The role of calcium in hypoxia-induced signal transduction and gene expression

Mammalian cells require a constant supply of oxygen in order to maintain adequate energy production, which is essential for maintaining normal function and for ensuring cell survival. Sustained hypoxia can result in cell death. Sophisticated mechanisms have therefore evolved which allow cells to respond and adapt to hypoxia. Specialized oxygen-sensing cells have the ability to detect changes in oxygen tension and transduce this signal into organ system functions that enhance the delivery of oxygen to tissue in a wide variety of different organisms. An increase in intracellular calcium levels is a primary response of many cell types to hypoxia/ischemia. The response to hypoxia is complex and involves the regulation of multiple signaling pathways and coordinated expression of perhaps hundreds of genes. This review discusses the role of calcium in hypoxia-induced regulation of signal transduction pathways and gene expression. An understanding of the molecular events initiated by changes in intracellular calcium will lead to the development of therapeutic approaches toward the treatment of hypoxic/ischemic diseases and tumors.     

The role of calcium in signal transduction processes in Sertoli cells

Sertoli cells play a pivotal role in regulation and maintenance of spermatogenesis. They are hormonally regulated predominantly by follicle-stimulating hormone (FSH) and testosterone (T). Although FSH and T have distinct mechanisms of action they act synergistically in promoting spermatogenesis. Stimulation of freshly isolated Sertoli cells with FSH evokes a prompt rise in cytosolic calcium which is quantitatively reproduced by cAMP. The cytosolic calcium response to FSH in Sertoli cells is predominantly attributable to serial signaling after the generation of endogenous cAMP. Calcium homeostasis of Sertoli cells may also be regulated by cAMP-independent metabolism. Vasoactive testicular paracrine hormones such as angiotensin II (AII) and vasopressin acting via inositol triphosphate generation induce cytosolic calcium rise predominantly derived from the thapsigargin-sensitive endoplasmic reticulum. Investigations involving androgens action on cytosolic calcium reveal a common mechanism of action between the peptide and steroid regulators of Sertoli cell function, indicating that cytosolic calcium ions may represent a unifying biochemical mechanism that could explain the synergism of FSH and T. Androgens rapidly and specifically increase cytosolic calcium, consistent with a plasma membrane site of action. This argues for the possible existence of a short term non-genomic signaling pathway in hormonal regulation of Sertoli cell function in addition to the classical longer term, slower genomic response.     

Evolution of signal transduction in intracellular symbiosis

Plant roots form intracellular symbioses with fungi and bacteria resulting in arbuscular mycorrhiza and nitrogen-fixing root nodules, respectively. A novel receptor like-kinase has been discovered that is required for the transduction of both bacterial and fungal symbiotic signals. This kinase defines an ancient signalling pathway that probably evolved in the context of arbuscular mycorrhiza and has been recruited subsequently for endosymbiosis with bacteria. An ancestral symbiotic interaction of roots with intracellular bacteria might have emerged from such a recruitment, in the progenitor of the nodulating clade of plants. Analysis of symbiotic mutants of host plants and bacterial microsymbionts has revealed that present-day endosymbioses require the coordinated induction of more than one signalling pathway for development.     

Cross-talk of parathyroid hormone-responsive dual signal transduction systems in osteoblastic osteosarcoma cells: Its role in PTH-induced homologous desensitization of intracellular calcium response

The present study was designed to characterize the cross-talk of parathyroid hormone (PTH)-responsive dual signal transduction systems (cAMP-dependent protein kinase (PKA) and calcium/protein kinase C [PKC]) and its participation in PTH-induced homologous desensitization of intracellular calcium ([Ca²⁺]i) in osteoblastic UMR-106 cells. Although our recent study revealed that prolonged (more than 2 h) pretreatment with PKC-activating phorbol ester, phorbol 12-myristate 13-acetate (PMA) significantly decreased the PTH-stimulated cAMP production, pretreatment with PMA (10^?7 and 10^?6 M) but not 10^?6 M 4alphaphorbol 12,13-didecanoate (PDD), incapable of activating PKC for 30 min significantly augmented 10^?7 M hPTH-(1-34)-stimulated cAMP production. H-7 (50 uM), a PKC inhibitor, significantly antagonized this PMA-induced effect. Pretreatment with 10^?6 M PMA for 30 min did not affect PTH receptor binding but significantly augmented a cAMP responsiveness to 10^?5 M forskolin and 1 ug/ml cholera toxin. Pertussis toxin (0.5 ug/ml) did not affect the PMA-induced augmentation of the PTH-stimulated cAMP production. PTH caused a complete homologous desensitization of [Ca²⁺]i response within 30 min. Pretreatment with 10^?4 M dibutyryl cAMP for 30 min and 6 h significantly reduced and completely blocked the PTH-induced increase in [Ca²⁺]i, respectively. Pretreatment with 10^?4 M Sp-cAMPS, a direct PKA activator, for 30 min completely blocked the PTH-induced increase in [Ca²⁺]i. Rp-cAMPS (10^?4 M), an antagonist of PKA, slightly but significantly antagonized the PTH-induced homologous desensitization of [Ca²⁺]i response. The present study indicates that the time of exposure to PKC activation is a critical determinant in modulating the cAMP system, while PKA activation counterregulatorily acts on the [Ca²⁺]i system, and that PKA activation is linked to the PTH-induced homologous desensitization of [Ca²⁺]i response. © 1994 Wiley-Liss, Inc.     

