Judith C. Williams

Plasma Membrane Ca2+ ATPase 4 Is Required for Sperm Motility and Male Fertility

Calcium and Ca2+-dependent signals play a crucial role in sperm motility and mammalian fertilization, but the molecules and mechanisms underlying these Ca2+-dependent pathways are incompletely understood. Here we show that homozygous male mice with a targeted gene deletion of isoform 4 of the plasma membrane calcium/calmodulin-dependent calcium ATPase (PMCA), which is highly enriched in the sperm tail, are infertile due to severely impaired sperm motility. Furthermore, the PMCA inhibitor 5-(and-6)-carboxyeosin diacetate succinimidyl ester reduced sperm motility in wild-type animals, thus mimicking the effects of PMCA4 deficiency on sperm motility and supporting the hypothesis of a pivotal role of the PMCA4 on the regulation of sperm function and intracellular Ca2+ levels.

The sarcolemmal calcium pump, alpha-1 syntrophin, and neuronal nitric-oxide synthase are parts of a macromolecular protein complex.

The main role of the plasma membrane Ca2+/calmodulin-dependent ATPase (PMCA) is in the removal of Ca2+ from the cytosol. Recently, we and others have suggested a new function for PMCA as a modulator of signal transduction pathways. This paper shows the physical interaction between PMCA (isoforms 1 and 4) and α-1 syntrophin and proposes a ternary complex of interaction between endogenous PMCA, α-1 syntrophin, and NOS-1 in cardiac cells. We have identified that the linker region between the pleckstrin homology 2 (PH2) and the syntrophin unique (SU) domains, corresponding to amino acids 399–447 of α-1 syntrophin, is crucial for interaction with PMCA1 and -4. The PH2 and the SU domains alone failed to interact with PMCA. The functionality of the interaction was demonstrated by investigating the inhibition of neuronal nitric-oxide synthase-1 (NOS-1); PMCA is a negative regulator of NOS-1-dependent NO production, and overexpression of α-1 syntrophin and PMCA4 resulted in strongly increased inhibition of NO production. Analysis of the expression levels ofα-1 syntrophin protein in the heart, skeletal muscle, brain, uterus, kidney, or liver of PMCA4–/– mice, did not reveal any differences when compared with those found in the same tissues of wild-type mice. These results suggest that PMCA4 is tethered to the syntrophin complex as a regulator of NOS-1, but its absence does not cause collapse of the complex, contrary to what has been reported for other proteins within the complex, such as dystrophin. In conclusion, the present data demonstrate for the first time the localization of PMCA1b and -4b to the syntrophin·dystrophin complex in the heart and provide a specific molecular mechanism of interaction as well as functionality.

Neuronal Nitric Oxide Synthase Signaling in the Heart Is Regulated by the Sarcolemmal Calcium Pump 4b

Background— Neuronal nitric oxide synthase (nNOS) has recently been shown to be a major regulator of cardiac contractility. In a cellular system, we have previously shown that nNOS is regulated by the isoform 4b of plasma membrane calcium/calmodulin-dependent ATPase (PMCA4b) through direct interaction mediated by a PDZ domain (PSD 95, Drosophilia Discs large protein and Zona occludens-1) on nNOS and a cognate ligand on PMCA4b. It remains unknown, however, whether this interaction has physiological relevance in the heart in vivo. Methods and Results— We generated 2 strains of transgenic mice overexpressing either human PMCA4b or PMCA ct120 in the heart. PMCA ct120 is a highly active mutant form of the pump that does not interact with or modulate nNOS function. Calcium was extruded normally from PMCA4b-overexpressing cardiomyocytes, but in vivo, overexpression of PMCA4b reduced the &bgr;-adrenergic contractile response. This attenuated response was not observed in ct120 transgenic mice. Treatment with a specific nNOS inhibitor (N&ohgr;-propyl-l-arginine) reduced the &bgr;-adrenergic response in wild-type and ct120 transgenic mice to levels comparable to those of PMCA4b transgenic animals. No differences in lusitropic response were observed in either transgenic strain compared with wild-type littermates. Conclusions— These data demonstrate the physiological relevance of the interaction between PMCA4b and nNOS and suggests its signaling role in the heart.

