Abstract
Osteoprotegerin (OPG) is a key regulator of bone remodeling. Mutations in OPG are involved in a variety of human diseases. We have shown that cochlear spiral ganglion cells secrete OPG at high levels and lack of OPG causes sensorineural hearing loss in addition to the previously described conductive hearing loss. In order to study the regulation of OPG expression, we conducted a database search on regulatory elements in the promoter region of the OPG gene, and identified two potential GATA-3 binding sites. Using luciferase assays and site directed mutagenesis, we demonstrate that these two elements are GATA-3 responsive and support GATA-3 transactivation in human HEK and HeLa cells. The expression of wild type GATA-3 activated OPG mRNA and protein expression, while the expression of a dominant negative mutant of GATA-3 or a GATA-3 shRNA construct reduced OPG mRNA and protein levels. GATA-3 deficient cells generated by expressing a GATA-3 shRNA construct were sensitive to apoptosis induced by etoposide and TNF-α. This apoptotic effect could be partly prevented by the co-treatment with exogenous OPG. Our results suggest new approaches to rescue diseases due to GATA-3 deficiency – such as in hypoparathyroidism, sensorineural deafness, and renal (HDR) syndrome – by OPG therapy.
Highlights
Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF) receptor superfamily and a key regulator of bone metabolism
We have focused on GATA-3 because mutations or deletions in the human GATA3 gene cause sensorineural hearing loss as a part of the syndrome of hypoparathyroidism, sensorineural deafness, and renal disease (HDR syndrome)[20,21,22,23]
The GATA-3 transactivated the OPG promoter 5.61 ± 0 .79 fold (n = 5, p = 0 .002), while the control transcriptional factors, retinoblastoma 1 (RB1) and general transcription factor II-I repeat domain-containing protein 1 (GTF2IRD1), which do not bind to the GATA-3 binding site, did not activate the OPG promoter (Fig. 1B)
Summary
Osteoprotegerin (OPG) is a member of the tumor necrosis factor (TNF) receptor superfamily and a key regulator of bone metabolism. Elevated OPG serum levels have been detected in patients with advanced malignancies such as prostate cancer[10], bladder carcinoma[11], and squamous cell head and neck cancer[12]. These observations confirm the role of OPG in tumor development. Others have demonstrated that the two Hox binding sites on human OPG promoter are responsible for bone morphogenesis protein (BMP) induced transcription of OPG gene and increased OPG secretion, inhibiting osteoclastogenesis[15]. Overexpression of OPG in human colorectal cancer cells is regulated by β -catenin and Tcf-4 bound to the human OPG promoter, mediating resistance to TRAIL-induced apoptosis in colon cancer[17]. Overexpression of GATA-3, in a mouse model of rheumatoid arthritis, protected against severe joint inflammation and bone erosion[25]
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