Transcriptional Basis of Ca2+ Remodeling Reversal Induced by Polyamine Synthesis Inhibition in Colorectal Cancer Cells.

Colorectal cancer (CRC) is associated with mutations in APC/Wnt leading to c-myc activation and the overexpression of ODC1, the limiting step in polyamine synthesis. CRC cells also display a remodeling of intracellular Ca2+ homeostasis that contributes to cancer hallmarks. As polyamines may modulate Ca2+ homeostasis during epithelial tissue repair, we investigated whether polyamine synthesis inhibition may reverse Ca2+ remodeling in CRC cells and, if so, the molecular basis for this reversal. To this end, we used calcium imaging and transcriptomic analysis in normal and CRC cells treated with DFMO, an ODC1 suicide inhibitor. We found that polyamine synthesis inhibition partially reversed changes in Ca2+ homeostasis associated with CRC, including a decrease in resting Ca2+ and SOCE along with an increased Ca2+ store content. We also found that polyamine synthesis inhibition reversed transcriptomic changes in CRC cells without affecting normal cells. Specifically, DFMO treatment enhanced the transcription of SOCE modulators CRACR2A; ORMDL3; and SEPTINS 6, 7, 8, 9, and 11, whereas it decreased SPCA2, involved in store-independent Orai1 activation. Therefore, DFMO treatment probably decreased store-independent Ca2+ entry and enhanced SOCE control. Conversely, DFMO treatment decreased the transcription of the TRP channels TRPC1 and 5, TRPV6, and TRPP1 while increasing TRPP2, thus probably decreasing Ca2+ entry through TRP channels. Finally, DFMO treatment enhanced the transcription of the PMCA4 Ca2+ pump and mitochondrial channels MCU and VDAC3 for enhanced Ca2+ extrusion through the plasma membrane and mitochondria. Collectively, these findings suggested the critical role of polyamines in Ca2+ remodeling in colorectal cancer.

Amyloid Beta Oligomers-Induced Ca2+ Entry Pathways: Role of Neuronal Networks, NMDA Receptors and Amyloid Channel Formation.

The molecular basis of amyloid toxicity in Alzheimer's disease (AD) remains controversial. Amyloid ß (Aß) oligomers promote Ca2+ influx, mitochondrial Ca2+ overload and apoptosis in hippocampal neurons in vivo and in vitro, but the primary Ca2+ entry pathways are unclear. We studied Ca2+ entry pathways induced by Aß oligomers in rat hippocampal and cerebellar neurons. Aß oligomers induce Ca2+ entry in neurons. Ca2+ responses to Aß oligomers are large after synaptic networking and prevented by blockers of synaptic transmission. In contrast, in neurons devoid of synaptic connections, Ca2+ responses to Aß oligomers are small and prevented only by blockers of amyloid channels (NA7) and NMDA receptors (MK801). A combination of NA7 and MK801 nearly abolished Ca2+ responses. Non-neuronal cells bearing NMDA receptors showed Ca2+ responses to oligomers, whereas cells without NMDA receptors did not exhibit Ca2+ responses. The expression of subunits of the NMDA receptor NR1/ NR2A and NR1/NR2B in HEK293 cells lacking endogenous NMDA receptors restored Ca2+ responses to NMDA but not to Aß oligomers. We conclude that Aß oligomers promote Ca2+ entry via amyloid channels and NMDA receptors. This may recruit distant neurons intertwisted by synaptic connections, spreading excitation and recruiting further NMDA receptors and voltage-gated Ca2+ channels, leading to excitotoxicity and neuron degeneration in AD.

Differential Ca2+ responses and store operated Ca2+ entry in primary cells from human brain tumors.

