Overexpression of cyclooxygenase-2 (COX-2) in articular tissues is an earmark of arthritis associated with increased chondrocyte apoptosis. Although high fluid shear induces chondrocyte apoptosis, the intracellular signaling pathways regulating this process remain largely unknown. In this application, we will test the hypothesis supported by compelling data that COX-2 activity is responsible for the decreased antioxidant capacity of chondrocytes and their apoptosis in response to shear stimulation. An extensive characterization of the signaling network regulating chondrocyte apoptosis may offer novel avenues for its control. We believe that the most judicious approach is to characterize the signaling mechanisms regulating COX-2 expression, and identify downstream targets of COX-2 activity which in turn regulate the activity of phase 2 antioxidant enzymes, and key pro-/anti- apoptotic genes. As has been argued in the literature, the signaling mechanisms are species-, cell- and stimulus- specific. Thus, Aim 1a will identify the cis-elements and their cognate trans- acting factors of shear-induced COX-2 expression in human chondrocytes. Using bioinformatics tools and cDNA microarrays coupled with selective, individual gene knockdowns via dominant negative/siRNA techno- logy, we will delineate the upstream signaling molecules of COX-2 expression (Aim 1b). This methodology will also enable us to define the downstream targets of COX-2 activity as well as other intermediate gene targets responsible for the differential expression of phase 2 genes (Aim 2).
Aim 3 will provide a mechanistic interpretation for the shear-induced chondrocyte apoptosis by identifying the key pro- and anti- apoptotic genes of the Bcl-2 family as well as their upstream regulatory genes. Comparative studies of the effects of fluid shear and cyclic strain on chondrocyte function will also be performed. This application integrates engineering principles with quantitative biology to reconstruct gene networks in shear-activated chondrocytes. The knowledge gained from these studies will provide a basis for the design of novel cell- based approaches for cartilage repair, and defining ideal bioreactor operating conditions for culturing artificial cartilage. Moreover, this approach will be vital for developing novel therapeutic strategies targeting molecular pathways relevant to arthritic pathogenesis and progression. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Type
Research Project (R01)
Project #
1R01AR053358-01A2
Application #
7199641
Study Section
Bioengineering, Technology and Surgical Sciences Study Section (BTSS)
Program Officer
Tyree, Bernadette
Project Start
2006-09-18
Project End
2010-08-31
Budget Start
2006-09-18
Budget End
2007-08-31
Support Year
1
Fiscal Year
2006
Total Cost
$370,293
Indirect Cost
Name
Johns Hopkins University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Wang, Pu; Guan, Pei-Pei; Guo, Chuang et al. (2013) Fluid shear stress-induced osteoarthritis: roles of cyclooxygenase-2 and its metabolic products in inducing the expression of proinflammatory cytokines and matrix metalloproteinases. FASEB J 27:4664-77
Wang, Pu; Zhu, Fei; Konstantopoulos, Konstantinos (2012) The antagonistic actions of endogenous interleukin-1? and 15-deoxy-?12,14-prostaglandin J2 regulate the temporal synthesis of matrix metalloproteinase-9 in sheared chondrocytes. J Biol Chem 287:31877-93
Grabias, Bryan M; Konstantopoulos, Konstantinos (2012) Epithelial-mesenchymal transition and fibrosis are mutually exclusive reponses in shear-activated proximal tubular epithelial cells. FASEB J 26:4131-41
Wang, Pu; Zhu, Fei; Konstantopoulos, Konstantinos (2011) Interleukin-6 synthesis in human chondrocytes is regulated via the antagonistic actions of prostaglandin (PG)E2 and 15-deoxy-?(12,14)-PGJ2. PLoS One 6:e27630
Wang, Pu; Zhu, Fei; Tong, Ziqiu et al. (2011) Response of chondrocytes to shear stress: antagonistic effects of the binding partners Toll-like receptor 4 and caveolin-1. FASEB J 25:3401-15
Zhu, Fei; Wang, Pu; Lee, Norman H et al. (2010) Prolonged application of high fluid shear to chondrocytes recapitulates gene expression profiles associated with osteoarthritis. PLoS One 5:e15174
Wang, Pu; Zhu, Fei; Konstantopoulos, Konstantinos (2010) Prostaglandin E2 induces interleukin-6 expression in human chondrocytes via cAMP/protein kinase A- and phosphatidylinositol 3-kinase-dependent NF-kappaB activation. Am J Physiol Cell Physiol 298:C1445-56
Zhu, F; Wang, P; Kontrogianni-Konstantopoulos, A et al. (2010) Prostaglandin (PG)D(2) and 15-deoxy-Delta(12,14)-PGJ(2), but not PGE(2), mediate shear-induced chondrocyte apoptosis via protein kinase A-dependent regulation of polo-like kinases. Cell Death Differ 17:1325-34
Wang, Pu; Zhu, Fei; Lee, Norman H et al. (2010) Shear-induced interleukin-6 synthesis in chondrocytes: roles of E prostanoid (EP) 2 and EP3 in cAMP/protein kinase A- and PI3-K/Akt-dependent NF-kappaB activation. J Biol Chem 285:24793-804
Healy, Zachary R; Zhu, Fei; Stull, Joshua D et al. (2008) Elucidation of the signaling network of COX-2 induction in sheared chondrocytes: COX-2 is induced via a Rac/MEKK1/MKK7/JNK2/c-Jun-C/EBPbeta-dependent pathway. Am J Physiol Cell Physiol 294:C1146-57

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