Several members of the CCN family of matricellular proteins are required for chondrocyte proliferation, survival, and ECM production in the growth plate. However, the mechanisms by which CCNs mediate these activities are unknown. Similarly, although some CCN proteins are expressed in adult articular cartilage and intervertebral discs (IVDs), their functions in these tissues are unknown. Thus there is a large gap in knowledge regarding the roles of CCN proteins in healthy adult cartilage and IVDs, and their roles in degenerative diseases such as osteoarthritis (OA) and degenerative disc disease (DDD), for which there are currently no therapies. The studies in this proposal address these fundamental issues. The central hypothesis is that CCNs 1, 2, and 4 have overlapping functions in and are required for chondrogenesis and postnatal maintenance of articular cartilage and nucleus pulposus (NP), and that they mediate their effects in part through regulation of HIF1a and HIF2a, and in part by acting as escorts/chaperones for ECM proteins. This hypothesis will be tested in three specific aims.
In Aim 1, the roles of CCNs 1, 2, and 4 in growth plate chondrogenesis and articular cartilage will be determined. Mice carrying floxed alleles of Ccns 1 and 2 have been generated, as have Ccn4-/- mice, and will be used to excise Ccn genes prenatally and postnatally. Preliminary studies support the hypothesis that CCNs 1 and 2 have overlapping functions in postnatal cartilage. Other studies in this aim build on preliminary data showing that loss of Ccn2 leads to decreased nuclear localization of NFkB (RelA), and decreased levels of HIF1a, both of which have pro-survival functions in cartilage. The hypothesis that CCNs induce HIF1a and HIF2a by activating NFkB /RelA is tested.
In Aim 2, the roles of CCNs in the formation and maintenance of the nucleus pulposus (NP) of the IVD are tested by generating mice in which excision of CCNs is induced.
This aim i s based on preliminary data showing that Ccn2 mutants have primary defects in formation of the NP, and these are exacerbated in Ccn1/2 double mutants. Other experiments in this aim test the hypothesis that CCNs mediate their effects on ECM production and cell survival in part through maintaining levels of HIF1a and HIF2a, as in growth plate cartilage.
In Aim 3, the ability of CCNs 1 and 2 to act directly as "chaperones" to facilitate ECM assembly is tested. Precedent for this mode of action comes from observations that other secreted proteins have this activity, and from the finding that CCN proteins contain CXXC motifs characteristic of such chaperones. The studies are innovative, because they utilize new mouse models to test two novel modes of action for CCNs: regulation of cell viability and matrix production by NFkB and HIFs, and direct roles in ECM assembly via chaperone-like activity. The proposed research is significant, because it is expected to demonstrate for the first time that CCNs are essential for the maintenance of articular cartilage and IVD. This knowledge has the potential to lead to new therapeutic approaches to the treatment of OA and DDD.

Public Health Relevance

Osteoarthritis (OA) is the most common form of arthritis and the leading cause of long-term disability in the US. Degenerative disc disease (DDD) is the most common form of low back pain, and as with OA, there are no effective therapies. CCN proteins are key regulators of cartilage formation;however, their roles in adult articular cartilage and intervertebral discs are unknown. The studies in this proposal investigate these functions and could lead to the development of sorely needed therapies for the treatment of OA and DDD.

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
5R01AR052686-09
Application #
8704235
Study Section
Special Emphasis Panel (ZRG1-MOSS-C (04))
Program Officer
Tyree, Bernadette
Project Start
2005-08-20
Project End
2016-06-30
Budget Start
2014-07-01
Budget End
2015-06-30
Support Year
9
Fiscal Year
2014
Total Cost
$395,728
Indirect Cost
$130,738
Name
University of California Los Angeles
Department
Orthopedics
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
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Cheung, Laurence C; Strickland, Deborah H; Howlett, Meegan et al. (2014) Connective tissue growth factor is expressed in bone marrow stromal cells and promotes interleukin-7-dependent B lymphopoiesis. Haematologica 99:1149-56
Tran, Cassie M; Fujita, Nobuyuki; Huang, Bau-Lin et al. (2013) Hypoxia-inducible factor (HIF)-1ýý and CCN2 form a regulatory circuit in hypoxic nucleus pulposus cells: CCN2 suppresses HIF-1ýý level and transcriptional activity. J Biol Chem 288:12654-66
Hall-Glenn, Faith; De Young, R Andrea; Huang, Bau-Lin et al. (2012) CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis. PLoS One 7:e30562
Nagashima, Takashi; Kim, Jaeyeon; Li, Qinglei et al. (2011) Connective tissue growth factor is required for normal follicle development and ovulation. Mol Endocrinol 25:1740-59
Hall-Glenn, Faith; Lyons, Karen M (2011) Roles for CCN2 in normal physiological processes. Cell Mol Life Sci 68:3209-17
Huang, Bau-Lin; Brugger, Sean M; Lyons, Karen M (2010) Stage-specific control of connective tissue growth factor (CTGF/CCN2) expression in chondrocytes by Sox9 and beta-catenin. J Biol Chem 285:27702-12
Maeda, Azusa; Nishida, Takashi; Aoyama, Eriko et al. (2009) CCN family 2/connective tissue growth factor modulates BMP signalling as a signal conductor, which action regulates the proliferation and differentiation of chondrocytes. J Biochem 145:207-16
Nishida, Takashi; Kondo, Seiji; Maeda, Azusa et al. (2009) CCN family 2/connective tissue growth factor (CCN2/CTGF) regulates the expression of Vegf through Hif-1alpha expression in a chondrocytic cell line, HCS-2/8, under hypoxic condition. Bone 44:24-31
Einhorn, Thomas A; Laurencin, Cato T; Lyons, Karen (2008) An AAOS-NIH symposium. Fracture repair: challenges, opportunities, and directions for future research. J Bone Joint Surg Am 90:438-42

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