Valeria Hansberg-Pastor

Estradiol increases cell growth in human astrocytoma cell lines through ERα activation and its interaction with SRC-1 and SRC-3 coactivators ☆

Estradiol (E2) regulates several cellular functions through the interaction with estrogen receptor subtypes, ERα and ERβ, which present different functional and regulation properties. ER subtypes have been identified in human astrocytomas, the most common and aggressive primary brain tumors. We studied the role of ER subtypes in cell growth of two human astrocytoma cell lines derived from tumors of different evolution grades: U373 and D54 (grades III and IV, respectively). E2 significantly increased the number of cells in both lines and the co-administration with an ER antagonist (ICI 182, 780) significantly blocked E2 effects. ERα was the predominant subtype in both cell lines. E2 and ICI 182, 780 down-regulated ERα expression. The number of U373 and D54 cells significantly increased after PPT (ERα agonist) treatment but not after DPN (ERβ agonist) one. To determine the role of SRC-1 and SRC-3 coactivators in ERα induced cell growth, we silenced them with RNA interference. Coactivator silencing blocked the increase in cell number induced by PPT. The content of proteins involved in proliferation and metastasis was also determined after PPT treatment. Western blot analysis showed that in U373 cells the content of PR isoforms (PR-A and PR-B), EGFR, VEGF and cyclin D1 increased after PPT treatment while in D54 cells only the content of EGFR was increased. Our results demonstrate that E2 induces cell growth of human astrocytoma cell lines through ERα and its interaction with SRC-1 and SRC-3 and also suggest differential roles of ERα on cell growth depending on astrocytoma grade.

Progesterone promotes cell migration, invasion and cofilin activation in human astrocytoma cells.

Astrocytomas are the most common and aggressive primary brain tumors in humans. Invasiveness of these tumors has been attributed in part to deregulation of cell motility-dependent cytoskeletal dynamics that involves actin-binding proteins such as cofilin. Progesterone (P4) has been found to induce migration and invasion of cells derived from breast cancer and endothelium. However, the role of P4 in migration and invasion of astrocytoma cells as well as its effects on astrocytomas cytoskeleton remodeling is not known. In this work we evaluated these aspects in D54 and U251 cells derived from human astrocytomas from the highest degree of malignancy (grade IV, glioblastoma). Our results showed that in scratch-wound assays P4 increased the number of D54 and U251 cells migrating from 3 to 48 h. Both RU486, a P4 receptor (PR) antagonist, and an oligonucleotide antisense against PR significantly blocked P4 effects. Transwell assays showed that P4 significantly increased the number of invasive cells at 24h. As in the case of migration, this effect was blocked by RU486. Finally, by Western blotting, an increase in the cofilin/p-cofilin ratio at 15 and 30 min and a decrease at 30 and 60 min in U251 and D54 cells, respectively, was observed after P4, P4+RU486 and RU486 treatments. These data suggest that P4 increases human astrocytoma cells migration and invasion through its intracellular receptor, and that cofilin activation by P4 is independent of PR action.

Sex Hormones Regulate Cytoskeletal Proteins Involved in Brain Plasticity

In the brain of female mammals, including humans, a number of physiological and behavioral changes occur as a result of sex hormone exposure. Estradiol and progesterone regulate several brain functions, including learning and memory. Sex hormones contribute to shape the central nervous system by modulating the formation and turnover of the interconnections between neurons as well as controlling the function of glial cells. The dynamics of neuron and glial cells morphology depends on the cytoskeleton and its associated proteins. Cytoskeletal proteins are necessary to form neuronal dendrites and dendritic spines, as well as to regulate the diverse functions in astrocytes. The expression pattern of proteins, such as actin, microtubule-associated protein 2, Tau, and glial fibrillary acidic protein, changes in a tissue-specific manner in the brain, particularly when variations in sex hormone levels occur during the estrous or menstrual cycles or pregnancy. Here, we review the changes in structure and organization of neurons and glial cells that require the participation of cytoskeletal proteins whose expression and activity are regulated by estradiol and progesterone.

