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Estrogen and phorbol esters regulate amphiregulin expression by two separate mechanisms in human breast cancer cell
I Martínez-Lacaci1, M Saceda, G D Plowman
1Vincent T. Lombardi Cancer Research Center, Georgetown University, Washington, D.C. 20007, USA.
Abstract:
The actions of 17 beta-estradiol (E2) and protein kinase C (PKC) appear to converge in the regulation of expression of certain growth modulatory genes, such as the growth factor amphiregulin (AR). AR is known to modulate cell growth by binding to the epidermal growth factor receptor. In the current report we established the mechanisms of the PKC-activating phorbol ester tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) and the steroid hormone E2 on the induction of AR expression in human breast carcinoma cell lines. TPA (100 nM) and E2 (1 nM) induce AR messenger RNA (mRNA) expression by 6- to 8-fold and 3- to 6-fold, respectively, in a time- and dose-dependent manner. In addition, immunoreactive AR protein is induced by both TPA and E2 by 6- to 8-fold and 2- to 4-fold, respectively. The PKC-modulating drugs, bryostatin and H-7, and antiestrogens (ICI 164,384 and 4-hydroxytamoxifen) interfere with AR induction by TPA and estrogen, respectively. The effects of TPA and E2 on the induction of AR mRNA were both closely associated with enhanced transcription of the AR gene. However, TPA had an additional effect at the posttranscriptional level by stabilizing the AR mRNA. The protein synthesis inhibitor, cycloheximide, prevented AR induction by TPA, suggesting that a component of the TPA induction of AR is indirect and dependent upon protein synthesis. Conversely, the E2 induction of AR transcription was found to be a direct response, independent of protein synthesis. The results presented herein thus demonstrate that TPA and E2 are able to stimulate AR gene transcription by two separate mechanisms.
Insights
17 beta-estradiol (E2) and protein kinase C (PKC) signaling pathways both increase amphiregulin (AR) expression in breast cancer cells. E2 directly enhances AR gene transcription, while TPA uses both transcriptional and post-transcriptional mechanisms.
Area of Science:
- Molecular Biology
- Cell Biology
- Endocrinology
Background:
- 17 beta-estradiol (E2) and protein kinase C (PKC) signaling pathways are crucial in regulating gene expression.
- Amphiregulin (AR), a growth factor, modulates cell growth via the epidermal growth factor receptor and is implicated in cancer progression.
- Understanding the interplay between E2, PKC, and AR is vital for cancer research.
Purpose of the Study:
- To elucidate the mechanisms by which the PKC-activating phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) and the steroid hormone E2 induce AR expression in human breast carcinoma cell lines.
- To investigate whether these inductions occur at the transcriptional or post-transcriptional level.
- To determine if the induction pathways are direct or indirect, involving protein synthesis.
Main Methods:
- Human breast carcinoma cell lines were treated with TPA and E2 at specific concentrations and time points.
- Messenger RNA (mRNA) and protein levels of AR were quantified using molecular biology techniques.
- The effects of PKC-modulating drugs, antiestrogens, and protein synthesis inhibitors were assessed.
Main Results:
- Both TPA and E2 significantly increased AR mRNA and protein expression in a time- and dose-dependent manner.
- TPA induced AR expression through enhanced gene transcription and mRNA stabilization, requiring protein synthesis.
- E2 induced AR gene transcription directly, independent of protein synthesis.
Conclusions:
- TPA and E2 stimulate AR gene transcription through distinct mechanisms.
- E2 acts directly on AR gene transcription, while TPA involves both transcriptional and post-transcriptional regulation, including protein synthesis.
- These findings highlight the complex regulation of AR by hormonal and signaling pathways in breast cancer cells.