TY - JOUR
T1 - Proteomic analysis and bioluminescent reporter gene assays to investigate effects of simulated microgravity on Caco-2 cells
AU - La Barbera, Giorgia
AU - Capriotti, Anna Laura
AU - Michelini, Elisa
AU - Piovesana, Susy
AU - Calabretta, Maria Maddalena
AU - Zenezini Chiozzi, Riccardo
AU - Roda, Aldo
AU - Laganà, Aldo
PY - 2017/8
Y1 - 2017/8
N2 - Microgravity is one of the most important features in spaceflight. Previous evidence from in-vitro studies has shown that significant changes occur under simulated microgravity. For this reason, human colon adenocarcinoma Caco-2 cells were selected as cell model of intestinal epithelial barrier and their response to altered gravity conditions was investigated, especially on the protein level. In this study, we combined label-free shotgun proteomics and bioluminescent reporter gene assays to identify key proteins and pathways involved in the response of Caco-2 cells under reference and microgravity conditions. A two-dimensional clinostat was modified with 3D-printed adaptors to hold conventional T25 culture flasks. The comparative proteome analysis led to identify 38 and 26 proteins differently regulated by simulated microgravity after 48 and 72 h, respectively. Substantial fractions of these proteins are involved in regulation, cellular and metabolic processes and localization. Bioluminescent reporter gene assays were carried out to investigate microgavity-induced alterations on the transcriptional regulation of key targets, such as NF-kB pathway and CYP27A1. While no significant difference was found in the basal transcription, a lower NF-kB basal activation in simulated microgravity conditions was reported, corroborating the hypothesis of reduced immunity in microgravity conditions.
AB - Microgravity is one of the most important features in spaceflight. Previous evidence from in-vitro studies has shown that significant changes occur under simulated microgravity. For this reason, human colon adenocarcinoma Caco-2 cells were selected as cell model of intestinal epithelial barrier and their response to altered gravity conditions was investigated, especially on the protein level. In this study, we combined label-free shotgun proteomics and bioluminescent reporter gene assays to identify key proteins and pathways involved in the response of Caco-2 cells under reference and microgravity conditions. A two-dimensional clinostat was modified with 3D-printed adaptors to hold conventional T25 culture flasks. The comparative proteome analysis led to identify 38 and 26 proteins differently regulated by simulated microgravity after 48 and 72 h, respectively. Substantial fractions of these proteins are involved in regulation, cellular and metabolic processes and localization. Bioluminescent reporter gene assays were carried out to investigate microgavity-induced alterations on the transcriptional regulation of key targets, such as NF-kB pathway and CYP27A1. While no significant difference was found in the basal transcription, a lower NF-kB basal activation in simulated microgravity conditions was reported, corroborating the hypothesis of reduced immunity in microgravity conditions.
KW - Bioluminescence
KW - Caco-2 cells
KW - Label-free proteomics
KW - Nuclear factor κ-B
KW - Reporter gene technology
KW - Simulated microgravity
UR - http://www.scopus.com/inward/record.url?scp=85027859865&partnerID=8YFLogxK
U2 - 10.1002/pmic.201700081
DO - 10.1002/pmic.201700081
M3 - Journal article
C2 - 28727291
AN - SCOPUS:85027859865
SN - 1615-9853
VL - 17
JO - Proteomics
JF - Proteomics
IS - 15-16
M1 - 1700081
ER -