SHIP-1, a target of miR-155, regulates endothelial cell responses in lung fibrosis

Abstract

Src Homology 2-containing Inositol Phosphatase-1 (SHIP-1) is a target of miR-155, a pro-inflammatory factor. Deletion of the SHIP-1 gene in mice caused spontaneous lung inflammation and fibrosis. However, the role and function of endothelial miR-155 and SHIP-1 in lung fibrosis remain unknown. Using whole-body miR-155 knockout mice and endothelial cell-specific conditional miR-155 (VEC-Cre-miR-155 or VEC-miR-155) or SHIP-1 (VEC-SHIP-1) knockout mice, we assessed endothelial-mesenchymal transition (EndoMT) and fibrotic responses in bleomycin (BLM) induced lung fibrosis models. Primary mouse lung endothelial cells (MLEC) and human umbilical vein endothelial cells (HUVEC) with SHIP-1 knockdown were analyzed in TGF-β1 or BLM, respectively, induced fibrotic responses. Fibrosis and EndoMT were significantly reduced in miR-155KO mice and changes in EndoMT markers in MLEC after TGF-β1 stimulation confirmed the in vivo findings. Furthermore, lung fibrosis and EndoMT responses were reduced in VEC-miR-155 mice but significantly enhanced in VEC-SHIP-1 mice after BLM challenge. SHIP-1 knockdown in HUVEC cells resulted in enhanced EndoMT induced by BLM. Meanwhile, these changes involved the PI3K/AKT, JAK/STAT3, and SMAD/STAT signaling pathways. These studies demonstrate that endothelial miR-155 plays an important role in fibrotic responses in the lung through EndoMT. Endothelial SHIP-1 is essential in controlling fibrotic responses and SHIP-1 is a target of miR-155. Endothelial cells are an integral part in lung fibrosis.

Keywords: endothelial-mesenchymal transition; lung fibrosis; miR-155; phosphatase SHIP-1.

Figure 1.. Endothelial-mesenchymal transition (EndoMT) was involved in BLM-induced lung fibrosis.

(A) Hematoxylin and eosin (H&E) and Masson’s Trichrome stained lung sections from WT mice with BLM challenge or NS control for 28 days (WT-NS, WT-BLM, n=7–8 mice/group). (B) Q-PCR analysis of α-SMA and VE-cadherin mRNA in the lung tissues of WT-NS and WT-BLM mice. Results are Means±SEM from triplicate samples of 3 repeats. (C) Western blot of α-SMA and VE-cadherin proteins in the lung tissues of WT-NS and WT-BLM mice. HSP-90 was used for sample loading control. Shown are representative blots of 3 independent experiments. (D) Immunofluorescence of CD31 and α-SMA in WT-NS and WT-BLM lung tissues and (E) Immunofluorescence of VE-Cadherin and α-SMA in WT-NS and WT-BLM lung tissues. Images were from different animals (n=7–8 mice/group). DAPI stained for nuclei (blue). *p<0.05, **p<0.01.

Figure 2.. Deletion of MiR-155 alleviated fibrotic and EndoMT responses.

(A) H&E and Trichrome stained lung sections from WT and miR-155^−/− mice with BLM stimulation for 28 days (WT-BLM, MiR-155^−/−-BLM, n=6–8 mice/group). (B) Hydroxyproline content in the lung tissues of WT and miR-155^−/− mice challenged with NS or BLM (n=6–8 mice/group). (C) Q-PCR of Collagen I mRNA in WT and MiR-155^−/− lung tissues after NS or BLM challenge. Results are presented as Means±SEM of triplicates of 3 repeats. (D, E) Western blots of α-SMA, Collagen I and VE-cadherin in the lung tissues of WT and miR-155^−/− mice with or without BLM. HSP-90 or β-Actin as loading controls. Shown are representative blots of 3 independent experiments. (F) Immunofluorescence of CD31 and α-SMA in WT-BLM and miR-155^−/−-BLM lung tissues. Images were from different aniamls (n=6–8 mice/group). (G) Immunofluorescence of VE-Cadherin and α-SMA in WT-BLM and miR-155^−/−-BLM lung tissues (n=6–8 mice/group). DAPI for nuclei (blue). *p<0.05; **p<0.01.

Figure 3.. Role of endothelial MiR-155 in lung fibrosis.

