In vivo real-time imaging of TGF-beta-induced transcriptional activation of the RANK ligand gene promoter in intraosseous prostate cancer.
BACKGROUND
Current animal models of prostate cancer (CaP) bone metastasis do not allow measurement of either tumor growth in bone over time or activation of gene promoters in intraosseous tumors. To develop these methods, we used bioluminescent imaging (BLI) to determine if expression of receptor activator of NF-kappaB ligand (RANKL), a pro-osteoclastogenic factor that promotes CaP bone metastases, is modulated by the bone matrix protein transforming growth factor-beta (TGF-beta) in vivo.


METHODS
C4-2B human CaP cells were treated with TGF-beta in vitro and RANKL mRNA and protein production were measured by polymerase chain reaction (PCR) and ELISA, respectively. Then C4-2B cells stably transfected with the RANKL promoter driving luciferase (lux) were injected intra-tibially into severe combined immundeficient (SCID) mice. Tumors were subjected to BLI every 2 weeks for 6 weeks and serum prostate specific antigen (PSA) was measured using ELISA. Vehicle (V), 1,25 dihydroxyvitamin D (VitD), or TGF-beta was administered to mice with established tumors and BLI to measure RANKL promoter activity was performed. Tumors were then subjected to immunohistochemistry for lux and assayed for RANKL mRNA levels.


RESULTS
TGF-beta induced RANKL protein and mRNA expression and activated the RANKL promoter activity in a dose-dependent manner in vitro. BLI demonstrated an increase in intraosseous tumor size over time, which correlated with serum PSA levels. Administration of TGF-beta and VitD to mice with established intraosseous tumors increased lux activity compared to V. Intratibial tumor RANKL mRNA expression paralleled the increased promoter activity. Immunohistochemistry confirmed the presence of lux in the intraosseous tumors.


CONCLUSIONS
These results demonstrate the ability to measure intraosseous tumor growth over time and gene promoter activation in an established intraosseous tumor in vivo and also demonstrate that TGF-beta induces activates the RANKL promoter. These results provide a novel method to explore the biology of CaP bone metastases.
Zhang
Jian
J
Department of Pathology and Unit for Laboratory Animal Medicine, School of Medicine, University of Michigan, Ann Arbor, Michigan, USA.
Lu
Yi
Y
Dai
Jinlu
J
Yao
Zhi
Z
Kitazawa
Riko
R
Kitazawa
Sohei
S
Zhao
Xinping
X
Hall
Daniel E
DE
Pienta
Kenneth J
KJ
Keller
Evan T
ET
D000818:Animals; D001859:Bone Neoplasms / Q000235:genetics / Q000503:physiopathology* / Q000556:secondary*; D002352:Carrier Proteins / Q000096:biosynthesis*; D004797:Enzyme-Linked Immunosorbent Assay; D008163:Luminescent Measurements; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D051379:Mice; D016513:Mice, SCID; D009374:Neoplasms, Experimental; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D017430:Prostate-Specific Antigen / Q000032:analysis; D011471:Prostatic Neoplasms / Q000235:genetics* / Q000473:pathology*; D053245:RANK Ligand; D012333:RNA, Messenger / Q000096:biosynthesis; D053246:Receptor Activator of Nuclear Factor-kappa B; D015533:Transcriptional Activation; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D053245:RANK Ligand; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; C505686:TNFRSF11A protein, human; C505688:TNFSF11 protein, human; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D016212:Transforming Growth Factor beta; D017430:Prostate-Specific Antigen