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. 2022 Jan;66(1):23-37.
doi: 10.1165/rcmb.2021-0112OC.

Bortezomib Inhibits Lung Fibrosis and Fibroblast Activation without Proteasome Inhibition

Loka Raghu Kumar Penke  1 , Jennifer Speth  1 , Scott Wettlaufer  1 , Christina Draijer  1 , Marc Peters-Golden  1   2
Affiliations

Bortezomib Inhibits Lung Fibrosis and Fibroblast Activation without Proteasome Inhibition

Loka Raghu Kumar Penke et al. Am J Respir Cell Mol Biol. 2022 Jan.

Abstract

The U.S. Food and Drug Administration-approved proteasomal inhibitor bortezomib (BTZ) has attracted interest for its potential antifibrotic actions. However, neither its in vivo efficacy in lung fibrosis nor its dependence on proteasome inhibition has been conclusively defined. In this study, we assessed the therapeutic efficacy of BTZ in a mouse model of pulmonary fibrosis, developed an in vitro protocol to define its actions on diverse fibroblast activation parameters, determined its reliance on proteasome inhibition for these actions in vivo and in vitro, and explored alternative mechanisms of action. The therapeutic administration of BTZ diminished the severity of pulmonary fibrosis without reducing proteasome activity in the lung. In experiments designed to mimic this lack of proteasome inhibition in vitro, BTZ reduced fibroblast proliferation, differentiation into myofibroblasts, and collagen synthesis. It promoted dedifferentiation of myofibroblasts and overcame their characteristic resistance to apoptosis. Mechanistically, BTZ inhibited kinases important for fibroblast activation while inducing the expression of DUSP1 (dual-specificity protein phosphatase 1), and knockdown of DUSP1 abolished its antifibrotic actions in fibroblasts. Collectively, these findings suggest that BTZ exhibits a multidimensional profile of robust inhibitory actions on lung fibroblasts as well as antifibrotic actions in vivo. Unexpectedly, these actions appear to be independent of proteasome inhibition, instead attributable to the induction of DUSP1.

Keywords: bortezomib; fibroblast activation; proteasome; pulmonary fibrosis.

