COX-2 expression mediated by calcium-TonEBP signaling axis under hyperosmotic conditions serves osmoprotective function in nucleus pulposus cells.

The nucleus pulposus (NP) of intervertebral discs experiences dynamic changes in tissue osmolarity because of diurnal loading of the spine. TonEBP/NFAT5 is a transcription factor that is critical in osmoregulation as well as survival of NP cells in the hyperosmotic milieu. The goal of this study was to investigate whether cyclooxygenase-2 (COX-2) expression is osmoresponsive and dependent on TonEBP, and whether it serves an osmoprotective role. NP cells up-regulated COX-2 expression in hyperosmotic media. The induction of COX-2 depended on elevation of intracellular calcium levels and p38 MAPK pathway, but independent of calcineurin signaling as well as MEK/ERK and JNK pathways. Under hyperosmotic conditions, both COX-2 mRNA stability and its proximal promoter activity were increased. The proximal COX-2 promoter (-1840/+123 bp) contained predicted binding sites for TonEBP, AP-1, NF-κB, and C/EBP-ß. While COX-2 promoter activity was positively regulated by both AP-1 and NF-κB, AP-1 had no effect and NF-κB negatively regulated COX-2 protein levels under hyperosmotic conditions. On the other hand, TonEBP was necessary for both COX-2 promoter activity and protein up-regulation in response to hyperosmotic stimuli. Ex vivo disc organ culture studies using hypomorphic TonEBP+/- mice confirmed that TonEBP is required for hyperosmotic induction of COX-2. Importantly, the inhibition of COX-2 activity under hyperosmotic conditions resulted in decreased cell viability, suggesting that COX-2 plays a cytoprotective and homeostatic role in NP cells for their adaptation to dynamically loaded hyperosmotic niches.

PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis.

The role of prolyl hydroxylase (PHD)-3 as a hypoxia inducible factor (HIF)-1α cofactor is controversial and remains unknown in skeletal tissues. We investigated whether PHD3 controls HIF-1 transcriptional activity in nucleus pulposus (NP) cells through the pyruvate kinase muscle (PKM)-2-Jumonji domain--containing protein (JMJD5) axis. PHD3-/- mice (12.5 mo old) showed increased incidence of intervertebral disc degeneration with a concomitant decrease in expression of the HIF-1α targets VEGF-A, glucose transporter-1, and lactate dehydrogenase A. PHD3 silencing decreased hypoxic activation of HIF-1α C-terminal transactivation domain (C-TAD), but not HIF-1α-N-terminal-(N)-TAD or HIF-2α-TAD. Moreover, PHD3 suppression in NP cells resulted in decreased HIF-1α enrichment on target promoters and lower expression of select HIF-1 targets. Contrary to other cell types, manipulation of PKM2 and JMJD5 levels had no effect on HIF-1 activity in NP cells. Likewise, stabilization of tetrameric PKM2 by a chemical approach had no effect on PHD3-dependent HIF-1 activity. Coimmunoprecipitation assays showed lack of association between HIF-1α and PKM2 in NP cells. Results support the role of the PHD3 as a cofactor for HIF-1, independent of PKM2-JMJD5.-Schoepflin, Z. R., Silagi, E. S., Shapiro, I. M., Risbud, M. V. PHD3 is a transcriptional coactivator of HIF-1α in nucleus pulposus cells independent of the PKM2-JMJD5 axis.

CCN2 suppresses catabolic effects of interleukin-1ß through α5ß1 and αVß3 integrins in nucleus pulposus cells: implications in intervertebral disc degeneration.

The objective of the study was to examine the regulation of CCN2 by inflammatory cytokines, IL-1ß, and TNF-α and to determine whether CCN2 modulates IL-1ß-dependent catabolic gene expression in nucleus pulposus (NP) cells. IL-1ß and TNF-α suppress CCN2 mRNA and protein expression in an NF-κB-dependent but MAPK-independent manner. The conserved κB sites located at -93/-86 and -546/-537 bp in the CCN2 promoter mediated this suppression. On the other hand, treatment of NP cells with IL-1ß in combination with CCN2 suppressed the inductive effect of IL-1ß on catabolic genes, including MMP-3, ADAMTS-5, syndecan 4, and prolyl hydroxylase 3. Likewise, silencing of CCN2 in human NP cells resulted in elevated basal expression of several catabolic genes and inflammatory cytokines like IL-6, IL-4, and IL-12 as measured by gene expression and cytokine protein array, respectively. Interestingly, the suppressive effect of CCN2 on IL-1ß was independent of modulation of NF-κB signaling. Using disintegrins, echistatin, and VLO4, peptide inhibitors to αvß3 and α5ß1 integrins, we showed that CCN2 binding to both integrins was required for the inhibition of IL-1ß-induced catabolic gene expression. It is noteworthy that analysis of human tissues showed a trend of altered expression of these integrins during degeneration. Taken together, these results suggest that CCN2 and inflammatory cytokines form a functional negative feedback loop in NP cells that may be important in the pathogenesis of disc disease.

