Differential Smad2/3 linker phosphorylation is a crosstalk mechanism of Rho/ROCK and canonical TGF-ß3 signaling in tenogenic differentiation.

The transforming growth factor (TGF)-ß3 is a well-known inducer for tenogenic differentiation, signaling via the Smad2/3 pathway. Furthermore, other factors like extracellular matrix or mechanical force can induce tenogenic differentiation and possibly alter the response to TGF-ß3 by signaling via the Rho/ROCK pathway. The aim of this study was to investigate the interplay of Rho/ROCK and TGF-ß3/Smad signaling in tenogenic differentiation, with the Smad2/3 molecule hypothesized as a possible interface. Cultured as monolayers or on collagen I matrices, mesenchymal stromal cells (MSC) were treated with the ROCK inhibitor Y-27632 (10 µM), TGF-ß3 (10 ng/ml) or both combined. Control cells were cultured accordingly, without Y-27632 and/or without TGF-ß3. At different time points, MSC were analyzed by real-time RT-PCR, immunofluorescence, and Western blot. Cultivation of MSC on collagen matrices and ROCK inhibition supported tenogenic differentiation and fostered the effect of TGF-ß3. The phosphorylation of the linker region of Smad2 was reduced by cultivation on collagen matrices, but not by ROCK inhibition. The latter, however, led to increased phosphorylation of the linker region of Smad3. In conclusion, collagen matrices and the Rho/ROCK signaling pathway influence the TGF-ß3/Smad2/3 pathway by regulating different phosphorylation sites of the Smad linker region.

Analysis of Membrane Type-1 Matrix Metalloproteinase (MT1-MMP, MMP14) in Eutopic and Ectopic Endometrium and in Serum and Endocervical Mucus of Endometriosis.

BACKGROUND: Membrane type-matrix metalloproteinases (MT-MMPs) are a subgroup of the matrix metalloproteinases (MMPs) family and are key molecules in the degradation of the extracellular matrix. Membrane type-1 matrix metalloproteinase (MT1-MMP, MMP14) is often deregulated in different cancer tissues and body fluids of human cancer patients; however, MT1-MMP levels in endometriosis and adenomyosis patients are currently unknown. MATERIALS AND METHODS: Tissue samples from patients with and without endometriosis or adenomyosis were analyzed with immunohistochemistry for the localization of MT1-MMP. Serum and endocervical mucus samples from patients with and without endometriosis or adenomyosis were investigated with MT1-MMP ELISAs. RESULTS: MT1-MMP was localized preferentially in the glands of eutopic and ectopic endometrium. MT1-MMP protein levels are significantly reduced in ovarian endometriosis (HSCORE = 31) versus eutopic endometrium (HSCORE = 91) and adenomyosis (HSCORE = 149), but significantly increased in adenomyosis (HSCORE = 149) compared to eutopic endometrium (HSCORE = 91). Similarly, analysis of the levels of MT1-MMP using enzyme-linked immune assays (ELISAs) demonstrated a significant increase in the concentrations of MT1-MMP in the serum of endometriosis patients (1.3 ± 0.8) versus controls (0.7 ± 0.2), but not in the endocervical mucus. Furthermore, MT1-MMP levels in the endocervical mucus of patients with endometriosis were notably reduced in patients using contraception (3.2 ± 0.4) versus those without contraception (3.8 ± 0.2). CONCLUSIONS: Taken together, our findings showed an opposite regulation of MT1-MMP in the tissue of ovarian endometriosis and adenomyosis compared to eutopic endometrium without endometriosis but increased serum levels in patients with endometriosis.

Transmigration of macrophages through primary adult rat Sertoli cells.

