The effect of low dose ionizing radiation on homeostasis and functional integrity in an organotypic human skin model.

Outside the protection of Earth's atmosphere, astronauts are exposed to low doses of high linear energy transfer (LET) radiation. Future NASA plans for deep space missions or a permanent settlement on the moon are limited by the health risks associated with space radiation exposures. There is a paucity of direct epidemiological data for low dose exposures to space radiation-relevant high LET ions. Health risk models are used to estimate the risk for such exposures, though these models are based on high dose experiments. There is increasing evidence, however, that low and high dose exposures result in different signaling events at the molecular level, and may involve different response mechanisms. Further, despite their low abundance, high LET particles have been identified as the major contributor to health risk during manned space flight. The human skin is exposed in every external radiation scenario, making it an ideal epithelial tissue model in which to study radiation induced effects. Here, we exposed an in vitro three dimensional (3-D) human organotypic skin tissue model to low doses of high LET oxygen (O), silicon (Si) and iron (Fe) ions. We measured proliferation and differentiation profiles in the skin tissue and examined the integrity of the skin's barrier function. We discuss the role of secondary particles in changing the proportion of cells receiving a radiation dose, emphasizing the possible impact on radiation-induced health issues in astronauts.

Integrated experimental and computational approach to understand the effects of heavy ion radiation on skin homeostasis.

The effects of low dose high linear energy transfer (LET) radiation on human health are of concern for space, occupational, and clinical exposures. As epidemiological data for such radiation exposures are scarce for making relevant predictions, we need to understand the mechanism of response especially in normal tissues. Our objective here is to understand the effects of heavy ion radiation on tissue homeostasis in a realistic model system. Towards this end, we exposed an in vitro three dimensional skin equivalent to low fluences of neon (Ne) ions (300 MeV u(-1)), and determined the differentiation profile as a function of time following exposure using immunohistochemistry. We found that Ne ion exposures resulted in transient increases in the tissue regions expressing the differentiation markers keratin 10, and filaggrin, and more subtle time-dependent effects on the number of basal cells in the epidermis. We analyzed the data using a mathematical model of the skin equivalent, to quantify the effect of radiation on cell proliferation and differentiation. The agent-based mathematical model for the epidermal layer treats the epidermis as a collection of heterogeneous cell types with different proliferation-differentiation properties. We obtained model parameters from the literature where available, and calibrated the unknown parameters to match the observed properties in unirradiated skin. We then used the model to rigorously examine alternate hypotheses regarding the effects of high LET radiation on the tissue. Our analysis indicates that Ne ion exposures induce rapid, but transient, changes in cell division, differentiation and proliferation. We have validated the modeling results by histology and quantitative reverse transcription polymerase chain reaction (qRT-PCR). The integrated approach presented here can be used as a general framework to understand the responses of multicellular systems, and can be adapted to other epithelial tissues.

Annexin A2 modulates radiation-sensitive transcriptional programming and cell fate.

We previously established annexin A2 as a radioresponsive protein associated with anchorage independent growth in murine epidermal cells. In this study, we demonstrate annexin A2 nuclear translocation in human skin organotypic culture and murine epidermal cells after exposure to X radiation (10-200 cGy), supporting a conserved nuclear function for annexin A2. Whole genome expression profiling in the presence and absence of annexin A2 [shRNA] identified fundamentally altered transcriptional programming that changes the radioresponsive transcriptome. Bioinformatics predicted that silencing AnxA2 may enhance cell death responses to stress in association with reduced activation of pro-survival signals such as nuclear factor kappa B. This prediction was validated by demonstrating a significant increase in sensitivity toward tumor necrosis factor alpha-induced cell death in annexin A2 silenced cells, relative to vector controls, associated with reduced nuclear translocation of RelA (p65) following tumor necrosis factor alpha treatment. These observations implicate an annexin A2 niche in cell fate regulation such that AnxA2 protects cells from radiation-induced apoptosis to maintain cellular homeostasis at low-dose radiation.

Quantitative phosphoproteomics identifies filaggrin and other targets of ionizing radiation in a human skin model.

Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low- and high-dose ionizing radiation-dependent signalling in a complex three-dimensional setting. Application of an isobaric labelling strategy using sham and three radiation doses (3, 10, 200 cGy) resulted in the identification of 1052 unique phosphopeptides. Statistical analyses identified 176 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1, 2 and 3) had altered phosphorylation levels following exposure to both low and high doses of radiation. Altered phosphorylation of multiple sites in profilaggrin linker domains coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to both low and high doses of ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain tissue integrity and mitigate effects of radiation exposure.

Using imaging methods to interrogate radiation-induced cell signaling.

There is increasing emphasis on the use of systems biology approaches to define radiation-induced responses in cells and tissues. Such approaches frequently rely on global screening using various high throughput 'omics' platforms. Although these methods are ideal for obtaining an unbiased overview of cellular responses, they often cannot reflect the inherent heterogeneity of the system or provide detailed spatial information. Additionally, performing such studies with multiple sampling time points can be prohibitively expensive. Imaging provides a complementary method with high spatial and temporal resolution capable of following the dynamics of signaling processes. In this review, we utilize specific examples to illustrate how imaging approaches have furthered our understanding of radiation-induced cellular signaling. Particular emphasis is placed on protein colocalization, and oscillatory and transient signaling dynamics.

Cell type-dependent gene transcription profile in a three-dimensional human skin tissue model exposed to low doses of ionizing radiation: implications for medical exposures.

The concern over possible health risks from exposures to low doses of ionizing radiation has been driven largely by the increase in medical exposures, the routine implementation of X-ray backscatter devices for airport security screening, and, most recently, the nuclear incident in Japan. Because of a paucity of direct epidemiological data at very low doses, cancer risk must be estimated from high dose exposure scenarios. However, there is increasing evidence that low and high dose exposures result in different signaling events and may have different response mechanisms than higher doses. We have examined the radiation-induced temporal response after exposure to 10 cGy of an in vitro three dimensional (3D) human skin tissue model using microarray-based transcriptional profiling. Cell type-specific analysis showed significant changes in gene expression with the levels of >1,400 genes altered in the dermis and >400 genes regulated in the epidermis. The two cell layers rarely exhibited overlapping responses at the mRNA level. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) measurements validated the microarray data in both regulation direction and value. Key pathways identified relate to cell cycle regulation, immune responses, hypoxia, reactive oxygen signaling, and DNA damage repair. The proliferation status as well as the expression of PCNA was examined in histological samples. We discuss in particular the role of proliferation, emphasizing how the disregulation of cellular signaling in normal tissue may impact progression toward radiation-induced secondary diseases.