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The Na+/K+ ATPase is a key regulator of the hepatocytes ionic homeostasis, which when altered may lead to many liver disorders. We demonstrated recently, a significant stimulation of the Na+/K+ ATPase in HepG2 cells treated with the S1P analogue FTY 720P, that was mediated through PGE2. The mechanism by which the prostaglandin exerts its effect was not investigated, and is the focus of this work. The type of receptors involved was determined using pharmacological inhibitors, while western blot analysis, fluorescence imaging of GFP-tagged Na+/K+ ATPase, and time-lapse imaging on live cells were used to detect changes in membrane abundance of the Na+/K+ ATPase. The activity of the ATPase was assayed by measuring the amount of inorganic phosphate liberated in the presence and absence of ouabain. The enhanced activity of the ATPase was not observed when EP4 receptors were blocked but still appeared in presence inhibitors of EP1, EP2 and EP3 receptors. The involvement of EP4 was confirmed by the stimulation observed with EP4 agonist. The stimulatory effect of PGE2 did not appear in presence of Rp-cAMP, an inhibitor of PKA, and was imitated by db-cAMP, a PKA activator. Chelating intracellular calcium with BAPTA-AM abrogated the effect of db-cAMP as well as that of PGE2, but PGE2 treatment in a calcium-free PBS medium did not, suggesting an involvement of intracellular calcium, that was confirmed by the results obtained with 2-APB treatment. Live cell imaging showed movement of GFP–Na+/K+ ATPase-positive vesicles to the membrane and increased abundance of the ATPase at the membrane after PGE2 treatment. It was concluded that PGE2 acts via EP4, PKA, and intracellular calcium.

Cisplatin (CDDP) is currently one of the most effective FDA-approved treatments for breast cancer. Previous studies have shown that CDDP-induced cell death in human breast cancer (MCF-7) cells is associated with disruption of calcium homeostasis. However, whether the sensitivity of breast cancer cells to cisplatin is associated with dysregulation of the expression of calcium-binding proteins (CaBPs) remains unknown. In this study, we evaluated the effect of the intracellular calcium chelator (BAPTA-AM) on viability of MCF-7 cells in the presence of toxic and sub-toxic doses of cisplatin. Furthermore, this study assessed the expression of CaBPs, calmodulin, S100A8, and S100A14 in MCF-7 cells treated with cisplatin. Cell viability was determined using MTT-based in vitro toxicity assay. Intracellular calcium imaging was done using Fluo-4 AM, a cell-permeant fluorescent calcium indicator. Expression of CaBPs was tested using real-time quantitative PCR. Exposure of cells to increasing amounts of CDDP correlated with increasing fluorescence of the intracellular calcium indicator, Fluo-4 AM. Conversely, treating cells with cisplatin significantly decreased mRNA levels of calmodulin, S100A8, and S100A14. Treatment of the cells with calcium chelator, BAPTA-AM, significantly enhanced the cytotoxic effects of sub-toxic dose of cisplatin. Our results indicated a statistically significant negative correlation between calmodulin, S100A8, and S100A14 expression and sensitivity of breast cancer cells to a sub-toxic dose of cisplatin. We propose that modulating the activity of calcium-binding proteins, calmodulin, S100A8, and S100A14, could be used to increase cisplatin efficacy, lowering its treatment dosage while maintaining its chemotherapeutic value., Cisplatin; Calcium-binding proteins; BAPTA-AM; Intracellular calcium.

Progesterone mediates its physiological functions through activation of both transcription-coupled nuclear receptors and seven-pass-transmembrane progesterone receptors (mPRs), which transduce the rapid non-genomic actions of progesterone by coupling to various signaling modules. However, the immediate mechanisms of action downstream of mPRs remain in question. Herein, we use an untargeted quantitative proteomics approach to identify mPR interactors to better define progesterone non-genomic signaling. Surprisingly, we identify the very-low-density lipoprotein receptor (VLDLR) as an mPRβ (PAQR8) partner that is required for mPRβ plasma membrane localization. Knocking down VLDLR abolishes non-genomic progesterone signaling, which is rescued by overexpressing VLDLR. Mechanistically, we show that VLDLR is required for mPR trafficking from the endoplasmic reticulum to the Golgi. Taken together, our data define a novel function for the VLDLR as a trafficking chaperone required for the mPR subcellular localization and, as such, non-genomic progesterone-dependent signaling.This article has an associated First Person interview with the first author of the paper.

