Crtc1 deficiency protects against sepsis-associated acute lung injury through activating akt signaling pathway

Abstract

Background: Interplay between systemic inflammation and programmed cell death contributes to the pathogenesis of acute lung injury (ALI). cAMP-regulated transcriptional coactivator 1 (CRTC1) has been involved in the normal function of the pulmonary system, but its role in ALI remains unclear.

Methods and results: We generated a Crtc1 knockout (KO; Crtc1^-/-) mouse line. Sepsis-induced ALI was established by cecal ligation and puncture (CLP) for 24 h. The data showed that Ctrc1 KO substantially ameliorated CLP-induced ALI phenotypes, including improved lung structure destruction, reduced pulmonary vascular permeability, diminished levels of proinflammatory cytokines and chemokines, compared with the wildtype mice. Consistently, in lipopolysaccharide (LPS)-treated RAW264.7 cells, Crtc1 knockdown significantly inhibited the expression of inflammatory effectors, including TNF-α, IL-1β, IL-6 and CXCL1, whereas their expressions were significantly enhanced by Crtc1 overexpression. Moreover, both Crtc1 KO in mice and its knockdown in RAW264.7 cells dramatically reduced TUNEL-positive cells and the expression of pro-apoptotic proteins. In contrast, Crtc1 overexpression led to an increase in the pro-apoptotic proteins and LPS-induced TUNEL-positive cells. Mechanically, we found that the phosphorylation of Akt was significantly enhanced by Crtc1 knockout or knockdown, but suppressed by Crtc1 overexpression. Administration of Triciribine, an Akt inhibitor, substantially blocked the protection of Crtc1 knockdown on LPS-induced inflammation and cell death in RAW264.7 cells.

Conclusions: Our study demonstrates that CRTC1 contribute to the pathological processes of inflammation and apoptosis in sepsis-induced ALI, and provides mechanistic insights into the molecular function of CRTC1 in the lung. Targeting CRTC1 would be a promising strategy to treat sepsis-induced ALI in clinic.

Keywords: Acute lung injury; Apoptosis; Crtc1; Inflammation; Sepsis.

Fig. 1

Crtc1 deficiency ameliorated CLP-induced ALI. (A, B) Scatter plot (A) and heat map (B) of Crtc1 mRNA changes in LPS-induced inflammation models from different datasets in the GEO database. (C) Crtc1 knockout was validated through DNA genotyping (upper) and Western blot (lower) in lung tissue. n = 3. (D, E) Representative images (D) and quantitation (E) of Evans blue albumin to evaluate pulmonary vascular permeability in Crtc1 KO mice, compared with WT mice. ^***P < 0.001 vs. Crtc1^+/+; ^###P < 0.001 vs. Sham. n = 6. (F, G) Representative H&E staining images (F) and ALI scoring (G) in lung tissue. ^*P < 0.05 vs. Crtc1^+/+; ^###P < 0.001 vs. Sham. n = 3. (H, I) Cytokine IL-6 (H) and TNF-α (I) in BALF were detected by ELISA. ^***P < 0.001 vs. Crtc1^+/+; ^###P < 0.001 vs. Sham. n = 6. (J-M) Impact of Crtc1 deficiency on CLP-induced Cytokines including TNF-α (J), IL-1β (K), IL-6 (L) and CXCL11 (M) in lung tissue detecting by qRT-PCR. ^*P < 0.05, ^**P < 0.01 vs. Crtc1^+/+; ^##P < 0.01 vs. Sham. n = 6

Fig. 2

Crtc1 knockout alleviated CLP-induced apoptosis in ALI.(A, B) Representative TUNEL staining images (A) and quantification data (B) showing the impact of Crtc1 KO on CLP-induced apoptosis in lung tissues. ^***P < 0.001 vs. Crtc1^+/+; ^###P < 0.001 vs. Sham. n = 6. (C-F) Western blot (C) and quantification (D-F) showing the protein level of Cleaved-caspase3/Caspase3 (D), Bcl-2 (E) and Bax (F) in mice lung tissues. ^*P < 0.05, ^**P < 0.01 vs. Crtc1^+/+; ^#P < 0.05, ^##P < 0.01 vs. Sham; n = 3

