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Bicarbonate Recycling by HIF-1-Dependent Carbonic Anhydrase Isoforms 9 and 12 Is Critical in Maintaining Intracellular pH and Viability of Nucleus Pulposus Cells.

Authors
  • Silagi, Elizabeth S1, 2
  • Schoepflin, Zachary R1, 2
  • Seifert, Erin L1, 3
  • Merceron, Christophe4
  • Schipani, Ernestina4, 5
  • Shapiro, Irving M1, 2
  • Risbud, Makarand V1, 2
  • 1 Program in Cell Biology and Regenerative Medicine, Jefferson College of Biomedical Sciences, Thomas Jefferson University, Philadelphia, PA, USA.
  • 2 Department of Orthopaedic Surgery, Sidney Kimmel Medical College, Thomas Jefferson University, Philadelphia, PA, USA.
  • 3 MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA.
  • 4 Department of Orthopaedic Surgery, University of Michigan Medical School, Ann Arbor, MI, USA.
  • 5 Department of Medicine, Division of Endocrinology, University of Michigan Medical School, Ann Arbor, MI, USA.
Type
Published Article
Journal
Journal of Bone and Mineral Research
Publisher
Wiley (John Wiley & Sons)
Publication Date
Feb 01, 2018
Volume
33
Issue
2
Pages
338–355
Identifiers
DOI: 10.1002/jbmr.3293
PMID: 28940640
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Intervertebral disc degeneration is a ubiquitous condition closely linked to chronic low-back pain. The health of the avascular nucleus pulposus (NP) plays a crucial role in the development of this pathology. We tested the hypothesis that a network comprising HIF-1α, carbonic anhydrase (CA) 9 and 12 isoforms, and sodium-coupled bicarbonate cotransporters (NBCs) buffer intracellular pH through coordinated bicarbonate recycling. Contrary to the current understanding of NP cell metabolism, analysis of metabolic-flux data from Seahorse XF analyzer showed that CO2 hydration contributes a significant source of extracellular proton production in NP cells, with a smaller input from glycolysis. Because enzymatic hydration of CO2 is catalyzed by plasma membrane-associated CAs we measured their expression and function in NP tissue. NP cells robustly expressed isoforms CA9/12, which were hypoxia-inducible. In addition to increased mRNA stability under hypoxia, we observed binding of HIF-1α to select hypoxia-responsive elements on CA9/12 promoters using genomic chromatin immunoprecipitation. Importantly, in vitro loss of function studies and analysis of discs from NP-specific HIF-1α null mice confirmed the dependency of CA9/12 expression on HIF-1α. As expected, inhibition of CA activity decreased extracellular acidification rate independent of changes in HIF activity or lactate/H+ efflux. Surprisingly, CA inhibition resulted in a concomitant decrease in intracellular pH that was mirrored by inhibition of sodium-bicarbonate importers. These results suggested that extracellular bicarbonate generated by CA9/12 is recycled to buffer cytosolic pH fluctuations. Importantly, long-term intracellular acidification from CA inhibition lead to compromised cell viability, suggesting that plasma-membrane proton extrusion pathways alone are not sufficient to maintain homeostatic pH in NP cells. Taken together, our studies show for the first time that bicarbonate buffering through the HIF-1α-CA axis is critical for NP cell survival in the hypoxic niche of the intervertebral disc. © 2017 American Society for Bone and Mineral Research. © 2017 American Society for Bone and Mineral Research.

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