Ca2+ is a ubiquitous intracellular messenger that transduces a variety of cellular responses downstream of the activation of G-protein-coupled or tyrosine kinase receptors. Depending on the agonist and cellular context, Ca2+ can mediate different responses in the same cell [1]. The specific cellular response transduced downstream of the particular Ca2+ transient is encoded in the spatial and temporal dynamics of the Ca2+ signal, leading to the activation of a subset of Ca2+-dependent effectors and the ensuing cellular response. As such, the duration, amplitude, frequency, and spatial localization of Ca2+ signals encode targeted signals that activate Ca2+-sensitive effectors to define a particular cellular response. To generate and fine-tune those Ca2+ signals, cells use two main Ca2+ sources: entry of extracellular Ca2+ and Ca2+ release from intracellular stores. The primary intracellular Ca2+ store is the endoplasmic reticulum (ER), which can concentrate Ca2+ in the hundreds of μM range [2]. In contrast, cytoplasmic Ca2+ concentration is kept at rest at extremely low levels (∼100 nM or lower), thus providing a low-noise background for detection of complex Ca2+ dynamics [3].The Ca2+-signaling machinery includes Ca2+ entry and extrusion pathways in the plasma membrane (PM), ER membrane Ca2+ release channels, and Ca2+ reuptake ATPases within the ER membrane [4]. These Ca2+ transport pathways, in addition to intracellular Ca2+ buffers and Ca2+ uptake and release through other intracellular organelles, primarily the mitochondria, combine to shape highly tuned and dynamic Ca2+ transients that regulate cellular functions [5].Under physiological conditions in non-excitable cells, Ca2+ transients are typically initiated downstream of agonist stimulation through the activation of the PLC-IP3 signal transduction cascade, which leads to the opening of intracellular Ca2+ channel inositol 1,4,5-trisphosphate receptors (IP3Rs) to release Ca2+ from intracellular stores [6]. Ca2+ release depletes the stores and activates a Ca2+ influx pathway in the PM termed store-operated Ca2+ entry (SOCE). SOCE is mediated by two key players: ER transmembrane Ca2+ sensors represented by the STIM family of proteins and PM Ca2+ channels of the Orai family that link directly to STIMs (see Chapters 1 through 3). The N-terminus of STIM1 faces the ER lumen and consists of two EF-hand domains that detect luminal Ca2+ concentration. The loss of STIM1 Ca2+ binding upon store depletion leads to conformational changes in the protein and its aggregation into clusters that translocate and stabilize into ER-PM junctions with very close apposition (∼20 nm) [7]. STIM1 within these ER-PM junctions binds to and recruits Orai1 through a diffusional trap mechanism, resulting in opening Orai1 channels and Ca2+ entry [8]. As such, the STIM-Orai clusters at ER-PM junctions define a specific microdomain at ER-PM junctions that also include the ER Ca-ATPase (SERCA) [9,10].The tightly regulated remodeling of the Ca2+-signaling machinery upon store depletion allows for specific Ca2+ signaling in the midrange between Ca2+ microdomains and global Ca2+ waves [10] (see Chapter 5). Spatially, Ca2+ signaling can occur in localized spatially restricted elementary Ca2+ release events that activate effectors located in the immediate proximity of the Ca2+ channel. Alternatively, Ca2+ signals/waves occur/spread through the entire cell resulting in a global spatially unrestricted signal.We have recently described a SOCE-dependent Ca2+-signaling modularity that signals in the midrange between these two spatial extremes [10]. Store depletion downstream of receptor activation and IP3 generation results in a localized Ca2+ entry point