PGE2 also stimulated marked increases in phospho-p38MAPK in COCs and granulosa cells within 515 min (data not shown). In addition, they and other groups have reported that LH induces expression of EGF-like factors, Areg, Btc, and Ereg in granulosa cells of preovulatory follicles during the ovulation process (31, 32). Additive antitumor effects using onapristone in combination with tamoxifen or the pure antiestrogen ICI 164384 have been demonstrated in several experimental models [28]. Hormonal induction of luteinization and prostaglandin endoperoxide synthase-2 involves multiple cellular signaling pathways. Although EGF-like factor mRNA was induced at 4 h in Ptgs2+/ mice, levels of Areg and Ereg but not Btc were reduced significantly at 4 h in Ptgs2/ mice. Furthermore, whereas FSH or forskolin-mediated phosphorylation of p38MAPK in COCs (Fig. Schematic Showing Potential Paracrine and Autocrine Pathways by Which Expression of EGF-Like Factors, Ptgs2, and Other Genes Expressed Are Regulated in Granulosa Cells and COCs of Ovulating Follicles The proposed sequence is as follows: 1) LH binds to its cognate receptor localized to granulosa cells stimulating cAMP production as well as 2) p38MAPK phosphorylation and activation (52, 53 ). 
Mice lacking progesterone receptor exhibit reproductive abnormalities. 2B, af). Cite this article. 1993, 28: 29-39. Breast Cancer Res Treat. Induction of Areg, Btc, and Ereg mRNA in Murine COCs and Granulosa Cells Total RNA was isolated from COCs and granulosa cells harvested from ovaries of mice treated with eCG for 48 h to stimulate growth of preovulatory follicles (0 h) followed by hCG to stimulate ovulation. Because Pgr/ (PRKO) mice exhibit impaired ovulation, the expression patterns of Areg, Ereg, and Btc mRNA were analyzed in RNA samples prepared from granulosa cells of mice primed with eCG and hCG. Although, at this time AREG did not impact expression of immune cell-related (Pdcd1, Runx1, and Cd52) genes, it did exert a small but nonsignificant increase in matrix-related (Has2, Ptgs2, and Tnfaip6) genes. Article Because cumulus cells of mouse and rat possess few if any LH receptors (27), the molecular and biochemical mechanisms by which LH impacts COC expansion have remained ill defined. Additionally, the PR antagonist RU486 has been shown to suppress cumulus expansion when porcine COCs were cultured with FSH and LH (26). Conti et al. Gene expression profiles of cumulus cell oocyte complexes during ovulation reveal cumulus cells express neuronal and immune-related genes: does this expand their role in the ovulation process? In colon cancer cells, PGE2 activated Areg gene promoter activity by cAMP-dependent regulation of factors binding to a critical cAMP response element (CRE) and/or guanine:cytosine-rich Sp1 binding domains (39, 40). Molecular events defining follicular developments and steroidogenesis in the ovary. COCs were incubated O/N at 4 C with primary antibodies. New signaling pathways for hormones and cyclic adenosine 3,5-monophosphate action in endocrine cells. 6B) and granulosa cells (not shown) was blocked completely by SB203580, it was not blocked by the EGF receptor tyrosine kinase inhibitor AG. Ovaries of immature mice primed with eCG for 48 h contain multiple preovulatory follicles. Specific bands were quantified by densitometric analyses using a Molecular Dynamics Personal Densitometer. Expression of PGRA alone did not induce EGF-like factor mRNA, nor did it enhance the effect of forskolin. Huggins C: Endocrine-induced regression of cancers. Hyaluronan and proteoglycans in ovarian follicles. Expression levels of Areg and Btc declined progressively thereafter, whereas expression levels of Ereg mRNA persisted until 12 h but declined significantly in the ovulated complexes. When nonexpanded COCs were isolated and cultured with AREG for 4 and 16 h, AREG stimulated not only genes involved in induction of cumulus expansion (Ptgs2, Has2, and Tnfaip6) but also genes regulating steroidogenesis and steroid hormone action (StAR, Cyp11a1, and Pgr) as well as immune-like functions. EMBO J. Regulated expression of ADAMTS family members in follicles and cumulus oocyte complexes: evidence for specific and redundant functions. Thus, no significant differences in the expression of these genes was noted in COCs at 8 h (Fig. The semiquantitative RT-PCR data are represented mean sd. 3A). Impaired cumulus mucification an female sterility in tumor necrosis factor-induced protein-6 deficient mice. Article