The blood testis barrier (BTB) is often studied with isolated immature Sertoli cells (SCs), transepithelial resistance (TER) measurements and FITC dextran diffusion assays. Recently, it was found that even in the absence of SCs, only few immune cells enter the seminiferous tubules. Thus, in this study, we evaluated the testicular immunological barrier (TIB) in vitro by transmigration of macrophages through SCs with and without peritubular cells (PCs) and with or without matrigel (MG). Primary PCs were isolated from adult rat testis and kept in mono- or co-cultures with the conditionally reprogrammed primary adult Sertoli cell line (PASC1) from rat that has been recently generated by our group. Rat monocytes isolated from fresh blood were differentiated into M0 macrophages, and after polarization to M1 or M2 macrophages characterized by gene expression of CXCL11 and TNF-α for M1, or CCL17 and CCL22 for M2. Transmigration of LeukoTracker-labeled M0, M1, and M2 macrophages through mono- and co-cultures of PCs/SCs with and without MG demonstrated that SCs are the main constituent of the TIB in vitro with only a negligible contribution of PCs or MG. Moreover, M2 macrophages showed less migration activity compared to M0 or M1. Treatment of SCs with testosterone (T) showed positive effects on the barrier in contrast to negative effects by interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α). The new transmigration model is suitable to evaluate transmigration of macrophages through a barrier consisting of testicular cells and can be applied to study the integrity of testicular barriers with respect to immunological aspects.

Impaired Expression of Membrane Type-2 and Type-3 Matrix Metalloproteinases in Endometriosis but Not in Adenomyosis.

Matrix metalloproteinases (MMPs) play an important role in menstruation and endometriosis; however, the membrane-type matrix metalloproteinases (MT-MMPs) are not well studied in endometriosis and adenomyosis. We analyzed MT2-MMP (MMP15) and MT3-MMP (MMP16) in eutopic endometrium with and without endometriosis and with and without adenomyosis and ectopic endometrium of deep infiltrating endometriosis (DIE), peritoneal endometriosis (PE), and ovarian endometriosis (Ov) by immunohistochemistry. Preferential expression of both proteins was observed in the glandular and luminal epithelial cells of the eutopic endometrium of patients with and without endometriosis with a ~2.5-fold stronger expression of MT3-MMP compared to MT2-MMP. We did not observe any differences during menstrual cycling and in eutopic endometrium of patients with and without endometriosis. Similarly, eutopic endometrium and adenomyotic tissue with and without endometriosis showed similar protein levels of MT2-MMP and MT3-MMP. In contrast, MT2-MMP and MT3-MMP protein was decreased in ectopic compared to eutopic endometrium and adenomyosis. The similar expression of MT2-MMP and MT3-MMP in eutopic endometrium in patients with and without endometriosis in contrast to the impaired expression in ectopic endometrium suggests that alterations occur after and not before endometrial implantation possibly by distinct interactions with the different environments. The differential protein expression of MT2/3-MMP in adenomyosis compared to endometriosis might suggest a different pathogenesis pathway for the two diseases.

DHEAS prevents pro-metastatic and proliferative effects of 17ß-estradiol on MCF-7 breast cancer cells.

It is generally assumed that circulating dehydroepiandrosterone sulfate (DHEAS) can be desulfated and further metabolized to estrogen, which is of concern for all patients with estrogen-responsive breast cancer. We addressed this issue by comparing the effects of DHEAS, its desulfated form DHEA, and 17ß-estradiol on human metastatic, estrogen-responsive MCF-7 breast cancer cells. Physiological concentrations of DHEAS promoted phosphorylation of Erk1/2, whereas DHEA and 17ß-estradiol failed to stimulate Erk1/2 phosphorylation, indicating that the sulfated steroid acts as an autonomous hormone. Exposure of MCF-7 cells to 17ß-estradiol stimulated cell proliferation and the expression of pro-metastatic and pro-invasive elements such as claudin-1, matrix metalloproteinase 9 (MMP9), and the CC chemokine ligand 2 (CCL2). In contrast, treatment with DHEAS did not stimulate these responses but prevented all of the actions of 17ß-estradiol, and as a consequence cell migration and invasion were completely inhibited. The results of this study not only challenge the assumption that DHEAS poses a danger as an endogenous source of estrogen, they rather favor the idea that keeping DHEAS levels within a physiological range might be supportive in treating estrogen-responsive breast cancer.

Betaglycan (TßRIII) is a Key Factor in TGF-ß2 Signaling in Prepubertal Rat Sertoli Cells.

