Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2023 Jan 24;16(2):177.
doi: 10.3390/ph16020177.

HSPB5 Inhibition by NCI-41356 Reduces Experimental Lung Fibrosis by Blocking TGF-β1 Signaling

Julie Tanguy  1   2   3   4 , Pierre-Marie Boutanquoi  1 , Olivier Burgy  1   2   3   4 , Lucile Dondaine  1   3   4 , Guillaume Beltramo  1   2   3   4 , Burhan Uyanik  1 , Carmen Garrido  1   3   4 , Philippe Bonniaud  1   2   3   4 , Pierre-Simon Bellaye  1   3   4   5 , Françoise Goirand  1   2   3   4
Affiliations

HSPB5 Inhibition by NCI-41356 Reduces Experimental Lung Fibrosis by Blocking TGF-β1 Signaling

Julie Tanguy et al. Pharmaceuticals (Basel). .

Abstract

Idiopathic pulmonary fibrosis is a chronic, progressive and lethal disease of unknown etiology that ranks among the most frequent interstitial lung diseases. Idiopathic pulmonary fibrosis is characterized by dysregulated healing mechanisms that lead to the accumulation of large amounts of collagen in the lung tissue that disrupts the alveolar architecture. The two currently available treatments, nintedanib and pirfenidone, are only able to slow down the disease without being curative. We demonstrated in the past that HSPB5, a low molecular weight heat shock protein, was involved in the development of fibrosis and therefore was a potential therapeutic _target. Here, we have explored whether NCI-41356, a chemical inhibitor of HSPB5, can limit the development of pulmonary fibrosis. In vivo, we used a mouse model in which fibrosis was induced by intratracheal injection of bleomycin. Mice were treated with NaCl or NCI-41356 (six times intravenously or three times intratracheally). Fibrosis was evaluated by collagen quantification, immunofluorescence and TGF-β gene expression. In vitro, we studied the specific role of NCI-41356 on the chaperone function of HSPB5 and the inhibitory properties of NCI-41356 on HSPB5 interaction with its partner SMAD4 during fibrosis. TGF-β1 signaling was evaluated by immunofluorescence and Western Blot in epithelial cells treated with TGF-β1 with or without NCI-41356. In vivo, NCI-41356 reduced the accumulation of collagen, the expression of TGF-β1 and pro-fibrotic markers (PAI-1, α-SMA). In vitro, NCI-41356 decreased the interaction between HSPB5 and SMAD4 and thus modulated the SMAD4 canonical nuclear translocation involved in TGF-β1 signaling, which may explain NCI-41356 anti-fibrotic properties. In this study, we determined that inhibition of HSPB5 by NCI-41356 could limit pulmonary fibrosis in mice by limiting the synthesis of collagen and pro-fibrotic markers. At the molecular level, this outcome may be explained by the effect of NCI-41356 inhibiting HSPB5/SMAD4 interaction, thus modulating SMAD4 and TGF-β1 signaling. Further investigations are needed to determine whether these results can be transposed to humans.

Keywords: HSPB5; TGF-β; anti-fibrotic therapy; pulmonary fibrosis (PF).

