Abstract
A physiological membrane-receptor agonist typically stimulates oscillations, of varying frequencies, in cytosolic Ca2+ concentration ([Ca2+]i). Whether and how [Ca2+]i oscillation frequency regulates agonist-stimulated downstream events, such as gene expression, in non-excitable cells remain unknown. By precisely manipulating [Ca2+]i oscillation frequency in histamine-stimulated vascular endothelial cells (ECs), we demonstrate that the gene expression of vascular cell adhesion molecule 1 (VCAM1) critically depends on [Ca2+]i oscillation frequency in the presence, as well as the absence, of histamine stimulation. However, histamine stimulation enhanced the efficiency of [Ca2+]i-oscillation-frequency-regulated VCAM1 gene expression, versus [Ca2+]i oscillations alone in the absence of histamine stimulation. Furthermore, a [Ca2+]i oscillation frequency previously observed to be the mean frequency in histamine-stimulated ECs was found to optimize VCAM1 mRNA expression. All the above effects were abolished or attenuated by blocking histamine-stimulated generation of intracellular reactive oxygen species (ROS), another intracellular signaling pathway, and were restored by supplementary application of a low level of H2O2. Endogenous NF-kappaB activity is similarly regulated by [Ca2+]i oscillation frequency, as well as its co-operation with ROS during histamine stimulation. This study shows that [Ca2+]i oscillation frequency cooperates with ROS to efficiently regulate agonist-stimulated gene expression, and provides a novel and general strategy for studying [Ca2+]i signal kinetics in agonist-stimulated downstream events.
Highlights
Cytosolic Ca2+ concentration ([Ca2+]i), the ubiquitous intracellular second messenger, couples stimuli and cell responses. [Ca2+]i oscillations, the repetitive increases and decreases in [Ca2+]i, were first demonstrated in hepatocytes (Woods et al, 1986) and have been observed in almost all non-excitable cells ever studied. [Ca2+]i oscillation frequency is profoundly modulated by a variety ofphysiological conditions and is very important in decoding Ca2+-signal-dependent downstream events
We found that the cooperation between [Ca2+]i oscillations and intracellular reactive oxygen species (ROS) enhanced the efficiency of [Ca2+]i-oscillation-frequency-regulated vascular cell adhesion molecule 1 (VCAM1) mRNA expression during histamine stimulation versus [Ca2+]i oscillations alone
To determine whether [Ca2+]i oscillation frequency regulates agonist-stimulated gene expression, endothelial cells (ECs) monolayers were subjected to conditions that generated [Ca2+]i oscillations with the same amplitude of ~0.9 μM and four different oscillation frequencies of 0.1, 0.3, 0.5 or 0.7 oscillations/minute in the presence or absence of 1 μM histamine stimulation
Summary
Cytosolic Ca2+ concentration ([Ca2+]i), the ubiquitous intracellular second messenger, couples stimuli and cell responses. [Ca2+]i oscillations, the repetitive increases and decreases in [Ca2+]i, were first demonstrated in hepatocytes (Woods et al, 1986) and have been observed in almost all non-excitable cells ever studied. [Ca2+]i oscillation frequency is profoundly modulated by a variety of (patho)physiological conditions and is very important in decoding Ca2+-signal-dependent downstream events. Experimental efforts in an ‘artificial’ [Ca2+]i-oscillation model created using the ‘calcium clamp’ in T lymphocytes showed that high-frequency [Ca2+]i oscillations activated both NFAT and NFκB, whereas low frequency activated only NF-κB (Dolmetsch et al, 1998). A more recent study using the ‘calcium clamp’ model further demonstrated that the frequency dependence of NFAT activation related to the kinetics of dephosphorylation and the translocation of cytoplasmic NFAT (Tomida et al, 2003). These seminal studies linking [Ca2+]i oscillations that were ‘artificially’ generated and regulated in the absence of a physiological membrane-receptor interaction and transcriptional activation raised the question: is frequencymodulated nuclear transcription or gene expression physiologically relevant? To fully investigate any physiological importance of [Ca2+]i oscillation frequency, it becomes necessary to precisely manipulate [Ca2+]i oscillation frequency during agonist stimulation
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