Cells 2013, 2, 224-243; doi:10.3390/cells2020224 OPEN ACCESS cells ISSN 2073-4409 www.mdpi.com/journal/cells Article Temporal Gene Expression Kinetics for Human Keratinocytes Exposed to Hyperthermic Stress Ibtissam Echchgadda 1,2, Caleb C. Roth 3, Cesario Z. Cerna 4 and Gerald J. Wilmink 1,* 1 Air Force Research Laboratory, 711th Human Performance Wing, Human Effectiveness Directorate, Bioeffects Division, Radio Frequency Bioeffects Branch, 4141 Petroleum Road, Bldg. 3260, Fort Sam Houston, TX 78234, USA; E-Mail: [email protected] 2 National Academy of Sciences NRC Research Associateship, Fort Sam Houston, TX 78234, USA 3 Department of Radiology, University of Texas Health Science Center San Antonio, San Antonio, TX 78229, USA; E-Mail: [email protected] 4 General Dynamics Information Technology, Fort Sam Houston, TX 78234, USA; E-Mail: [email protected] * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +1-210-539-8004; Fax: +1-210-539-7945. Received: 28 February 2013; in revised form: 29 March 2013 / Accepted: 2 April 2013 / Published: 10 April 2013 Abstract: The gene expression kinetics for human cells exposed to hyperthermic stress are not well characterized. In this study, we identified and characterized the genes that are differentially expressed in human epidermal keratinocyte (HEK) cells exposed to hyperthermic stress. In order to obtain temporal gene expression kinetics, we exposed HEK cells to a heat stress protocol (44 °C for 40 min) and used messenger RNA (mRNA) microarrays at 0 h, 4 h and 24 h post-exposure. Bioinformatics software was employed to characterize the chief biological processes and canonical pathways associated with these heat stress genes. The data shows that the genes encoding for heat shock proteins (HSPs) that function to prevent further protein denaturation and aggregation, such as HSP40, HSP70 and HSP105, exhibit maximal expression immediately after exposure to hyperthermic stress. In contrast, the smaller HSPs, such as HSP10 and HSP27, which function in mitochondrial protein biogenesis and cellular adaptation, exhibit maximal expression during the “recovery phase”, roughly 24 h post-exposure. These data suggest that the temporal expression kinetics for each particular HSP appears to correlate with the cellular function that is required at each time point. In summary, these data provide additional insight regarding the expression kinetics of genes that are triggered in HEK cells exposed to hyperthermic stress. Cells 2013, 2 225 Keywords: keratinocytes; heat shock; gene expression; cellular stress response; bioinformatics 1. Introduction Human beings frequently encounter a wide variety of hyperthermic stressors. Common examples include exposure to hot conducting objects, elevated environmental temperatures, as well as electromagnetic radiation from the sun and numerous man-made sources, such as lasers, communication devices and imaging systems [1–10]. The heat generated by hyperthermic stressors can be harmful to organisms, because it can temporarily or permanently alter the conformation and function of vital intracellular biomacromolecules (i.e., lipids, proteins and nucleic acids). Such modifications can result in an array of damaging effects, including cellular morphological alterations (i.e., shape, size, irregularities and roughness), cytoskeleton restructuring, organelle reorganization and/or fragmentation and direct interference with intracellular transport processes [11]. Hyperthermic effects can interfere with an organism’s normal homeostasis, and thus, if they are not adequately counteracted, they can threaten its survival. In order to survive and minimize the effects of hyperthermic stress, virtually all cells in living organisms have evolved sophisticated sensing, response and adaptation mechanisms. The crux of these mechanisms is the molecular defense reaction, most commonly referred to as the cellular stress response (CSR). The CSR is immediately activated in response to stress stimuli, and a hallmark of this response is the robust transcriptional expression of hundreds of diverse genes. These genes encode for a multitude of proteins involved in many important functions, including cellular survival, deoxyribonucleic acid (DNA) sensing and repair, redox regulation, proteolysis, molecular chaperoning, cell cycle control and apoptosis [12,13]. Furthermore, each CSR gene exhibits unique expression profiles, which differ in magnitude, onset time and duration of expression [4,10]. The epidermis, the outermost layer of an organism’s skin, regularly encounters a wide variety of stressors. In fact, the primary function of the epidermis is to give organisms a physiological protection barrier against environmental stimuli, such as hyperthermic stressors. This protective role is mediated primarily by keratinocytes, the predominant epithelial cell type (~95%). Due to their anatomical location, keratinocytes are habitually exposed to environmental temperature variations, and thus, they are the primary and frequent target of hyperthermic stress. In addition, their frequent