source at the SOCE clusters that induces Ca2+ entry into the cytoplasm, which is readily taken up into the ER lumen through SERCA activity only to be released again through open IP3Rs distally to the SOCE entry site and gate Ca2+-activated Cl- channels as downstream Ca2+ effectors. This mechanism, referred to as “Ca2+ teleporting,” allows for specific activation of Ca2+ effectors that are distant from the point source Ca2+ channel without inducing a global Ca2+ wave, thus providing a novel module in the Ca2+-signaling repertoire. A cartoon summary of Ca2+ teleporting is found in Figure 12.1.The relationship between Ca2+ signaling and cellular proliferation is complex with Ca2+ transients detected at various stages of the cell cycle [2]. These transients are thought to activate a multitude of Ca2+ effectors downstream of the initial Ca2+ signal, which were shown to be important for cellular proliferation, including, for example, calmodulin (CaM) and Ca2+-CaM-dependent protein kinase II (CaMKII).However, there are a few cases where Ca2+ signals have been shown directly to be critical for cell cycle progression, including in mitosis for nuclear envelope breakdown and for chromosome disjunction [11]. In contrast, nuclear envelope breakdown during meiosis in vertebrate oocytes occurs independently of Ca2+, but Ca2+ is required for the completion of meiosis I in vertebrate oocytes [12–14]. Interestingly, multiple Ca2+ signaling pathways are modified during M-phase of the cell cycle, with the best defined example being Xenopus oocyte maturation [15].Several Ca2+ influx pathways have been implicated in cell proliferation and cell cycle progression, including TRP channels, voltage-gated Ca2+ channels (CaV), purinergic P2X receptors, ionotropic glutamate receptors, and SOCE [16]. Blockers of voltage-gated Ca2+ channels were shown to slow down cell growth, arguing for a role for these channels in cell cycle progression [16–18]. Experimental manipulation of the expression levels of members of the TRPC, TRPV, and TRPM families of cation channels, which are Ca2+ permeable, was linked to cell proliferation with differential effects depending on the particular channel studied [16]. However, some of these studies are difficult to interpret because channel knockdown or overexpression could have significantly broader effects on Ca2+ signaling than affecting Ca2+ influx through the specific channel in question, as it may lead to changes in expression of other Ca2+-signaling pathways as a compensatory mechanism. Furthermore, the majority of TRP channels conduct cations with some Ca2+ permeability and are not Ca2+ selective like Orai1 or CaV channels, with the exception of TRPV5 and TRPV6 (see Chapter 13). Hence, changes in their expression is likely to affect the ionic balance across the cell membrane with effects on resting membrane potential, which may in turn affect cell proliferation.The relationship between SOCE and cell proliferation is an intimate one that goes beyond the well-recognized roles of Ca2+ signaling in cellular growth and proliferation. SOCE is dramatically downregulated during the division phase of the cell cycle through mechanisms that have not been fully elucidated. This is in line with the significant remodeling of the Ca2+-signaling machinery during M-phase, which has been well characterized during oocyte meiosis. Furthermore, there is mounting evidence from multiple neoplasms for an important role for SOCE in metastasis.This chapter presents a brief overview of our current knowledge as to the mechanisms regulating SOCE during cell cycle from cellular proliferation to metastasis with an emphasis on SOCE regulation during cell division (mitosis and meiosis).

