Human Freud-2/CC2D1B: A Novel Repressor of Postsynaptic Serotonin-1A Receptor Expression
Received 20 August 2008; received in revised form 11 February 2009; accepted 28 February 2009. published online 08 May 2009.
Background
Altered expression of serotonin-1A (5-HT1A) receptors, both presynaptic in the raphe nuclei and post-synaptic in limbic and cortical target areas, has been implicated in mood disorders such as major depression and anxiety. Within the 5-HT1A receptor gene, a powerful dual repressor element (DRE) is regulated by two protein complexes: Freud-1/CC2D1A and a second, unknown repressor. Here we identify human Freud-2/CC2D1B, a Freud-1 homologue, as the second repressor.
Methods
Freud-2 distribution was examined with Northern and Western blot, reverse transcriptase polymerase chain reaction, and immunohistochemistry/immunofluorescence; Freud-2 function was examined by electrophoretic mobility shift, reporter assay, and Western blot.
Results
Freud-2 RNA was widely distributed in brain and peripheral tissues. Freud-2 protein was enriched in the nuclear fraction of human prefrontal cortex and hippocampus but was weakly expressed in the dorsal raphe nucleus. Freud-2 immunostaining was co-localized with 5-HT1A receptors, neuronal and glial markers. In prefrontal cortex, Freud-2 was expressed at similar levels in control and depressed male subjects. Recombinant hFreud-2 protein bound specifically to 5′ or 3′ human DRE adjacent to the Freud-1 site. Human Freud-2 showed strong repressor activity at the human 5-HT1A or heterologous promoter in human HEK-293 5-HT1A-negative cells and neuronal SK-N–SH cells, a model of postsynaptic 5-HT1A receptor-positive cells. Furthermore, small interfering RNA knockdown of endogenous hFreud-2 expression de-repressed 5-HT1A promoter activity and increased levels of 5-HT1A receptor protein in SK-N–SH cells.
Conclusions
Human Freud-2 binds to the 5-HT1A DRE and represses the human 5-HT1A receptor gene to regulate its expression in non-serotonergic cells and neurons.
aOttawa Health Research Institute (Neuroscience), University of Ottawa, Ottawa, Ontario, Canada
bDepartment of Psychiatry, Human Behavior, University of Mississippi Medical Center, Jackson, Mississippi
cInstitute of Pharmacology Polish, Academy of Sciences, Krakow, Poland
dDepartment of Psychiatry, Case Western Reserve University, Cleveland, Ohio
Address reprint requests to Paul R. Albert, Ph.D., OHRI (Neuroscience), University of Ottawa, Neuroscience, 451 Smyth Road, Ottawa, Ontario, Canada K1H-8M5
ABSTRACT