Transforming growth factor-ßs (TGF-ßs) signal after binding to the TGF-ß receptors TßRI and TßRII. Recently, however, betaglycan (BG) was identified as an important co-receptor, especially for TGF-ß2. Both proteins are involved in several testicular functions. Thus, we analyzed the importance of BG for TGF-ß1/2 signaling in Sertoli cells with ELISAs, qRT-PCR, siRNA silencing and BrdU assays. TGF-ß1 as well as TGF-ß2 reduced shedding of membrane-bound BG (mBG), thus reducing the amount of soluble BG (sBG), which is often an antagonist to TGF-ß signaling. Treatment of Sertoli cells with GM6001, a matrix metalloproteinases (MMP) inhibitor, also counteracted BG shedding, thus suggesting MMPs to be mainly involved in shedding. Interestingly, TGF-ß2 but not TGF-ß1 enhanced secretion of tissue inhibitor of metalloproteinases 3 (TIMP3), a potent inhibitor of MMPs. Furthermore, recombinant TIMP3 attenuated BG shedding. Co-stimulation with TIMP3 and TGF-ß1 reduced phosphorylation of Smad3, while a combination of TIMP3/TGF-ß2 increased it. Silencing of BG as well as TIMP3 reduced TGF-ß2-induced phosphorylation of Smad2 and Smad3 significantly, once more highlighting the importance of BG for TGF-ß2 signaling. In contrast, this effect was not observed with TIMP3/TGF-ß1. Silencing of BG and TIMP3 decreased significantly Sertoli cell proliferation. Taken together, BG shedding serves a major role in TGF-ß2 signaling in Sertoli cells.

The Sialic Acid-Dependent Nematocyst Discharge Process in Relation to Its Physical-Chemical Properties Is A Role Model for Nanomedical Diagnostic and Therapeutic Tools.

Formulas derived from theoretical physics provide important insights about the nematocyst discharge process of Cnidaria (Hydra, jellyfishes, box-jellyfishes and sea-anemones). Our model description of the fastest process in living nature raises and answers questions related to the material properties of the cell- and tubule-walls of nematocysts including their polysialic acid (polySia) dependent target function. Since a number of tumor-cells, especially brain-tumor cells such as neuroblastoma tissues carry the polysaccharide chain polySia in similar concentration as fish eggs or fish skin, it makes sense to use these findings for new diagnostic and therapeutic approaches in the field of nanomedicine. Therefore, the nematocyst discharge process can be considered as a bionic blue-print for future nanomedical devices in cancer diagnostics and therapies. This approach is promising because the physical background of this process can be described in a sufficient way with formulas presented here. Additionally, we discuss biophysical and biochemical experiments which will allow us to define proper boundary conditions in order to support our theoretical model approach. PolySia glycans occur in a similar density on malignant tumor cells than on the cell surfaces of Cnidarian predators and preys. The knowledge of the polySia-dependent initiation of the nematocyst discharge process in an intact nematocyte is an essential prerequisite regarding the further development of target-directed nanomedical devices for diagnostic and therapeutic purposes. The theoretical description as well as the computationally and experimentally derived results about the biophysical and biochemical parameters can contribute to a proper design of anti-tumor drug ejecting vessels which use a stylet-tubule system. Especially, the role of nematogalectins is of interest because these bridging proteins contribute as well as special collagen fibers to the elastic band properties. The basic concepts of the nematocyst discharge process inside the tubule cell walls of nematocysts were studied in jellyfishes and in Hydra which are ideal model organisms. Hydra has already been chosen by Alan Turing in order to figure out how the chemical basis of morphogenesis can be described in a fundamental way. This encouraged us to discuss the action of nematocysts in relation to morphological aspects and material requirements. Using these insights, it is now possible to discuss natural and artificial nematocyst-like vessels with optimized properties for a diagnostic and therapeutic use, e.g., in neurooncology. We show here that crucial physical parameters such as pressure thresholds and elasticity properties during the nematocyst discharge process can be described in a consistent and satisfactory way with an impact on the construction of new nanomedical devices.

Cardiotonic steroid ouabain stimulates steroidogenesis in Leydig cells via the α3 isoform of the sodium pump.

