Transcriptional analysis of normal human fibroblast responses to microgravity stress

Abstract

To understand the molecular mechanism(s) of how spaceflight affects cellular signaling pathways, quiescent normal human WI-38 fibroblasts were flown on the STS-93 space shuttle mission. Subsequently, RNA samples from the space-flown and ground-control cells were used to construct two cDNA libraries, which were then processed for suppression subtractive hybridization (SSH) to identify spaceflight-specific gene expression. The SSH data show that key genes related to oxidative stress, DNA repair, and fatty acid oxidation are activated by spaceflight, suggesting the induction of cellular oxidative stress. This is further substantiated by the up-regulation of neuregulin 1 and the calcium-binding protein calmodulin 2. Another obvious stress sign is that spaceflight evokes the Ras/mitogen-activated protein kinase and phosphatidylinositol-3 kinase signaling pathways, along with up-regulating several G1-phase cell cycle traverse genes. Other genes showing up-regulation of expression are involved in protein synthesis and pro-apoptosis, as well as pro-survival. Interactome analysis of functionally related genes shows that c-Myc is the "hub" for those genes showing significant changes. Hence, our results suggest that microgravity travel may impact changes in gene expression mostly associated with cellular stress signaling, directing cells to either apoptotic death or premature senescence.

Fig. 1

Three total RNA samples independently extracted from equal amounts of culture derived from each of four independent cartridges. Shown in this figure are two examples of the four CCMs of three such extracted samples, either space-flown (F3 and F4) or ground controls (G3 and G4), using Trizol reagent. The quality of the total RNA samples was visualized on 1% agarose gel, stained with ethidium bromide and exposed to ultra-violet light. Note that more total RNA was extracted from space-flown than from ground-control cells.

Fig. 2

Schematic flow diagram of SSH procedure and identification of genes either up- or down-regulated by spaceflight.

Fig. 3

Functional categories of genes either up- or down-regulated by spaceflight compared with ground controls.

Fig. 4

A putative interactomic network of normal WI-38 fibroblasts after spaceflight. The directions of the arrows indicate one element acting on another. Without an attached abbreviation sign, red arrows denote “up-regulate”, while black arrows mean “down-regulate”. With attached signs, “P”, “dP”, “M”, and “I” stand for “phosphorylate”, “dephosphorlate”, “methylate”, and “inhibit”, respectively. The solid lines illustrate a physical binding between two elements; dashed lines and arrows suggest a likely but unproven relationship. In addition, red- or black-lettered genes are expected to be up- or down-regulated by spaceflight, respectively. Finally, green- and blank-background genes are identified by our SSH experiment or by speculation, respectively.