Christos Stournaras

The human prostate cancer cell line LNCaP bears functional membrane testosterone receptors that increase PSA secretion and modify actin cytoskeleton

Recent findings have shown that, in addition to the genomic action of steroids, through intracellular receptors, short‐time effects could be mediated through binding to membrane sites. In the present study of prostate cancer LNCaP cells, we report that dihydrotestosterone and the non‐internalizable analog testosterone‐BSA increase rapidly the release of prostate‐specific antigen (PSA) in the culture medium. Membrane testosterone binding sites were identified through ligand binding on membrane preparations, flow cytometry, and confocal laser microscopy of the non‐internalizable fluorescent analog testosterone‐BSA‐FITC, on whole cells. Binding on these sites is time‐ and concentration‐dependent and specific for testosterone, presenting a Kd of 10.9 nM and a number of 144 sites/mg protein (~13000 sites/cell). Membrane sites differ immunologically for intracellular androgen receptors. The secretion of PSA after membrane testosterone receptor stimulation was inhibited after pretreatment with the actin cytoskeleton disrupting agent cytochalasin B. In addition, membrane testosterone binding modifies the intracellular dynamic equilibrium of monomeric to filamentous actin and remodels profoundly the actin cytoskeleton organization. These results are discussed in the context of a possible involvement of these sites in cancer chemotherapy.

Cell responses regulated by early reorganization of actin cytoskeleton.

Microfilaments exist in a dynamic equilibrium between monomeric and polymerized actin and the ratio of monomers to polymeric forms is influenced by a variety of extracellular stimuli. The polymerization, depolymerization and redistribution of actin filaments are modulated by several actin‐binding proteins, which are regulated by upstream signalling molecules. Actin cytoskeleton is involved in diverse cellular functions including migration, ion channels activity, secretion, apoptosis and cell survival. In this review we have outlined the role of actin dynamics in representative cell functions induced by the early response to extracellular stimuli.

Phosphorylated EGFR and PI3K/Akt signaling kinases are expressed in circulating tumor cells of breast cancer patients

IntroductionThe phosphoinositide-3 kinase (PI3K)/Akt pathway, operating downstream of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor (HER)2, is implicated in cell migration and survival. EGFR and HER2 are expressed in circulating tumor cells, but the activation status of downstream signaling molecules has not yet been reported.MethodsTo investigate expression levels of EGFR, HER2, PI3K, and Akt in circulating tumor cells, we used peripheral blood mononuclear cells from 32 cytokeratin-19 mRNA-positive patients with early (n = 16) and metastatic (n = 16) breast cancer.Peripheral blood mononuclear cell cytospins were double stained with cytokeratin antibody along with one of the following: EGFR, phospho-EGFR, HER2, phospho-PI3K, or phospho-Akt antibodies.ResultsEGFR and HER2 were expressed in circulating tumor cells of 38% and 50% patients with early and 44% and 63% patients with metastatic disease, respectively. Interestingly, phospho-PI3K and phospho-Akt expression levels were similar at 88% (14 out of 16) and 81% (13 out of 16), respectively, in circulating tumor cells of patients with early and metastatic disease. Phospho-EGFR was observed in circulating tumor cells of two (33%) early and six (86%) metastatic EGFR-positive patients. Immunomagnetic separation of peripheral blood mononuclear cells, using EpCAM antibody, and subsequent double-staining experiments of circulating tumor cells showed that EGFR was co-expressed with HER2, phospho-Akt and phospho-PI3K kinases, indicating activation of the corresponding survival signaling pathway.ConclusionsOur findings demonstrate that circulating tumor cells express receptors and activated signaling kinases of the EGFR/HER2/PI3K/Akt pathway, which could be used as targets for their effective elimination.

Neurosteroids as Endogenous Inhibitors of Neuronal Cell Apoptosis in Aging

Abstract:  The neuroactive steroids dehydroepiandrosterone (DHEA), its sulfate ester DHEAS, and allopregnanolone (Allo) are produced in the adrenals and the brain. Their production rate and levels in serum, brain, and adrenals decrease gradually with advancing age. The decline of their levels was associated with age‐related neuronal dysfunction and degeneration, most probably because these steroids protect central nervous system (CNS) neurons against noxious agents. Indeed, DHEA(S) protects rat hippocampal neurons against NMDA‐induced excitotoxicity, whereas Allo ameliorates NMDA‐induced excitotoxicity in human neurons. These steroids exert also a protective role on the sympathetic nervous system. Indeed, DHEA, DHEAS, and Allo protect chromaffin cells and the sympathoadrenal PC12 cells (an established model for the study of neuronal cell apoptosis and survival) against serum deprivation–induced apoptosis. Their effects are time‐ and dose‐dependent with EC50 1.8, 1.1, and 1.5 nM, respectively. The prosurvival effect of DHEA(S) appears to be NMDA‐, GABAA‐ sigma1‐, or estrogen receptor‐independent, and is mediated by G‐protein‐coupled‐specific membrane binding sites. It involves the antiapoptotic Bcl‐2 proteins, and the activation of prosurvival transcription factors CREB and NF‐κB, upstream effectors of the antiapoptotic Bcl‐2 protein expression, as well as prosurvival kinase PKCα/β, a posttranslational activator of Bcl‐2. Furthermore, they directly stimulate biosynthesis and release of neuroprotective catecholamines, exerting a direct transcriptional effect on tyrosine hydroxylase, and regulating actin depolymerization and submembrane actin filament disassembly, a fast‐response cellular system regulating trafficking of catecholamine vesicles. These findings suggest that neurosteroids may act as endogenous neuroprotective factors. The decline of neurosteroid levels during aging may leave the brain unprotected against neurotoxic challenges.

