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Integrative genome-wide expression analysis bears evidence of estrogen receptor-independent transcription in heregulin-stimulated MCF-7 cells.

Nagashima T, Suzuki T, Kondo S, Kuroki Y, Takahashi K, Ide K, Yumoto N, Hasegawa A, Toyoda T, Kojima T, Konagaya A, Suzuki H, Hayashizaki Y, Sakaki Y, Hatakeyama M - PLoS ONE (2008)

Bottom Line: Through application of various threshold settings, 333 (326 up-regulated and 7 down-regulated) HRG-regulated genes were detected.However, with respect to enriched upstream TFs, 4 common TFs were identified in the 14 HRG-regulated (28.6%) and 13 E2-regulated (30.8%) gene groups.These results indicated that while E2 and HRG may induce common TFs, the regulatory mechanisms that govern HRG- and E2-induced gene expression differ.

View Article: PubMed Central - PubMed

Affiliation: Computational and Experimental Systems Biology Group, RIKEN Genomic Sciences Center, RIKEN Yokohama Institute, Yokohama, Japan.

ABSTRACT
Heregulin beta-1 (HRG) is an extracellular ligand that activates mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI3K)/Akt signaling pathways through ErbB receptors. MAPK and Akt have been shown to phosphorylate the estrogen receptor (ER) at Ser-118 and Ser-167, respectively, thereby mimicking the effects of estrogenic activity such as estrogen responsive element (ERE)-dependent transcription. In the current study, integrative analysis was performed using two tiling array platforms, comprising histone H3 lysine 9 (H3K9) acetylation and RNA mapping, together with array comparative genomic hybridization (CGH) analysis in an effort to identify HRG-regulated genes in ER-positive MCF-7 breast cancer cells. Through application of various threshold settings, 333 (326 up-regulated and 7 down-regulated) HRG-regulated genes were detected. Prediction of upstream transcription factors (TFs) and pathway analysis indicated that 21% of HRG-induced gene regulation may be controlled by the MAPK cascade, while only 0.6% of the gene expression is controlled by ERE. A comparison with previously reported estrogen (E2)-regulated gene expression data revealed that only 12 common genes were identified between the 333 HRG-regulated (3.6%) and 239 E2-regulated (5.0%) gene groups. However, with respect to enriched upstream TFs, 4 common TFs were identified in the 14 HRG-regulated (28.6%) and 13 E2-regulated (30.8%) gene groups. These results indicated that while E2 and HRG may induce common TFs, the regulatory mechanisms that govern HRG- and E2-induced gene expression differ.

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Estrogen receptor phosphorylation induced by HRG and E2.Phosphorylation levels of estrogen receptor alpha at Ser-118 and Ser-167 treated with HRG (10 nM) or E2 (10 nM) were measured at the designated time period. Three-independent western blots were performed for each ligand. Representative figures are shown on the left. Levels of phosphorylation were quantified using a densitometer and are shown graphically on the right.
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pone-0001803-g001: Estrogen receptor phosphorylation induced by HRG and E2.Phosphorylation levels of estrogen receptor alpha at Ser-118 and Ser-167 treated with HRG (10 nM) or E2 (10 nM) were measured at the designated time period. Three-independent western blots were performed for each ligand. Representative figures are shown on the left. Levels of phosphorylation were quantified using a densitometer and are shown graphically on the right.

Mentions: Modes of ER activation have been categorized largely on the basis of steroid (E2)-dependent and steroid-independent mechanisms. E2 binds directly to ER thereby facilitating ER dimerization and subsequent high affinity binding to ERE (referred to as steroid-dependent ER activation) [13]. Growth hormones such as epidermal growth factor (EGF) and insulin-like growth factor (IGF)-I have been shown to activate ER by phosphorylating ER at Ser-118 through activation of MAPK [6]. On the other hand, Akt and pp90rsk1 activated by EGF have been shown to phosphorylate ER at Ser-167 [7], [8], [14]. These steroid-independent activations of ER are also capable of leading to ER- and ERE-mediated transcription events. HRG is a growth hormone ligand that strongly activates MAPK and PI3K/Akt through ErbB receptor activation [1]–[4]. We first assessed an assay to confirm that HRG induces ER phosphorylation at Ser-118 and Ser-167 in MCF-7 cells in our culture condition. The results clearly indicated that HRG treatment immediately induced phosphorylation at both serine residues (Figure 1). These results are consistent with an earlier report showing HRG-dependent ER activation [15]. On the other hand, E2 induced weak phosphorylation at Ser-118 and no phosphorylation at Ser-167. We assumed that ER is strongly activated by HRG under the assay conditions used in this study.


