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ABSTRACT Compared with our extensive understanding of the cell cycle, we have limited knowledge of how the cell quiescence–proliferation decision is regulated. Using a zebrafish epithelial model, we report a novel signaling mechanism governing the cell quiescence–proliferation decision. Zebrafish Ca2+-transporting epithelial cells, or ionocytes, maintain high cytoplasmic Ca2+ concentration ([Ca2+]c) due to the expression of Trpv6. Genetic deletion or pharmacological inhibition of Trpv6, or reduction of external Ca2+ concentration, lowered the [Ca2+]c and reactivated these cells. The ionocyte reactivation was attenuated by chelating intracellular Ca2+ and inhibiting calmodulin (CaM), suggesting involvement of a Ca2+ and CaM-dependent mechanism. Long-term imaging studies showed that after an initial decrease, [Ca2+]c gradually returned to the basal levels. There was a concomitant decease in endoplasmic reticulum (ER) Ca2+ levels. Lowering the ER Ca2+ store content or inhibiting ryanodine receptors impaired ionocyte reactivation. Further analyses suggest that CaM-dependent protein kinase kinase (CaMKK) is a key molecular link between Ca2+ and Akt signaling. Genetic deletion or inhibition of CaMKK abolished cell reactivation, which could be rescued by expression of a constitutively active Akt. These results suggest that the quiescence–proliferation decision in zebrafish ionocytes is regulated by Trpv6-mediated Ca2+ and CaMKK–Akt signaling.
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Nanomolar tetrabromopyrrole alters Ca2+ dynamics in cortical neuronal networks by selective modification of ryanodine receptors and micromolar is neurotoxic due to SERCA pump inhibition.
Naturally synthesized marine organohalogens (MOH) and their anthropo- genic homologs produced as disinfection byproducts (DBP) are an emerging environmental health concern because several have been identified to exhibit potent biological activities in model systems, including cytotoxicity, genotox- icity, carcinogenicity and developmental toxicity. The molecular mechanisms mediating toxicity are poorly understood. Recently we discovered that several specific MOH and DBP measured in environmental and biological samples, including halopyrroles, halobipyrroles, haloindoles, and hydroxylated poly- brominated diphenylethers directly modify ryanodine receptors and SERCA pump activity, two key proteins anchored within sarcoplasmic/endoplasmic reticulum (SR/ER) that work in physiological opposition to tightly regulate net ER/SR Ca2+ dynamics and thereby shape meaningful Ca2+-dependent cel- lular processes. Using intact HEK293 cells null for ryanodine receptors (RyRs) expression and those that stably express RyR1, we demonstrate that tetra- bromopyrrole (TBP) selectively sensitizes RyR1 channels to caffeine-triggered Ca2+ release only in RyR1-expressing cells. TBP at higher concentrations also depletes of SR/ER Ca2+ stores in both null and RyR1 expressing cells com- mensurate with its lower potency to inhibitory SERCA in biochemical assays. Exposure of primary neuronal/glial co-cultures derived from newborn mice shows that TBP inhibits the frequency and amplitude of spontaneous Ca2+ oscillations (IC50=246 and 426nM, respectively), whereas >1μM produces a sustained rise in cytoplasmic Ca2+. Subchronic (24HR) exposure to TBP caused loss of neuronal/glial viability using the MTT assay (EC50=12.4μM). These re- sults show that nM TBP selectively targets RyR-mediated Ca2+ dynamics in a manner that has been shown to affect neurodevelopment, whereas low-μM exposures causes overt neurotoxicity, likely mediated by the combination of RyR activation and SERCA inhibition.
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- Award ID(s):
- 1840842
- PAR ID:
- 10096551
- Date Published:
- Journal Name:
- The Toxicologist
- Volume:
- 168
- ISSN:
- 0731-9193
- Page Range / eLocation ID:
- 80
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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