Cardiotonic steroids such as ouabain are potent inhibitors of the sodium pump and have been widely used for centuries in the treatment of congestive heart failure. In recent decades, however, they have also been identified as hormone-like molecules that trigger signaling cascades of physiological relevance by using the various sodium pump α subunit isoforms as receptors. The murine Leydig cell line MLTC-1 expresses both the ubiquitous, relatively ouabain-insensitive α1 isoform of the sodium pump and the ouabain-sensitive α3 isoform that is normally found in neuronal cells. The physiological relevance of the simultaneous presence of the two isoforms in Leydig cells has not been previously addressed. MLTC-1 Leydig cells contain lipid droplets (LDs) and are capable of progesterone biosynthesis when stimulated by luteinizing hormone (LH). When exposed to low nanomolar concentrations of ouabain, they respond with stimulation of Erk1/2, CREB, and ATF-1 phosphorylation, LD enlargement, and perilipin2 mobilization to the LDs. As a result, progesterone biosynthesis is augmented. Abrogation of α3 isoform expression by siRNA prevents all of the above responses, indicating that it is the hormone/receptor-like interaction of ouabain exclusively with this isoform that triggers the signaling events that normally occur when LH binds to its receptor. Considering that ouabain is produced endogenously and is found in seminal fluid, one can speculate that effects of this substance on germ and somatic cells of the testis might play a role in male reproductive physiology.

ZIP9 but not the androgen receptor mediates testosterone-induced migratory activity of metastatic prostate cancer cells.

LNCaP cells are derived from a metastatic lesion of human prostate adenocarcinoma. They express the classical androgen receptor (AR) and ZIP9, a Zn2+ transporter that also binds testosterone and mediates signaling by interacting with G-proteins. Our results show that LNCaP cells respond to testosterone by mobilizing their migratory machinery. Their exposure to testosterone triggers the formation of lamellipodia, reorganization of the actin cytoskeleton, phosphorylation of focal adhesion kinase (FAK) at Tyr925 and of paxillin at Tyr118, expression of matrix metalloproteinase 2 (MMP-2), and cell migration. Silencing ZIP9 expression by means of siRNA does not affect the responsiveness of the classical AR to testosterone; however, it prevents all of the testosterone effects described above: formation of lamellipodia cannot be induced, stimulation of FAK or paxillin phosphorylation or MMP-2 expression is prevented, and cell migration does not take place in the absence of ZIP9. The data presented show that testosterone/ZIP9 interactions might have not only physiological but also pathophysiological relevance. The fact that the migratory machinery of a metastatic prostate cancer cell line is activated exclusively through testosterone/ZIP9 and not through testosterone/AR interactions suggests that targeting specific inhibition of testosterone/ZIP9-mediated events might help in developing new therapeutic strategies against androgen-induced progression of prostate cancer.

Impairment of the Gnα11-controlled expression of claudin-1 and MMP-9 and collective migration of human breast cancer MCF-7 cells by DHEAS.

Although dehydroepiandrosterone sulfate (DHEAS) constitutes the most abundant steroid in humans, in-depth investigations of its effects are rather scarce. We address here DHEAS effects on the estrogen receptor-positive metastatic human breast cancer cell line MCF-7. We focus on DHEAS-mediated signaling that might influence expression of claudin-1 and matrix metalloproteinase-9 (MMP-9), both known to be critical factors for migration and invasiveness of various cancers, including breast cancer cells. Physiological concentrations of DHEAS trigger persistent phosphorylation of Erk1/2 in MCF-7 cells. Exposure of these cells for 24 h to 1 µM DHEAS also leads to a significant reduction of claudin-1 expression that cannot be prevented by high concentrations of the steroid sulfatase inhibitor STX64, indicating that desulfation and further conversion of DHEAS to some other steroid hormone is not required for this action. In addition, exposure of MCF-7 cells to the same concentration of DHEAS completely abolishes MMP-9 expression and considerably impairs cell migratory behavior. Abrogation of Gnα11 expression by siRNA prevents the stimulatory effect of DHEAS on Erk1/2 phosphorylation, consistent with a G-protein-coupled receptor being involved in the DHEAS-induced signaling. Nevertheless, Gnα11 also has direct effects that do not depend on DHEAS; thus, when Gnα11 expression is suppressed, expression of claudin-1 and MMP-9 as well as cell migration are significantly reduced. This is the first report demonstrating direct involvement of DHEAS and Gnα11 in the regulation of claudin-1 and MMP-9 expression and migration of MCF-7 cells.

