Definition and function

The term “immediate early gene” (IEG) or “early response gene” was originally used during the mid 1970s and early 1980s for viral genes that were rapidly transcribed following invasion of a host (SIMON et al., 2006). Characteristically, these genes can be expressed under conditions of protein synthesis inhibition such as treatment with cycloheximide, ruling out that other newly synthesized gene products are responsible for their induction. IEG encoded mRNAs and proteins rapidly accumulate after stimulation, but also have a short half-life due to a limited stability of their mRNAs and a rapid turn-over of their proteins. Many IEGs act as third messengers in an intracellular signal transduction cascade between cell surface receptors, cytoplasmic second messengers, and specific target genes in the nucleus (KIESSLING and GASS, 1993). With the discovery of the human homologs to retroviral oncogenes the concept of IEGs as key transcription factors initiating the subsequent expression of so-called “late response genes”, mainly involved in the regulation of cell growth and differentiation, was transferred to humans (SIMON et al., 2006). Late response genes

18 Introduction

are only expressed after the production of immediate early genes acting as transcription factors on their promoter regions.

Transcription factors are characterized by a DNA binding domain that binds gene specific regulatory sites and a second domain that exhibits transcriptional activation potential. These site-specific transcription factors recruit numerous coactivators to the transcription machinery to initiate gene-specific transcription. More than 2000 transcription factors and around 200 to 300 coactivators are encoded in the human genome (BRIVANLOU and DARNELL, 2002). In addition, several corepressors, chromatin remodelers, histone acetylases, deacetylases, kinases, and methylases are assembled into the RNA polymerase complex. The combination of approximately six to eight different proteins out this vast array of regulators seems to be unique for the activation of each gene (BRIVANLOU and DARNELL, 2002). Consequently, ensuring the right amount of the right protein in the right cell has to be critical in all physiological and pathological processes such as CNS development and inflammation.

Intensity, duration, and type of different biophysical and biochemical stimuli influence the transcriptional activity of IEGs on target genes in a cell-type specific manner (MURPHY and BLENIS, 2006; SIMON et al., 2006). A transient IEG induction mediates a short-term cell adaptation to the initial stimulus by protein neosynthesis, whereas sustained IEG induction seems to be involved in developmental processes, cell cycle control, cell survival, synaptic plasticity, learning, and immune functions (LANAHAN and WORLEY, 1998; MURPHY and BLENIS, 2006; SIMON et al., 2006).

In addition, overexpression of IEGs such as c-jun and c-fos are hallmarks of several malignancies (MURPHY and BLENIS, 2006). Interestingly, the IEG response in neurons seems to be limited to approximately 30 to 40 genes, of which perhaps 10 to 15 are transcription factors (LANAHAN and WORLEY, 1998).

It has been shown that IEG function also interferes with posttranscriptional processes such as cytoplasmic polyadenylation, degradation by adenosine- and uracil-rich element binding factors, and even expression of related non-coding RNAs to speed up transcriptional responses (SIMON et al., 2006). However, IEGs encode not only for transcription factors, but also for secreted and cytoskeletal proteins, growth

Introduction 19

factors, chemo-attractants, metabolic enzymes, and proteins involved in signal transduction (LANAHAN and WORLEY, 1998; SNG et al., 2004 ; Fig. 1.2).

Figure 1-2: Pathway of immediate early gene induction.

Stimulus

A distinct and specific IEG expression pattern depending on the cell type and condition is rapidly induced by a variety of physiological and pathological stimuli including somatosensory perception, metabolic and mechanical stress, free radicals, hormones, cytokines, ischemia, and seizures (KIESSLING and GASS, 1993;

LANAHAN and WORLEY, 1998; SIMON et al., 2006). All these higly interactive stimuli induce mitogen-activated protein kinase (MAPK) pathways in combination with other signal transduction cascades such the nuclear factor-κB (NF-κB) pathway (PAHL, 1999; SIMON et al., 2006). The exact extent and time-course of activation of each MAPK member, namely the extracellular-signal-regulated kinase (ERK), the c-JUN N-terminal kinase (JNK), and the 38 kDa mitogen-activated protein kinase (p38), by a particular stimulus determines the specific induction of a distinct set of IEGs and their target genes (MURPHY and BLENIS, 2006). ERK activates Ets-1 and c-fos, JNK triggers c-jun and p53, and Max protein activation is induced by p38 (Fig. 1-3).

20 Introduction

Max protein plays a central and essential role as an obligate dimerization-DNA-binding partner for Myc oncoproteins and Mad transcriptional repressors (AMATI et al., 1992; BLACKWOOD and EISENMAN et al., 1991; BLACKWOOD et al., 1992;

HURLIN et al., 1995; REDDY et al., 1992; VÄSTRIK et al., 1995). All these IEG proteins can act as transcription factors and are thereby implicated in fundamental processes including cell proliferation, differentiation, and growth, cytokine production, and apoptosis (DANG, 1999; ROSENBERG, 2002).

Figure 1-3: Signal transduction cascades including the NF-κB and MAPK pathways after TNF-α stimulation.

Legend: AP-1, activator protein-1; EBS, ETS binding site; ERK, extracellular-signal-regulated kinase; IκB, Inhibitor of NF-κB; IKK, IκB kinase; JNK, c-JUN N-terminal kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; MEKK, MEK kinase; NEMO, NF-κB essential modulator; NIK, NF-κB inducing kinase; p38, 38 kDa MAPK; RIP, receptor interacting protein; TNF-α, tumor necrosis factor-α; TNF-R1, TNF-α receptor 1; TRADD, TNF-R associated death domain; TRAF-2, TNF-R associated factor-2.

Introduction 21

Interestingly, two possible feedback loops exist between the ERK pathway and IEG-encoded proteins products during sustained ERK signalling. In the first, C-terminal phosphorylation increases the stability of IEG-products such as c-fos and allows the docking of ERK to specific protein interaction domains of IEG-encoded proteins. In the second, stabilized nuclear IEG-products retain activated ERK in the nucleus to promote their continuous activation by ERK.

While the downstream cascades that follow IEG expression are highly complex interactive networks, IEG expression itself may only show limited variation in its response patterns (SIMON et al., 2006). Major technical problems still encumber the study of the transcriptome. Therefore, it might be appropriate to focus on the relatively simple IEG level at the current stage (SIMON et al., 2006). However, studies quantifying IEG expression in developmental and inflammatory processes in order to investigate their molecular mechanisms are rare.