Fig. 3

Crtc1 Silencing attenuates LPS-induced inflammation and apoptosis in RAW264.7 cells. (A) Validation of Crtc1 knockdown in in RAW264.7 cells treated with Crtc1-specific or negative control siRNAs using qRT-PCR. *P < 0.05; n = 3. (B-E) qRT-PCR showing the impact of Crtc1 knockdown on LPS-induced cytokines, such as TNF-α (B), IL-1β (C), IL-6 (D), and CXCL1 (E) in RAW264.7 cells. ^*P < 0.05, ^**P < 0.01 vs. siNeg;^##P < 0.01, ^###P < 0.001 vs. Control; n = 6. (F, G) Representative TUNEL staining images (F) and quantification data (G) showing the impact of Crtc1 knockdown on LPS-induced apoptosis in RAW264.7 cells. ^**P < 0.01 vs. siNeg; ^###P < 0.001 vs. Control; n = 3. (H-K) Western blot (H) and quantification (I-K) showing the impact of Crtc1 knockdown on LPS-induced alternations in Cleaved-caspase3/Caspase3 (I), Bcl-2 (J) and Bax (K) in RAW264.7 cells. ^**P < 0.01, ^***P < 0.001 vs. siNeg; ^#P < 0.05, ^###P < 0.001 vs. Control; n = 3

Fig. 4

Crtc1 overexpression increases LPS-induced inflammation and apoptosis in RAW264.7 cells. (A, B) Western blot (A) and quantification (B) of Crtc1 overexpression in RAW264.7 cells. **P < 0.01; n = 3. (C-F) qRT-PCR showing the impact of Crtc1 overexpression on LPS-induced expression of TNF-α (C), IL-1β (D), IL-6 (E), and CXCL1 (F) in RAW264.7 cells. ^*P < 0.05, ^***P < 0.001 vs. EGFP;^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. Control; n = 6. (G, H) Representative TUNEL staining images (G) and quantification (H) showing the impact of Crtc1 overexpression on LPS-induced apoptosis in RAW264.7 cells. ^***P < 0.001 vs. EGFP; ^###P < 0.001 vs. Control; n = 3. (I-L) Western blot (I) and quantification (J-L) showing the impact of Crtc1 overexpression on LPS-induced alternations in Cleaved-caspase3/Caspase3 (J), Bcl-2 (K) and Bax (L) in RAW264.7 cells. ^*P < 0.05, ^**P < 0.01 vs. EGFP; ^#P < 0.05, ^###P < 0.001 vs. Control; n = 3

Fig. 5

Akt signaling pathway mediates the pro-apoptosis role Crtc1 in ALI. (A-D) Western blot (A) and quantification (B-D) showing the role of Crtc1 gene manipulation on p-Akt/Akt. ^*P < 0.05, ^**P < 0.01 vs. Crtc1^+/+; ^#P < 0.05, ^###P < 0.001 vs. Sham; n = 3. (E, F) Representative TUNEL staining images (E) and quantification (F) showed Triciribine (10µM) blocked the inhibition of Crtc1 knockdown on LPS-induced TUNEL-positive cells. ^**P < 0.01 vs. LPS group; ^#P < 0.05 vs. siCrtc1 + LPS group; n = 3. (G-J) Western blot (G) and quantification (H-J) showing Triciribine reversed the protective effects of Crtc1 silencing on apoptosis-associated proteins, such as Cleaved-caspase3/Caspase3 (H), Bcl-2 (I) and Bax (J) in RAW264.7 cells. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. LPS group; ^#P < 0.05 vs. siCrtc1 + LPS group; n = 3. (K-N) Triciribine blocked the inhibition of Crtc1 knockdown on LPS-induced inflammation factors including TNF-α (K), IL-1β (L), IL-6 (M) and CXCL1 (N). ^*P < 0.05, ^***P < 0.001 vs. LPS group; ^#P < 0.05 vs. siCrtc1 + LPS group; n = 6