The role of phosphotyrosine phosphatases in haematopoietic cell signal transduction

Phosphotyrosine phosphatases (PTPases) are the enzymes which remove phosphate groups from protein tyrosine residues. An enormous number of phosphatases have been cloned and sequenced during the past decade, many of which are expressed in haematopoietic cells. This review focuses on the biochemistry and cell biology of three phosphatases, the transmembrane CD45 and the cytosolic SH2-domain-containing PTPases SHP-1 and SHP-2, to illustrate the diverse ways in which PTPases regulate receptor signal transduction. The involvement of these and other PTPases has been demonstrated in haematopoietic cell development, apoptosis, activation and non-responsiveness. A common theme in the actions of many haematopoietic cell PTPases is the way in which they modulate the thresholds for receptor signalling, thereby regulating critical events in the positive and negative selection of lymphocytes. There is growing interest in haematopoietic PTPases and their associated regulatory proteins as targets for pharmaceutical intervention and in the involvement of these enzymes in human disease.     

Regulation of cGMP synthesis in photoreceptors: role in signal transduction and congenital diseases of the retina

Calcium feedback in vertebrate photoreceptors regulates synthesis of cGMP, a second messenger in phototransduction. The decrease in the free intracellular Ca(2+) concentrations caused by illumination stimulates two isoforms of retinal membrane guanylyl cyclase (RetGC) via Ca(2+)-sensor proteins and thus contributes to photoreceptor recovery and light adaptation. Unlike other members of the membrane guanylyl cyclase family, retinal guanylyl cyclases do not have identified extracellular peptide ligands. Recoverin-like proteins, GCAP-1 and GCAP-2, interact with the intracellular portion of the cyclases and stimulate its activity through dimerization of the cyclase subunits. Several mutations that affect the function of photoreceptor guanylyl cyclase and the activator protein have been linked to various forms of congenital human retinal diseases, such as Leber congenital amaurosis, cone and cone-rod dystrophy.     

The role of calcium in follicle-stimulating hormone signal transduction in Sertoli cells.

Sertoli cells are hormonally regulated by follicle-stimulating hormone (FSH) acting upon a G-protein-linked cell surface FSH receptor. FSH increases intracellular cyclic AMP but the involvement of other signal transduction mechanisms including intracellular calcium in FSH action are not proven. Using freshly isolated rat Sertoli cells we measured cytosolic free ionized calcium levels by dual-wavelength fluorescence spectrophotometry using the calcium-sensitive fluorescent dye Fura2-AM. The cytosolic calcium concentration in unstimulated Sertoli cells was 89 +/- 2 nM (n = 151 experiments) and was markedly increased by either calcium channel ionophores (ionomycin, Bay K8644) or plasma membrane depolarization consistent with the presence of voltage-sensitive and -independent calcium channel in Sertoli cell membranes. Ovine FSH stimulated a specific, sensitive (ED50, 5.0 ng of S-16/ml), and dose-dependent (maximal at 20 ng/ml) rise in cytosolic calcium commencing within 60 s to reach levels of 192 +/- 31 nM after 180 s and lasting for at least 10 min. The effect of FSH was replicated by forskolin, cholera toxin, and dibutyryl cyclic AMP, suggesting that cyclic AMP may mediate the FSH-induced rise in cytosolic calcium. The FSH-induced rise in cytosolic calcium required extracellular calcium and was abolished by calcium channel blockers specific for dihydropyridine (verapamil, nicardipine), nonvoltage-gated (ruthenium red) or all calcium channels (cobalt). Thus FSH action on Sertoli cells involves a specific, rapid, and sustained increase in cytosolic calcium which requires extracellular calcium and involves both dihydropyridine-sensitive, voltage-gated calcium channels and voltage-independent, receptor-gated calcium channels in the plasma membranes of rat Sertoli cells. The replication by cyclic AMP of the effects of FSH suggests that calcium may be a signal-amplification or -modulating mechanism rather than an alternate primary signal transduction system for FSH in Sertoli cells.     