The sarcolemmal calcium pump inhibits the calcineurin/nuclear factor of activated T-cell pathway via interaction with the calcineurin A catalytic subunit

The calcineurin/nuclear factor of activated T-cell (NFAT) pathway represents a crucial transducer of cellular function. There is increasing evidence placing the sarcolemmal calcium pump, or plasma membrane calcium/calmodulin ATPase pump (PMCA), as a potential modulator of signal transduction pathways. We demonstrate a novel interaction between PMCA and the calcium/calmodulin-dependent phosphatase, calcineurin, in mammalian cells. The interaction domains were located to the catalytic domain of PMCA4b and the catalytic domain of the calcineurin A subunit. Endogenous calcineurin activity, assessed by measuring the transcriptional activity of its best characterized substrate, NFAT, was significantly inhibited by 60% in the presence of ectopic PMCA4b. This inhibition was notably reversed by the co-expression of the PMCA4b interaction domain, demonstrating the functional significance of this interaction. PMCA4b was, however, unable to confer its inhibitory effect in the presence of a calcium/calmodulin-independent constitutively active mutant calcineurin A suggesting a calcium/calmodulin-dependent mechanism. The modulatory function of PMCA4b is further supported by the observation that endogenous calcineurin moves from the cytoplasm to the plasma membrane when PMCA4b is overexpressed. We suggest recruitment by PMCA4b of calcineurin to a low calcium environment as a possible explanation for these findings. In summary, our results offer strong evidence for a novel functional interaction between PMCA and calcineurin, suggesting a role for PMCA as a negative modulator of calcineurin-mediated signaling pathways in mammalian cells. This study reinforces the emerging role of PMCA as a molecular organizer and regulator of signaling transduction pathways.

Construction and characterization of multiple human colon cancer cell lines for inducibly regulated gene expression

Validation of targets for cancer drug discovery requires robust experimental models. Systems based on inducible gene expression are well suited to this purpose but are difficult to establish in several epithelial cell types. Using the recently discovered transcriptional transactivator (rtTA2S‐M2), we developed a strategy for fast and efficient generation of Tet On cells. Multiple clones of HCT116, SW480, and HT29 human colon cancer cells for doxycycline‐regulated gene expression were constructed that constitutively express green fluorescent protein (GFP) for selection/maintenance purposes. The cell lines displayed good fold inducibility (49–124× HCT116; 178–621× SW480; 261–787× HT29) and minimal leakiness after transient transfection with a luciferase reporter or with vectors driving inducible expression of red fluorescent protein (dsRed2), constitutively active c‐Src or dominant negative K‐Ras4B. The clones preserved their transformed phenotype as demonstrated by comparing their properties to respective wild type cells, in terms of growth in vitro and in vivo (as tumor xenografts), cell cycle traverse, and sensitivity to drugs used in chemotherapy. These engineered cell lines enabled tightly controlled inducible gene expression both in vitro and in vivo, and proved well suited for construction of double‐stable cell lines inducibly expressing a protein of interest. As such they represent a useful research tool for example, to dissect oncogene function(s) in colon cancer. Supplementary material for this article be found at http://www.mrw.interscience.wiley.com/suppmat/0730‐2312/suppmat/94/suppmat_welman.doc.

Adenovirus vector-mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane.

Carboxypeptidase G2 (CPG2) is a powerful prodrug-converting enzyme. Without a requirement for endogenous enzymes or cofactors, it can directly activate mustard alkylating prodrugs to cytotoxic species, killing both quiescent and dividing cells. This paper provides the first report of its use in the context of a clinically relevant delivery vehicle using adenovirus vectors. To strengthen the efficacy of the prodrug-activating system, the enzyme has been engineered to be secreted or glycosylphosphatidylinositol (GPI) anchored to the extracellular membrane of tumor cells, resulting in an enhanced bystander effect by facilitating diffusion of the active drug through extracellular, rather than intracellular, activation. Using the vectors, we have achieved expression of functional secreted or GPI-anchored CPG2 in a panel of tumor cell lines demonstrating no loss in efficacy as a result of GPI anchor retention. Despite variable transduction efficiencies inherent to these vectors, greater than 50% cell kill was achievable in all of the cell lines tested following only a single exposure to the prodrug ZD2767P. Even in cell lines refractive to infection with the vectors, substantial cell death was recorded, indicative of the enhanced bystander effect generated following extracellular prodrug activation. A direct evaluation of the efficacy of our system has been made against adenoviral delivery of herpes simples virus thymidine kinase plus ganciclovir (GCV), a suicide gene therapy approach already in the clinic. In a short-term human glioma culture (IN1760) resistant to the clinical chemotherapeutic drug CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea), thymidine kinase/GCV effected no cell killing compared to 70% cell killing with our system.

