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. 2016 Nov;14(5):4496-4504.
doi: 10.3892/mmr.2016.5841. Epub 2016 Oct 12.

Label-free LC-MS/MS shotgun proteomics to investigate the anti-inflammatory effect of rCC16

Min Pang  1 , Xin-Yan Bai  2 , Yan Li  3 , Ji-Zhong Bai  4 , Li-Rong Yuan  1 , Shou-An Ren  1 , Xiao-Yun Hu  1 , Xin-Ri Zhang  1 , Bao-Feng Yu  2 , Rui Guo  2 , Hai-Long Wang  2
Affiliations

Label-free LC-MS/MS shotgun proteomics to investigate the anti-inflammatory effect of rCC16

Min Pang et al. Mol Med Rep. 2016 Nov.

Abstract

Clara cell protein (CC16) is an anti-inflammatory protein, which is expressed in the airway epithelium. It is involved in the development of airway inflammatory diseases, including chronic obstructive pulmonary disease and asthma. However, the exact molecular mechanism underlying its anti‑inflammatory action remains to be fully elucidated. The aim of the present study was to define the protein profiles of the anti‑inflammatory effect of CC16 in lipopolysaccharide (LPS)‑treated rat tracheal epithelial (RTE) cells using shotgun proteomics. Protein extracts were obtained from control RTE cells, RTE cells treated with LPS and RTE cells treated with LPS and recombinant CC16 (rCC16). Subsequent label‑free quantification and bioinformatics analyses identified 12 proteins that were differentially expressed in the three treatment groups as a cluster of five distinct groups according to their molecular functions. Five of the twelve proteins were revealed to be associated with the cytoskeleton: Matrix metalloproteinase‑9, myosin heavy chain 10, actin‑related protein‑3 homolog, elongation factor 1‑α‑1 (EF‑1‑α‑1), and acidic ribosomal phosphoprotein P0. Five of the twelve proteins were associated with cellular proliferation: DNA‑dependent protein kinase catalytic subunit, EF‑1‑α‑1, tyrosine 3‑monooxygenase, caspase recruitment domain (CARD) protein 12 and adenosylhomocysteinase (SAHH) 3. Three proteins were associated with gene regulation: EF‑1‑α‑1, SAHH 3 and acidic ribosomal phosphoprotein P0. Three proteins were associated with inflammation: Tyrosine 3‑monooxygenase, CARD protein 12 and statin‑related protein. ATPase (H+‑transporting, V1 subunit A, isoform 1) was revealed to be associated with energy metabolism, and uridine diphosphate glycosyltransferase 1 family polypeptide A8 with drug metabolism and detoxification. The identified proteins were further validated using reverse transcription‑quantitative polymerase chain reaction. These protein profiles, and their interacting protein network, may facilitate the elucidation of the molecular mechanisms underlying the anti‑inflammatory effects of CC16.

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Figures

Figure 1.
Figure 1.
Venn diagram presenting the differential expression patterns of the identified proteins. Comparisons of the differential expression patterns of the identified proteins were made as follows: (A) LPS vs. control, low expression in LPS-treated cells; (B) LPS vs. control, high expression in LPS-treated cells; (C) rCC16 + LPS vs. LPS, low expression in rCC16 + LPS-treated cells; and (D) rCC16 + LPS vs. LPS, high expression in rCC16 + LPS-treated cells. LPS, lipopolysaccharide; rCC16, recombinant Clara cell protein.
Figure 2.
Figure 2.
Protein expression profiles of RTE cells cultured in the presence or absence of LPS and rCC16. Seven proteins were downregulated by LPS only (LPS) and upregulated by additional rCC16 treatment (LPS+rCC16), and five proteins were upregulated by LPS only (LPS) and downregulated by further rCC16 treatment (LPS+rCC16). Data are presented as fold changes (≤0.83 for downregulated proteins or ≥1.2 for upregulated proteins) averaged from three independent experiments. RTE, rat tracheal epithelial; LPS, lipopolysaccharide; rCC16, recombinant Clara cell protein. Ugt 1a8, uridine diphosphate glycosyltransferase 1 family polypeptide A8; EF-1-α-1, elongation factor 1-α 1.
Figure 3.
Figure 3.
Gene ontology analysis of differentially expressed proteins based on (A) their molecular functions and (B) their associated biological processes, using PANTHER classification. Over-representation statistics were performed using the hypergeometric analysis and Benjamini & Hochberg False Discovery Rate correction.
Figure 4.
Figure 4.
A network of interacting proteins associated with anti-inflammatory action of rCC16, as revealed by Search Tool for the Retrieval of Interacting Genes/Proteins analysis. rCC16, recombinant Clara cell protein.
Figure 5.
Figure 5.
Reverse transcription-quantitative polymerase chain reaction analysis of genes coding for the differentially expressed proteins. mRNA expression levels followed a similar pattern to protein expression levels. *P<0.05. Ugt 1a8, uridine diphosphate glycosyltransferase 1 family polypeptide A8.
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References

    1. Vestbo J, Hurd SS, Agustí AG, Jones PW, Vogelmeier C, Anzueto A, Barnes PJ, Fabbri LM, Martinez FJ, Nishimura M, et al. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary. Am J Respir Crit Care Med. 2013;187:347–365. doi: 10.1164/rccm.201204-0596PP. - DOI - PubMed
    1. Gosens R, Zaagsma J, Meurs H, Halayko AJ. Muscarinic receptor signaling in the pathophysiology of asthma and COPD. Respir Res. 2006;7:73. doi: 10.1186/1465-9921-7-73. - DOI - PMC - PubMed
    1. Chen LC, Zhang Z, Myers AC, Huang SK. Cutting edge: Altered pulmonary eosinophilic inflammation in mice deficient for Clara cell secretory 10-kDa protein. J Immunol. 2001;167:3025–3028. doi: 10.4049/jimmunol.167.6.3025. - DOI - PubMed
    1. Pilette C, Godding V, Kiss R, Delos M, Verbeken E, Decaestecker C, De Paepe K, Vaerman JP, Decramer M, Sibille Y. Reduced epithelial expression of secretory component in small airways correlates with airflow obstruction in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 2001;163:185–194. doi: 10.1164/ajrccm.163.1.9912137. - DOI - PubMed
    1. Shijubo N, Itoh Y, Yamaguchi T, Imada A, Hirasawa M, Yamada T, Kawai T, Abe S. Clara cell protein-positive epithelial cells are reduced in small airways of asthmatics. Am J Respir Crit Care Med. 1999;160:930–933. doi: 10.1164/ajrccm.160.3.9803113. - DOI - PubMed

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