PKCα and PKCδ Activation Regulates Transcriptional Activity and Degradation of Progesterone Receptor in Human Astrocytoma Cells

Progesterone regulates cancer cell proliferation and invasion through its receptors (PR-A and PR-B), whose phosphorylation modifies their transcriptional activity and induce their degradation. We identified by in silico analysis a putative residue (Ser400) in PR that might be phosphorylated by protein kinase C (PKC), a family of enzymes involved in the proliferation and infiltration of astrocytomas, the most frequent and aggressive brain tumors. A grade III human astrocytoma-derived cell line was used to study the role of PKC in PR phosphorylation, transcriptional activity, and degradation. Treatment with PKC activator [tetradecanoyl phorbol acetate (TPA)] increased PR phosphorylation in Ser400 after 5 minutes, which in turn induced PR transcriptional activity and its subsequent degradation by the 26S proteasome 3-5 hours after treatment. Silencing or inhibition of PKCα and PKCδ blocked PR phosphorylation and degradation induced by TPA. Both PR isoforms were associated with PKCα and reached the maximum association after 5 minutes of TPA addition. These data correlated with immunnofluorescence assays in which nuclear colocalization of PKCα with PR increased after TPA treatment. We observed a 2-fold increase in cell proliferation after PKC activation with TPA that was reduced with the PR antagonist, RU486. The PR S400A mutant revealed that this residue is essential for PKC-mediated PR phosphorylation and degradation. Our results show a key participation of PKCα and PKCδ in PR regulation and function.

Expression and hormonal regulation of membrane progesterone receptors in human astrocytoma cells.

Progesterone (P) participates in the regulation of the growth of several tumors, including astrocytomas, the most common and malignant human brain tumors. It has been reported that P induces astrocytomas growth in part by its interaction with its intracellular receptors (PR). Recently, it has been reported that membrane progesterone receptors (mPRs) are expressed in ovarian and breast cancer cells, and that P could exert some actions through these receptors, however, it is unknown whether mPRs are expressed in astrocytomas. In this work, U251 and U87 cell lines derived from human astrocytomas grade IV were used to study the expression, localization and hormonal regulation of three mPRs subtypes. Using RT-qPCR and Western blot techniques, we found that mPRα and mPRβ are clearly expressed at mRNA and protein levels in astrocytoma cells whereas mPRγ was barely expressed in these cells. Immunofluorescence staining showed that mPRα and mPRβ were mainly located in the cell surface. Flow cytometry assays demonstrated that in U251 and U87 cells, mPRβ is expressed by a higher percentage of both permeabilized and non-permeabilized cells as compared with mPRα. The percentage of cells expressing mPRγ was very low. P and estradiol (E) (10, 100 nM and 1 μM) decreased mPRα protein content at 12 h. In contrast, both P (100 nM and 1 μM) and E (10 and 100 nM) increased mPRβ content. Finally, by in silico analysis, we identified that mPRα, mPRβ and mPRγ promoters contain several progesterone and estrogen response elements. Our results indicate that mPRs are expressed in human astrocytoma cells, exhibiting a differential regulation by E and P. These data suggest that some P actions in astrocytoma cells may be mediated by mPRs.

Allopregnanolone promotes proliferation and differential gene expression in human glioblastoma cells

&NA; Allopregnanolone (3&agr;‐THP) is one of the main reduced progesterone (P4) metabolites that is recognized as a neuroprotective and myelinating agent. 3&agr;‐THP also induces proliferation of different neural cells. It has been shown that P4 favors the progression of glioblastomas (GBM), the most common and aggressive primary brain tumors. However, the role of 3&agr;‐THP in the growth of GBMs is unknown. Here, we studied the effects of 3&agr;‐THP on the number of cells, proliferation and gene expression in U87 cell line derived from a human GBM. 3&agr;‐THP (10, 100 nM and 1 &mgr;M) increased the number of U87 cells, and at 10 nM exerted a similar increase in both the number of total and proliferative U87 cells as compared with P4 (10 nM). Interestingly, finasteride (F; 100 nM), an inhibitor of 5&agr;‐reductase (5&agr;R), an enzyme necessary to metabolize P4 and produce 3&agr;‐THP, blocked the increase in the number of U87 cells induced by P4. By using RT‐qPCR, we determined that U87 cells express 5&agr;‐R isoenzymes 1 and 2 (5&agr;R1 and 5&agr;R2), being 5&agr;R1 the predominant one in these cells. 3&agr;‐THP (10 nM) increased the expression of TGF&bgr;1, EGFR, VEGF and cyclin D1 genes. P4 increased TGF&bgr;1 and EGFR expression, and this effect was blocked by F. These data provide evidence that P4, through its metabolite 3&agr;‐THP, can promote in part cell proliferation of human GBM cells by changing the expression of genes involved in tumor progression. Highlights3&agr;‐THP and progesterone (P4) promote proliferation of U87 human glioblastoma cells.U87 cells express higher levels of 5&agr;R1 than those of 5&agr;R2.P4 increases TGF&bgr;1 and EGFR expression, and finasteride blocks this effect.3&agr;‐THP promotes the expression of TGF&bgr;1, EGFR, VEGF and cyclin D1 in U87 cells.

