Faculty Bibliography

Taxol (Paclitaxel) is an important natural product for the treatment of solid tumors. Despite a well documented tubulin-stabilizing effect, many side effects of taxol therapy cannot be explained by cytoskeletal mechanisms. In the present study submicromolar concentrations of taxol, mimicking concentrations found in patients, induced cytosolic calcium (Ca(2+)) oscillations in a human neuronal cell line. These oscillations were independent of extracellular and mitochondrial Ca(2+) but dependent on intact signaling via the phosphoinositide signaling pathway. We identified a taxol binding protein, neuronal Ca(2+) sensor 1 (NCS-1), a Ca(2+) binding protein that interacts with the inositol 1,4,5-trisphosphate receptor from a human brain cDNA phage display library. Taxol increased binding of NCS-1 to the inositol 1,4,5-trisphosphate receptor. Short hairpin RNA-mediated knockdown of NCS-1 in the same cell line abrogated the response to taxol but not to other agonists stimulating the phosphoinositide signaling pathway. These findings are important for studies involving taxol as a research tool in cell biology and may help to devise new strategies for the management of side effects induced by taxol therapy.

In both nonexcitable and excitable cells, the inositol 1,4,5-trisphosphate receptor (IP(3)R) is the primary cytosolic target responsible for the initiation of intracellular calcium (Ca(2+)) signaling. To fulfill this function, the IP(3)R depends on interaction with accessory subunits and regulatory proteins. These include proteins that reside in the lumen of the endoplasmic reticulum (ER), such as chromogranin A and B and ERp44, and cytosolic proteins, such as neuronal Ca(2+) sensor 1, huntingtin, cytochrome c, IP(3)R-binding protein released with inositol 1,4,5-trisphosphate, Homer, and 4.1N. Specific interactions between these modulatory proteins and the IP(3)R have been described, making it clear that the controlled modulation of the IP(3)R by its binding partners is necessary for physiological cell regulation. The functional coupling of these modulators with the IP(3)R can control apoptosis, intracellular pH, the initiation and regulation of neuronal Ca(2+) signaling, exocytosis, and gene expression. The pathophysiological relevance of IP(3)R modulation is apparent when the functional interaction of these proteins is enhanced or abolished by mutation or overexpression. The subsequent deregulation of the IP(3)R leads to pathological changes in Ca(2+) signaling, signal initiation, the amplitude and frequency of Ca(2+) signals, and the duration of the Ca(2+) elevation. Consequences of this deregulation include abnormal growth and apoptosis. Complex regulation of Ca(2+) signaling is required for the cell to live and function, and this difficult task can only be managed when the IP(3)R teams up and acts properly with its numerous binding partners.

Testosterone plays a crucial role in neuronal function, but elevated concentrations can have deleterious effects. Here we show that supraphysiological levels of testosterone (micromolar range) initiate the apoptotic cascade. We used three criteria, annexin V labeling, caspase activity, and DNA fragmentation, to determine that apoptotic pathways were activated by testosterone. Micromolar, but not nanomolar, testosterone concentrations increased the response in all three assays of apoptosis. In addition, testosterone induced different concentration-dependent Ca2+ signaling patterns: at low concentrations of testosterone (100 nm), Ca2+ oscillations were produced, whereas high concentrations (1-10 microm) induced a sustained Ca2+ increase. Elevated testosterone concentrations increase cell death, and this effect was abolished in the presence of either inhibitors of caspases or the inositol 1,4,5-trisphosphate receptor (InsP3R)-mediated Ca2+ release. Knockdown of InsP3R type 1 with specific small interfering RNA also abolished the testosterone-induced cell death and the prolonged Ca2+ signals. In contrast, knockdown of InsP3R type 3 modified neither the apoptotic response nor the Ca2+ signals. These results support our hypothesis that elevated testosterone alters InsP3R type 1-mediated intracellular Ca2+ signaling and that the prolonged Ca2+ signals lead to apoptotic cell death. These effects of testosterone on neurons will have long term effects on brain function.

