The role and mechanism of progesterone receptor activation of extra-nuclear signaling pathways in regulating gene transcription and cell cycle progression

doi:10.1016/j.steroids.2008.01.010

Steroids

Volume 73, Issues 9–10, October 2008, Pages 922-928

https://doi.org/10.1016/j.steroids.2008.01.010 Get rights and content

Abstract

Human progesterone receptor (PR) contains a polyproline motif in the amino-terminal domain that interacts with the SH3 domain of Src and mediates rapid activation of c-Src and downstream MAPK (Erk-1/-2) independent of the transcriptional activity of PR. Forcedly target PR to different locations in the cell by use of mutations or tags for different cell compartments showed that progestin activation of Src/MAPK is mediated by PR outside the nucleus. No distinction could be made between the cytoplasm and cell membrane as the site of PR activation of Src. Therefore we can only conclude that this is an extra-nuclear action of PR. Interestingly, the B isoform of PR which is naturally distributed between cytoplasm and nucleus mediated progestin activation of Src/MAPK, whereas PR-A that is predominantly nuclear failed to do so indicating that the two PR isoforms have distinct abilities to mediate rapid activation of signaling pathways. Due to distinct cellular locations, progestin activation of Src/MAPK signaling can regulate selected target genes such as cyclin D1 (CCND1) that lack direct PR binding response elements (PREs). Progestin induction of CCND1 was observed in cells expressing PR-B but not PR-BΔSH3 or PR-A and induction in the presence of PR-B was dramatically reduced in the presence of inhibitors of Src or MAPK. In contrast progestin induction of Sgk (serum and glucocorticoid regulated kinase) gene, which contains a classical PRE, was observed with both PR isoforms as well as PR-BΔSH3 and was unaffected by Src and MAPK inhibitors. PR bound to enhancer region of Sgk in a progestin dependent manner as detected by chromatin co-immunoprecipitation (ChIP) whereas no PR binding to CCDN1 was observed. Consistent with CCND1 data, progestin stimulation of cell cycle progression was only observed in cells expressing PR-B but not cells expressing PR-BΔSH3 or PR-A. These results demonstrate the importance of PR activation of extra-nuclear signaling pathways in regulating selected target genes and cell cycle progression.

Section snippets

Extra-nuclear functions of human progesterone receptor

In addition to direct transcriptional effects mediated by nuclear PR, we and others have shown that progestins can rapidly activate the Src/Ras/MAPK, PI3 kinase/Akt and JAK2/Stat3 signaling pathway in breast cancer and mammary epithelial cells [3], [8], [9], [10], [11], [12], [13], [14], [15]. These effects of progestins on cell signaling pathways in the absence of transcription are dependent on conventional PR, suggesting PR has dual functions as a nuclear transcription factor and as a

Progestin activation of Src is an extra-nuclear function of PR

Although PR-A and -B have the identical SH3 domain interaction motif in their NTDs and both isoforms are capable of efficiently binding and activating Src kinases in cell-free assay [16], [17], PR-A was unable to mediate progestin-induced activation of Src and downstream MAPK in cells [16]. Analysis of intracellular localization of PR-A and PR-B in different cell types and conditions revealed that PR-B was distributed between the cytoplasm and nucleus, whereas PR-A was predominantly nuclear [16]

Progestin activation of Src is sufficient to induce activation of a MAPK dependent, Elk-1 transcription factor

Since MAPK is capable of phosphorylating and activating nuclear transcription factors raised the possibility that PR activation of Src/MAPK pathway may provide alternative means for progesterone regulation of gene transcription, independent of the direct nuclear transcription activity of PR. To test this possibility, we determined the effect of progestin on activation of a MAPK target, the Ets family transcription factor, Elk-1. Using an Elk reporter system, consisted of the C-terminal

Progestin induction of endogenous cyclin D1 gene and cell cycle progression is dependent on the SH3-domain binding motif of PR

To further determine the contribution of signaling pathways activated by PR extra-nuclear function on endogenous target genes, two known PR targets, cyclin D1 (CCND1) and Sgk (serum glucocorticoid regulated kinase) were analyzed in T47D breast cancer cells. CCND1 lacks a progesterone response element (PRE) [18], [19] while Sgk contains a characterized PRE/GRE ∼1100 bp upstream of the transcription start site [20]. In cells expressing PR-B, progestin-induced a 4–5 fold induction of CCND1