FIH-1-Mint3 axis does not control HIF-1 transcriptional activity in nucleus pulposus cells.

The objective of this study was to determine the role of FIH-1 in regulating HIF-1 activity in the nucleus pulposus (NP) cells and the control of this regulation by binding and sequestration of FIH-1 by Mint3. FIH-1 and Mint3 were both expressed in the NP and were shown to strongly co-localize within the cell nucleus. Although both mRNA and protein expression of FIH-1 decreased in hypoxia, only Mint3 protein levels were hypoxiasensitive. Overexpression of FIH-1 was able to reduce HIF-1 function, as seen by changes in activities of hypoxia response element-luciferase reporter and HIF-1-C-TAD and HIF-2-TAD. Moreover, co-transfection of either full-length Mint3 or the N terminus of Mint3 abrogated FIH-1-dependent reduction in HIF-1 activity under both normoxia and hypoxia. Nuclear levels of FIH-1 and Mint3 decreased in hypoxia, and the use of specific nuclear import and export inhibitors clearly showed that cellular compartmentalization of overexpressed FIH-1 was critical for its regulation of HIF-1 activity in NP cells. Interestingly, microarray results after stable silencing of FIH-1 showed no significant changes in transcripts of classical HIF-1 target genes. However, expression of several other transcripts, including those of the Notch pathway, changed in FIH-1-silenced cells. Moreover, co-transfection of Notch-ICD could restore suppression of HIF-1-TAD activity by exogenous FIH-1. Taken together, these results suggest that, possibly due to low endogenous levels and/or preferential association with substrates such as Notch, FIH-1 activity does not represent a major mechanism by which NP cells control HIF-1-dependent transcription, a testament to their adaptation to a unique hypoxic niche.

ALK Deletion Exons 2 to 19: Case Report of a Rare ALK Inhibitor-Responsive Lung Cancer Driver Oncogene.

ALK internal deletions of nonkinase domain exons occur in 0.01% of lung cancers with ALK genomic aberrations. We report a lung adenocarcinoma with a previously undescribed somatic ALK deletion of exons 2 to 19 with dramatic and sustained (>23 mo) response to alectinib. Our and other reported cases with ALK nonkinase domain deletions (between introns and exons 1-19) can display positive results in nonsequencing-based lung cancer diagnostic tests (such as immunohistochemistry) used to screen for more common ALK rearrangements. This case report emphasizes that "ALK-driven" lung cancers should be expanded to encompass those harboring not only ALK rearrangements with other genes but also ALK nonkinase domain deletions.

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

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.

Class I and IIa HDACs Mediate HIF-1α Stability Through PHD2-Dependent Mechanism, While HDAC6, a Class IIb Member, Promotes HIF-1α Transcriptional Activity in Nucleus Pulposus Cells of the Intervertebral Disc.

