Submitted on November 29, 2005
Accepted on February 2, 2006
Whole Genome Microarray Analysis of Growth Hormone Induced Gene Expression in Bone: T-box3, a Novel Transcription Factor, Regulates Osteoblast Proliferation
Kristen E Govoni1, Seong Keun Lee1, Robert B Chadwick2, Hongrun Yu1, Yuji Kasukawa1, David J Baylink2, and Subburaman Mohan3*
1 Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, CA, USA
2 Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, CA, USA; Department of Medicine, Loma Linda University, Loma Linda, CA, USA
3 Musculoskeletal Disease Center, Jerry L. Pettis Memorial Veterans Affairs Medical Center, Loma Linda, CA, USA; Department of Medicine, Loma Linda University, Loma Linda, CA, USA; Department of Biochemistry, Loma Linda University, Loma Linda, CA, USA
* To whom correspondence should be addressed. E-mail: subburaman.mohan{at}med.va.gov.
Growth hormone (GH) is important in the development and maintenance of bone; however, the IGF-dependent and -independent molecular pathways involved remain to be established. We used microarray analysis to evaluate GH signaling pathways in four week old GH-deficient mice following a single injection of GH (4 mg/kg body wt) or PBS (n = 6/group) at 6 or 24 hours after treatment. 6,160 genes were differentially expressed at P 
0.05, and 17% of these genes were identified at both time points. Several of the genes differentially expressed were ESTs and the remaining genes fell into 49 GO categories. For subsequent studies, we focused on T-box (Tbx) 3, a novel transcription factor, which increased > 2 fold at both time points. Real-time reverse transcriptase (RT)-PCR analysis determined that pre-treatment with IGF binding protein-4 did not block GH-induced Tbx3 expression in vitro. Pretreatment with TNF
blocked GH-induced Tbx3 expression. Tbx3 expression increased during osteoblast differentiation and following BMP-7 and Wnt3a treatment (P 0.05). Blocking Tbx3 expression by small interfering (si) RNA decreased cell number and [3H]Thymidine incorporation (P < 0.01). In conclusion: 1) GH caused acute changes in several novel genes, suggesting that many GH-induced signaling pathways and target genes remain to be discovered. 2) Since Tbx3 expression is regulated in osteoblasts and blockage of Tbx3 expression decreased cell number and DNA synthesis, we propose that Tbx3 is an important determinant of osteoblast cell number.