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Intravenous administration of NCI-41356 reduces pulmonary fibrosis in bleomycin treated mice. (A). Mice experimental injection plan between day 0 (D0), the day of bleomycin (BLM) injection (1.5 mg/kg) and the day of sacrifice (D21). Mice were treated with NaCl 0.9% (n = 6) or NCI-41356 (n = 6) (15 mg/kg) 3 times a week between day 9 and 21; (B). Collagen quantification by Sircol assay (expressed as µg of collagen per mg of lung) of bleomycin treated mice lungs treated or not with NCI-41356 (* p = 0.0317); (C). Collagen semi-quantification by Sirius Red staining performed on lung sections of mice treated by bleomycin with or without NCI-41356 (* p = 0.0159, control = 1); (D). Quantification of α-SMA staining by immunofluorescence performed on lung sections of mice treated by bleomycin with or without NCI-41356 (* p = 0.0159); (E). TGF-β1 mRNA expression (brown color, black arrow) on lung tissues of mice treated by bleomycin with or without NCI-41356 (**** p < 0.0001)(×20). Data are expressed as means ± SEM and compared using nonparametric Mann–Whitney test (Figure 1B–D) or Welch’s test (Figure 1E).
Figure 2
Figure 2
Intratracheal administration of NCI-41356 reduces pulmonary fibrosis in bleomycin-treated mice. (A). Mouse experimental injection plan between day 0 (D0), the day of bleomycin (BLM) injection (1.5 mg/kg) and the day of sacrifice (D21). Mice were treated with NaCl 0.9% (n = 6) or NCI-41356 (n = 6) (15 mg/kg) at D12, D14 and D17; (B). Collagen quantification by Sircol assay (expressed as µg of collagen per mg of lung) of mice lungs treated with bleomycin with or without NCI-41356 (** p = 0.0079); (C). Collagen semi-quantification by Sirius Red staining performed on lung sections of mice treated by bleomycin with or without NCI-41356 (** p = 0.0079, control = 1) and representative staining; (D). Quantification of α-SMA staining by immunofluorescence performed on lung tissues of mice treated by bleomycin with or without NCI-41356 (** p = 0.0079); (E). Relative PAI-1 mRNA expression in the lung of mice treated by bleomycin with or without NCI-41356 (* p = 0.0159); (F). TGF-β1 mRNA expression (brown color, black arrow) on lung tissues from mice treated by bleomycin with or without NCI-41356 (*** p = 0.0002) (×20). Data are expressed as means ± SEM and compared using nonparametric Mann–Whitney test (Figure 2B–E) or Welch’s test (Figure 2F).
Figure 3
Figure 3
NCI-41356 decreases SMAD4/HSPB5 interaction. (A). Anti-aggregation assay on luciferase exposed to heat shock (HS) in presence or absence of recombinant chaperone HSPB1/HSPB5 (1.5 μM) and NCI-41356 (100 µM) (n = 3) (**** p < 0.0001); (B). Interaction measurement by thermophoresis between HSPB5 and SMAD4 proteins in presence or not of NCI-41356, tested at 100 µM and 1 mM (* p = 0.0107); KD (dissociation constant); (C). Proximity ligation assay between SMAD4/HSPB5 on A549 cells treated by either NCI-41356 (100 µM) or TGF-β1 (10 ng/mL) or both compounds (quantification one the left and representative image on the right) (×40); One red spot matches with one interaction between these two proteins (n = 3) (*** p = 0.0003; **** p < 0.0001). Data are expressed as means ± SEM and compared using nonparametric Mann–Whitney (anti-aggregation assay) and Kruskal–Wallis tests (thermophoresis assay) or one-way ANOVA (PLA assay).
Figure 4
Figure 4
NCI-41356 modulates TGF-β1 signaling. (A). Immunofluorescence staining of SMAD4 protein (red) on A549 cells treated with TGF-β1 (10 ng/mL) with or without NCI-41356 (100 µM) (n = 3) (×40). Nuclear staining: 4′,6-diamidino-2-phenylindole (blue); (B). Protein expression os Smad4, pSMAD3, GAPDH and PARP studied by Western blot of nuclear or cytoplasmic fractions of A549 cells treated with TGF-β1 (10 ng/mL) with or without NCI-41356 (100 µM). One representative experiment out of three is shown; (C). Relative PAI-1 mRNA expression compared to control L32 in the lung of mice treated by bleomycin with or without NCI-41356 by intravenous route (* p = 0.0159). Data are expressed as means ± SEM and compared using nonparametric Mann–Whitney test.
Figure 5
Figure 5
Proposed mechanical pathway involved in inhibitory effects of NCI-41356. NCI-41356 inhibits the interaction between HSPB5 and SMAD4, thus hampering the translocation of the SMAD4/pSMAD2/pSMAD3 complex into the nucleus. As a consequence, NCI-41356 limits the synthesis of collagen, pro-fibrotic markers and subsequent fibrosis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Raghu G., Remy-Jardin M., Richeldi L., Thomson C.C., Inoue Y., Johkoh T., Kreuter M., Lynch D.A., Maher T.M., Martinez F.J., et al. Idiopathic Pulmonary Fibrosis (an Update) and Progressive Pulmonary Fibrosis in Adults: An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline. Am. J. Respir. Crit. Care Med. 2022;205:e18–e47. doi: 10.1164/rccm.202202-0399ST. - DOI - PMC - PubMed
    1. Raghu G., Remy-Jardin M., Myers J.L., Richeldi L., Ryerson C.J., Lederer D.J., Behr J., Cottin V., Danoff S.K., Morell F., et al. Diagnosis of Idiopathic Pulmonary Fibrosis. An Official ATS/ERS/JRS/ALAT Clinical Practice Guideline. Am. J. Respir. Crit. Care Med. 2018;198:e44–e68. doi: 10.1164/rccm.201807-1255ST. - DOI - PubMed
    1. Maher T.M., Bendstrup E., Dron L., Langley J., Smith G., Khalid J.M., Patel H., Kreuter M. Global Incidence and Prevalence of Idiopathic Pulmonary Fibrosis. Respir. Res. 2021;22:197. doi: 10.1186/s12931-021-01791-z. - DOI - PMC - PubMed
    1. Burgy O., Bellaye P.-S., Beltramo G., Goirand F., Bonniaud P. Pathogenesis of Fibrosis in Interstitial Lung Disease. Curr. Opin. Pulm. Med. 2020;26:429–435. doi: 10.1097/MCP.0000000000000706. - DOI - PubMed
    1. Bonniaud P., Kolb M., Galt T., Robertson J., Robbins C., Stampfli M., Lavery C., Margetts P.J., Roberts A.B., Gauldie J. Smad3 Null Mice Develop Airspace Enlargement and Are Resistant to TGF-Beta-Mediated Pulmonary Fibrosis. J. Immunol. 2004;173:2099–2108. doi: 10.4049/jimmunol.173.3.2099. - DOI - PubMed
  NODES
admin 6
twitter 2