The objective of this study was to determine the role of histone deacetylases (HDACs) in regulating HIF-1α protein stability and activity in nucleus pulposus (NP) cells. Treatment of NP cells with pan-HDAC inhibitor TSA resulted in decreased HIF-1α levels under both normoxia and hypoxia in a dose-dependent fashion. TSA-mediated HIF-1α degradation was rescued by concomitant inhibition of not only the 26S proteasome but also PHD2 function. Moreover, TSA treatment of PHD2(-/-) cells had little effect on HIF-1α levels, supporting the notion that inhibition of PHD2 function by HDACs contributed to HIF-1α stabilization. Surprisingly, class-specific HDAC inhibitors did not affect HIF-1α protein stability, indicating that multiple HDACs controlled HIF-1α stability by regulating HIF-1α-PHD2 interaction in NP cells. Interestingly, lower-dose TSA that did not affect HIF-1α stability decreased its activity and target gene expression. Likewise, rescue of TSA-mediated HIF-1α protein degradation by blocking proteasomal or PHD activity did not restore HIF-1 activity, suggesting that HDACs independently regulate HIF-1α stability and activity. Noteworthy, selective inhibition of HDAC6 and not of class I and IIa HDACs decreased HIF-1-mediated transcription under hypoxia to a similar extent as lower-dose TSA, contrasting the reported role of HDAC6 as a transcriptional repressor in other cell types. Moreover, HDAC6 inhibition completely blocked TSA effects on HIF-1 activity. HDAC6 associated with and deacetylated HSP90, an important cofactor for HIF-1 function in NP cells, and HDAC6 inhibition decreased p300 transactivation in NP cells. Taken together, these results suggest that although multiple class I and class IIa HDACs control HIF-1 stability, HDAC6, a class IIb HDAC, is a novel mediator of HIF-1 activity in NP cells possibly through promoting action of critical HIF-1 cofactors. © 2016 American Society for Bone and Mineral Research.

Defining the phenotype of young healthy nucleus pulposus cells: recommendations of the Spine Research Interest Group at the 2014 annual ORS meeting.

Low back pain is a major physical and socioeconomic problem. Degeneration of the intervertebral disc and especially that of nucleus pulposus (NP) has been linked to low back pain. In spite of much research focusing on the NP, consensus among the research community is lacking in defining the NP cell phenotype. A consensus agreement will allow easier distinguishing of NP cells from annulus fibrosus (AF) cells and endplate chondrocytes, a better gauge of therapeutic success, and a better guidance of tissue-engineering-based regenerative strategies that attempt to replace lost NP tissue. Most importantly, a clear definition will further the understanding of physiology and function of NP cells, ultimately driving development of novel cell-based therapeutic modalities. The Spine Research Interest Group at the 2014 Annual ORS Meeting in New Orleans convened with the task of compiling a working definition of the NP cell phenotype with hope that a consensus statement will propel disc research forward into the future. Based on evaluation of recent studies describing characteristic NP markers and their physiologic relevance, we make the recommendation of the following healthy NP phenotypic markers: stabilized expression of HIF-1α, GLUT-1, aggrecan/collagen II ratio >20, Shh, Brachyury, KRT18/19, CA12, and CD24.

Design of a novel mobility device controlled by the feet motion of a standing child.

Self-generated mobility is a major contributor to the physical, emotional, cognitive, and social development of infants and toddlers. When young children have disorders that hinder self locomotion, their development is at risk for delay. Independent mobility via traditional power mobility devices may prevent this delay, but do little to encourage the child's development of gross motor skills. This research aims to develop a bio-driven mobile-assistive device that is controlled and driven by moving the feet, which may encourage the development of gross motor skills. In this study, system feasibility is shown by experiments on five typically developing toddlers and one special needs toddler with spastic cerebral palsy. Children were placed in the bio-driven device and instructed to navigate through a maze. All subjects were able to successfully complete the maze in multiple trials. Additionally, two toddlers showed evidence of improved driving skill by completing the maze in shorter times in successive trials on a given testing day. The results suggest that such a device is feasible for purposeful driving. Recommendations are given for the device and protocol redesign for related future testing.

Design of a novel mobility device controlled by the feet motion of a standing child: a feasibility study.

Self-generated mobility is a major contributor to the physical, emotional, cognitive, and social development of infants and toddlers. When young children have disorders that hinder self locomotion, their development is at risk for delay. Independent mobility via traditional power mobility devices may prevent this delay, but do little to encourage the child's development of gross motor skills. This research aims to develop a bio-driven mobile-assistive device that is controlled and driven by moving the feet, which may encourage the development of gross motor skills. In this study, system feasibility is shown by experiments on five typically developing toddlers and one special needs toddler with spastic cerebral palsy. Children were placed in the bio-driven device and instructed to navigate through a maze. All subjects were able to successfully complete the maze in multiple trials. In addition, two toddlers showed evidence of improved driving skill by completing the maze in shorter times in successive trials on a given testing day. The results suggest that such a device is feasible for purposeful driving. Recommendations are given for the device and protocol redesign for related future testing.