Work in the Lassar lab has established that forced expression of Runx2 in somites can activate expression of chondrocyte hypertrophy markers (i.e., Ihh and Collagen X) only if these cells are first induced to become chondrocytes by a variety of treatments, suggesting that a chondrogenic co-factor allows Runx2 to activate expression of markers of chondrocyte hypertrophy. Consistent with this notion, the Lassar lab has established that co-transfection of ectopic Runx2 together with the BMP signal tranducer, Smad 1, can induce expression of a luciferase construct driven by Collagen X regulatory sequences only in chondrocytes and not in fibroblasts. By employing a systematic electrophoretic mobility shift assay (EMSA) with the chicken collagen X regulatory sequences, the Lassar lab has identified a DNA binding activity (termed Fast Mobility Complex) specifically in hypertrophic chondrocytes that contains the transcription factor FoxA2 and interacts with a sequence that is necessary to drive chondrocyte-specific expression of linked reporter genes. Recent work in the Lassar lab has established: (1) that FoxA factors are specifically expressed in differentiated chondrocytes, (2) that FoxA binding sites are conserved in both avian and mammalian collagen X enhancers, (3) that mutation of FoxA binding sites in the avian collagen X enhancer severely decreases Runx2/Smad1-mediated activation of this enhancer in chicken sternal chondrocytes, (4) that while FoxA1 and FoxA2 are induced during chondrogenesis in micromass cultures of chicken limb bud mesenchymal cells, FoxA2 and FoxA3 are specifically expressed during murine chondrogenic differentiation, (5) that forced expression of either FoxA1, FoxA2 or FoxA3 robustly activates the expression of collagen X-reporter constructs in either chondrocytes or fibroblasts, and (6) that shRNA-mediated knockdown of FoxA factors in chicken sternal chondrocytes both inhibits the ability of Runx2/Smad1 to activate the expression of a collagen X-luciferase reporter and inhibits the expression of endogenous collagen X, Ihh, and MMP13. In light of both these new findings, and published findings of others indicating that FoxA2/3 are expressed in murine growth plate chondrocytes, I hypothesize that both FoxA2 and FoxA3 may play an important role in promoting chondrocyte maturation in mice. In this proposal I propose to determine whether FoxA2 and FoxA3 play a role in regulating chondrocyte maturation in mice. The goal of this proposal is to elucidate the factors that control chondrocyte hypertrophy in the hope that this knowledge will lead to the development of reagents that could block this program in articular cartilage and thereby slow the progression of osteoarthritis.

Public Health Relevance

The course of cartilage degradation in osteoarthritis (OA) involves an activation of the chondrocyte hypertrophy program in the affected cartilage, suggesting that activation of the chondrocyte maturation program could play a key role in progression of cartilage destruction in OA. The goal of this proposal is to elucidate the factors that control chondrocyte hypertrophy in the hope that this knowledge will lead to the development of reagents that could block this program in articular cartilage and thereby slow the progression of OA.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR055552-03
Application #
8248083
Study Section
Skeletal Biology Structure and Regeneration Study Section (SBSR)
Program Officer
Tyree, Bernadette
Project Start
2010-04-01
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
3
Fiscal Year
2012
Total Cost
$366,120
Indirect Cost
$150,120
Name
Harvard University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047006379
City
Boston
State
MA
Country
United States
Zip Code
02115
Ogawa, Hiroyasu; Kozhemyakina, Elena; Hung, Han-Hwa et al. (2014) Mechanical motion promotes expression of Prg4 in articular cartilage via multiple CREB-dependent, fluid flow shear stress-induced signaling pathways. Genes Dev 28:127-39
Kozhemyakina, Elena; Ionescu, Andreia; Lassar, Andrew B (2014) GATA6 is a crucial regulator of Shh in the limb bud. PLoS Genet 10:e1004072
Daoud, Georges; Kempf, Hervé; Kumar, Deepak et al. (2014) BMP-mediated induction of GATA4/5/6 blocks somitic responsiveness to SHH. Development 141:3978-87
Kumar, Deepak; Lassar, Andrew B (2014) Fibroblast growth factor maintains chondrogenic potential of limb bud mesenchymal cells by modulating DNMT3A recruitment. Cell Rep 8:1419-31