Cadmium-induced insulin release does not involve changes in intracellular handling of calcium

A possible interaction between Cd2+ and Ca2+ as a component in Cd2+-induced insulin release was investigated in beta cells isolated from obese hyperglycemic mice. The glucose stimulated Cd2+ uptake was dependent on the concentration of sugar. This uptake was sigmoidal with a Km for glucose of about 5 mM and was suppressed by both 50 microM of the voltage-activated Ca2+ channel blocker D-600 and 12 mM Mg2+. In the presence of 8 mM glucose 5 microM Cd2+ evoked a prompt and sustained stimulatory response, corresponding to about 3-fold of the insulin release obtained in the absence of the ion. Whereas 5 microM Cd2+ was without effect on the glucose-stimulated 45Ca efflux in the presence of extracellular Ca2+, 40 microM inhibited it. At a concentration of 5 microM, Cd2+ had no effect on the resting membrane potential or the depolarization evoked by either glucose or K+. In the absence of extracellular Ca2+ there was only a modest stimulation of 45Ca efflux by 5 microM Cd2+. Studies of the ambient free Ca2+ concentration maintained by permeabilized cells also indicate that 5 microM Cd2+ do not mobilize intracellularly bound Ca2+ to any great extent. On the contrary, at this concentration, Cd2+ even suppressed inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release. The present study suggests that Cd2+ stimulates insulin release by a direct mechanism which does not involve an increase in cytoplasmic free Ca2+ concentration.     

Endoglin promotes endothelial cell proliferation and TGF-beta/ALK1 signal transduction

Endoglin is a transmembrane accessory receptor for transforming growth factor-beta (TGF-beta) that is predominantly expressed on proliferating endothelial cells in culture and on angiogenic blood vessels in vivo. Endoglin, as well as other TGF-beta signalling components, is essential during angiogenesis. Mutations in endoglin and activin receptor-like kinase 1 (ALK1), an endothelial specific TGF-beta type I receptor, have been linked to the vascular disorder, hereditary haemorrhagic telangiectasia. However, the function of endoglin in TGF-beta/ALK signalling has remained unclear. Here we report that endoglin is required for efficient TGF-beta/ALK1 signalling, which indirectly inhibits TGF-beta/ALK5 signalling. Endothelial cells lacking endoglin do not grow because TGF-beta/ALK1 signalling is reduced and TGF-beta/ALK5 signalling is increased. Surviving cells adapt to this imbalance by downregulating ALK5 expression in order to proliferate. The ability of endoglin to promote ALK1 signalling also explains why ectopic endoglin expression in endothelial cells promotes proliferation and blocks TGF-beta-induced growth arrest by indirectly reducing TGF-beta/ALK5 signalling. Our results indicate a pivotal role for endoglin in the balance of ALK1 and ALK5 signalling to regulate endothelial cell proliferation.     

An intracellular calcium increase and protein kinase C activation fail to initiate T cell proliferation in the absence of a costimulatory signal

Resting T lymphocytes proliferate in response to a combination of a calcium ionophore and a phorbol ester. This observation suggests that an increase in intracellular calcium free ion concentration [Ca2+]i and activation of protein kinase C (PKC) are sufficient signaling events for the initiation of T cell proliferation. In contrast, an accessory cell-generated costimulatory signal, acting independently of the rise in [Ca2+]i and PKC activation, is required for Ag-induced proliferation of type I T cell clones. We now report that this costimulatory signal is unexpectedly also being delivered via a cell-cell interaction during the response to ionomycin and phorbol ester. In the absence of this signal (at limiting cell numbers), T cells fail to divide. We also demonstrate that proliferation in response to immobilized anti-CD3 mAb requires the cell-cell interaction. These results suggest a model of T cell stimulation in which activation of a costimulatory signaling pathway is important in the regulation of the IL-2 gene, and only in the presence of this (third) signal can an increase in [Ca2+]i and PKC activity induce T cell proliferation. Such a model predicts that IL-2-dependent expansion of T cell clones in vivo in response to Ag receptor occupancy requires the delivery of an independent accessory cell-derived co-stimulatory signal.     