Discussion

An important issue that remains unresolved with regard to rapid signaling mediated by the classical steroid receptors is the intra-cellular site of receptor interaction with signaling molecules. Studies have shown localization of a subpopulation of steroid receptors with the plasma membrane either by biochemical fractionation or immunocytochemistry [22], [23], [24], [25], [26]. In fact, isolated cell membranes of endothelial cells that contain ER are able to support rapid estrogen activation of

References (41)

D. Mangelsdorf et al.
The nuclear receptor superfamily: the second decade
Cell
(1995)
X. Li et al.
Unfolding the action of progesterone receptor
J Biol Chem
(2003)
V. Boonyaratanakornkit et al.
Progesterone receptor contains a proline-rich motif that directly interacts with SH3 domains and activates c-Src family tyrosine kinases
Mol Cell
(2001)
E.R. Levin
Cellular functions of plasma membrane estrogen receptors
Steroids
(2002)
A. Pedram et al.
A conserved mechanism for steroid receptor translocation to the plasma membrane
J Biol Chem
(2007)
S. Schuh et al.
A 90,000-dalton binding protein common to both steroid receptors and the Rous sarcoma virus transforming protein, pp60v-src
J Biol Chem
(1985)
E. Sandilands et al.
RhoB and actin polymerization coordinate Src activation with endosome-mediated delivery to the membrane
Dev Cell
(2004)
T. Hitosugi et al.
Epidermal growth factor directs sex-specific steroid signaling through Src activation
J Biol Chem
(2007)
N.L. Weigel et al.
Phosphorylation and progesterone receptor function
J Steroid Biochem Mol Biol
(1995)
D.P. Edwards
Regulation of signal transduction pathways by estrogen and progesterone
Annu Rev Physiol
(2005)

P. Kastner et al.

Two distinct estrogen-regulated promoters generate transcripts encoding two functionally different human progesterone receptor forms A and B

EMBO J

(1990)

P.H. Giangrande et al.

The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding

Mol Cell Biol

(2000)

E. Vegeto et al.

Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function

Mol Endocrinol

(1993)

O.M. Conneely et al.

Progesterone receptors in mammary gland development and tumorigenesis

J Mammary Gland Biol Neoplasia

(2003)

G. Castoria et al.

Non-transcriptional action of estradiol and progestin triggers DNA synthesis

EMBO

(1999)

A. Migliaccio et al.

Activation of Src/p21ras/Erk pathway by progesterone via cross-talk with estrogen receptor

EMBO

(1998)

A. Skildum et al.

Progesterone receptors induce cell cycle progression via activation of mitogen-activated protein kinases

Mol Endocrinol

(2005)

Faivre EJ, Lange CA. Progesterone receptor upregulated Wnt-1 to induce EGFR-transactivation of c-Src dependent...

C. Ballare et al.

Two domains of the progesterone receptor interact with the estrogen receptor and are required for progesterone activation of the c-Src/Erk pathway in mammalian cells

Mol Cell Biol

(2003)

R.P. Carnevale et al.

Progestin effects on breast cancer cell proliferation, proteases activation, and in vivo development of metastatic phenotype all depend on progesterone receptor capacity to activate cytoplasmic signaling pathways

Mol Endocrinol

(2007)

Cited by (113)