Testosterone/bicalutamide antagonism at the predicted extracellular androgen binding site of ZIP9.

ZIP9 is a Zn2+ transporter, testosterone receptor, and mediator of signaling events through G-proteins. Despite these pivotal properties, however, its physiological and pathophysiological significance has not yet been comprehensively addressed. Using a cell line that lacks the classical androgen receptor we show that ZIP9-mediated phosphorylation of Erk1/2, CREB, or ATF-1 and expression of claudin-5 and zonula occludens-1 by testosterone can be completely antagonized by bicalutamide (Casodex), an anti-androgen of significant clinical impact. Computational modeling and docking experiments with ZIP9 reveal typical characteristics of ZIP transporters and an extracellular binding site for testosterone capable of accommodating bicalutamide. The presence of this site is verified by our demonstration that the membrane-impermeable testosterone analogue T-BSA-FITC labels the membrane only when ZIP9 is expressed and that this labeling is completely prevented by bicalutamide. The study connects structural features of ZIP9 to its functions and indicates a possible relevance of ZIP9 as a pharmacological target.

Signaling events associated with gonadotropin releasing hormone-agonist-induced hormonal castration and its reversal in canines.

A gonadotropin-releasing hormone agonist (GnRH-A) implant induces hormonal castration in dogs that is associated with reduced prostate and testes size. We address the molecular events associated with hormonal castration by examining GnRH-A effects on expression and phosphorylation of a number of key signaling proteins. Male beagles were treated for 5 months with a GnRH-A implant, and then surgically castrated at 0, 3, 6, 12, and, 24 weeks after implant removal; untreated animals served as controls. GnRH-A treatment led to activation of c-Raf, Erk1/2, and, p53 in the testes. Phosphorylation of p53 occurred at Ser15, consistent with activation of the c-Raf-Erk1/2-p53 signaling cascade that triggers growth arrest or apoptosis. GnRH-A also suppressed the anti-apoptotic protein Bcl-xL; reduced phosphorylation of the transcription factors CREB and ATF1; and down-regulated expression of StAR and P450scc, proteins involved in steroidogenesis. Although androgen receptor expression was little affected by GnRH-A treatment, levels of ZIP9, a membrane-bound Zn2+ transporter that mediates non-classical signaling of testosterone, were abrogated. All of these effects were reversed within 24 weeks after implant removal. Thus, molecular signatures of implant-dependent hormonal castration include reversible cell cycle arrest and apoptosis, loss of steroidogenesis, and reduced transcriptional activity. Mol. Reprod. Dev. 83: 1092-1101, 2016. © 2016 Wiley Periodicals, Inc.

Ouabain interactions with the α4 isoform of the sodium pump trigger non-classical steroid hormone signaling and integrin expression in spermatogenic cells.

In addition to the ubiquitous α1 isoform of the sodium pump, sperm cells also express a male-specific α4 isoform whose function has been associated with sperm motility, fertility, and capacitation. Here we investigate in the murine spermatogenic cell line GC-2 interactions of the α4 isoform with the cardiotonic steroid ouabain in signaling cascades involved in the non-classical action of steroid hormones. Exposure of GC-2 cells to low concentrations of ouabain stimulates the phosphorylation of Erk1/2 and of the transcription factors CREB and ATF-1. As a consequence of this signaling cascade, ouabain stimulates on the mRNA level the expression of integrins αv, ß3 and α5, whose expression is also modulated by the cAMP response element. Increased expression of integrins αv and ß3 is also seen in cultures of seminiferous tubules exposed to 10nM ouabain. At the protein level we observed a significant stimulation of ß3 integrin expression by ouabain. Abrogation of α4 isoform expression by siRNA leads to the complete suppression of all ouabain-induced signaling mentioned above, including its stimulatory effect on the expression of ß3 integrin. The results presented here demonstrate for the first time the induction of signaling cascades through the interaction of ouabain with the α4 isoform in a germ-cell derived cell line. The novel finding that these interactions lead to increased expression of integrins in GC-2 cells and the confirmation of these results in the ex vivo experiments indicate that hormone/receptor-like interactions of ouabain with the α4 isoform might be of significance for male physiology.