(A, B) Q-PCR of Fibronectin and VE-cadherin mRNA in isolated MLEC from WT and MiR-155KO mice. Results are Mean±SEM of triplicates with 3 repeats. (C) Western blot of α-SMA, Vimentin and VE-cadherin in isolated WT and MiR-155KO MLEC stimulated with TGF-β1 for 72 hrs. Shown are representative blots from 3 independent experiments. (D, E) Immunofluorescence of CD31, Vimentin, VE-Cadherin, and α-SMA in MLEC after stimulation with TGF-β1. Results reprensent 3 independent experiments. (F) H&E and Trichrome staining of lung sections of WT and VEC-miR155 mice after BLM for 28 days (n=6–8 mice/group). (G) Q-PCR of Collagen I mRNA in WT and VEC-miR155 mouse lungs with or without BLM. Results are Mean±SEM of triplicates with 3 repeats. (H) Western blot of Collagen I, α-SMA and VE-cadherin in WT and VEC-miR155 mouse lungs. Shown are representative blots of 3 independent experiments. (I) Immunofluorescence of VE-Cadherin and α-SMA in WT and VEC-miR155 mouse lungs (n=6–8 mice/group). DAPI for nuclei (blue). *p<0.05; **p<0.01.

Figure 4.. Lack of endothelial SHIP-1 facilitated lung fibrosis and EndoMT in vivo.

(A) Western blot of SHIP-1 in miR-155^−/−, VEC-miR-155 and WT mouse lungs before and after BLM challenge for 28 days (n=6 mice/group). (B) Immunoprecipitation (IP) and Western blot (IB) of SHIP-1 protein in HUVEC. Shown are representative results of 3 independent experiments. (C) Q-PCR of SHIP-1 mRNA in mouse lung endothelial cells (MLECs) from WT and VEC-miR155 mice before TGF-β1 stimulation. Results are Mean±SEM of triplicates with 3 repeats. (D) Western blot of SHIP-1 in MLECs from miR-155^−/−, WT and VEC-SHIP-1 mice before and after TGF-β1 stimulation for 72 hrs. Results represent 3 independent experiments. (E) H&E and Trichrome stained lung sections from WT and VEC-Cre-SHIP-1 (VEC-SHIP-1) mice after BLM challenge for 28 days (n=7–8 mice/group). (F) Q-PCR of Collagen I and VE-cadherin mRNA in WT and VEC- SHIP-1 mouse lungs. Results are Mean±SEM of triplicates with 3 repeats. (G) Western blot of Collagen I and α-SMA in WT and VEC-SHIP-1 mouse lung tissues and (H) Western blot of α-SMA and VE-cadherin in WT and VEC-SHIP-1 mouse lung tissues. Blots are representative results of 3 independent experiments. (I) Immunofluorescence of VE-Cadherin and α-SMA in WT and VEC-SHIP-1 mouse lung sections. Images were from different animals (n=6–8 mice/group). DAPI for nuclei (blue). *p<0.05; **p<0.01.

Figure 5.. Effect of endothelial SHIP-1 on TGF-β1-induced EndoMT in vitro.

(A) Flow cytometry analysis of CD31 and SHIP-1 expression in isolated lung cells from WT and VEC-SHIP-1 mice. Upper right panels show the percentage of CD31 and SHIP-1 double positive cells. Results represent 3 independent experiments. (B) Western blot of SHIP-1 expression in WT and VEC-SHIP-1 mouse lung endothelial cells (MLECs). Shown is a representative blot of 3 separate experiments. (C) Immunofluorescence of VE-Cadherin and α-SMA in WT and VEC-SHIP-1 MLECs and (D) Immunofluorescence of CD31 and Vimentin in WT and VEC-SHIP-1 MLECs. Images were taken from 3 independent experiments. (E) Western blot of VE-Cadherin, α-SMA and Vimentin in WT and VEC-SHIP-1 MLECs with or without TGF-β1 stimulation for 72 hrs. Shown are representative results of 3 independent experiments. (F) Western blot of CD31 and α-SMA in primary HUVECs treated with siRNA to SHIP-1 or scrambled siRNA and with/without BLM for 72 hrs. Shown are representative results of 3 independent experiments.

Figure 6.. Endothelial miR-155 and SHIP-1 in pulmonary fibrosis involving PI3K/AKT, STAT3 and SMAD2/3 signaling pathways.

(A) Western blot of PI3K, p-AKT-1, Twist and Snail in WT and miR-155KO mouse lung tissues after BLM challenge for 28 days (n=6–8/group). (B) Expression of STAT3, Smad2/3 as well as activation of p-STAT3, p-Smad2, and p-Smad3 were detected by Western blot in WT and miR-155KO mice lung tissues (n=6–8/group). (C) Western blot of PI3K, p-PI3K, p-AKT-1, Twist and Snail in WT and VEC-miR-155 mice lung tissues (n=6–8/group). (D) Expression of STAT3 as well as activation of p-STAT3, p-Smad2, and p-Smad3 in WT and VEC-miR-155 mouse lung tissues were analyzed by Western blot (n=6–8/group). (E) Protein expression of PI3K, Twist, Snail as well as activation of p-PI3K, and p-AKT1 in WT and VEC-SHIP-1 mouse lung tissues (n=6–8/group). (F) Western blot of p-STAT3, STAT3, p-SMAD2 and p-SMAD3 in WT and VEC-SHIP-1 mouse lung tissues (n=6–8/group).