Alveolar epithelial cell growth hormone releasing hormone receptor in alveolar epithelial inflammation

Abstract

Purpose: Growth hormone-releasing hormone (GHRH) is a 44-amino acid peptide that regulates growth hormone (GH) secretion. We hypothesized that GHRH receptor (GHRH-R) in alveolar type 2 (AT2) cells could modulate pro-inflammatory and possibly subsequent pro-fibrotic effects of lipopolysaccharide (LPS) or cytokines, such that AT2 cells could participate in lung inflammation and fibrosis. Methods: We used human alveolar type 2 (iAT2) epithelial cells derived from induced pluripotent stem cells (iPSC) to investigate how GHRH-R modulates gene and protein expression. We tested iAT2 cells’ gene expression in response to LPS or cytokines, seeking whether these mechanisms caused endogenous production of pro-inflammatory molecules or mesenchymal markers. Quantitative real-time PCR (RT-PCR) and Western blotting were used to investigate differential expression of epithelial and mesenchymal markers. Result: Incubation of iAT2 cells with LPS increased expression of IL1-β and TNF-α in addition to mesenchymal genes, including ACTA2, FN1 and COL1A1. Alveolar epithelial cell gene expression due to LPS was significantly inhibited by GHRH-R peptide antagonist MIA-602. Incubation of iAT2 cells with cytokines like those in fibrotic lungs similarly increased expression of genes for IL1-β, TNF-α, TGFβ-1, Wnt5a, smooth muscle actin, fibronectin and collagen. Expression of mesenchymal proteins, such as N-cadherin and vimentin, were also elevated after prolonged exposure to cytokines, confirming epithelial production of pro-inflammatory molecules as an important mechanism that might lead to subsequent fibrosis. Conclusion: iAT2 cells clearly expressed the GHRH-R. Exposure to LPS or cytokines increased iAT2 cell production of pro-inflammatory factors. GHRH-R antagonist MIA-602 inhibited pro-inflammatory gene expression, implicating iAT2 cell GHRH-R signaling in lung inflammation and potentially in fibrosis.

Keywords:

• Inflammation
• growth hormone releasing hormone
• MIA-602
• alveolar epithelial cells
• mesenchymal
• post-inflammatory fibrosis

Acknowledgements

The authors thank Fei Tang, PhD for biostatistical assistance in experimental design and analysis of the data; and, they thank Irving Vidaurre for technical assistance.

Authors’ contributions

Cui, T planned and executed experiments, analyzed data, prepared figures, wrote and edited the manuscript. Wangpaichitr, M conducted experiments, analyzed data and assisted with writing the manuscript. Schally, A suggested testing and provided MIA-602 and MR-409, assisted in experimental design and thoroughly edited the manuscript. Griswold, A analyzed RNA-seq data and prepared figures for the manuscript. Vidaurre, I planned and executed experiments, analyzed data, prepared figures and edited the manuscript. Sha, W synthesized and prepared peptides for use in experiments. Jackson, R planned, supervised and assisted in experiments, reviewed experimental and statistical data and drafted the manuscript.

Disclosure statement

A.V.S. and R.J. are listed as co-inventors on patents of GHRH analogs, which were assigned to the University of Miami and Department of Veterans Affairs.

Additional information

Funding

This work was supported by the Miami VAHS Research Service, a Distinguished Medical Research Scientist Award (AVS) and Merit Review grant (MW) from the VA Office of Research and Development.