High doses of dexamethasone induce endoplasmic reticulum stress-mediated apoptosis by promoting calcium ion influx-dependent CHOP expression in osteoblasts

Background The long-term use of dexamethasone (Dex), a well-known immunosuppressant, leads to an imbalance in bone metabolism and rapid decline of bone mineral density due to apoptosis of osteoblasts. The molecular mechanisms by which Dex induces osteoblast apoptosis remain unclear. Materials and methods MC3T3-E1 cells were treated with 0, 10−8, 10−6, and 10−4 M Dex for 24 h. ATF6, phosphorylated PERK, PERK, phosphorylated IRE1, and IRE1 expression, cell apoptosis, and caspase-12 and caspase-3 activity were measured. CHOP expression and calcium ion influx rate were measured in cells treated with 0 and 10−4 M Dex for 24 h. The effect of 2-APB treatment was assessed in cells treated with 0 or 10−4 M Dex. Results Levels of ATF6 and phosphorylated PERK and IRE1 increased in a dose-dependent manner in MC3T3-E1 cells treated with 10−8, 10−6, and 10−4 M Dex, compared to the control group (P < 0.05). Cells treated with 10−6 and 10−4 M Dex had significantly increased apoptotic rates and caspase-12 and caspase-3 activities (P < 0.05). Cells treated with 10−4 M Dex had significantly increased CHOP levels and calcium ion influx rates (P < 0.05). Combined treatment with 10−4 M Dex and 2-APB abrogated the observed increases in cell apoptosis and caspase-12 and caspase-3 activities (P < 0.05). Conclusions High doses of Dex induce CHOP expression by promoting calcium ion influx-dependent induction of ATF6, phosphorylated PERK and phosphorylated IRE1, which induce endoplasmic reticulum stress-mediated apoptosis in osteoblasts. 2-APB protects the osteoblasts from the effects of Dex, preventing endoplasmic reticulum stress-mediated apoptosis.

P2K1 Receptor, Heterotrimeric Gα Protein and CNGC2/4 Are Involved in Extracellular ATP-Promoted Ion Influx in the Pollen of Arabidopsis thaliana

As an apoplastic signal, extracellular ATP (eATP) is involved in plant growth and development. eATP promotes tobacco pollen germination (PG) and pollen tube growth (PTG) by stimulating Ca2+ or K+ absorption. Nevertheless, the mechanisms underlying eATP-stimulated ion uptake and their role in PG and PTG are still unclear. Here, ATP addition was found to modulate PG and PTG in 34 plant species and showed a promoting effect in most of these species. Furthermore, by using Arabidopsis thaliana as a model, the role of several signaling components involved in eATP-promoted ion (Ca2+, K+) uptake, PG, and PTG were investigated. ATP stimulated while apyrase inhibited PG and PTG. Patch-clamping results showed that ATP promoted K+ and Ca2+ influx into pollen protoplasts. In loss-of-function mutants of P2K1 (dorn1-1 and dorn1-3), heterotrimeric G protein α subunit (gpa1-1, gpa1-2), or cyclic nucleotide gated ion channel (cngc2, cngc4), eATP-stimulated PG, PTG, and ion influx were all impaired. Our results suggest that these signaling components may be involved in eATP-promoted PG and PTG by regulating Ca2+ or K+ influx in Arabidopsis pollen grains.

High Doses of Dexamethasone Induce Endoplasmic Reticulum Stress-Mediated Apoptosis by Promoting Calcium Ion Influx-Dependent CHOP Expression in Osteoblasts