Regulation and dysregulation of intracellular calcium (Ca2+) signaling via the inositol 1,4,5-trisphosphate receptor (InsP3R) has been linked to many cellular processes and pathological conditions. In the present study, addition of neuronal calcium sensor-1 (NCS-1), a high-affinity, low-capacity, calcium-binding protein, to purified InsP3R type 1 (InsP3R1) increased the channel activity in both a calcium-dependent and -independent manner. In intact cells, enhanced expression of NCS-1 resulted in increased intracellular calcium release upon stimulation of the phosphoinositide signaling pathway. To determine whether InsP3R1/NCS-1 interaction could be functionally relevant in bipolar disorders, conditions in which NCS-1 is highly expressed, we tested the effect of lithium, a salt widely used for treatment of bipolar disorders. Lithium inhibited the enhancing effect of NCS-1 on InsP3R1 function, suggesting that InsP3R1/NCS-1 interaction is an essential component of the pathomechanism of bipolar disorder.

Glucose sensing is essential for the ability of pancreatic beta-cells to produce insulin in sufficient quantities to maintain blood glucose within the normal range. Stress causes the release of adrenergic hormones that increase circulating glucose by promoting glucose production and inhibiting insulin release. We have shown that extracellular signal-regulated kinases 1 and 2 (ERK1/2) are responsive to glucose in pancreatic beta-cells and that glucose activates ERK1/2 by mechanisms independent of insulin. Here we show that glucose-induced activation of ERK1/2 is inhibited by epinephrine through the alpha2-adrenergic receptor. Epinephrine and the selective alpha2-adrenergic agonist UK14304 reduced insulin secretion and glucose-stimulated ERK1/2 activation in a pertussis toxin-sensitive manner, implicating the alpha subunit of a Gi family member. Alpha2-adrenergic agonists also reduced stimulation of ERK1/2 by glucagon-like peptide 1 and KCl, but not by phorbol ester or nerve growth factor. Our findings suggest that alpha2-adrenergic agonists act via a Gi family member on early steps in ERK1/2 activation, supporting the idea that ERK1/2 are regulated in a manner that reflects insulin demand.

Testosterone has short- and long-term roles in regulating neuronal function. Here, we show rapid intracellular androgen receptor-independent effects of testosterone on intracellular Ca2+ in neuroblastoma cells. We identified testosterone-induced Ca2+ signals that began primarily at the neurite tip, followed by propagation towards the nucleus, which was then repeated to create an oscillatory pattern. The initial transient depended upon production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], but subsequent transients required both extracellular Ca2+ influx and Ca2+ release from intracellular stores. Inhibition of pertussis toxin-sensitive G-protein receptors or the use of siRNA for the Ins(1,4,5)P3 receptor type 1 blocked the Ca2+ response, whereas inhibition or knock-down of the intracellular androgen receptor was without effect. Cytosolic and nuclear Ca2+ were buffered with parvalbumin engineered to be targeted to the cytosol or nucleus. Cytoplasmic parvalbumin blocked Ca2+ signaling in both compartments; nuclear parvalbumin blocked only nuclear signals. Expression of a mutant parvalbumin did not modify the testosterone-induced Ca2+ signal. Neurite outgrowth in neuroblastoma cells was enhanced by the addition of testosterone. This effect was inhibited when cytosolic Ca2+ was buffered and was attenuated when parvalbumin was targeted to the nucleus. Our results are consistent with a fast effect of testosterone, involving Ins(1,4,5)P3-mediated Ca2+ oscillations and support the notion that there is synergism in the pathways used for neuronal cell differentiation involving rapid non-genomic effects and the classical genomic actions of androgens.

Gain-of-function mutations in SHP-2/PTPN11 cause Noonan syndrome, a human developmental disorder. Noonan syndrome is characterized by proportionate short stature, facial dysmorphia, increased risk of leukemia, and congenital heart defects in approximately 50% of cases. Congenital heart abnormalities are common in Noonan syndrome, but the signaling pathway(s) linking gain-of-function SHP-2 mutants to heart disease is unclear. Diverse cell types coordinate cardiac morphogenesis, which is regulated by calcium (Ca2+) and the nuclear factor of activated T-cells (NFAT). It has been shown that the frequency of Ca2+ oscillations regulates NFAT activity. Here, we show that in fibroblasts, Ca2+ oscillations in response to FGF-2 require the phosphatase activity of SHP-2. Conversely, gain-of-function mutants of SHP-2 enhanced FGF-2-mediated Ca2+ oscillations in fibroblasts and spontaneous Ca2+ oscillations in cardiomyocytes. The enhanced frequency of cardiomyocyte Ca2+ oscillations induced by a gain-of-function SHP-2 mutant correlated with reduced nuclear translocation and transcriptional activity of NFAT. These data imply that gain-of-function SHP-2 mutants disrupt the Ca2+ oscillatory control of NFAT, suggesting a potential mechanism for congenital heart defects in Noonan syndrome.