Apoptosis is a regulated form of cell death that proceeds by defined biochemical pathways. Most apoptosis is controlled by interactions between pro-survival and pro-apoptotic Bcl-2 family proteins in which death is often the consequence of permeabilization of the mitochondrial outer membrane. Many drugs affect this equilibrium to favor apoptosis but this process is not completely understood. We show that the chemotherapeutic drug cisplatin initiates an apoptotic pathway by phosphorylation of a pro-survival Bcl-2 family member, Bcl-xL, by cyclin-dependent kinase 2. The phosphorylation occurred at a previously unreported site and its biologic significance was demonstrated by a phosphomimetic modification of Bcl-xL that was able to induce apoptosis without addition of cisplatin. The mechanism of cell death induction was similar to that initiated by pro-apoptotic Bcl-2 family proteins, that is, phosphorylated Bcl-xL translocated to the mitochondrial membrane, and formed pores in the membrane. This initiated cytochrome c release and caspase activation that resulted in cell death.

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. We found that an 18-kDa protein identified by mass spectrometry as p21WAF1/Cip1was phosphorylated by Cdk2 starting 12 h after cisplatin exposure. The analysis showed it was phosphorylated at serine 78, a site not previously identified. The adenoviral transduction of p21 before cisplatin exposure protects from cytotoxicity by inhibiting Cdk2. Although cisplatin causes induction of endogenous p21, the protection is inefficient. We hypothesized that phosphorylation of p21 at serine 78 could affect its role as a Cdk inhibitor, and thereby lessen its ability to protect from cisplatin cytotoxicity. To investigate the effect of serine 78 phosphorylation on p21 activity, we replaced serine 78 with aspartic acid, creating the phosphomimic p21S78D. Mutant p21S78Dwas an inefficient inhibitor of Cdk2 and was inefficient at protecting TKPTS cells from cisplatin-induced cell death. We conclude that phosphorylation of p21 by Cdk2 limits the effectiveness of p21 to inhibit Cdk2, which is the mechanism for continued cisplatin cytotoxicity even after the induction of a protective protein.

Cisplatin cytotoxicity is dependent on cyclin-dependent kinase 2 (Cdk2) activity in vivo and in vitro. A Cdk2 mutant (Cdk2-F80G) was designed in which the ATP-binding pocket was altered. When expressed in mouse kidney cells, this protein was kinase inactive, did not inhibit endogenous Cdk2, but protected from cisplatin. The mutant was localized in the cytoplasm, but when coexpressed with cyclin A, it was activated, localized to the nucleus, and no longer protected from cisplatin cytotoxicity. Cells exposed to cisplatin in the presence of the activated mutant had an apoptotic phenotype, and endonuclease G was released from mitochondria similar to that mediated by endogenous Cdk2. But unlike apoptosis mediated by wild-type Cdk2, cisplatin exposure of cells expressing the activated mutant did not cause cytochrome c release or significant caspase-3 activation. We conclude that cisplatin likely activates both caspase-dependent and -independent cell death, and Cdk2 is required for both pathways. The mutant-inactive Cdk2 protected from both death pathways, but after activation by excess cyclin A, caspase-independent cell death predominated.

In contrast to the well-defined role of Ca 2+ signals during mitosis, the contribution of Ca 2+ signaling to meiosis progression is controversial, despite several decades of investigating the role of Ca 2+ and its effectors in vertebrate oocyte maturation. We have previously shown that during Xenopus oocyte maturation, Ca 2+ signals are dispensable for entry into meiosis and for germinal vesicle breakdown. However, normal Ca 2+ homeostasis is essential for completion of meiosis I and extrusion of the first polar body. In this study, we test the contribution of several downstream effectors in mediating the Ca 2+ effects during oocyte maturation. We show that calmodulin and calcium-calmodulin-dependent protein kinase II (CAMK2) are not critical downstream Ca 2+ effectors during meiotic maturation. In contrast, accumulation of Aurora kinase A (AURKA) protein is disrupted in cells deprived of Ca 2+ signals. Since AURKA is required for bipolar spindle formation, failure to accumulate AURKA may contribute to the defective spindle phenotype following Ca 2+ deprivation. These findings argue that Ca 2+ homeostasis is important in establishing the oocyte’s competence to undergo maturation in preparation for fertilization and embryonic development.