Differential regulation of the two RhoA-specific GEF isoforms Net1/Net1A by TGF-β and miR-24: role in epithelial-to-mesenchymal transition

In the present study we analyzed the regulation of the two isoforms of the RhoA-specific guanine nucleotide exchange factor Net1 by transforming growth factor-β (TGF-β) in keratinocytes. We report that short-term TGF-β treatment selectively induced Net1 isoform2 (Net1A) but not Net1 isoform1. This led to upregulation of cytoplasmic Net1A protein levels that were necessary for TGF-β-mediated RhoA activation. Smad signaling and the MAPK/ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway were involved in Net1A upregulation by TGF-β. Interestingly, long-term TGF-β treatment resulted in Net1 mRNA downregulation and Net1A protein degradation by the proteasome. Furthermore, we identified the microRNA miR-24 as a novel post-transcriptional regulator of Net1A expression. Silencing of Net1A resulted in disruption of E-cadherin- and zonula occludens-1 (ZO-1)-mediated junctions, as well as expression of the transcriptional repressor of E-cadherin, Slug and the mesenchymal markers N-cadherin, plasminogen activator inhibitor-1 (PAI-1) and fibronectin, indicating that late TGF-β-induced downregulation of Net1A is involved in epithelial-to-mesenchymal transition (EMT). Finally, miR-24 was found to be implicated in the regulation of the EMT program in response to TGF-β and was shown to be directly involved in the TGF-β-induced breast cancer cell invasiveness through Net1A regulation. Our results emphasize the importance of Net1 isoform2 in the short- and long-term TGF-β-mediated regulation of EMT.

Control of transforming growth factor β signal transduction by small GTPases

The integrated roles of small GTPases in executing the transforming growth factor β (TGFβ) signaling pathway have attracted increasing attention in recent years. In this review, we summarize recent findings on TGFβ signaling during receptor endocytosis, Smad trafficking and actin cytoskeleton remodeling, and emphasize the role of small GTPases in these processes. First, we give an overview of the different endocytic routes taken by TGFβ receptors, their impact on active TGFβ signaling versus degradation and their regulation by the small GTPases Rab, RalA/Ral‐binding protein 1 and Rap2. Second, we focus on the mechanisms and regulation of Smad trafficking in the cytoplasm, through the nuclear pores and into the nucleus, and the contribution of Ran GTPase to these events. Third, we summarize the role of Rho small GTPases in early and late cytoskeleton remodeling in various cell models and diseases, and the positive and negative cross‐talk between Rho GTPases and the TGFβ/Smad pathway. The biological significance of this exciting research field, the perspectives and critical open questions are discussed.

Hepatocyte swelling leads to rapid decrease of the G‐/total actin ratio and increases actin mRNA levels

Exposure of isolated rat hepatocytes to hypotonic (190 mosinol/1) incubation media lowered the cellular G‐actin level without affecting the total actin content: here the G‐/total actin ratio decreased by 15.5 ± 1.4% (n=7). Similar effects were observed following isotonic cell swelling by either addition of glutamine (10 mM) or insulin (100 nM), resulting in a decrease of the G‐/total actin ratios by 13.5 ± 2.1 % (n = 5) and 14.1 ± 1.1% (n = 11), respectively. The effects of hypotonic exposure, glutamine and insulin on the G‐/total actin ratio largely occurred within 1 min and persisted for at least 2 h in presence of the respective effectors. After a 120 min exposure to hypotonic media, glutamine or insulin the actin mRNA levels were increased 2.4‐, 2.0‐ and 3.6‐fold, respectively. Hypertonic exposure lowered the G‐/total actin ratio by only 4.9 ± 2.5% (n = 4) and increased actin mRNA levels only 1.2‐fold. There was a close relationship between glutamine‐ and hypotonicity‐induced cell swelling and the decrease of G‐/total actin ratios. The data suggest that cell swelling exerts rapid and marked effects on the state of actin polymerization and increases actin mRNA levels. Thus, cytoskeletal alterations in response to cell swelling may be involved in the regulation of hepatic metabolism by cell volume.