Novel signaling pathways that control ovarian follicular development, follicular growth and ovulation. 8A). 2). Regulated Expression of Areg and Ereg mRNA by FSH and AREG in Cultured COCs Is Blocked by Inhibition of EGF Receptor Tyrosine Kinase Activity or PTGS2 Activity A, COCs were cultured with FSH or AREG as in panel A with or without inhibitors of EGF-receptor tyrosine kinase (AG; 10 m) and PTGS2 (NS; NS398, 10 m). In COCs, AREG not only induced genes that impact matrix formation but also genes involved in steroidogenesis (StAR, Cyp11a1) and immune cell-like functions (Pdcd1, Runx1, Cd52). On the other hand, induction of Ptgs2 expression by AREG was suppressed by the MEK1 inhibitor but not the p38MAPK inhibitor. Lanari, C., Molinolo, A.A. Progesterone receptors - animal models and cell signaling in breast cancer Diverse activation pathways for the progesterone receptor - possible implications for breast biology and cancer. A possible role for EGF or EGF-like factors in the ovulation process has been strengthened by recent observations showing that LH induces in mouse and rat preovulatory follicles the expression mRNAs encoding the EGF-like factors amphiregulin (AREG), epiregulin (EREG) (3133), and betacellulin (BTC) (33) in temporal and spatial patterns similar to those of Ptgs2 and Pgr (32, 33). Oligonucleotide primers for RT-PCRs were from Sigma-Genosys (Houston, TX). The molecular bridges that link the LH surge with functional changes in cumulus cells that possess few LH receptors are being unraveled. Expression pattern and cellular localization of two critical non-nuclear progesterone receptors in the ovine corpus luteum during the estrous cycle and early pregnancy. Thus, the p38MAPK and ERK1/2 pathways act synergistically and sequentially and are essential for cumulus cell functions, such as cumulus expansion during ovulation process. The other sites (Ser 102, Ser 294 and Ser 345) are hormone inducible, and 12 hours of treatment are required to reach maximal phosphorylation. Moreover, recently available clinical data demonstrate a higher risk of breast cancer in patients under hormone replacement therapy using a combination of estrogens and progestins, as compared with those using estrogens alone [2, 3]. TNFAIP6 binds to hyaluronan backbone and interacts with the serum-derived factor II heavy chain to stabilize matrix formation (19). The PR complex will bind a specific DNA sequence, the progesterone-responsive element, and will initiate the transcription of target genes. These data document that the rapid induction of Areg and Ereg mRNA and protein expression by hCG in vivo is dependent on expression of Ptgs2. Hyaluronate binding properties of versican. Lamb C, Simian M, Molinolo A, Pazos P, Lanari C: Regulation of cell growth of a progestin-dependent murine mammary carcinoma in vitro: progesterone receptor involvement in serum or growth factor-induced cell proliferation. Giangrande PH, McDonnell DP: The A and B isoforms of the human progesterone receptor: two functionally different transcription factors encoded by a single gene. A complete review of the classical mechanism by which PRs are activated by their natural ligands is beyond the scope of the present review, and has been extensively described elsewhere [19]. Lastly, ovulated COCs were isolated from oviducts of mice 16 h after hCG and cultured for an additional 8 h without or with AREG to determine whether these factors remain critical for stability of expanded COCs after ovulation. 1999, 59: 4276-4284. 10.1210/me.13.6.829. As with the estrogen receptor (ER) signaling pathway, blocking the progesterone/PR system, alone or in combination with other treatment modalities, has arisen as a logical therapeutic possibility in breast cancer [4]. More recently Park et al. However, in this study, we also provide the novel observation that mRNAs encoding these EGF-like factors are induced markedly in cumulus cells in vivo and in vitro.
