Bortezomib Inhibits Hypoxia-Induced Proliferation by Suppressing Caveolin-1/SOCE/[Ca2+]i Signaling Axis in Human PASMCs

Background. Previous studies have demonstrated the ubiquitin-proteasome inhibitor bortezomib (BTZ) can effectively alleviate hypoxia-induced pulmonary hypertension (HPH) by suppressing the intracellular calcium homeostasis in pulmonary arterial smooth muscle cells (PASMCs). Further evaluation showed that the antiproliferation roles of BTZ are mainly mediated by inhibition of the intracellular calcium homeostasis. Caveolin-1 belongs to one of the key regulators of the intracellular calcium homeostasis in PASMCs, which can regulate the store-operated calcium entry (SOCE). However, the effects of BTZ on Caveolin-1 remain unclear. Methods. Primarily cultured human PASMCs were used as the cell model. CCK-8 assay was performed to assess the PASMCs proliferation. Western blotting and real-time qPCR were used to detect the mRNA and protein expressions. Fura-2-based fluorescence imaging experiments were used to determine the intracellular calcium concentration ([Ca2+]i). The protein synthesis inhibitor cycloheximide (CHX) was utilized to determine the protein degradation process. Results. Firstly, in cultured human PASMCs, treatment of BTZ for 24 or 60 hours significantly downregulates Caveolin-1 at both mRNA and protein levels. Secondly, in the presence CHX, BTZ treatment also leads to downregulated protein expression and fastened protein degradation of Caveolin-1, indicating that BTZ can promote the Caveolin-1 protein degradation, other than the BTZ on Caveolin-1 mRNA transcription. Then, BTZ significantly attenuates the hypoxia-elevated baseline [Ca2+]i, SOCE, and cell proliferation. Conclusion. We firstly observed that the ubiquitin-proteasome inhibitor BTZ can inhibit the Caveolin-1 expression at both mRNA transcription and protein degradation processes, providing new mechanistic basis of BTZ on PASMC proliferation.

Neuronal-Specific Inhibition of Endoplasmic Reticulum Mg2+/Ca2+ ATPase Ca2+ Uptake in a Mixed Primary Hippocampal Culture Model of Status Epilepticus

Loss of intracellular calcium homeostasis is an established mechanism associated with neuronal dysfunction and status epilepticus. Sequestration of free cytosolic calcium into endoplasmic reticulum by Mg2+/Ca2+ adenosinetriphosphatase (ATPase) is critical for maintenance of intracellular calcium homeostasis. Exposing hippocampal cultures to low-magnesium media is a well-accepted in vitro model of status epilepticus. Using this model, it was shown that endoplasmic reticulum Ca2+ uptake was significantly inhibited in homogenates from cultures demonstrating electrophysiological seizure phenotypes. Calcium uptake was mainly neuronal. However, glial Ca2+ uptake was also significantly inhibited. Viability of neurons exposed to low magnesium was similar to neurons exposed to control solutions. Finally, it was demonstrated that Ca2+ uptake inhibition and intracellular free Ca2+ levels increased in parallel with increasing incubation in low magnesium. The results suggest that inhibition of Mg2+/Ca2+ ATPase-mediated endoplasmic reticulum Ca2+ sequestration contributes to loss of intracellular Ca2+ homeostasis associated with status epilepticus. This study describes for the first time inhibition of endoplasmic reticulum Mg2+/Ca2+ ATPase in a mixed primary hippocampal model of status epilepticus. In combination with animal models of status epilepticus, the cell culture model provides a powerful tool to further elucidate mechanisms that result in inhibition of Mg2+/Ca2+ ATPase and downstream consequences of decreased enzyme activity.