Brain tumors comprise a large series of tumor cancer from benign to highly malignant gliomas and metastases from primary tumors outside the brain. Intracellular Ca2+ homeostasis is involved in a large series of cell functions including cell proliferation, migration, and cell death. Store-operated Ca2+ entry (SOCE), the most important Ca2+ entry pathway in non-excitable cells, is involved in cell proliferation and migration and enhanced in tumor cells from breast cancer, colon cancer and cell lines derived from glioblastoma but there are almost no studies in human primary glioblastoma cells or other brain tumors. We have developed a single procedure to obtain primary cells from a large series (n = 49) of human brain tumors including schwannomas, meningiomas, oligodendrogliomas, astrocytomas, glioblastomas and brain metastases from ovary, breast and lung. Cells were characterized by immunofluorescence and subjected to Ca2+ imaging to investigate resting intracellular Ca2+ levels, Ca2+ responses to physiological agonists as well as voltage-operated Ca2+ entry and SOCE. We found significant differences in resting intracellular Ca2+ and Ca2+ responses to plasma membrane depolarization and ATP among the different tumor cells. Only malignant tumor cells, displayed Ca2+ responses to ATP. SOCE is significantly increased in malignant gliomas whereas voltage-gated Ca2+ entry is decreased. In addition, SOCE is significantly larger in high grade gliomas than in low grade gliomas suggesting that SOCE increases with glioma progression. These data may provide new insights on the role of intracellular Ca2+ and purinergic signalling in brain tumors.

Remodeling of Intracellular Ca2+ Homeostasis in Rat Hippocampal Neurons Aged In Vitro.

Aging is often associated with a cognitive decline and a susceptibility to neuronal damage. It is also the most important risk factor for neurodegenerative disorders, particularly Alzheimer's disease (AD). AD is related to an excess of neurotoxic oligomers of amyloid ß peptide (Aßo); however, the molecular mechanisms are still highly controversial. Intracellular Ca2+ homeostasis plays an important role in the control of neuronal activity, including neurotransmitter release, synaptic plasticity, and memory storage, as well as neuron cell death. Recent evidence indicates that long-term cultures of rat hippocampal neurons, resembling aged neurons, undergo cell death after treatment with Aßo, whereas short-term cultures, resembling young neurons, do not. These in vitro changes are associated with the remodeling of intracellular Ca2+ homeostasis with aging, thus providing a simplistic model for investigating Ca2+ remodeling in aging. In vitro aged neurons show increased resting cytosolic Ca2+ concentration, enhanced Ca2+ store content, and Ca2+ release from the endoplasmic reticulum (ER). Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria is also enhanced. Aged neurons also show decreased store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway related to memory storage. At the molecular level, in vitro remodeling is associated with changes in the expression of Ca2+ channels resembling in vivo aging, including changes in N-methyl-D-aspartate NMDA receptor and inositol 1,4,5-trisphosphate (IP3) receptor isoforms, increased expression of the mitochondrial calcium uniporter (MCU), and decreased expression of Orai1/Stim1, the molecular players involved in SOCE. Additionally, Aßo treatment exacerbates most of the changes observed in aged neurons and enhances susceptibility to cell death. Conversely, the solely effect of Aßo in young neurons is to increase ER-mitochondria colocalization and enhance Ca2+ transfer from ER to mitochondria without inducing neuronal damage. We propose that cultured rat hippocampal neurons may be a useful model to investigate Ca2+ remodeling in aging and in age-related neurodegenerative disorders.

Amyloid ß Oligomers Increase ER-Mitochondria Ca2+ Cross Talk in Young Hippocampal Neurons and Exacerbate Aging-Induced Intracellular Ca2+ Remodeling.