Progesterone receptor-Grb2 interaction is associated with better outcomes in breast cancer
2024, Journal of Steroid Biochemistry and Molecular Biology
In addition to mediating nuclear transcription, PR mediates extranuclear functions mainly through the PR polyproline domain (PPD) interaction with the SH3 domain of cytoplasmic signaling molecules. PR-PPD-SH3 interaction inhibits EGF-mediated signaling and decreases lung cancer cell proliferation. Grb2 is an essential adaptor molecule with an SH2 domain flanked by two SH3 domains. In this study, we examined whether PR, through interaction between PR-PPD and Grb2-SH3, can interact with Grb2 in cells and breast cancer tissues. Our previous study shows that interaction between PR-PPD and Grb2 could interfere with cytoplasmic signaling and lead to inhibition of EGF-mediated signaling. GST-pulldown analysis shows that PR-PPD specifically interacts with the SH3 domains of Grb2. Immunofluorescence staining shows colocalization of PR and Grb2 in both the nucleus and cytoplasm in BT-474 breast cancer cells. Using Bimolecular Fluorescence Complementation (BiFC) analysis, we show that PR and Grb2 interact in breast cancer cells through the Grb2-SH3 domain. Proximity Ligation Assay (PLA) analysis of 43 breast cancer specimens shows that PR-Grb2 interaction is associated with low histological stage and negatively correlates with lymph node invasion and metastasis in breast cancer. These results, together with our previous findings, suggest that PR-PPD interaction with Grb2 plays an essential role in PR-mediated growth factor signaling inhibition and could contribute significantly to better prognosis in PR- and Grb2-positive breast cancer. Our finding provides a basis for additional studies to explore a novel therapeutic strategy for cancer treatment.
Aberrant upregulation of CDK1 contributes to medroxyprogesterone acetate (MPA) resistance in cancer-associated fibroblasts of the endometrium
2022, Biochemical and Biophysical Research Communications
The response to medroxyprogesterone acetate (MPA) decreases as endometrial disease progresses from the benign to malignancy. In a mouse model, progesterone receptor (PR) expression in normal fibroblasts is accountable for the MPA's inhibitory effects in cancer cells. However, it is still unclear, if and how, fibroblasts from human tumors respond to MPA. In this study, three benign-associated fibroblasts (BAFs) and four cancer-associated fibroblasts (CAFs) were isolated from human benign and cancerous endometrial tissues, respectively, to examine MPA activation on PR signaling. PR-B protein expression were heterogeneously expressed in both CAFs and BAFs, despite a lower mRNA expression in the former. In a luciferase reporter assay, MPA treatment stimulated some PR DNA-binding activity in BAFs but not in CAFs. Yet, activation of PR target gene was generally more pronounced in MPA-treated CAFs compared to BAFs. Cyclin-dependent kinase 1 (CDK1) was exclusively upregulated by 10 nM MPA in CAFs (5.1-fold vs. 1.1-fold in BAFs, P < 0.05), leading to a higher CDK1 protein expression. Subsequently in a dose-response study, CAFs showed an average of ∼20% higher cell viability when compared to BAFs, indicative of drug resistance to MPA. MPA resistance was also observed in EC-CAFs co-culture, when MPA-treated cells showed greater tumor spheroid formation than in EC-BAFs co-culture (2-fold, P < 0.01). The increased cell viability observed in CAFs was reversed with mifepristone (RU486), a PR antagonist which suppressed MPA-induced CDK1 expression. This indicates that MPA-induced abnormal upregulation of CDK1 may contribute to the enhanced CAFs cell proliferation, suggesting a new mechanism of MPA resistance within endometrial cancer microenvironment.
Progesterone decreases ovarian cancer cells migration and invasion
2020, Steroids
Citation Excerpt :
This study demonstrated that n-PR-B, but not n-PR-A, could selectively increase FAK expression in the absence of ligand, leading to increased migration of breast cancer MDA-MB-231 cells [25]. In addition, n-PR-A is located primarily in the nucleus, while n-PR-B moves continuously between nuclear and cytoplasmic compartments [26,27]. Thus, n-PR-B could mediate direct transcriptional events or rapid cytoplasmic changes by interacting with non-nuclear signaling pathways.
The progression of cancer depends on the interaction between the cells and their microenvironment. Progesterone is a steroid and progestogen sex hormone produced by the corpus luteum, which is a transitory endocrine gland in female mammals and prepares the endometrium for implantation. Also, progesterone is involved in antitumorigenic process in different types of cancer. Our goal is to investigate the role of progesterone in cell invasion and migration. Ovarian cells were treated with different concentrations of progesterone. 500 nM or 1 μM progesterone decreased the migration of the cells in 24 h or less without affecting the viability. Immunoblot showed that treatment with 1 μM progesterone decreased the phosphorylated forms of Src and FAK, and the cells were less polarized. Our results suggest that progesterone interferes with migration and invasion of ovarian cells. Inhibitory experiments inferred the progesterone receptor playing a role in migration and invasion. Decreased phosphorylation of molecules involved in these processes was also found.
Sexual dimorphism in the immune system
2019, The Autoimmune Diseases
Sexual dimorphism is the term that refers to differences between males and females of the same species and is most obvious as differences in external appearances. However, there can also be sexual dimorphism of internal organs and biological functions, including the immune system. Sexual dimorphism in the immune system is important in medicine because it can lead to sex differences in the responses to infection and vaccination and sex differences in the development of autoimmune disease. This chapter focuses on the effects of sexual dimorphism on immunocompetence, and on some of the possible mechanisms underlying these effects, including the effects of the sex hormones, sex chromosomes, and sexually dimorphic cultural and environmental effects on immunity. The possible consequences for autoimmunity of sexual dimorphism in the immune system are also considered.
Activation of membrane progesterone receptor-alpha increases proliferation, migration, and invasion of human glioblastoma cells
2018, Molecular and Cellular Endocrinology
Glioblastoma is the most frequent and aggressive brain tumor due to its high capacity to migrate and invade normal brain tissue. The steroid hormone progesterone (P4) contributes to the progression of glioblastoma by promoting proliferation, migration, and cellular invasion through the activation of its intracellular receptor (PR). However, the use of PR antagonist RU486 partially blocks the effects of P4, suggesting the participation of signaling pathways such as those mediated by membrane receptors to P4 (mPRs). Therefore, this study aimed to investigate the effects of mPRα subtype activation on proliferation, migration, and invasion of human glioblastoma cells.
We treated human glioblastoma cell lines U87 and U251 with the specific mPRα agonist Org OD 02-0, and evaluated its effects on cell number, proliferation, migration, and invasion. Additionally, we measured the phosphorylation of the kinases Src and Akt in both cell lines upon Org OD 02-0 treatment.
Org OD 02-0 (100 nM) augmented the number of U87 and U251 cells by increasing cell proliferation. The treatment with this agonist also increased U87 and U251 cell migration and invasion. Both proliferation and cell invasion decreased when mPRα expression was silenced. Finally, we observed that Org OD 02-0 increased the content of p-Src and p-Akt in both cell lines.
Our data suggest that P4 produces its effects in human glioblastoma progression not only by PR interaction but also through cell signaling pathways activated by mPRα.
Extranuclear signaling by sex steroid receptors and clinical implications in breast cancer
2018, Molecular and Cellular Endocrinology
Estrogen and progesterone play essential roles in the development and progression of breast cancer. Over 70% of breast cancers express estrogen receptors (ER) and progesterone receptors (PR), emphasizing the need for better understanding of ER and PR signaling. ER and PR are traditionally viewed as transcription factors that directly bind DNA to regulate gene networks. In addition to nuclear signaling, ER and PR mediate hormone-induced, rapid extranuclear signaling at the cell membrane or in the cytoplasm which triggers downstream signaling to regulate rapid or extended cellular responses. Specialized membrane and cytoplasmic proteins may also initiate hormone-induced extranuclear signaling. Rapid extranuclear signaling converges with its nuclear counterpart to amplify ER/PR transcription and specify gene regulatory networks. This review summarizes current understanding and updates on ER and PR extranuclear signaling. Further investigation of ER/PR extranuclear signaling may lead to development of novel targeted therapeutics for breast cancer management.