1. In addition, the induction by FSH of mRNAs for Ptgs2 and Areg was dependent on p38MAPK and MEK1 activation but not PKA. The stimulatory effect of MPA could be mimicked by basic fibroblast growth factor [15], and these effects could be blocked by antiprogestins or by PR anti-sense oligonucleotides. These results suggested that FSH mediated induction of Areg mRNA precedes AREG induction of Ptgs2 and PGE2 production that, via its receptors in cumulus cells, provides an secondary, autocrine pathway to regulate expression of Areg in cumulus cells (Fig. Radiolabeled [32P]deoxy (d)-CTP was purchased from ICN (Los Angeles, CA). Specifically, they put forward the notion that LH induction of Areg, Ereg, and Btc in granulosa cells of PO follicles provides ligands that activate EGF receptors in cumulus cells leading to the induction of factors essential for COC expansion; namely, Has2, Ptgs2, and Tnfaip6 mRNA. Copyright 2006 by The Endocrine Society, Editorial: Final Musings on the Impact of Molecular Endocrinology, Editorial: Reflections on the Impact of Molecular Endocrinology on a Scientific Career, The Journal of Clinical Endocrinology & Metabolism, Receive exclusive offers and updates from Oxford Academic. J Biol Chem. No tumors were observed in untreated controls or in virgin female BALB/c mice of our colony. FSH stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-induced kinase (Sgk): evidence for A-kinase independent signaling in granulosa cells. 1993, 27: 41-56. 2001, 98: 8-10. These results indicated that cumulus cells, as well as granulosa cells, produce these EGF-like ligands, and therefore expression of these factors in cumulus cells might provide an autocrine mechanism to regulate cumulus cell function. Mifepristone demonstrated dose-dependent growth inhibitory effects in vitro in PR-positive human breast cancer cell lines, and its antiproliferative effects were evident even in the total absence of estrogens [26]. Lanari C, Molinolo AA, Pasqualini CD: Inhibitory effect of medroxyprogesterone acetate on foreign body tumorigenesis in mice. Lange CA, Richer JK, Horwitz KB: Hypothesis: progesterone primes breast cancer cells for cross-talk with proliferative or antiproliferative signals. 2002, 23: 749-757. 4 with or without inhibitors of PKA (KT; KT5720, 10 m), p38MAPK (SB203580, 20 m) or MEK1 (PD; PD98059, 20 m). Phosphorylation may not serve as a regulatory onoff switch for transcriptional activity, but rather functions to either amplify or attenuate activity [20]. Data from these gene profiling studies showed that mRNAs encoding the EGF-like factors Areg, Ereg, and Btc were induced markedly in intact COCs isolated 8 h after hCG in vivo (37). Activated PRs would recruit a series of important regulatory proteins, which can serve as coactivators or corepressors, such as SRC-1, SRC-2 and SRC-3, CBP/p300 and others. Thus, LH induction of PGE2- and progesterone-dependent pathways in granulosa cells mediate critical events during the ovulation process. Phosphorylation of p38MAPK by FSH in cumulus cells has also been documented and linked to expansion of porcine and murine COCs (20, 54). THE SURGE OF LH induces marked functional (endocrine, biochemical, and molecular) changes in the preovulatory follicle. The effects by which the progesterone/PR signaling pathway can be modulated are many, and the reviewed evidence from both experimental results and clinical trials clearly demonstrate that this is a promising strategic target for breast cancer therapy.
