In the intervertebral disc, the cells of the nucleus pulposus (NP) function in a unique environmental niche, characterized by a very limited vascular supply that imposes metabolic constraints on the disc cells and a proteoglycan-rich extracellular matrix. We have shown that the oxygen tension of the NP is very low and unlike all other tissues, cells of the NP exhibit constitutive expression of HIF-1a. We advance the new hypothesis that constitutive expression of HIF-1a serves to adapt the NP cells to their avascular environment and that changes in HIF-1 activity is required for their survival and function. The first Specific Aims is to test the hypotheses that high constitutive HIF-1a expression is required for survival of the NP cells in the hypoxic disc;to investigate mechanisms regulating constitutive expression and activity of HIF-1a;using tissue derived from human degenerative discs to correlate NP HIF-1 activity with degeneration and susceptibility to apoptosis. We will transiently and stably suppress HIF-1a and assess the impact of HIF-1a downregulation on cell survival and death. We will then restore HIF-1a expression in suppressed cells and evaluate survival, apoptosis and cell phenotype. We will also determine if changes in expression of pVHL, FIH and PHDs regulate HIF-1a expression and transcriptional activity in NP cells. We will use cells isolated form clinical human disc samples, with varying degree of degeneration, to determine if susceptibility to apoptogen treatment is linked to endogenous HIF-1a activity and to the level of degeneration. The second specific aim is to test the hypothesis that constitutive HIF-1a expression regulates proteoglycan synthesis and GAG chain formation and sulfation;to correlate HIF-1 activity to proteoglycan synthesis in human degenerative disc samples. We will determine the role of constitutive HIF-1a in the expression and synthesis of aggrecan core protein, a molecule that has a HIF-1 responsive promoter element. In addition, we will evaluate the role of HIF-1a in regulating expression of enzymes required for GAG synthesis (glucuronosyltransferase I;GlcAT-I) and chondroitin sulfation (chondroitin-4-O-sulfosyl transferase 2;C4ST-2). We will investigate if proteoglycan synthesis by cells isolated form clinical human disc samples is linked to HIF-1a activity. The last Specific Aim is to develop a rabbit model with suppression of HIF-1a in NP cells using lentiviral HIF-1a-SiRNA;to learn if inactivation of HIF-1a promotes a loss of cellularity, a decrease in proteoglycan synthesis and degeneration of the intervertebral disc. To complement the in vivo studies, and to investigate early cellular events in NP degeneration, we will perform disc organ culture studies using floxed-HIF-1a mouse. Inactivation of the HIF-1a gene in organ cultured discs will be achieved by injecting adenovirus expressing cre recombinase. The information provided by proposed studies will provide insights into mechanisms of early degenerative changes in the NP cells and provide foundation for development of interventional strategies to prevent degenerative disc disease.
The incidence of low back pain, which is often linked to degenerative changes in the intervertebral disc, is extraordinary high with annual costs to the US health care industry exceeding 33 billion dollars. None of the current therapies can completely restore the function of the degenerative intervertebral disc and thereby prevent further deterioration of the compromised spine. The proposed studies will provide insights into mechanisms of early degenerative changes in the disc and may permit development of interventional strategies to prevent degenerative disease.
|Schoepflin, Zachary R; Shapiro, Irving M; Risbud, Makarand V (2016) Class I and IIa HDACs Mediate HIF-1Î± Stability Through PHD2-Dependent Mechanism, While HDAC6, a Class IIb Member, Promotes HIF-1Î± Transcriptional Activity in Nucleus Pulposus Cells of the Intervertebral Disc. J Bone Miner Res 31:1287-99|
|Suyama, Kaori; Silagi, Elizabeth S; Choi, Hyowon et al. (2016) Circadian factors BMAL1 and RORÎ± control HIF-1Î± transcriptional activity in nucleus pulposus cells: implications in maintenance of intervertebral disc health. Oncotarget 7:23056-71|
|Tian, Ye; Yuan, Wen; Li, Jun et al. (2016) TGFÎ² regulates Galectin-3 expression through canonical Smad3 signaling pathway in nucleus pulposus cells: implications in intervertebral disc degeneration. Matrix Biol 50:39-52|
|Binch, Abbie L A; Shapiro, Irving M; Risbud, Makarand V (2016) Syndecan-4 in intervertebral disc and cartilage: Saint or synner? Matrix Biol 52-54:355-62|
|Choi, Hyowon; Johnson, Zariel I; Risbud, Makarand V (2015) Understanding nucleus pulposus cell phenotype: a prerequisite for stem cell based therapies to treat intervertebral disc degeneration. Curr Stem Cell Res Ther 10:307-16|
|Ye, Wei; Zhou, Jie; Markova, Dessislava Z et al. (2015) Xylosyltransferase-1 expression is refractory to inhibition by the inflammatory cytokines tumor necrosis factor Î± and IL-1Î² in nucleus pulposus cells: novel regulation by AP-1, Sp1, and Sp3. Am J Pathol 185:485-95|
|Shapiro, Irving M; Landis, William J; Risbud, Makarand V (2015) Matrix vesicles: Are they anchored exosomes? Bone 79:29-36|
|Johnson, Z I; Schoepflin, Z R; Choi, H et al. (2015) Disc in flames: Roles of TNF-Î± and IL-1Î² in intervertebral disc degeneration. Eur Cell Mater 30:104-16; discussion 116-7|
|Johnson, Zariel I; Gogate, Shilpa S; Day, Rebecca et al. (2015) Aquaporin 1 and 5 expression decreases during human intervertebral disc degeneration: Novel HIF-1-mediated regulation of aquaporins in NP cells. Oncotarget 6:11945-58|
|Risbud, Makarand V; Schoepflin, Zachary R; Mwale, Fackson et al. (2015) Defining the phenotype of young healthy nucleus pulposus cells: recommendations of the Spine Research Interest Group at the 2014 annual ORS meeting. J Orthop Res 33:283-93|
Showing the most recent 10 out of 48 publications