Ca(2+) signals control DNA synthesis and repair, gene transcription, and other cell functions that occur within the nucleus. The nuclear envelope can store Ca(2+) and release it into the nucleus via either the inositol 1,4,5-trisphosphate receptor (InsP3R) or the ryanodine receptor (RyR). Furthermore, many cell types have a reticular network within their nuclei and InsP3Rs on this nucleoplasmic reticulum permit local subnuclear control of Ca(2+) signals and Ca(2+)-dependent intranuclear events. However, it is unknown whether RyR similarly is expressed on the nucleoplasmic reticulum and can control subnuclear Ca(2+) signals. Here we report that the type 1 RyR is expressed on intranuclear extensions of the sarcoplasmic reticulum of C2C12 cells, a skeletal muscle derived cell line. In addition, two-photon photorelease of caged Ca(2+) in the region of the nucleoplasmic reticulum evoked Ca(2+)-induced Ca(2+) release (CICR) within the nucleus, which could be suppressed by the RyR inhibitor dantrolene. These results show that intranuclear extensions of the nuclear envelope have functional RyR and provide a possible mechanism whereby cells expressing RyR can regulate Ca(2+) signals in discrete regions within the nucleus.

Polycystin-2 (PC2), a member of the transient receptor potential family of ion channels (TRPP2), forms a calcium-permeable cation channel. Mutations in PC2 lead to polycystic kidney disease. From the primary sequence and by analogy with other channels in this family, PC2 is modeled to have six transmembrane domains. However, most of the structural features of PC2, such as how large the channel is and how many subunits make up the pore of the channel, are unknown. In this study, we estimated the pore size of PC2 from the permeation properties of the channel. Organic cations of increasing size were used as current carriers through the PC2 channel after PC2 was incorporated into lipid bilayers. We found that dimethylamine, triethylamine, tetraethylammonium, tetrabutylammonium, tetrapropylammonium, and tetrapentylammonium were permeable through the PC2 channel. The slope conductance of the PC2 channel decreased as the ionic diameter of the organic cation increased. For each organic cation tested, the currents were inhibited by gadolinium and anti-PC2 antibody. Using the dimensions of the largest permeant cation, the minimum pore diameter of the PC2 channel was estimated to be at least 11 A. The large pore size suggests that the primary state of this channel found in vivo is closed to avoid rundown of cation gradients across the plasma membrane and excessive calcium leak from endoplasmic reticulum stores.

Autoreactive T cell activation is a consistent feature of murine lupus; however, the mechanism of such activation remains unclear. We hypothesized that naive CD4+ T cells in lupus have a lower threshold of activation through their TCR-CD3 complex that renders them more susceptible to stimulation with self-Ags. To test this hypothesis, we compared proliferation, IL-2 production, and single cell calcium signaling of naive CD4+ T cells isolated from Fas-intact MRL/+(Fas-lpr) mice with H-2k-matched B10.BR and CBA/CaJ controls, following anti-CD3 stimulation in the presence or absence of anti-CD28. We also assessed the responsiveness of naive CD4+ T cells isolated from Fas-intact MRL and control mice bearing a rearranged TCR specific for amino acids 88-104 of pigeon cytochrome c to cognate and low affinity peptide Ags presented by bone marrow-matured dendritic cells. TCR transgenic and wild-type CD4+ T cells from MRL mice displayed a lower threshold of activation than control cells, a response that was class II MHC dependent. The rise in intracellular calcium in MRL vs controls was enhanced and prolonged following anti-CD3 triggering, suggestive of proximal defects in TCR-engendered signaling as the mechanism for the observed hyperactivity. These findings were observed as early as 1-2 mo postweaning and, based on analysis of F1 T cells, appeared to be dominantly expressed. This genetically altered threshold for activation of MRL T cells, a consequence of a proximal defect in CD3-mediated signal transduction, may contribute to the abrogation of T cell tolerance to self-Ags in lupus.