The immune-related genes, Pdcd1, Runx1, and Cd52, were selectively expressed in cumulus cells just before and after ovulation in vivo (37). Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP2. Conversely, AREG but not FSH induced Ptsg2 mRNA at 0.5 h with peak expression of Ptgs2 and Areg mRNAs at 4 h, processes blocked by the EGF receptor tyrosine kinase inhibitor AG1478 (AG), PD98059, and NS398. EGF-like growth factors as mediators of LH action in the ovulatory follicle. Aldaz CM, Liao QY, Paladugu A, Rehm S, Wang H: Allelotypic and cytogenetic characterization of chemically induced mouse mammary tumors: high frequency of chromosome 4 loss of heterozygosity at advanced stages of progression.
Mice lacking progesterone receptor exhibit reproductive abnormalities. 2B, af). Cite this article. 1993, 28: 29-39. Breast Cancer Res Treat. Induction of Areg, Btc, and Ereg mRNA in Murine COCs and Granulosa Cells Total RNA was isolated from COCs and granulosa cells harvested from ovaries of mice treated with eCG for 48 h to stimulate growth of preovulatory follicles (0 h) followed by hCG to stimulate ovulation. Because Pgr/ (PRKO) mice exhibit impaired ovulation, the expression patterns of Areg, Ereg, and Btc mRNA were analyzed in RNA samples prepared from granulosa cells of mice primed with eCG and hCG. Although, at this time AREG did not impact expression of immune cell-related (Pdcd1, Runx1, and Cd52) genes, it did exert a small but nonsignificant increase in matrix-related (Has2, Ptgs2, and Tnfaip6) genes. Article Because cumulus cells of mouse and rat possess few if any LH receptors (27), the molecular and biochemical mechanisms by which LH impacts COC expansion have remained ill defined. Additionally, the PR antagonist RU486 has been shown to suppress cumulus expansion when porcine COCs were cultured with FSH and LH (26). Conti et al. Gene expression profiles of cumulus cell oocyte complexes during ovulation reveal cumulus cells express neuronal and immune-related genes: does this expand their role in the ovulation process? In colon cancer cells, PGE2 activated Areg gene promoter activity by cAMP-dependent regulation of factors binding to a critical cAMP response element (CRE) and/or guanine:cytosine-rich Sp1 binding domains (39, 40). Molecular events defining follicular developments and steroidogenesis in the ovary. COCs were incubated O/N at 4 C with primary antibodies. New signaling pathways for hormones and cyclic adenosine 3,5-monophosphate action in endocrine cells. 6B) and granulosa cells (not shown) was blocked completely by SB203580, it was not blocked by the EGF receptor tyrosine kinase inhibitor AG. Ovaries of immature mice primed with eCG for 48 h contain multiple preovulatory follicles. Specific bands were quantified by densitometric analyses using a Molecular Dynamics Personal Densitometer. Expression of PGRA alone did not induce EGF-like factor mRNA, nor did it enhance the effect of forskolin. Huggins C: Endocrine-induced regression of cancers. Hyaluronan and proteoglycans in ovarian follicles. Expression levels of Areg and Btc declined progressively thereafter, whereas expression levels of Ereg mRNA persisted until 12 h but declined significantly in the ovulated complexes. When nonexpanded COCs were isolated and cultured with AREG for 4 and 16 h, AREG stimulated not only genes involved in induction of cumulus expansion (Ptgs2, Has2, and Tnfaip6) but also genes regulating steroidogenesis and steroid hormone action (StAR, Cyp11a1, and Pgr) as well as immune-like functions. EMBO J. Regulated expression of ADAMTS family members in follicles and cumulus oocyte complexes: evidence for specific and redundant functions. Thus, no significant differences in the expression of these genes was noted in COCs at 8 h (Fig. The semiquantitative RT-PCR data are represented mean sd. 3A). Impaired cumulus mucification an female sterility in tumor necrosis factor-induced protein-6 deficient mice. Article Novel signaling pathways that control ovarian follicular development, follicular growth and ovulation. 