Mycobacterium tuberculosis phagosomes exhibit altered calmodulin-dependent signal transduction: contribution to inhibition of phagosome-lysosome fusion and intracellular survival in human macrophages

Mycobacterium tuberculosis successfully parasitizes macrophages by disrupting the maturation of its phagosome, creating an intracellular compartment with endosomal rather than lysosomal characteristics. We have recently demonstrated that live M. tuberculosis infect human macrophages in the absence of an increase in cytosolic Ca(2+) ([Ca(2+)](c)), which correlates with inhibition of phagosome-lysosome fusion and intracellular viability. In contrast, killed M. tuberculosis induces an elevation in [Ca(2+)](c) that is coupled to phagosome-lysosome fusion. We tested the hypothesis that defective activation of the Ca(2+)-dependent effector proteins calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) contributes to the intracellular pathogenesis of tuberculosis. Phagosomes containing live M. tuberculosis exhibited decreased levels of CaM and the activated form of CaMKII compared with phagosomes encompassing killed tubercle bacilli. Furthermore, ionophore-induced elevations in [Ca(2+)](c) resulted in recruitment of CaM and activation of CaMKII on phagosomes containing live M. tuberculosis. Specific inhibitors of CaM or CaMKII blocked Ca(2+) ionophore-induced phagosomal maturation and enhanced the bacilli's intracellular viability. These results demonstrate a novel role for CaM and CaMKII in the regulation of phagosome-lysosome fusion and suggest that defective activation of these Ca(2+)-activated signaling components contributes to the successful parasitism of human macrophages by M. tuberculosis.     

Role of raf isoforms in signal transduction pathways relevant to leukemia cell proliferation

The invited commentary is addressed to the paper of Shelton et al published in this issue of Cell Cycle. The intracellular pathways that control cell growth constitute a complex nexus of signaling interactions that serve to regulate cell proliferation, differentiation and apoptosis. Dysregulation of these processes leads to the loss of control of cell growth that is characteristic of malignancy. Understanding cell signaling is a major challenge for modern biological research. One way to begin to unravel the intricate web of signaling pathways is to investigate the effects of mutant forms of key component proteins. One such protein is the serine/threonine-specific protein kinase, Raf.     

2-Cys peroxiredoxin function in intracellular signal transduction: therapeutic implications

H(2)O(2) is a reactive oxygen species that has drawn much interest because of its role as a second messenger in receptor-mediated signaling. Mammalian 2-Cys peroxiredoxins have been shown to eliminate efficiently the H(2)O(2) generated in response to receptor stimulation. 2-Cys peroxiredoxins are members of a novel peroxidase family that catalyze the H(2)O(2) reduction reaction in the presence of thioredoxin, thioredoxin reductase and NADPH. Several lines of evidence suggest that 2-Cys peroxiredoxins have dual roles as regulators of the H(2)O(2) signal and as defenders of oxidative stress. In particular, 2-Cys peroxiredoxin appears to provide selective, specific and localized control of receptor-mediated signal transduction. Thus, the therapeutic potential of 2-Cys peroxiredoxins is clear for diseases, such as cancer and cardiovascular diseases, that involve reactive oxygen species.     

Resonant activation of calcium signal transduction in neurons

The relevant parameters of calcium fluxes mediating activation of immediate-early genes and the collapse of growth cones in mouse DRG neurons in response to action potentials delivered in different temporal patterns were measured in a multicompartment cell culture preparation using digital flourescence videomicroscopy. Growth cone collapse was produced by trains of action potentials causing a large rise in [Ca²⁺]_i, but after chronic exposure to patterned stimulation growth cones regenerated and became insensitive to the stimulus-induced increase in [Ca²⁺]_i. Calcium reached similar peak concentrations, but the [Ca²⁺]_i increased more slowly than in naive growth cones (time constant of 6.0 s versus 1.4 s in naive growth cones). Semiquantitative PCR measurements of gene expression showed that pulsed stimulation delivered at 1-min intervals for 30 min induced expression of c-fos, but the same total number of action potentials delivered at 2-min intervals failed to induce c-fos expression, even though this stimulus induces a larger peak [Ca²⁺]_i than the effective stimulus pattern. The experiments suggest that the kinetics of calcium fluxes produced by different patterns of stimulation, and changes in the kinetics of calcium flux in neurons under different states of activation, are critical in determining the effects of action potentials on growth cone motility or expression of IE genes during development of neuronal circuits. We propose that differences in kinetics of individual reactions in the stimulus–response pathway may lead to resonance of activation in the neuron, such that certain processes will be selectively activated by particular temporal patterns of stimulation. 1994 John Wiley & Sons, Inc.