Bortezomib Inhibits The Hypoxia-induced Proliferation Of PASMCs By Downregulating Caveolin-1 Expression At Both MRNA Transcription And Protein Degradation Levels

Pulmonary hypertension(PH)is a clinical syndrome characterized by increased pulmonary arterial pressure,and is defined as a mean pulmonary arterial pressure(m PAP)? 20 mm Hg under resting state at sea level.In the early stage of the PH pathognesis,only increased pulmonary vascular resistance was observed,while in the later stage,reduced pulmonary blood flow will be oberved,which progressively induces right heart failure and eventually death.The disease is characterized by numerous etiology,high prevalence,hidden onset,and high mortality.The main pathological changes are endothelial cell apoptosis,vascular remodeling,and distal pulmonary vascular occlusion.The vascular remodeling is mainly caused by the excessive proliferation and migration of smooth muscle cells.The traditional treatment is mainly developed to enhance the myocardial contractility,diuresis,dilate pulmonary blood vessels,relieve the symptoms and enhance the exercise capacity of the patients,which cannot cure the disease from the basis.At present,targeted treatment medications for are mainly used for the treatment of type I of pulmonary arterial hypertension and type IV of chronic thromboembolic pulmonary hypertension,which can reduce symptoms and prolong survival time,but also has disadvantages such as high price and many reported side effects.Therefore,to develop novel,better treatments that targeting to the improvement of endothelial function,reducing the thickening and remodeling of the pulmonary vasculature.Our previous research has uncovered found that bortezomib(BTZ)exerts significant therapeutic effect on PH in a chronic hypoxia-induced PH rat model.BTZ can significantly reduce the right ventricular pressure,and normalize the pulmonary vascular remodeling.Moreover,the underlying molecular mechanism including an inhibition on the expression of classical transient receptor protein 1,6(TRPC1,6)in the pulmonary arterial smooth muscle cells(PASMCs),thereby inhibiting the store-operated calcium entry(SOCE)and then reducethe basal calcium in PASMCs.However,the detail regulatory mechanism remains largely unknown.In recent years,emerging literatures have reported that Caveolin-1 is an important SOCE regulatory protein and an important participant of the intracellular calcium homeostasis in PASMCs.Studies have found that hypoxia upregulates Caveolin-1,which accounts for the hypoxia-enhanced SOCE and intracellular calcium free concentration and proliferation in PASMCs.Therefore,in this study,we further explored whether BTZ also targets Caveolin-1 to regulate the calcium homeostasis of PASMCs and further regulate cell proliferation.?Rationale?A primarily cultured rat PASMCs model was established,and the effects of BTZ on Caveolin-1 transcription level and protein expression were observed by RT-q PCR and Western blot,respectively.Cycloheximide(CHX),a protein synthesis inhibitor,was used to block the protein synthesis and the effects of BTZ on Caveolin-1 degradation pathway was observed thereafter;Given the fact that Caveolin-1 protein degradates via the lysosomal pathway,the effects of both BTZ and lysosomal inhibitor Chloroquine(CHQ)on the hypoxia-upregulated Caveolin-1 expression and hypoxia-induced proliferation were also studied.?Method?1.Primarilly culture of rat distal pulmonary artery smooth muscle cellsMale Sprague-Dawley(SD)rats(about 250g)were anesthetized with 3% sodium pentobarbital,then the distal pulmonary arteries were isolated from the rat.Carefully remove the adventitia,longitudinally cut the pulmonary arteries,scrape the endothelium by using a cotton swab.The vessel tissues were digested with collagenase after strain filtration,inoculated in a 10 cm petri dish,the identified cells were inoculated in a 35 mm petri dish containing a slide,and cultured for 5-6 days with DMEM containing 10% FBS.The cell purity was assessed by using a positive rate of cells expressing smooth muscle alpha actin by using immunofluorescence staining.2.Immunoblotting experimentsDiscard the culture medium of different experimental groups,rinse twice with pre-chilled PBS,add RIPA lysate containing PMSF,lyse on ice for 30 minutes on a shaker,and collect cells from the culture dish with a cell scraper.The protein supernatant was collected by centrifugation at 4 degrees.The protein concentration was determined by the BCA method.After the same amount of protein was loaded,the protein in the gel was transferred to a PVDF membrane using 10% SDS-PAGE gel.The membrane was blocked by 5% nonfat milk and incubated in primary antibodies against Caveolin-1 at 4degree for overnight.Afterall,the membrane was washed for 3 times with TBST,10-15 min / time,and subjected for 1-hour exposure of secondary antibody at room temperature,discard the secondary antibody,wash 3 times with TBST,10-15 min / time,and add luminous solution for exposure.3.Real-time PCRThe total RNA of PASMCs was extracted by Trizol method,and the RNA was reverse transcribed into c DNA according to the RT kit of TAKARA company.The quantitative PCR reaction was performed according to the fluorescent quantitative PCR kit of TAKARA company.The specificity of the product was judged by the single peak dissolution curve.4.Cell proliferation experimentCells were cultured in a 96-well petri dish,10 ?l of CCK8 reagent was added to each experimental well,and incubated at 37 ° C in a normal oxygen incubator for 4 hours.Cell absorbance was measured at 450 nm using a microplate reader.5.Determination of intracellular calcium ionPrimary pulmonary artery smooth muscle cells were cultured on a 25 mm prototype cover glass.fura-2 fluorescent dye was added after treatment.The cells were incubated in an incubator for 1 hour.The changes of 340/380 fluorescence intensity and calcium ion concentration in the cells were measured by fluorescence microscope.?Results?1.Short-term treatment(3,6,12 hours)of BTZ does not significantly regulate the protein expression of Caveolin-1,but a 24 and 60 hours treatment of BTZ markedlydownregulates the hypoxic upregulation of Caveolin-1 protein and m RNA expression(P<0.05).2.The protein synthesis inhibitor CHX was used to block the nascent protein synthesis.Then,BTZ treatment can reduce the protein expression and accelerate the protein degradation of Caveolin-1 in rat PASMCs.3.Given the fact that Caveolin-1 protein is degraded via the lysosomal pathway.We further determined that a short-term treatment with CHQ(3,6,12 hours)had no significant effects on the protein expression of Caveolin-1,while a 24 or 60 hours treatment of CHQ could significantly decrease the Caveolin-1 expression at both protein and m RNA levels,likely due to the inhibition of Caveolin-1 transcription.4.Both BTZ and CHQ can significantly inhibit the hypoxia-induced proliferation of PASMCs,while the combined use of the two exerts no further add-on effects than the single use of each of the two.5.In the cell calcium ion determination experiment,the basic calcium and SOCE in the distal PASMCs of hypoxic rats are higher than that of normal oxygen.BTZ and CHQ can reduce the basic calcium and SOCE,but the combined use does not further reduce them.?Conclusion?The proteasome inhibitor BTZ can downregulate the hypoxia-induced expression of Caveolin-1 by both inhibiting the m RNA transcription and promoting the protein degradation,thereby suppressing the hypoxia-induced proliferation of PASMCs.