exposure to heat stress has also been reported to render them with specialized cellular responses, which make them more resistant to stress than virtually all other cell types [14,15]. Clearly, keratinocytes carry a disproportionate burden in maintaining homeostasis for organisms; thus, they are vital players in sensing, responding and adapting to the burdens of hyperthermic stress. Over the past decade, numerous microarray experiments have been performed to better understand the stress response of human epidermal keratinocytes (HEK) [15–19]. These studies have been particularly fruitful, because they have resulted in the identification of hundreds of stress-inducible genes. However, previous gene expression studies have failed to provide a comprehensive picture of the cellular stress response for several reasons. First, the microarray gene chips utilized in most of the previous studies were incomplete and, thus, provided expression kinetics for only a fraction of the genes Cells 2013, 2 226 in the human genome. Second, many previous studies made gene expression measurements at a single time point; as a result, temporal gene expression kinetics could not be ascertained. Finally, the majority of past studies examined the response of skin cells to ultraviolet stress; however, few studies have specifically analyzed the transcriptional response of HEK to hyperthermic stress [10]. In this study, we sought to identify and characterize the genes that are differentially expressed in HEK exposed to hyperthermic stress. To obtain temporal gene expression kinetics, we exposed HEK to a heat stress protocol (44 °C for 40 min) and used mRNA microarrays at three time points: 0 h, 4 h and 24 h post-heat treatment, as outlined in Figure 1A. Then, we used bioinformatics software to characterize the chief biological processes and canonical pathways associated with these genes. These data provide valuable new insights that give us a much clearer picture of the genes and intracellular signaling pathways that are triggered in human cells exposed to hyperthermic stress. Figure 1. (A) A schematic of the heat shock exposure protocol and post analysis techniques. (B) Cellular MTT viability for keratinocytes exposed to hyperthermic stress. Viability percentage data (relative to untreated samples) was measured for wells with concentration densities ranging from 10,000 to 100,000 cells per well. Viability was measured at 0, 4 and 24 h post-heat treatment. (C) Microarray global gene expression profiles. The data shows that 762, 1,422 and 1,478 total genes were differentially expressed at 0, 4 and 24 h post-exposure, respectively. (D) Gene expression for minimal stress protein, heat shock protein 70 (HSPA6) using qRT-PCR. The mRNA expression fold values were measured for sham and heat treatment groups. For each time point, three samples (S1, S2 and S3) were assessed, each in triplicates. Values were calculated in relation to β-actin and normalized to a separate RNA calibrator. A B s 160 y s a l s l s e a c e T 120 l T b a i M v h t f i 80 o w e g d a e t 0 h post-heat (mean = 120 +/- 13) n n i e 40 4 h post-heat (mean = 113 +/- 10) m c r r e 24 h post-heat (mean = 108 +/- 11) e t P e d 0 0 20,000 40,000 60,000 80,000 100,000 Total number of cells plated per well C D 30.0 25.0 20.0 change 15.0 : Fold 10.0 qPCR 5.0 0.0 Cells 2013, 2 227 2. Results and Discussion 2.1. Keratinocytes Induce an Appreciable Stress Response When Exposed To Hyperthermic Stress We conducted an initial set of experiments to investigate the impact that our selected hyperthermia stress protocol had on cellular viability. For these studies, HEKs were exposed to hyperthermic stress and cellular metabolic activity was measured at 0, 4 and 24 h post-exposure using MTT assays (Figure 1B). For all cell densities tested, the data shows that greater than 90% of the cells survive after being exposed to our hyperthermic stress protocol. This data confirms that the HEKs cells were viable post-heat exposure. After confirming that the HEK cells survived post-exposure, we then sought to test whether our selected hyperthermic stress protocol induced an appreciable transcriptional stress response To investigate the transcriptional stress response, we exposed HEK cells to hyperthermic stress and used microarray gene chips to quantify the expression level of genes at 0, 4 and 24 h post-exposure (Figure 1A). Since a signature feature of an appreciable stress response is the rapid and marked upregulation of minimal stress genes [12], we conducted microarray and PCR analyses to examine the genes that HEKs express when exposed to hyperthermia (Figure 1C,D). Using Ingenuity Pathway Analysis (IPA) software, we filtered the microarray data using the following cut-offs: (i) an absolute value of expression magnitude (log2 ratio relative to control) greater or equal to 1.5; and (ii) a p-value less than or equal to 0.05. Of the 54,675 probe sets tested on each gene chip, we found that 762, 1,422 and 1,478 genes were differentially expressed at the 0, 4 and 24 h time points, respectively (Figure 1C).