View all citing articles on Scopus

View full text

The role and mechanism of progesterone receptor activation of extra-nuclear signaling pathways in regulating gene transcription and cell cycle progression

Abstract

Section snippets

Extra-nuclear functions of human progesterone receptor

Progestin activation of Src is an extra-nuclear function of PR

Progestin activation of Src is sufficient to induce activation of a MAPK dependent, Elk-1 transcription factor

Progestin induction of endogenous cyclin D1 gene and cell cycle progression is dependent on the SH3-domain binding motif of PR

Discussion

Cell

J Biol Chem

Mol Cell

Steroids

J Biol Chem

J Biol Chem

Dev Cell

J Biol Chem

J Steroid Biochem Mol Biol

Regulation of signal transduction pathways by estrogen and progesterone

Annu Rev Physiol

Two distinct estrogen-regulated promoters generate transcripts encoding two functionally different human progesterone receptor forms A and B

EMBO J

The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding

Mol Cell Biol

Human progesterone receptor A form is a cell- and promoter-specific repressor of human progesterone receptor B function

Mol Endocrinol

Progesterone receptors in mammary gland development and tumorigenesis

J Mammary Gland Biol Neoplasia

Non-transcriptional action of estradiol and progestin triggers DNA synthesis

EMBO

Activation of Src/p21ras/Erk pathway by progesterone via cross-talk with estrogen receptor

EMBO

Progesterone receptors induce cell cycle progression via activation of mitogen-activated protein kinases

Mol Endocrinol

Two domains of the progesterone receptor interact with the estrogen receptor and are required for progesterone activation of the c-Src/Erk pathway in mammalian cells

Mol Cell Biol

Progestin effects on breast cancer cell proliferation, proteases activation, and in vivo development of metastatic phenotype all depend on progesterone receptor capacity to activate cytoplasmic signaling pathways

Mol Endocrinol