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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Synopsis Homeothermic endotherms defend their body temperature in cold environments using a number of behavioral and physiological mechanisms. Maintaining a stable body temperature primarily requires heat production through shivering or non-shivering thermogenesis (NST). Although the use of NST is well established in mammalian systems, the mechanisms and extent to which NST is used in birds are poorly understood. In mammals, one well-characterized mechanism of NST is through uncoupling of Ca2+ transport from ATP hydrolysis by sarco/endoplasmic reticulum ATPase (SERCA) in the skeletal muscle, which generates heat and may contribute to Ca2+ signaling for fatigue resistance and mitochondrial biogenesis. Two small proteins—sarcolipin (SLN) and phospholamban (PLN)—are known to regulate SERCA in mammals, but recent work shows inconsistent responses of SLN to cold acclimation in birds. In this study, we measured SERCA uncoupling in the pectoralis flight muscle of control (18°C) and cold-acclimated (−8°C) dark-eyed juncos (Junco hyemalis) that exhibited suppressed SLN transcription in the cold. We measured SERCA activity and Ca2+ uptake rates for the first time in cold-acclimated birds and found greater SERCA uncoupling in the muscle of juncos in the cold. However, SERCA uncoupling was not related to SLN or PLN transcription or measures of mitochondrial biogenesis. Nonetheless, SERCA uncoupling reduced an individual’s risk of hypothermia in the cold. Therefore, while SERCA uncoupling in the cold could be indicative of NST, it does not appear to be mediated by known regulatory proteins in these birds. These results prompt interesting questions about the significance of SLN and PLN in birds and the role of SERCA uncoupling in response to environmental conditions.
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It is unclear that how subcellular organelles respond to external mechanical stimuli. Here, we investigated the molecular mechanisms by which mechanical force regulates Ca2+ signaling at endoplasmic reticulum (ER) in human mesenchymal stem cells. Without extracellular Ca2+, ER Ca2+ release is the source of intracellular Ca2+ oscillations induced by laser-tweezer-traction at the plasma membrane, providing a model to study how mechanical stimuli can be transmitted deep inside the cell body. This ER Ca2+ release upon mechanical stimulation is mediated not only by the mechanical support of cytoskeleton and actomyosin contractility, but also by mechanosensitive Ca2+ permeable channels on the plasma membrane, specifically TRPM7. However, Ca2+ influx at the plasma membrane via mechanosensitive Ca2+ permeable channels is only mediated by the passive cytoskeletal structure but not active actomyosin contractility. Thus, active actomyosin contractility is essential for the response of ER to the external mechanical stimuli, distinct from the mechanical regulation at the plasma membrane.
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Frog virus 3 (FV3) is the type species of the genus Ranavirus (family Iridoviridae). FV3 and FV3-like viruses are globally distributed infectious agents with the capacity to replicate in three vertebrate classes (teleosts, amphibians, and reptiles). At the cellular level, FV3 and FV3-like viruses can infect cells from virtually all vertebrate classes. To date, the cellular receptors that are involved in the FV3 entry process are unknown. Class A scavenger receptors (SR-As) are a family of evolutionarily conserved cell-surface receptors that bind a wide range of chemically distinct polyanionic ligands and can function as cellular receptors for other DNA viruses, including vaccinia virus and herpes simplex virus. The present study aimed to determine whether SR-As are involved in FV3 cellular entry. By using well-defined SR-A competitive and non-competitive ligand-blocking assays and absolute qPCR, we demonstrated that the SR-A competitive ligands drastically reduced the quantities of cell-associated viral loads in frog cells. Moreover, inducing the expression of a human SR-AI in an SR-A null cell line significantly increased FV3–cell association. Together, our results indicate that SR-As are utilized by FV3 during the cellular entry process.more » « less
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Abstract Multiple therapies have been studied to ameliorate the neuroinhibitory cues present after traumatic injury to the central nervous system. Two previous in vitro studies have demonstrated the efficacy of the Food and Drug Administration‐approved cardiovascular therapeutic, protamine (PRM), to overcome neuroinhibitory cues presented by chondroitin sulfates; however, the effect of a wide range of PRM concentrations on neuronal and glial cells has not been evaluated. In this study, we investigate the therapeutic efficacy of PRM with primary cortical neurons, hippocampal neurons, mixed glial cultures, and astrocytes. The threshold for PRM toxicity is shown to be at or above 10 µg mL−1depending on the cell population, that 10 µg mL−1PRM enables neurons to overcome the inhibitory cues presented by chondroitin sulfate type A, and that soluble PRM allows neurons to more effectively overcome inhibition compared to a PRM coating. Changes in gene expression of reactive astrocytes is also assessed with soluble PRM and it is determined that PRM does not increase their neurotoxic phenotype and that PRM may reduce brevican and serpin transcription in cortical and spinal cord astrocytes. This is the first study to thoroughly assess the toxicity threshold of PRM with neural cells and study astrocyte response after acute exposure to PRM in vitro.
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Accepted Manuscript1.0
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