This Supplement is in response to the American Reinvestment and Recovery Act of 2009, specifically in response to Recovery Act Funds for Competitive Revision Applications (NOT-OD-09-058). It is a Supplement to our funded parent grant R01 AR 050847 (NIH/NIAMS) titled """"""""Cis Regulatory Motifs in Adult Articular Chondrocytes"""""""".
The specific aims of the parent grant are (1) Investigate the role of Site-1 protease (S1P) in chondrogenesis and maintenance of the chondrocyte phenotype in vivo;and (2) Investigate the role of S1P on hypertrophic chondrocyte differentiation and matrix synthesis in vitro. S1P plays a fundamental role in the processing of endoplasmic reticulum (ER) membrane-bound latent transcription factors to their free and active nuclear form. It activates the transcription factor SREBP that plays a role in maintaining lipid homeostasis;it also activates ATF6, a transcription factor with a role in ER stress response. A study of the S1Pcko mice (mice with cartilage-specific deletion of S1P) phenotype show that the mice exhibit chondrodysplasia as the cartilage suffers from lack of the major cartilage protein, collagen type IIB (Col IIB). The chondrocytes also exhibit ER stress coupled with a down-regulation of important lipogenic pathways and entrapment of Col IIB in the ER. This Supplement is designed to investigate ER stress response regulatory pathways in chondrocytes, provide newer employment opportunities, and will involve the collaborative efforts of Washington University, Vanderbilt University, and Harvard Medical School.
In SPECIFIC AIM 1, we will investigate whether down-regulation of lipogenic pathways in chondrocytes causes ER stress and the entrapment of Col IIB in the ER. We will measure the cholesterol/fatty acid composition of the chondrocytic ER membrane by gas chromatography/mass spectrometry and use electron paramagnetic resonance spectroscopy to analyze ER membrane fluidity. The ER will be obtained from chondrocytes from S1Pcko mice, from tamoxifen-injected S1Pf/f;CreERT mice, and from chondrocytes from S1Pf/f mice treated with Cre recombinase-expressing adenoviruses to delete S1P in vitro.
In SPECIFIC AIM 2, we will analyze the importance of a functional ER stress response pathway to cartilage and skeletal development. For this analysis we will construct Xbp1f/f;Col2-Cre mice to delete Xbp1 (X-box-binding protein 1) function (which is involved in ER stress response) specifically in chondrocytes. The Xbp1f/f;Col2-Cre mice will be systematically studied through morphological, histological, and immunofluorescence studies to study the impact of Xbp1 deletion in cartilage and skeletal development.
In SPECIFIC AIM 3, we will use a tibial fracture-healing model developed in our laboratory, to study the requirement of S1P and Xbp1 for cartilage and skeletal development in postnatal mice. A time-line has been developed to complete all three specific aims within two years.

Public Health Relevance

An inability to respond to endoplasmic reticulum stress has been shown in recent years to be linked to debilitating human diseases such as diabetes, the neurodegenerative diseases retinitis pigmentosa and alzheimer's, atherosclerosis, and cancer. This proposal will focus on understanding the causes of endoplasmic reticulum stress in chondrocytes and analyze the importance of an endoplasmic reticulum stress response to cartilage and skeletal development. The knowledge gained from this study may allow for better strategies in designing tissue-engineered cartilage that would allow for the replacement of degenerate joint cartilage as seen during osteoarthritis.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-MOSS-A (96))
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Tyree, Bernadette
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Washington University
Schools of Medicine
Saint Louis
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