Alzheimer's disease (AD) is the most common neurodegenerative disorder and strongly associated to aging. AD has been related to excess of neurotoxic oligomers of amyloid ß peptide (Aßo), loss of intracellular Ca2+ homeostasis and mitochondrial damage. However, the intimate mechanisms underlying the pathology remain obscure. We have reported recently that long-term cultures of rat hippocampal neurons resembling aging neurons are prone to damage induced by Aß oligomers (Aßo) while short-term cultured cells resembling young neurons are not. In addition, we have also shown that aging neurons display critical changes in intracellular Ca2+ homeostasis including increased Ca2+ store content and Ca2+ transfer from the endoplasmic reticulum (ER) to mitochondria. Aging also promotes the partial loss of store-operated Ca2+ entry (SOCE), a Ca2+ entry pathway involved in memory storage. Here, we have addressed whether Aßo treatment influences differentially intracellular Ca2+ homeostasis in young and aged neurons. We found that Aßo exacerbate the remodeling of intracellular Ca2+ induced by aging. Specifically, Aßo exacerbate the loss of SOCE observed in aged neurons. Aßo also exacerbate the increased resting cytosolic Ca2+ concentration, Ca2+ store content and Ca2+ release as well as increased expression of the mitochondrial Ca2+ uniporter (MCU) observed in aging neurons. In contrast, Aßo elicit none of these effects in young neurons. Surprisingly, we found that Aßo increased the Ca2+ transfer from ER to mitochondria in young neurons without having detrimental effects. Consistently, Aßo increased also colocalization of ER and mitochondria in both young and aged neurons. However, in aged neurons, Aßo suppressed Ca2+ transfer from ER to mitochondria, decreased mitochondrial potential, enhanced reactive oxygen species (ROS) generation and promoted apoptosis. These results suggest that modulation of ER-mitochondria coupling in hippocampal neurons may be a novel physiological role of Aßo. However, excess of Aßo in the face of the remodeling of intracellular Ca2+ homeostasis associated to aging may lead to loss of ER-mitochondrial coupling and AD.

Store-operated Ca2+ entry and Ca2+ responses to hypothalamic releasing hormones in anterior pituitary cells from Orai1-/- and heptaTRPC knockout mice.

Store operated Ca2+ entry (SOCE) is the most important Ca2+ entry pathway in non-excitable cells. However, SOCE can also play a pivotal role in excitable cells such as anterior pituitary (AP) cells. The AP gland contains five different cell types that release six major AP hormones controlling most of the entire endocrine system. AP hormone release is modulated by Ca2+ signals induced by different hypothalamic releasing hormones (HRHs) acting on specific receptors in AP cells. TRH and LHRH both induce Ca2+ release and Ca2+ entry in responsive cells while GHRH and CRH only induce Ca2+ entry. SOCE has been shown to contribute to Ca2+ responses induced by TRH and LHRH but no molecular evidence has been provided. Accordingly, we used AP cells isolated from mice devoid of Orai1 channels (noted as Orai1-/- or Orai1 KO mice) and mice lacking expression of all seven canonical TRP channels (TRPC) from TRPC1 to TRPC7 (noted as heptaTRPC KO mice) to investigate contribution of these putative channel proteins to SOCE and intracellular Ca2+ responses induced by HRHs. We found that thapsigargin-evoked SOCE is lost in AP cells from Orai1-/- mice but unaffected in cells from heptaTRPC KO mice. Conversely, while spontaneous intracellular Ca2+-oscillations related to electrical activity were not affected in the Orai1-/- mice, these responses were significantly reduced in heptaTRPC KO mice. We also found that Ca2+ entry induced by TRH and LHRH is decreased in AP cells isolated from Orai1-/-. In addition, Ca2+ responses to several HRHs, particularly TRH and GHRH, are decreased in the heptaTRPC KO mice. These results indicate that expression of Orai1, and not TRPC channel proteins, is necessary for thapsigargin-evoked SOCE and is required to support Ca2+ entry induced by TRH and LHRH in mouse AP cells. In contrast, TRPC channel proteins appear to contribute to spontaneous Ca2+-oscillations and Ca2+ responses induced by TRH and GHRH. We conclude that expression of Orai1 and TRPC channels proteins may play differential and significant roles in AP physiology and endocrine control.