously distributed in food plants and plant food products, have been documented to exhibit anti-tumor properties. Therefore, it was of much interest to explore the effects of flavonoid antioxidants on the metastatic activity of A431-III cells. Exposure of A431-III cells to two potent dietary flavonoids, namely luteolin (Lu) and quercetin (Qu), caused inhibition of invadopodia formation and decrement in ECM degradation. We conclude that Lu and Qu attenuate the phosphorylation of cortactin and Src in A431-III cells. As a consequence, there ensues a disruption of invadopodia generation and the suppression of MMP secretion. These changes, in concert, bring about a reduction in metastasis.