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

Abstract

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.

Figure 1

Effects of short-term treatments of bortezomib (BTZ) or chloroquine (CHQ) on Caveolin-1 protein expression in normoxic and hypoxic (4% O₂) human pulmonary arterial smooth muscle cells (PASMCs). Western blots (a) and bar graph (b) representing the protein expression level of Caveolin-1, p21, and LC3B upon treatment with BTZ (10 nM) or CHQ (μM) for 3, 6, and 12 hours in human PASMCs under normoxic and hypoxic conditions. Beta tubulin was used as house-keeping protein. The bar graph represents the mean ± SEM, n = 5 in each group; ^∗p < 0.05, ^&p < 0.05 as indicated.

Figure 2

Effects of long-term treatments of BTZ and/or CHQ on Caveolin-1 protein expression in normoxic and hypoxic human PASMCs. Western blots (a) and bar graph (b) representing the protein expression level of Caveolin-1, p21, and LC3B upon treatment with BTZ (10 nM) or CHQ (μM) for 24 and 60 hours in human PASMCs under normoxic and hypoxic conditions. Beta tubulin was used as house-keeping protein. The bar graph represents the mean ± SEM, n = 5 in each group; ^∗p < 0.05, ^&p < 0.05 as indicated.

Figure 3

Effects of BTZ and CHX on the Caveolin-1 protein expression in the presence of cycloheximide (CHX) in human PASMCs under normoxia and hypoxia. Western blot (a) and bar graph (b) representing the protein expression level of Caveolin-1 and p21 upon treatment with BTZ and/or CHQ in the presence of CHX (20 μg/ml) for 24 hours in human PASMCs under normoxic and hypoxic conditions. The bar graph represents the mean ± SEM, n = 4 in each group; ^∗p < 0.05 vs. Hyp+CHX.

Figure 4

Effects of BTZ on the protein degradation of Caveolin-1 protein in human PASMCs under normoxia and hypoxia. Western blot (a) and line chart (b) representing the protein expression level of Caveolin-1 and p21 upon treatment with/without BTZ in the presence of CHX within a 24-hour time window in human PASMCs under normoxic and hypoxic conditions. The bar graph represents the mean ± SEM, n = 5 in each group.

Figure 5

Effects of long-term treatments of BTZ and/or CHQ on Caveolin-1 mRNA expression in human PASMCs under normoxia and hypoxia. Bar graph representing the mRNA expression level of Caveolin-1 upon treatment with BTZ and/or CHQ for 24 and 60 hours in human PASMCs under normoxic and hypoxic conditions. 18S was used as house-keeping gene. The bar graph represents the mean ± SEM, n = 4 in each group; ^∗p < 0.05, ^&p < 0.05 as indicated.

Figure 6

Effects of BTZ and/or CHQ treatment on the proliferation and intracellular calcium homeostasis of human PASMCs under normoxia and hypoxia. Bar graphs representing the normalized proliferation (a), baseline [Ca²⁺]_i, and SOCE (b) of PASMCs upon treatment with BTZ and/or CHQ for 60 hours under normoxic and hypoxic conditions. The bar graph represents the mean ± SEM, n = 6 in (a) and n = 3 in (b); ^∗p < 0.05, ^&p < 0.05 as indicated.