Non-classical testosterone signaling mediated through ZIP9 stimulates claudin expression and tight junction formation in Sertoli cells.

In the classical signaling pathway, testosterone regulates gene expression by activating the cytosolic/nuclear androgen receptor. In the non-classical pathway, testosterone activates cytosolic signaling cascades that are normally triggered by growth factors. The nature of the receptor involved in this signaling pathway is a source of controversy. In the Sertoli cell line 93RS2, which lacks the classical AR, we determined that testosterone stimulates the non-classical signaling pathway, characterized by the phosphorylation of Erk1/2 and transcription factors CREB and ATF-1. We also demonstrated that testosterone increases the expression of the tight junction (TJ) proteins claudin-1 and claudin-5. Both of these proteins are known to be essential constituents of TJs between Sertoli cells, and as a consequence of their increased expression transepithelial resistance across Sertoli cell monolayers is increased. ZIP9 is a Zn(2+)transporter that was recently shown to be a membrane-bound testosterone receptor. Silencing its expression in 93RS2 Sertoli cells by siRNA completely prevents Erk1/2, CREB, and ATF-1 phosphorylation as well the stimulation of claudin-1 and -5 expression and TJ formation between neighboring cells. The study presented here demonstrates for the first time that in Sertoli cells testosterone acts through the receptor ZIP9 to trigger the non-classical signaling cascade, resulting in increased claudin expression and TJ formation. Since TJ formation is a prerequisite for the maintenance of the blood-testis barrier, the testosterone/ZIP9 effects might be significant for male physiology. Further assessment of these interactions will help to supplement our knowledge concerning the mechanism by which testosterone plays a role in male fertility.

Cardiac glycoside ouabain induces activation of ATF-1 and StAR expression by interacting with the α4 isoform of the sodium pump in Sertoli cells.

Sertoli cells express α1 and α4 isoforms of the catalytic subunit of Na(+),K(+)-ATPase (sodium pump). Our recent findings demonstrated that interactions of the α4 isoform with cardiotonic steroids (CTS) like ouabain induce signaling cascades that resemble the so-called non-classical testosterone pathway characterized by activation of the c-Src/c-Raf/Erk1/2/CREB signaling cascade. Here we investigate a possible physiological significance of the activated cascade. The results obtained in the current investigation show that the ouabain-induced signaling cascade also leads to the activation of the CREB-related activating transcription factor 1 (ATF-1) in the Sertoli cell line 93RS2 in a concentration- and time-dependent manner, as demonstrated by detection of ATF-1 phosphorylated on Ser63 in western blots. The ouabain-activated ATF-1 protein was found to localize to the cell nuclei. The sodium pump α4 isoform mediates this activation, as it is ablated when cells are incubated with siRNA to the α4 isoform. Ouabain also leads to increased expression of steroidogenic acute regulator (StAR) protein, which has been shown to be a downstream consequence of CREB/ATF-1 activation. Taking into consideration that CTS are most likely produced endogenously, the demonstrated induction of StAR expression by ouabain establishes a link between CTS, the α4 isoform of the sodium pump, and steroidogenesis crucial for male fertility and reproduction.

Cardiotonic steroids trigger non-classical testosterone signaling in Sertoli cells via the α4 isoform of the sodium pump.

