Negative regulation of the osteoblast function in multiple myeloma through the repressor gene E4BP4 activated by malignant plasma cells.
OBJECTIVE
To explore the pathogenetic mechanisms that suppress the osteoblast function in multiple myeloma because osteogenesis results in defective new bone formation and repair.


METHODS
Microarray gene analysis revealed the overexpression of E4BP4, a transcriptional repressor gene, in normal osteoblasts cocultured with myeloma cells that were releasing the parathyroid hormone-related protein (PTHrP). Thus, the effect of E4BP4 was assessed in PTHrP-stimulated osteoblasts by measuring the RNA levels of both Runx2 and Osterix as major osteoblast transcriptional activators. Because E4BP4 is a negative regulator of the cyclooxygenase-2 (COX-2) pathway that drives the expression of both Runx2 and Osterix, these factors were investigated after prostaglandin E(2) treatment to overcome the COX-2 defect as well as in E4BP4-silenced osteoblasts. Finally, E4BP4, PTHrP, Osterix, and osteocalcin levels were measured in vivo in patients with bone disease together with the E4BP4 protein in bone biopsies.


RESULTS
E4BP4 was specifically induced by PTHrP and inhibited both Runx2 and Osterix, whereas E4BP4-silenced osteoblasts expressed functional levels of both factors. The prostaglandin E(2) treatment of E4BP4-up-regulated osteoblasts promptly restored Runx2 and Osterix activities, suggesting that integrity of COX-2 pathway is essential for their transcription. Down-regulation of Osterix by E4BP4 was confirmed in vivo by its inverse levels in osteoblasts from myeloma patients with increased serum PTHrP, whose bone biopsies expressed the E4BP4 protein.


CONCLUSIONS
Our data support the role of E4BP4 as osteoblast transcriptional repressor in inhibiting both Runx2 and Osterix in myeloma bone disease and correlate its effect with the increased PTHrP activity.
Silvestris
Franco
F
Department of Internal Medicine and Clinical Oncology, University of Bari Medical School, Piazza Giulio Cesare 11, Bari, Italy. f.silvestris@dimo.uniba.it
Cafforio
Paola
P
De Matteo
Monica
M
Calvani
Nicola
N
Frassanito
Maria A
MA
Dammacco
Franco
F
D000328:Adult; D050976:Basic-Leucine Zipper Transcription Factors / Q000096:biosynthesis*; D050659:Core Binding Factor Alpha 1 Subunit / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D051546:Cyclooxygenase 2 / Q000378:metabolism; D015232:Dinoprostone / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D020868:Gene Silencing; D006801:Humans; D009101:Multiple Myeloma / Q000378:metabolism* / Q000473:pathology; D020411:Oligonucleotide Array Sequence Analysis; D010006:Osteoblasts / Q000166:cytology* / Q000378:metabolism; D010012:Osteogenesis; D044162:Parathyroid Hormone-Related Protein / Q000378:metabolism; D010950:Plasma Cells / Q000378:metabolism*; D000076182:Sp7 Transcription Factor; D014157:Transcription Factors / Q000037:antagonists & inhibitors / Q000096:biosynthesis
D050976:Basic-Leucine Zipper Transcription Factors; D050659:Core Binding Factor Alpha 1 Subunit; C497440:NFIL3 protein, human; D044162:Parathyroid Hormone-Related Protein; C493709:RUNX2 protein, human; D000076182:Sp7 Transcription Factor; C478163:SP7 protein, human; D014157:Transcription Factors; D051546:Cyclooxygenase 2; D015232:Dinoprostone