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Figures

Figure 1.
Figure 1.
Bortezomib (BTZ) administration improves bleomycin-induced fibrosis in mice. (A) Scheme illustrating the timelines for in vivo administration of bleomycin and BTZ for determination of experimental endpoints at Day 21 and for the pertinent phases of the pulmonary response in the bleomycin model of pulmonary fibrosis. (B) Digital images of Masson’s trichrome staining for collagen deposition (blue) at Day 21 in mice treated ± bleomycin and BTZ. Scale bars, 500 μm. (C–F) Effect of BTZ treatment in mice treated ± bleomycin as reflected by changes in lung hydroxyproline content (C) and the mRNA expression of fibrotic markers Col1α1 (D), Ctgf (E), and Tgf-β1 (F); in C–F, each symbol represents an individual mouse, and horizontal lines represent mean values. Values in each group represent results from two pooled independent experiments with a total of 8–9 mice per group. *P < 0.05, two-way ANOVA. Col1α1 = collagen type I α 1; Ctgf = connective tissue growth factor; Tgf-β1 = transforming growth factor β-1.
Figure 2.
Figure 2.
Influence of BTZ on proteasome activity in vivo and in vitro. (A) Lung tissues from in vivo groups in Figure 1 were assessed for proteasome activity via 20S proteasome activity assay 1 hour after harvest. Purified 20S proteasome and its activity inhibitor lactacystin were used as experimental positive and negative controls, respectively. (B) Densitometric analysis of ubiquitinated protein bands in lung tissue harvested at Day 21 from mice treated with saline, bleomycin, or bleomycin followed by treatment with BTZ 0.1 mg/kg or 0.25 mg/kg; total density of the lane for each mouse is expressed relative to the density of the GAPDH band for that lane. The densitometry value of each bleomycin sample from Figure E2B (see data supplement) (mice 1, 2, and 3) was then normalized to the densitometric value of the same sample run on a separate gel shown in Figure E2A in order to merge the data into a single graph. (C) Design of experiment to assess the effect of BTZ dose and incubation protocol on proteasome activity in fibroblasts (Fibs) and in myofibroblasts (MyoFibs). (D) Fibs and MyoFibs were treated with BTZ at 10 or 100 nM, and medium was either changed at 30 minutes or not changed; 20S proteasome activity was assessed immediately or at 30 minutes, 1 hour, 2 hours, 6 hours, or 24 hours later. In A, each symbol represents an individual mouse and horizontal lines represent mean values. Each symbol in B represents individual mice with mean values. Symbols in D represent mean values (±SE) from three independent experiments. For A and D, *P < 0.05 (two-way ANOVA) and, for B, *P < 0.01 (Student’s t test, unpaired). a.u. = arbitrary units; Pos. Cont. = positive control.
Figure 3.
Figure 3.
BTZ inhibits FGF-2 (fibroblast growth factor-2)–induced Fib proliferation and TGF-β–induced Fib differentiation. (A) Design of experiments to evaluate BTZ effects on FGF-2–induced Fib proliferation endpoints depicted in B and C. (B) Cells were pretreated ±BTZ (10 nM) for 30 minutes, after which the medium was replaced and cells were stimulated ±FGF-2. Cells were harvested and proliferation was quantified at 72 hours. Control value represents fluorescence of DMSO-treated Fibs. (C) Cells were pretreated ±BTZ (10 nM) for 30 minutes, after which the medium was replaced and cells were stimulated ±FGF-2. Cells were harvested and assessed for the expression of FOXM1 and CYCB1 proteins by Western blot at 48 hours; left panel, representative blot; right panel, mean densitometric analysis of blots from three experiments. (D) Design of experiments to assess the capacity of BTZ to attenuate Fib differentiation using a “prevention protocol.” (E and F) Fibs were treated ±BTZ (10 nM) for 30 minutes, after which the medium was replaced and cells were stimulated ±TGF-β. Cells were harvested and assessed for the expression of differentiation-associated genes α-SMA and COL1α2 mRNA by quantitative real-time PCR (qPCR) at 24 hours (E) and their proteins by Western blot at 48 hours (F). In F, the left panel depicts a representative blot, and the right panel provides densitometric analysis of blots from three experiments. All data represent mean values (±SE) from three independent experiments. *P < 0.05, two-way ANOVA.
Figure 4.
Figure 4.
BTZ dedifferentiates both established TGF-β–elicited MyoFibs and idiopathic pulmonary fibrosis (IPF) Fibs. (A) Design of experiments to assess the capacity of BTZ to reduce MyoFib phenotype in a “dedifferentiation protocol.” (B and C) Fibs treated for 48 hours with TGF-β to elicit differentiation into MyoFibs were then treated ±BTZ (10 nM) for 30 minutes, after which the medium was changed. Cells were harvested and assessed for the expression of α-SMA and COL1α2 mRNA by qPCR at 24 hours (B) and protein by Western blot at 48 hours (C). In C, the left panel presents a representative blot and the right panel provides mean densitometric values (±SE) of Western blots from three independent experiments. For B and C, GAPDH mRNA and protein were used to normalize α-SMA and COL1α2 expression by qPCR and Western blot, respectively. (D) Design of experiments to assess BTZ effects on IPF Fibs using a combination “dedifferentiation” and “prevention” protocol. (E and F) IPF Fibs were treated ±BTZ (10 nM) for 30 minutes, after which the medium was replaced and they were stimulated ±TGF-β. Cells were harvested and analyzed for the expression of α-SMA and COL1α2 mRNA by qPCR at 24 hours (E) and protein by Western blot at 48 hours (F). For B and C and for E and F, GAPDH mRNA and protein were used to normalize α-SMA and COL1α2 expression by qPCR and Western blot, respectively. In C and F, the left panel presents a representative blot, and the middle and right panels depict mean densitometric analysis of α-SMA and COL1α2 from Western blots from three experiments; the dashed line in the left panel indicates that the lanes were from the same blot but noncontiguous. All data represent mean values (±SE) from three independent experiments. *P < 0.05, two-way ANOVA.