Background: The molecular mechanisms by which dexamethasone (Dex) induces apoptosis in osteoblasts remain unclear.Materials and Methods: MC3T3-E1 cells were treated with 0, 10-8, 10-6, and 10-4 M Dex for 24 h. The expression of ATF6, and phosphorylated PERK and IRE1, cell apoptosis, and the activity of caspase-12 and caspase-3 were measured. The expression of CHOP and the rate of influx of calcium ions were also measured in cells treated with 0 and 10-4 M Dex for 24 h. The effect of 2-APB treatment was assessed in cells treated with 0 or 10-4 M Dex.Results: The levels of ATF6 and phosphorylated PERK and IRE1 increased in a dose-dependent manner in MC3T3-E1 cells treated with 10-8, 10-6, and 10-4 M Dex, compared to in cells treated with 0 M Dex (P <0.05). Cells treated with 10-6 and 10-4 M Dex had significantly increased cell apoptosis rates and caspase-12 and caspase-3 activity compared to the control (P <0.05). Cells treated with 10-4 M Dex had significantly increased levels of CHOP and calcium ion influx rates compared to in the control (P <0.05). Combined treatment with 10-4 M Dex and 2-APB abrogated the observed increases in cell apoptosis and the activity of caspase-12 and caspase-3 (P>0.05). Conclusion: High doses of Dex induce endoplasmic reticulum stress-mediated apoptosis by promoting calcium ion influx-dependent expression of CHOP, and the activation of caspase-12 and caspase-3 in osteoblasts. Combined treatment with 2-APB protects the cells from the effects of Dex, preventing endoplasmic reticulum stress-mediated apoptosis.

Inflammasome-Induced Osmotic Pressure and the Mechanical Mechanisms Underlying Astrocytic Swelling and Membrane Blebbing in Pyroptosis

Cell swelling and membrane blebbing are characteristic of pyroptosis. In the present study, we explored the role of intracellular tension activity in the deformation of pyroptotic astrocytes. Protein nanoparticle-induced osmotic pressure (PN-OP) was found to be involved in cell swelling and membrane blebbing in pyroptotic astrocytes, and was associated closely with inflammasome production and cytoskeleton depolymerization. However, accumulation of protein nanoparticles seemed not to be absolutely required for pyroptotic permeabilization in response to cytoskeleton depolymerization. Gasdermin D activation was observed to be involved in modification of typical pyroptotic features through inflammasome-induced OP upregulation and calcium increment. Blockage of nonselective ion pores can inhibit permeabilization, but not inflammasome production and ion influx in pyroptotic astrocytes. The results suggested that the inflammasomes, as protein nanoparticles, are involved in PN-OP upregulation and control the typical features of pyroptotic astrocytes.

Putative spanner function of the Vibrio PomB plug region in the stator rotation model for flagellar motor

Bacterial flagella are the best-known rotational organelles in the biological world. The spiral-shaped flagellar filaments that extending from the cell surface rotate like a screw to create a propulsive force. At the base of the flagellar filament lies a protein motor that consists of a stator and a rotor embedded in the membrane. The stator is composed of two types of membrane subunits, PomA(MotA) and PomB(MotB), which are energy converters that assemble around the rotor to couple rotation with the ion flow. Recently, stator structures, where two MotB molecules are inserted into the center of a ring made of five MotA molecules, were reported. This structure inspired a model in which the MotA ring rotates around the MotB dimer in response to ion influx. Here, we focus on the Vibrio PomB plug region, which is involved in flagellar motor activation. We investigated the plug region using site-directed photo-crosslinking and disulfide crosslinking experiments. Our results demonstrated that the plug interacts with the extracellular short loop region of PomA, which is located between transmembrane helices 3 and 4. Although the motor stopped rotating after crosslinking, its function recovered after treatment with a reducing reagent that disrupted the disulfide bond. Our results support the hypothesis, which has been inferred from the stator structure, that the plug region terminates the ion influx by blocking the rotation of the rotor as a spanner. Importance The biological flagellar motor resembles a mechanical motor. It is composed of a stator and a rotor. The force is transmitted to the rotor by the gear-like stator movements. It has been proposed that the pentamer of MotA subunits revolves around the axis of the B subunit dimer in response to ion flow. The plug region of the B subunit regulates the ion flow. Here, we demonstrated that the ion flow was terminated by crosslinking the plug region of PomB with PomA. These findings support the rotation hypothesis and explain the role of the plug region in blocking the rotation of the stator unit.