Activation of FAK/PI3K/Rac1 Signaling Controls Actin Reorganization and Inhibits Cell Motility in Human Cancer Cells

We have recently identified a specific signaling pathway that regulates actin reorganization in malignant human breast and prostate epithelial cells associated with FAK, PI-3K and Rac1 activation. Here we report that this pathway operates in MCF7 cells upon activation of membrane androgen receptors (mAR). Stimulation of mAR by the non-permeable testosterone-BSA conjugate resulted in early actin reorganization documented by quantitative measurements of actin dynamics and morphological analysis of microfilament organization. This effect was regulated by early phosphorylation of FAK and subsequent PI-3K and Rac1 activation. The functional role of this pathway was further shown in A375 melanoma cells. Treatment with the opioid antagonist αs1 casomorphin resulted in rapid and potent actin remodeling in A375 cells, regulated by rapid activation of the FAK/PI-3K/Rac1 signaling. Pretreatment of both cell lines with the specific PI-3K inhibitor wortmannin blocked actin reorganization. Interestingly, wound healing assays revealed that testosterone-BSA and α s1 casomorphin significantly inhibited MCF7 and A375 cell motility respectively. These effects were abrogated through blockade of PI-3K signaling by wortmannin. The results presented here indicate that actin reorganization through FAK/PI3-K/Rac-1 activation operates in various human cancer cell systems supporting a functional role for FAK/PI-3K/Rac1/actin signaling in controlling cell motility.

Dexamethasone alters rapidly actin polymerization dynamics in human endometrial cells: Evidence for nongenomic actions involving cAMP turnover

Glucocorticoids, in addition to their well characterized effects on the genome, may affect cell function in a manner not involving genomic pathways. The mechanisms by which the latter is achieved are not yet clear. A possible means for this action may involve the actin cytoskeleton, since the dynamic equilibrium of actin polymerization changes rapidly following exposure to several stimuli, including hormones. The aim of the present work was to find out if glucocorticoids exert rapid, nongenomic effects on actin polymerization in Ishikawa human endometrial cells, which represent a well characterized in vitro cell model expressing functional glucocorticoid receptors. Short term exposure of the cells to the synthetic glucocorticoid dexamethasone resulted in an overall decrease of the G/total‐actin ratio in a time‐ and dose‐dependent manner. Specifically, in untreated Ishikawa cells the G/total‐actin ratio was 0.48 ± 0.01 (n = 26). It became 0.35 ± 0.01 (n = 13, P < 0.01) following exposure to 10‐7 M dexamethasone for 15 min. This was induced by a significant decrease of the cellular G‐actin level, without affecting the total actin content, indicating a rapid actin polymerization. This conclusion was fully confirmed by direct fluorimetry measurements, that showed a significant increase of the F‐actin content by 44% (n = 6, P < 0.001) in cells treated with dexamethasone (10‐7 M, 15 min). The rapid dexamethasone‐induced alterations of the state of actin polymerization were further supported by fluorescence microscopy. The latter studies showed that the microfilaments of cells pretreated with 10‐7 M dexamethasone for 15 min were more resistant to various concentrations of the antimicrofilament drug cytochalasin B, compared to untreated cells, implying microfilament stabilization. The action of dexamethasone on actin polymerization seems to be mediated via specific glucocorticoid binding sites, since the addition of the glucocorticoid antagonist RU486 completely abolished its effect. Moreover, it appears to act via non‐transcriptional pathways, since actinomycin D did not block the dexamethasone‐induced actin polymerization. In addition, cell treatment with 10‐7 M dexamethasone for 15 min fully reversed the forskolin‐, but not the 8‐bromo‐cAMP‐induced actin depolymerization. In line with these findings, the cAMP content of Ishikawa cells was decreased by 29.2% after a 15 min treatment with 10‐7 M dexamethasone (n = 4, P < 0.01). In conclusion, our results showed that dexamethasone induces rapid, time‐, and dose‐dependent changes in actin polymerization dynamics in Ishikawa cells. This action seems to be mediated via cAMP, involving probably nongenomic pathways. The above findings offer new perspectives for the understanding of the early cellular responses to glucocorticoids.

Actin Cytoskeleton: A Signaling Sensor in Cell Volume Regulation

The actin microfilaments are well known dynamic structures that support and organize the cell membrane and functions associated with the membrane such as ion channels and transporters. In addition, many aspects of cellular physiology seem to be actively modulated by changes in actin cytoskeleton dynamics, which involve reorganization and restructuring of the filaments. For both of these reasons, the actin cytoskeleton has attracted special attention since the early days of cell volume regulation research. Mechanisms controlling the actin equilibrium in response to external stimuli were studied and the signaling cascades leading to the regulation of actin cytoskeleton dynamics have been partially elucidated. They include: a) activation of specific actin binding proteins that regulate actin polymerization dynamics, b) activation of protein kinases or phosphatases regulating phosphorylation of specific cytoskeletal proteins and c) activation of signal transduction pathways leading from membrane receptor activation to actin reorganization involving small GTPases of the Rho and Rac families. These intracellular signal transducers are activated by extracellular stimuli that include hormones, growth factors, cytokines, or ions, many of them in turn are partially known to participate in cell volume regulation. These findings provide strong evidence that the actin cytoskeleton is involved in cell volume regulation by sensing and mediating extracellular signals.