8 research outputs found
GLUT1 Is Redundant in Hypoxic and Glycolytic Nucleus Pulposus Cells of the Intervertebral Disc
Glycolysis is central to homeostasis of nucleus pulposus (NP) cells in the avascular intervertebral disc. Since the glucose transporter, GLUT1, is a highly enriched phenotypic marker of NP cells, we hypothesized that it is vital for the development and postnatal maintenance of the disc. Surprisingly, primary NP cells treated with 2 well-characterized GLUT1 inhibitors maintained normal rates of glycolysis and ATP production, indicating intrinsic compensatory mechanisms. We showed in vitro that NP cells mitigated GLUT1 loss by rewiring glucose import through GLUT3. Of note, we demonstrated that substrates, such as glutamine and palmitate, did not compensate for glucose restriction resulting from dual inhibition of GLUT1/3, and inhibition compromised long-term cell viability. To investigate the redundancy of GLUT1 function in NP, we generated 2 NP-specific knockout mice: Krt19CreERT Glut1fl/fl and Foxa2Cre Glut1fl/fl. There were no apparent defects in postnatal disc health or development and maturation in mutant mice. Microarray analysis verified that GLUT1 loss did not cause transcriptomic alterations in the NP, supporting that cells are refractory to GLUT1 loss. These observations provide the first evidence to our knowledge of functional redundancy in GLUT transporters in the physiologically hypoxic intervertebral disc and underscore the importance of glucose as the indispensable substrate for NP cells
The Role of HIF-2α in the Intervertebral Disc
Low back pain, and associated intervertebral disc degeneration, is the leading cause of disability and has a substantial economic burden. Identifying links between disc degeneration and low back pain is essential. However, understanding basic intervertebral disc biology is critical to this endeavor. The intervertebral disc is a unique, avascular, and thus physiologically hypoxic organ comprising three major compartments. The innermost nucleus pulposus (NP) is encompassed by the annulus fibrosus and capped superiorly and inferiorly by cartilaginous end plates. Cells within this hypoxic niche have adapted to survive through the unique regulation of genes controlled by hypoxia inducible factors (HIFs). Our prior work has found that hypoxia inducible factor 1α (HIF-1α) expression is required for the development and maintenance of the NP and overall disc structure. Although the function of HIF-1α in the disc has been extensively explored, neither the role of HIF-2α (a homologue of HIF-1α) nor its transcriptome is thoroughly investigated in the disc context. Additionally, glucose transporter 1 (GLUT1/Slc2a1), a target of HIF-1α and a phenotypic marker of NP cells, is the main glucose transporter critical to the function of cartilage and bone. Even though GLUT1 is highly enriched in the NP, we do not know the importance of GLUT1 in the adult intervertebral disc. Accordingly, this work had two main aims: (1) to examine and elucidate the role of HIF-2α in NP cells, and (2) to evaluate the importance and the role of GLUT1 in the adult disc. Using an NP-specific HIF-2α conditional knockout mouse model, we show that HIF-2α is not necessary for NP compartment development, and rather than compromising NP compartment, like loss of HIF-1α, loss of HIF-2α provides transient protection against age-related disc generation. These are novel observations that support the notion that HIF-2α may be independent of the functions of HIF-1α. Furthermore, using an NP-specific overexpression HIF-2α mouse model, we show a very mild but significant increase in the NP grades of degeneration in 24-month-old HIF-2α overexpressing mice. Microarray analysis from the NP of HIF-2α overexpressing mice suggests HIF-2α to be important for regulating genes and associated with CompBio themes linked to cilia. Both conditional knockout and overexpression NP-specific HIF-2α mouse models explored here provide strong evidence for the differences in roles of HIF-2α vs. HIF-1α in the disc. Furthermore, in contrast to many cell types, we show using an adult NP-specific conditional knockout mouse model of GLUT1 that loss of GLUT1 does not impact overall disc morphology nor alter NP transcriptomic profile. We observed that the maintenance of mRNA and protein levels of alternative glucose transporters (i.e., GLUT3) in the NP of GLUT1 knockout mice shows that these alternative glucose transporters can provide NP with sufficient glucose concentrations. Overall, the studies presented here add insights into the biological role of HIF-2α, confirms the redundancy of glucose transporters, and reaffirm the importance of glucose utilization in NP cells. These results contribute to a new understanding of the specific role of HIF-2α and GLUT1 in NP cells, which help advance our understanding of the unique biological functions of the intervertebral disc
DataSheet1_Increased HIF-2α activity in the nucleus pulposus causes intervertebral disc degeneration in the aging mouse spine.pdf
Hypoxia-inducible factors (HIFs) are essential to the homeostasis of hypoxic tissues. Although HIF-2α, is expressed in nucleus pulposus (NP) cells, consequences of elevated HIF-2 activity on disc health remains unknown. We expressed HIF-2α with proline to alanine substitutions (P405A; P531A) in the Oxygen-dependent degradation domain (HIF-2αdPA) in the NP tissue using an inducible, nucleus pulposus-specific K19CreERT allele to study HIF-2α function in the adult intervertebral disc. Expression of HIF-2α in NP impacted disc morphology, as evident from small but significantly higher scores of degeneration in NP of 24-month-old K19CreERT; HIF-2αdPA (K19-dPA) mice. Noteworthy, comparisons of grades within each genotype between 14 months and 24 months indicated that HIF-2α overexpression contributed to more pronounced changes than aging alone. The annulus fibrosus (AF) compartment in the 14-month-old K19-dPA mice exhibited lower collagen turnover and Fourier transform-infrared (FTIR) spectroscopic imaging analyses showed changes in the biochemical composition of the 14- and 24-month-old K19-dPA mice. Moreover, there were changes in aggrecan, chondroitin sulfate, and COMP abundance without alterations in NP phenotypic marker CA3, suggesting the overexpression of HIF-2α had some impact on matrix composition but not the cell phenotype. Mechanistically, the global transcriptomic analysis showed enrichment of differentially expressed genes in themes closely related to NP cell function such as cilia, SLIT/ROBO pathway, and HIF/Hypoxia signaling at both 14- and 24-month. Together, these findings underscore the role of HIF-2α in the pathogenesis of disc degeneration in the aged spine.</p
Long-term treatment with senolytic drugs Dasatinib and Quercetin ameliorates age-dependent intervertebral disc degeneration in mice
Intervertebral disc degeneration is a leading cause of chronic back pain and disability. Here the authors show that long term treatment with senolytic compounds Dasatinib and Quercetin reduces disc senescence burden and ameliorates age-dependent degeneration in mice