The α4 isoform of the Na(+),K(+)-ATPase (sodium pump) is known to be expressed in spermatozoa and to be critical for their motility. In the investigation presented here, we find that the rat-derived Sertoli cell line 93RS2 also expresses considerable amounts of the α4 isoform in addition to the α1 isoform. Since Sertoli cells are not motile, one can assume that the function of the α4 isoform in these cells must differ from that in spermatozoa. Thus, we assessed a potential involvement of this isoform in signaling pathways that are activated by the cardiotonic steroid (CTS) ouabain, a highly specific sodium pump ligand. Treatment of 93RS2 cells with ouabain leads to activation of the c-Src/c-Raf/Erk1/2 signaling cascade. Furthermore, we show for the first time that the activation of this cascade by ouabain results in phosphorylation and activation of the transcription factor CREB. This signaling cascade is induced at low nanomolar concentrations of ouabain, consistent with the involvement of the α4 isoform. This is further supported by experiments involving siRNA: silencing of α4 expression entirely blocks ouabain-induced activation of Erk1/2 whereas silencing of α1 has no effect. The findings of this study unveil new aspects in CTS/sodium pump interactions by demonstrating for the first time ouabain-induced signaling through the α4 isoform. The c-Src/c-Raf/Erk1/2/CREB cascade activated by ouabain is identical to the so-called non-classical signaling cascade that is normally triggered in Sertoli cells by testosterone. Taking into consideration that CTS are produced endogenously, our results may help to gain new insights into the physiological mechanisms associated with male fertility and reproduction.

Sodium pump alpha1 and alpha3 subunit isoforms mediate distinct responses to ouabain and are both essential for survival of human neuroblastoma.

Using SK-N-AS human neuroblastoma cells, which co-express the alpha1 and alpha3 isoforms of the sodium pump alpha subunit, we selectively silenced either the alpha1 or alpha3 subunit by means of transfection with small interfering RNA, and investigated cell survival and the cellular response to ouabain. We found that both of the alpha subunits are essential for cell survival, indicating that substitution of one subunit for the other is not sufficient. In the presence of both alpha subunits, ouabain causes sustained activation of extracellular signal-regulated kinases 1 and 2 (Erk1/2). This activation is not affected when the alpha1 subunit is silenced. However, when alpha3 expression is silenced, ouabain-induced activation of Erk1/2 does not occur, even at a high concentration of ouabain (1 microM). Thus, ouabain-induced Erk1/2 activation is mediated in SK-N-AS cells by alpha3 only, and alpha1 does not participate in this event. This is a clear demonstration of selective involvement of a specific sodium pump alpha subunit isoform in ouabain-induced signaling.

Isoform specificity of cardiac glycosides binding to human Na+,K+-ATPase alpha1beta1, alpha2beta1 and alpha3beta1.

Cardiac glycosides inhibit the Na(+),K(+)-ATPase and are used for the treatment of symptomatic heart failure and atrial fibrillation. In human heart three isoforms of Na(+),K(+)-ATPase are expressed: alpha(1)beta(1), alpha(2)beta(1) and alpha(3)beta(1). It is unknown, if clinically used cardiac glycosides differ in isoform specific affinities, and if the isoforms have specific subcellular localization in human cardiac myocytes. Human Na(+),K(+)-ATPase isoforms alpha(1)beta(1), alpha(2)beta(1) and alpha(3)beta(1) were expressed in yeast which has no endogenous Na(+),K(+)-ATPase. Isoform specific affinities of digoxin, digitoxin, beta-acetyldigoxin, methyldigoxin and ouabain were assessed in [(3)H]-ouabain binding assays in the absence or presence of K(+) (each n=5). The subcellular localizations of the Na(+),K(+)-ATPase isoforms were investigated in isolated human atrial cardiomyocytes by immunohistochemistry. In the absence of K(+), methyldigoxin (alpha(1)>alpha(3)>alpha(2)) and ouabain (alpha(1)=alpha(3)>alpha(2)) showed distinct isoform specific affinities, while for digoxin, digitoxin and beta-acetyldigoxin no differences were found. In the presence of K(+), also digoxin (alpha(2)=alpha(3)>alpha(1)) and beta-acetyldigoxin (alpha(1)>alpha(3)) had isoform specificities. A comparison between the cardiac glycosides demonstrated highly different affinity profiles for the isoforms. Immunohistochemistry showed that all three isoforms are located in the plasma membrane and in intracellular membranes, but only alpha(1)beta(1) and alpha(2)beta(1) are located in the T-tubuli. Cardiac glycosides show distinct isoform specific affinities and different affinity profiles to Na(+),K(+)-ATPase isoforms which have different subcellular localizations in human cardiomyocytes. Thus, in contrast to current notion, different cardiac glycoside agents may significantly differ in their pharmacological profile which could be of hitherto unknown clinical relevance.