8A). 2). Regulated Expression of Areg and Ereg mRNA by FSH and AREG in Cultured COCs Is Blocked by Inhibition of EGF Receptor Tyrosine Kinase Activity or PTGS2 Activity A, COCs were cultured with FSH or AREG as in panel A with or without inhibitors of EGF-receptor tyrosine kinase (AG; 10 m) and PTGS2 (NS; NS398, 10 m). In COCs, AREG not only induced genes that impact matrix formation but also genes involved in steroidogenesis (StAR, Cyp11a1) and immune cell-like functions (Pdcd1, Runx1, Cd52). On the other hand, induction of Ptgs2 expression by AREG was suppressed by the MEK1 inhibitor but not the p38MAPK inhibitor. Lanari, C., Molinolo, A.A. Progesterone receptors - animal models and cell signaling in breast cancer Diverse activation pathways for the progesterone receptor - possible implications for breast biology and cancer. A possible role for EGF or EGF-like factors in the ovulation process has been strengthened by recent observations showing that LH induces in mouse and rat preovulatory follicles the expression mRNAs encoding the EGF-like factors amphiregulin (AREG), epiregulin (EREG) (3133), and betacellulin (BTC) (33) in temporal and spatial patterns similar to those of Ptgs2 and Pgr (32, 33). Oligonucleotide primers for RT-PCRs were from Sigma-Genosys (Houston, TX). The molecular bridges that link the LH surge with functional changes in cumulus cells that possess few LH receptors are being unraveled. Expression pattern and cellular localization of two critical non-nuclear progesterone receptors in the ovine corpus luteum during the estrous cycle and early pregnancy. Thus, the p38MAPK and ERK1/2 pathways act synergistically and sequentially and are essential for cumulus cell functions, such as cumulus expansion during ovulation process. The other sites (Ser 102, Ser 294 and Ser 345) are hormone inducible, and 12 hours of treatment are required to reach maximal phosphorylation. Moreover, recently available clinical data demonstrate a higher risk of breast cancer in patients under hormone replacement therapy using a combination of estrogens and progestins, as compared with those using estrogens alone [2, 3]. TNFAIP6 binds to hyaluronan backbone and interacts with the serum-derived factor II heavy chain to stabilize matrix formation (19). The PR complex will bind a specific DNA sequence, the progesterone-responsive element, and will initiate the transcription of target genes. These data document that the rapid induction of Areg and Ereg mRNA and protein expression by hCG in vivo is dependent on expression of Ptgs2. Hyaluronate binding properties of versican. Lamb C, Simian M, Molinolo A, Pazos P, Lanari C: Regulation of cell growth of a progestin-dependent murine mammary carcinoma in vitro: progesterone receptor involvement in serum or growth factor-induced cell proliferation. Giangrande PH, McDonnell DP: The A and B isoforms of the human progesterone receptor: two functionally different transcription factors encoded by a single gene. A complete review of the classical mechanism by which PRs are activated by their natural ligands is beyond the scope of the present review, and has been extensively described elsewhere [19]. Lastly, ovulated COCs were isolated from oviducts of mice 16 h after hCG and cultured for an additional 8 h without or with AREG to determine whether these factors remain critical for stability of expanded COCs after ovulation. 1999, 59: 4276-4284. 10.1210/me.13.6.829. As with the estrogen receptor (ER) signaling pathway, blocking the progesterone/PR system, alone or in combination with other treatment modalities, has arisen as a logical therapeutic possibility in breast cancer [4]. More recently Park et al. However, in this study, we also provide the novel observation that mRNAs encoding these EGF-like factors are induced markedly in cumulus cells in vivo and in vitro.