Integrative genome-wide expression analysis bears evidence of estrogen receptor-independent transcription in heregulin-stimulated MCF-7 cells.

Nagashima T, Suzuki T, Kondo S, Kuroki Y, Takahashi K, Ide K, Yumoto N, Hasegawa A, Toyoda T, Kojima T, Konagaya A, Suzuki H, Hayashizaki Y, Sakaki Y, Hatakeyama M - PLoS ONE (2008)

Estrogen receptor phosphorylation induced by HRG and E2.Phosphorylation levels of estrogen receptor alpha at Ser-118 and Ser-167 treated with HRG (10 nM) or E2 (10 nM) were measured at the designated time period. Three-independent western blots were performed for each ligand. Representative figures are shown on the left. Levels of phosphorylation were quantified using a densitometer and are shown graphically on the right.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2266794&req=5

pone-0001803-g001: Estrogen receptor phosphorylation induced by HRG and E2.Phosphorylation levels of estrogen receptor alpha at Ser-118 and Ser-167 treated with HRG (10 nM) or E2 (10 nM) were measured at the designated time period. Three-independent western blots were performed for each ligand. Representative figures are shown on the left. Levels of phosphorylation were quantified using a densitometer and are shown graphically on the right.
Mentions: Modes of ER activation have been categorized largely on the basis of steroid (E2)-dependent and steroid-independent mechanisms. E2 binds directly to ER thereby facilitating ER dimerization and subsequent high affinity binding to ERE (referred to as steroid-dependent ER activation) [13]. Growth hormones such as epidermal growth factor (EGF) and insulin-like growth factor (IGF)-I have been shown to activate ER by phosphorylating ER at Ser-118 through activation of MAPK [6]. On the other hand, Akt and pp90rsk1 activated by EGF have been shown to phosphorylate ER at Ser-167 [7], [8], [14]. These steroid-independent activations of ER are also capable of leading to ER- and ERE-mediated transcription events. HRG is a growth hormone ligand that strongly activates MAPK and PI3K/Akt through ErbB receptor activation [1]–[4]. We first assessed an assay to confirm that HRG induces ER phosphorylation at Ser-118 and Ser-167 in MCF-7 cells in our culture condition. The results clearly indicated that HRG treatment immediately induced phosphorylation at both serine residues (Figure 1). These results are consistent with an earlier report showing HRG-dependent ER activation [15]. On the other hand, E2 induced weak phosphorylation at Ser-118 and no phosphorylation at Ser-167. We assumed that ER is strongly activated by HRG under the assay conditions used in this study.

Bottom Line: Through application of various threshold settings, 333 (326 up-regulated and 7 down-regulated) HRG-regulated genes were detected.However, with respect to enriched upstream TFs, 4 common TFs were identified in the 14 HRG-regulated (28.6%) and 13 E2-regulated (30.8%) gene groups.These results indicated that while E2 and HRG may induce common TFs, the regulatory mechanisms that govern HRG- and E2-induced gene expression differ.

View Article: PubMed Central - PubMed

Affiliation: Computational and Experimental Systems Biology Group, RIKEN Genomic Sciences Center, RIKEN Yokohama Institute, Yokohama, Japan.

ABSTRACT
Heregulin beta-1 (HRG) is an extracellular ligand that activates mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-OH kinase (PI3K)/Akt signaling pathways through ErbB receptors. MAPK and Akt have been shown to phosphorylate the estrogen receptor (ER) at Ser-118 and Ser-167, respectively, thereby mimicking the effects of estrogenic activity such as estrogen responsive element (ERE)-dependent transcription. In the current study, integrative analysis was performed using two tiling array platforms, comprising histone H3 lysine 9 (H3K9) acetylation and RNA mapping, together with array comparative genomic hybridization (CGH) analysis in an effort to identify HRG-regulated genes in ER-positive MCF-7 breast cancer cells. Through application of various threshold settings, 333 (326 up-regulated and 7 down-regulated) HRG-regulated genes were detected. Prediction of upstream transcription factors (TFs) and pathway analysis indicated that 21% of HRG-induced gene regulation may be controlled by the MAPK cascade, while only 0.6% of the gene expression is controlled by ERE. A comparison with previously reported estrogen (E2)-regulated gene expression data revealed that only 12 common genes were identified between the 333 HRG-regulated (3.6%) and 239 E2-regulated (5.0%) gene groups. However, with respect to enriched upstream TFs, 4 common TFs were identified in the 14 HRG-regulated (28.6%) and 13 E2-regulated (30.8%) gene groups. These results indicated that while E2 and HRG may induce common TFs, the regulatory mechanisms that govern HRG- and E2-induced gene expression differ.

Show MeSH
Related in: MedlinePlus