Figure 5.
Figure 5.
BTZ sensitizes established MyoFibs to FAS-mediated apoptosis. (A) Fibs were treated with activating anti-FAS antibody at 100 ng/ml for 14 hours. Cells were harvested, phosphatidylserine on early apoptotic cells was detected by Pacific Blue-Annexin V staining, and the percentage of apoptotic cells was quantified by flow cytometry. (B) Design of experiments to assess BTZ effect on TGF-β–elicited MyoFib apoptosis. (C–F) TGF-β–elicited MyoFibs were treated ±BTZ (10 nM) for 30 minutes, after which the medium was changed, and they were then stimulated ±anti-FAS antibody, as in A. Cells were harvested and apoptotic cells quantified either by flow cytometric analysis of phosphatidylserine staining at 14 hours (C) or by caspase 3/7 activity assay (D). (E) Effect of BTZ on expression of the pro- and antiapoptotic genes BIRC5, APAF1, and BID measured by qPCR at 24 hours. (F) Effect of BTZ on expression of FAS mRNA by qPCR and FAS protein analysis by Western blot at the time points indicated. (G) Design of experiments to assess BTZ effect on IPF Fib apoptosis sensitivity. (H) IPF Fibs (n = 6) were treated ±BTZ (10 nM) for 30 minutes, after which the medium was changed and cells were stimulated ±anti-FAS antibody. Cells were harvested at 24 hours, and apoptosis was quantified by caspase 3/7 activity. In E and F, GAPDH mRNA and protein were used to normalize apoptosis genes or FAS expression by qPCR and Western blot, respectively. All data represent mean values (±SE) from three independent experiments. *P < 0.05, two-way ANOVA.
Figure 6.
Figure 6.
BTZ inhibits key kinases activated by TGF-β and FGF-2. (A) Design of experiments to assess BTZ effects on phosphorylation of key kinases activated by TGF-β or FGF-2. (B) Fibs were treated ±BTZ (10 nM) for 30 minutes, after which the medium was replaced and cells were stimulated with TGF-β or FGF-2 for 30 minutes. Cells were harvested, and phosphorylation status of P38 by TGF-β and AKT by FGF-2 was assessed by Western blot. Total P38 and AKT proteins were used to normalize phospho-P38 and phospho-AKT, respectively. Graphs represent mean densitometric analysis of phospho-P38 and phospho-AKT Western blots. (C) Design of experiments to assess the ability of 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) to prevent BTZ modulation of TGF-β–induced MyoFib differentiation. (D) Cells were treated ±DRB (25 μM) for 30 minutes and then treated ±BTZ (10 nM) for an additional 30 minutes. Medium was replaced, and cells were stimulated with TGF-β to analyze the expression of α-SMA mRNA by qPCR at 48 hours. All data represent mean values (±SE) from four independent experiments. *P < 0.05, two-way ANOVA.
Figure 7.
Figure 7.
The antifibrotic actions of BTZ require de novo induction of DUSP1 (dual-specificity protein phosphatase 1). (A) Design of experiment to assess the expression of phosphatase genes by BTZ (upper); Fibs were treated ±BTZ (10 nM) for 30 minutes, and cells were harvested immediately to assess the expression of various cellular phosphatase mRNAs by qPCR (lower). (B) Cells were treated ±BTZ (10 nM) for 30 minutes, harvested, and assessed for the expression of DUSP1 mRNA by qPCR (lower) and protein by Western blot (upper). (C) Mice were treated with bleomycin followed by treatment at Day 15 with or without BTZ 0.1 mg/kg, and lungs were harvested at 2 hours, 4 hours, and 6 hours after BTZ treatment for determination of DUSP1 protein expression by Western blot. (D) Design of experiment to assess the requirement for new transcription of the induction of DUSP1 by BTZ (upper); cells were treated ±DRB (25 μM) for 30 minutes and then ±BTZ (10 nM) for an additional 30 minutes; cells were harvested immediately to assess the expression of DUSP1 mRNA by qPCR (lower). (E) Fibs were treated ±BTZ (10 nM) for 10 or 30 minutes and harvested, after which the phosphorylation status of P65 and DUSP1 was assessed by Western blot. (F) Cells were treated ±TPCA-1 (5 μM) for 30 minutes and then ±BTZ (10 nM) for an additional 30 minutes; cells were harvested immediately to assess the expression of DUSP1 mRNA by qPCR. (G and H) DUSP1 Cont and DUSP1 KD cells were treated ±BTZ (10 nM) for 30 minutes, after which the medium was replaced and cells were stimulated ±TGF-β (G) or ±FGF-2 (H) and harvested at 48 hours to analyze the expression of α-SMA or FOXM1 mRNA, respectively, by qPCR. All data represent mean values (±SE) from three independent experiments. *P < 0.05, two-way ANOVA. KD = knockdown; TPCA-1 = 2-[(aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide
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