ECTS2013 Poster Presentations Cell biology: osteoblasts and bone formation (50 abstracts)
Nuclear translocation of oxytocin receptor mediates increased gene expression in osteoblasts
Adriana Di Benedetto 1 , Concetta Cuscito 1 , Graziana Colaianni 1 , Roberto Tamma 1 , Beatrice Nico 1 , Damiana Calvano 2 , Carlo Zambonin 2 , Michelangelo Corcelli 1 & Alberta Zallone 1
1Department of Basic Medical Sciences, Neurosciences and Organs of Senses, University of Bari, Bari, Italy; 2Department of Chemistry, University of Bari, Bari, Italy.
The neuro-hypophiseal hormone oxytocin (OT) is a novel anabolic regulator of bone mass (Tamma et al. PNAS, 2009), upregulating expression of critical osteoblast transcription factors. These effects are mediated by oxytocin receptor, a GPCR expressed by osteoblasts. Recently an increasing number of reports indicates that GPCRs could be targeted to the nuclear membrane; prostaglandin receptors, endothelin receptors and β-adrenergic receptors among others (Boivin et al. 2008). Accordingly we found OTRs in osteoblast nuclear extracts after OT stimulation (15–30 min). Confocal microscopy performed on intact cells either transfected with OTR-GFP or stained after fixation indicated a nuclear localization after OT stimulation, data confirmed by immunogold staining. Exogenous OTR-GFP, transfected in primary osteoblasts, colocalized with β-arrestin1/2 within 2–3 min after OT treatment, thereafter was found in RAB5-positive endosomal vesicles and then colocalized with transportin-1. Eventually, at least a part of the receptors was sorted to the nucleus where OTR-GFP was evident by confocal microscopy both in intact cells and in isolated nuclei. MALDI-TOF analysis of nuclear proteins immunoprecipitated with anti-OTR confirmed this finding; the spectra analyzed with FindPept Database revealed the presence of four peptides corresponding to OTR intracellular loops. We hypothesized as possible role for OTR in nuclei the regulation of transcription and/or transcription factors. Indeed, in response to OT stimulus, a physical interaction of native OTRs with the osteoblast transcription factor Runx-2 and with the transcription co-activator Schnurri-2 was found by immunoprecipitation. The blockage of OTR endocytosis by β-arrestins silencing, prevented OT induced up-regulation of Osx, ATF-4, BSP, and osteocalcin. Similarly by transportin-1 silencing, OTR nuclear localization as well as the up-regulation of Osx, ATF-4, osteocalcin, and BSP were impaired. Taken together these data suggest that OT anabolic effect on bone could be dependent upon a novel mechanism initiated by β-arrestin-mediated OTR internalization and followed by transportin-1 dependent nuclear translocation.
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Bone Abstracts
ISSN 2052-1219 (online)
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