1. In addition, the induction by FSH of mRNAs for Ptgs2 and Areg was dependent on p38MAPK and MEK1 activation but not PKA. The stimulatory effect of MPA could be mimicked by basic fibroblast growth factor [15], and these effects could be blocked by antiprogestins or by PR anti-sense oligonucleotides. These results suggested that FSH mediated induction of Areg mRNA precedes AREG induction of Ptgs2 and PGE2 production that, via its receptors in cumulus cells, provides an secondary, autocrine pathway to regulate expression of Areg in cumulus cells (Fig. Radiolabeled [32P]deoxy (d)-CTP was purchased from ICN (Los Angeles, CA). Specifically, they put forward the notion that LH induction of Areg, Ereg, and Btc in granulosa cells of PO follicles provides ligands that activate EGF receptors in cumulus cells leading to the induction of factors essential for COC expansion; namely, Has2, Ptgs2, and Tnfaip6 mRNA. Copyright 2006 by The Endocrine Society, Editorial: Final Musings on the Impact of Molecular Endocrinology, Editorial: Reflections on the Impact of Molecular Endocrinology on a Scientific Career, The Journal of Clinical Endocrinology & Metabolism, Receive exclusive offers and updates from Oxford Academic. J Biol Chem. No tumors were observed in untreated controls or in virgin female BALB/c mice of our colony. FSH stimulates phosphorylation and activation of protein kinase B (PKB/Akt) and serum and glucocorticoid-induced kinase (Sgk): evidence for A-kinase independent signaling in granulosa cells. 1993, 27: 41-56. 2001, 98: 8-10. These results indicated that cumulus cells, as well as granulosa cells, produce these EGF-like ligands, and therefore expression of these factors in cumulus cells might provide an autocrine mechanism to regulate cumulus cell function. Mifepristone demonstrated dose-dependent growth inhibitory effects in vitro in PR-positive human breast cancer cell lines, and its antiproliferative effects were evident even in the total absence of estrogens [26]. Lanari C, Molinolo AA, Pasqualini CD: Inhibitory effect of medroxyprogesterone acetate on foreign body tumorigenesis in mice. Lange CA, Richer JK, Horwitz KB: Hypothesis: progesterone primes breast cancer cells for cross-talk with proliferative or antiproliferative signals. 2002, 23: 749-757. 4 with or without inhibitors of PKA (KT; KT5720, 10 m), p38MAPK (SB203580, 20 m) or MEK1 (PD; PD98059, 20 m). Phosphorylation may not serve as a regulatory onoff switch for transcriptional activity, but rather functions to either amplify or attenuate activity [20]. Data from these gene profiling studies showed that mRNAs encoding the EGF-like factors Areg, Ereg, and Btc were induced markedly in intact COCs isolated 8 h after hCG in vivo (37). Activated PRs would recruit a series of important regulatory proteins, which can serve as coactivators or corepressors, such as SRC-1, SRC-2 and SRC-3, CBP/p300 and others. Thus, LH induction of PGE2- and progesterone-dependent pathways in granulosa cells mediate critical events during the ovulation process. Phosphorylation of p38MAPK by FSH in cumulus cells has also been documented and linked to expansion of porcine and murine COCs (20, 54). THE SURGE OF LH induces marked functional (endocrine, biochemical, and molecular) changes in the preovulatory follicle. The effects by which the progesterone/PR signaling pathway can be modulated are many, and the reviewed evidence from both experimental results and clinical trials clearly demonstrate that this is a promising strategic target for breast cancer therapy.
The immune-related genes, Pdcd1, Runx1, and Cd52, were selectively expressed in cumulus cells just before and after ovulation in vivo (37). Abortive expansion of the cumulus and impaired fertility in mice lacking the prostaglandin E receptor subtype EP2. Conversely, AREG but not FSH induced Ptsg2 mRNA at 0.5 h with peak expression of Ptgs2 and Areg mRNAs at 4 h, processes blocked by the EGF receptor tyrosine kinase inhibitor AG1478 (AG), PD98059, and NS398. EGF-like growth factors as mediators of LH action in the ovulatory follicle. Aldaz CM, Liao QY, Paladugu A, Rehm S, Wang H: Allelotypic and cytogenetic characterization of chemically induced mouse mammary tumors: high frequency of chromosome 4 loss of heterozygosity at advanced stages of progression.