Adenovirus expression of IL-1 and NF-κB inhibitors does not inhibit acute adenoviral-induced brain inflammation, but delays immune system-mediated elimination of transgene expression

Despite their ability to provide long-term transgene expression in the central nervous system of naïve hosts, the use of first-generation adenovirus (Ad) vectors for the treatment of chronic neurological disorders is limited by peripheral immunization, which stimulates anti-adenovirus immune responses and causes severe inflammation in the central nervous system (CNS) and elimination of transgene expression. The purpose of this study was to investigate the roles of NF-kappaB and interleukin-1 (IL-1) during inflammatory responses to Ads in the CNS of naïve and preimmunized rats. We assessed activation of macrophages/microglia, up-regulation of MHC I expression, infiltration of leukocytes, and transgene expression following delivery of Ads to the rat striatum. After delivery of increasing doses of adenoviral vectors expressing various anti-inflammatory agents (e.g., NF-kappaB or IL-1 inhibitors) to naïve rats, no reduction in Ad-mediated CNS inflammation was seen 1 week after delivery of Ads, compared to a control Ad.hCMV.beta-galactosidase (RAd.35) virus. We then assessed CNS inflammation and transgene expression at a time when control transgene expression would be completely eliminated, i.e., 1 month post-vector injection into the brain. This would optimize the assessment of an anti-inflammatory agent expressed by an adenoviral vector that could either delay or diminish immune system-mediated elimination of transgene expression. As expected, at 1 month postinfection, control preimmunized rats receiving Ad.mCMV.beta-galactosidase (RAd.36)/saline or RAd.36/Ad.null (RAd.0) showed complete elimination of beta-galactosidase expression in the brain and levels of inflammation comparable to those of naïve animals. However, animals injected with RAd.36 in combination with Ads expressing NF-kappaB or IL-1 inhibitors showed a delayed elimination of beta-galactosidase compared to controls. As predicted, the extended presence of transgene expression was accompanied by increased levels of CNS inflammation. This suggests that blocking NF-kappaB or IL-1 delays, albeit partially, transgene elimination in the presence of a preexisting systemic immune response. Prolonged transgene expression is predicted to extend concurrent brain inflammation, as noted earlier. Taken together these data demonstrate a role for NF-kappaB and IL-1 in immune system-mediated elimination of Ad-mediated CNS transgene expression.

CELL TYPE SPECIFIC AND INDUCIBLE TRANSGENESIS IN THE ANTERIOR PITUITARY GLAND

With the recent emergence of the sequence for the human genome, and the inevitable wealth of DNA sequences that will be gathered from it, the use of genetic manipulation as a tool in uncovering the molecular basis of many physiological processes within the AP gland will become more apparent. The use of knockout and transgenic technologies has already been extremely useful in elucidating roles for many gene products. It is now well established that these approaches have their drawbacks, i.e. although the introduction or deletion of a particular gene may be useful for uncovering a particular rolein vivothe compensatory changes that are likely to occur during development may alter the animals phenotype. This would in turn affect the physiological process, which might occur in wild type animals. Viral vectors offer the possibility of creating transgenics in specific organs, tissues or even regions within a larger organ (i.e. brain nuclei) during adulthood. This offers a very powerful tool to not only express genes of interest, but also to ablate specific cell populations by expressing toxins or depleting specific mRNAs by expressing ribozymes. The possibilities of this technology are enormous both to uncover physiological effects, also for the development of experimental therapeutic strategies, for the treatment of human disease. In this chapter we will explore the possibility of expressing transgenes within specific cell populations in the anterior pituitary gland and the switching “on” and “off” of these exogenous genes.