Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors
Creators: name: Bedini, Andrea <1979>
Publisher: name: Alma Mater Studiorum - Università di Bologna
Year: 2008
Subjects: BIO/14 Farmacologia

description: The organization of the nervous and immune systems is characterized by obvious differences and
striking parallels. Both systems need to relay information across very short and very long
distances.
The nervous system communicates over both long and short ranges primarily by means of more
or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange
communication in the immune system occurs mainly via the ordered and guided migration
of immune cells and systemically acting soluble factors such as antibodies, cytokines, and
chemokines. Its short-range communication either is mediated by locally acting soluble factors or
transpires during direct cell–cell contact across specialized areas called “immunological synapses”
(Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented
by a complex array of factors that induce cell growth and differentiation: these factors in the
immune system are called cytokines; in the nervous system, they are called neurotrophic factors.
Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either
system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most
potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived
neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et
al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth
factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000).
These factors may allow the two systems to “cross-talk” and eventually may provide a molecular
explanation for the reports that inflammation after central nervous system (CNS) injury has
beneficial effects (Moalem et al., 1999).
In order to shed some more light on such a cross-talk, therefore, transcription factors modulating
mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated.
More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell
receptor induction of MOPr expression in T-lymphocytes.
Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the
G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated
exogenous opioids or endogenous opioid peptides, and contribute to the regulation of
several functions including pain transmission, respiration, cardiac and gastrointestinal functions,
and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer
and Hollt 2006).
Recently, induction of MOPr expression in different immune cells induced by cytokines has been
reported (Kraus et al., 2001; Kraus et al., 2003).
The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has
clusters of potential binding sites for different transcription factors (Law et al. 2004).
Several studies, primarily focused on the upstream region of the OPRM1 promoter, have
investigated transcriptional regulation of MOPr expression. Presently, however, it is still not
completely clear how positive and negative transcription regulators cooperatively coordinate cellor
tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its
expression.
IGF-I and its receptors are widely distributed throughout the nervous system during
development, and their involvement in neurogenesis has been extensively investigated
(Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such
neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al.,
2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor
which, after ligand binding-induced autophosphorylation, associates with specific adaptor
proteins and activates different second messengers (Bondy and Cheng 2004). These include:
phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di
Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al.
2000; Yadav et al. 2005).
REST plays a complex role in neuronal cells by differentially repressing target gene expression
(Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during
neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated
in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al.
2006). Thus, the REST concentration seems to influence its function and the expression of
neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al.
2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural
induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes
not yet required by the differentiation program, but its expression decreases after five days of
treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in
which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the
disappearance of REST (Di Toro et al. 2005).
The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the
repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional
repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth
factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates
OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A
series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell
models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels
of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated
OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with
constructs deficient in the REST DNA binding element, or when REST was down-regulated in
retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of
transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3
DNA element located in the promoter, increases OPRM1 transcription.
T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major
histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular
signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the
genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat
CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study
whether its signalling influence OPRM1 expression. Results were that TCR engagement
determined a significant induction of OPRM1 expression through the activation of transcription
factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been
expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation
and recycling, whereas morphine did not.
Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a
critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how
human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates
OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as
well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune
cells suggest an important role for MOPr as a molecular bridge between neurons and immune
cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous
system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli
and analgesic and neuroprotective effects.