Proliferative and Invasive Effects of Progesterone-Induced Blocking Factor in Human Glioblastoma Cells

Progesterone-induced blocking factor (PIBF) is a progesterone (P4) regulated protein expressed in different types of high proliferative cells including astrocytomas, the most frequent and aggressive brain tumors. It has been shown that PIBF increases the number of human astrocytoma cells. In this work, we evaluated PIBF regulation by P4 and the effects of PIBF on proliferation, migration, and invasion of U87 and U251 cells, both derived from human glioblastomas. PIBF mRNA expression was upregulated by P4 (10 nM) from 12 to 24 h. Glioblastoma cells expressed two PIBF isoforms, 90 and 57 kDa. The content of the shorter isoform was increased by P4 at 24 h, while progesterone receptor antagonist RU486 (10 μM) blocked this effect. PIBF (100 ng/mL) increased the number of U87 cells on days 4 and 5 of treatment and induced cell proliferation on day 4. Wound-healing assays showed that PIBF increased the migration of U87 (12–48 h) and U251 (24 and 48 h) cells. Transwell invasion assays showed that PIBF augmented the number of invasive cells in both cell lines at 24 h. These data suggest that PIBF promotes proliferation, migration, and invasion of human glioblastoma cells.

The interplay between intracellular progesterone receptor and PKC plays a key role in migration and invasion of human glioblastoma cells.

Intracellular progesterone receptors (PRs) and protein kinases C (PKCs) are known regulators of cancer cell proliferation and metastasis. Both PRs and PKCs are found overexpressed in grade IV human astrocytomas, also known as glioblastomas, which are the most frequent and aggressive brain tumors. In the present study, we investigated whether PR activation by PKC induces the migration and invasion of glioblastoma derived cell lines and if PKCα and δ isoforms are involved in PR activation. We observed that PKC activation with tetradecanoylphorbol acetate (TPA) increases the migration and invasion capacity of two human glioblastoma derived human cell lines (U251 MG and U87) and that the treatment with the PR receptor antagonist RU486 blocks these processes. Interestingly, the pharmacological inhibition of the isoenzymes PKCα and PKCδ also resulted in a blocked PR transcriptional activity. Also, TPA-dependent PR activation increases the expression of progesterone-induced blocking factor (PIBF), a known PR target gene. These results hint to an existing cross-talk between PKCs and PRs in regulating the infiltration process of human glioblastomas.

DNA Methylation Analysis of Steroid Hormone Receptor Genes

Steroid hormone receptors (SHR) are important transcription factors for regulating different physiological and pathological processes. Their altered expression has been strongly associated to cancer progression. Epigenetic marks such as DNA methylation have been proposed as one of the regulatory mechanisms for SHR expression in cancer. DNA methylation occurs at CpG dinucleotides, which form clusters known as CpG islands. These islands are mostly observed at promoter regions of housekeeping genes, and their aberrant methylation in cancer cells is associated with silencing of tumor-suppressor gene expression. SHR genes are characterized for presenting alternative promoters with different CpG island content, which are prone to be methylated. The method of choice for studying DNA methylation is bisulfite sequencing, since it provides information about the methylation pattern at single-nucleotide level. The method is based on the deamination of cytosine residues to uracil after treatment with sodium bisulfite. The converted DNA is amplified by a polymerase chain reaction, cloned, and sequenced. Here, we describe a protocol for bisulfite sequencing suitable for analyzing different CpG regions in SHR genes.

3.10 – Mechanism of Progesterone Action in the Brain

Progesterone is a pleiotropic hormone that regulates a wide range of physiological and pathological processes in the central nervous system. The actions of progesterone are mediated by classical and nonclassical mechanisms, and many of them depend on its intracellular receptor (PR). PR expresses two main isoforms (PR-B and PR-A), whose actions are driven by the participation of specific coregulators, posttranslational modifications, and epigenetic mechanisms among other events, that in turn are influenced by the cellular context and developmental stage. In the present chapter, we address in depth about the regulation and molecular actions of PR in the brain.