An effect of PGE2 on water and chloride absorption was already established in a previous work. This study is an attempt to find the mechanism of action of the prostaglandin by investigating the involvement of three major transporters namely the Na+ -K+ ATPase, the Na+/H+ exchanger and the Na+ K+ 2Cl- cotransporter. Rats were injected with PGE2 and 15 min later, the colon was perfused in situ with Krebs Ringer buffer, and net water and chloride absorption were determined. When the involvement of the cotransporter and/or the exchanger was investigated, animals were injected with, respectively, furosemide and amiloride 10 min before PGE2. Superficial and crypt colonocytes were then isolated and the protein expression of the Na+ -K+ ATPase and the Na+ K+ 2Cl- was determined by western blot analysis. The effect of PGE2 on the pump activity in presence or absence of the transporters' inhibitors was also studied. PGE2 decreased net water and chloride absorption from the colon, increased the Na+ -K+ ATPase activity in superficial cells and reduced it in crypt cells. The prostaglandin was found to stimulate secretion in superficial cells by targeting the Na+ K+ 2Cl- symporter, and reduce absorption in crypt cells by targeting the Na+/H+ antiporter. Changes in the activity of the pump are secondary to changes in the activity of the other transporters.

This work investigated the effect of different doses of PGE2 on net water and Cl− absorption from the rat colon, using an in situ perfusion technique. PGE2 exerted opposite effects at different concentrations. Net water and Cl− absorption was significantly reduced at low doses with a minimum at 0.4 μg/100 g BW, and significantly elevated at high doses with an observed maximal effect at 21 μg/100 g BW. At low doses, PGE2 increased in superficial cells, the activity of the Na+–K+ ATPase and the protein expression of the Na+K+2Cl− cotransporter, but reduced them in crypt cells. Thus, the reduction in net water and Cl− absorption was ascribed to an increase in secretion by surface cells that masked absorptive processes. At high doses, PGE2 increased significantly the activity of the Na+–K+ ATPase in superficial cells only, and was without any effect on the protein expression of the cotransporter and the pump in both surface and crypt cells. The observed increase in net water and Cl− absorption was attributed in this case to an increase in absorptive processes with no effect on secretion.

Endoplasmic reticulum (ER) stress contributes to acute kidney injury induced by several causes. Kidney dysfunction was shown to be influenced by gender differences. In this study we observed differences in the severity of kidney injury between male and female mice in response to tunicamycin, an ER stress agent. Tunicamycin-treated male mice showed a severe decline in kidney function and extensive kidney damage of proximal tubules in the kidney outer cortex (S1 and S2 segments). Interestingly, female tunicamycin-treated mice did not show a decline in kidney function, and their kidneys showed damage localized primarily to proximal tubules in the inner cortex (S3 segment). Protein markers of ER stress, glucose-regulated protein, and X-box binding protein 1 were also more elevated in male mice. Similarly, the induction of apoptosis was higher in tunicamycin-treated male mice, as measured by the activation of Bax and caspase-3. Testosterone administered to female mice before tunicamycin resulted in a phenotype similar to male mice with a comparable decline in renal function, tissue morphology, and induction of ER stress markers. We conclude that kidneys of male mice are much more susceptible to ER stress-induced acute kidney injury than those of females. Moreover, this sexual dimorphism could provide an interesting model to study the relation between kidney function and injury to a specific nephron segment.

Vertebrate oocytes are maintained in meiotic arrest for prolonged periods of time before undergoing oocyte maturation in preparation for fertilization. Cyclic AMP (cAMP) signaling plays a crucial role in maintaining meiotic arrest, which is released by a species-specific hormonal signal. Evidence in both frog and mouse argues that meiotic arrest is maintained by a constitutively active G-protein coupled receptor (GPCR) leading to high cAMP levels. Because activated GPCRs are typically targeted for endocytosis as part of the signal desensitization pathway, we were interested in determining the role of trafficking at the cell membrane in maintaining meiotic arrest. Here we show that blocking exocytosis, using a dominant-negative SNAP25 mutant in Xenopus oocytes, releases meiotic arrest independently of progesterone. Oocyte maturation in response to the exocytic block induces the MAPK and Cdc25C signaling cascades, leading to MPF activation, germinal vesicle breakdown and arrest at metaphase of meiosis II with a normal bipolar spindle. It thus replicates all tested aspects of physiological maturation. Furthermore, inhibiting clathrin-mediated endocytosis hinders the effectiveness of progesterone in releasing meiotic arrest. These data show that vesicular traffic at the cell membrane is crucial in maintaining meiotic arrest in vertebrates, and support the argument for active recycling of a constitutively active GPCR at the cell membrane.