Transcriptional profiling of the nucleus pulposus: say yes to notochord

This editorial addresses the debate concerning the origin of adult nucleus pulposus cells in the light of profiling studies by Minogue and colleagues. In their report of several marker genes that distinguish nucleus pulposus cells from other related cell types, the authors provide novel insights into the notochordal nature of the former. Together with recently published work, their work lends support to the view that all cells present within the nucleus pulposus are derived from the notochord. Hence, the choice of an animal model for disc research should be based on considerations other than the cell loss and replacement by non-notochordal cells

Lack of evidence for involvement of TonEBP and hyperosmotic stimulus in induction of autophagy in the nucleus pulposus.

Nucleus pulposus (NP) cells reside in a physiologically hyperosmotic environment within the intervertebral disc. TonEBP/NFAT5 is an osmo-sensitive transcription factor that controls expression of genes critical for cell survival under hyperosmotic conditions. A recent report on NP and studies of other cell types have shown that hyperosmolarity triggers autophagy. However, little is known whether such autophagy induction occurs through TonEBP. The goal of this study was to investigate the role of TonEBP in hyperosmolarity-dependent autophagy in NP. Loss-of-function studies showed that autophagy in NP cells was not TonEBP-dependent; hyperosmolarity did not upregulate autophagy as previously reported. NP tissue of haploinsufficient TonEBP mice showed normal pattern of LC3 staining. NP cells did not increase LC3-II or LC3-positive puncta under hyperosmotic conditions. Bafilomycin-A1 treatment and tandem mCherry-EGFP-LC3B reporter transfection demonstrated that the autophagic flux was unaffected by hyperosmolarity. Even under serum-free conditions, NP cells did not induce autophagy with increasing osmolarity. Hyperosmolarity did not change the phosphorylation of ULK1 by mTOR and AMPK. An ex vivo disc organ culture study supported that extracellular hyperosmolarity plays no role in promoting autophagy in the NP. We conclude that hyperosmolarity does not play a role in autophagy induction in NP cells

Absence of room temperature ferromagnetism in bulk Mn-doped ZnO

Structural and magnetic properties have been studied for polycrystalline Zn_1-xMn_xO (x=0.02, 0.03, 0.05). Low-temperature (~500 oC) synthesis leaves unreacted starting ZnO and manganese oxides. Contrary to a recent report, no bulk ferromagnetism was observed for single-phase materials synthesized in air at temperatures above 900 oC. Single-phase samples show paramagnetic Curie-Weiss behavior.Comment: Accepted for publication in J. Appl. Phys., RevTeX, 3 pages, 4 figure

Understanding nucleus pulposus cell phenotype: a prerequisite for stem cell based therapies to treat intervertebral disc degeneration.

Intervertebral disc (IVD) degeneration and associated low back pain (LBP) remains a major burden to our society without significant improvements in treatment strategies or patient\u27s quality of life. While the recent cell-transplantation studies for treatment of degenerative disc disease have shown promising results, to better gauge the success and functional outcomes of these therapies, it is crucial to understand if transplanted cells give rise to healthy nucleus pulposus (NP) tissue. NP cell phenotype is unique and is defined by expression of a characteristic set of markers that reflect specialized physiology and function. This review summarizes phenotypic markers that mirror the unique physiology and function of NP cells and their progenitors and should be considered to when measuring outcomes of cell-based therapies to treat disc degeneration

The cGAS-STING Pathway Affects Vertebral Bone but Does Not Promote Intervertebral Disc Cell Senescence or Degeneration

The DNA-sensing cGAS-STING pathway promotes the senescence-associated secretory phenotype (SASP) and mediates type-I interferon inflammatory responses to foreign viral and bacterial DNA as well as self-DNA. Studies of the intervertebral disc in humans and mice demonstrate associations between aging, increased cell senescence, and disc degeneration. Herein we assessed the role of STING in SASP promotion in STING gain- (N153S) and loss-of-function mouse models. N153S mice evidenced elevated circulating levels of proinflammatory markers including IL-1β, IL-6, and TNF-α, showed elevated monocyte and macrophage abundance in the vertebral marrow, and exhibited a mild trabecular and cortical bone phenotype in caudal vertebrae. Interestingly, despite systemic inflammation, the structural integrity of the disc and knee articular joint remained intact, and cells did not show a loss of their phenotype or elevated SASP. Transcriptomic analysis of N153S tissues demonstrated an upregulated immune response by disc cells, which did not closely resemble inflammatory changes in human tissues. Interestingly, STING-/- mice also showed a mild vertebral bone phenotype, but the absence of STING did not reduce the abundance of SASP markers or improve the age-associated disc phenotype. Overall, the analyses of N153S and STING-/- mice suggest that the cGAS-STING pathway is not a major contributor to SASP induction and consequent disc aging and degeneration but may play a minor role in the maintenance of trabecular bone in the vertebrae. This work contributes to a growing body of work demonstrating that systemic inflammation is not a key driver of disc degeneration

Hypoxia promotes noncanonical autophagy in nucleus pulposus cells independent of MTOR and HIF1A signaling

Nucleus pulposus (NP) cells reside in the avascular and hypoxic microenvironment of intervertebral discs. Importantly, many activities related to survival and function of NP cells are controlled by the HIF-family of transcription factors. We hypothesize that NP cells adapt to their hypoxic niche through modulation of macroautophagy/autophagy. In various cell types, hypoxia induces autophagy in a HIF1A-dependent fashion; however, little is known about hypoxic regulation of autophagy in NP cells. Hypoxia increases the number of autophagosomes as seen by TEM analysis and LC3-positive puncta in NP cells. Hypoxic induction of autophagy was also demonstrated by a significantly higher number of autophagosomes and smaller change in autolysosomes in NP cells expressing tandem-mCherry-EGFP-LC3B. Increased LC3-II levels were not accompanied by a concomitant increase in BECN1 or the ATG12-ATG5 complex. In addition, ULK1 phosphorylation at Ser757 and Ser777 responsive to MTOR and AMPK, respectively, was not affected in hypoxia. Interestingly, when MTOR activity was inhibited by rapamycin or Torin1, LC3-II levels did not change, suggesting a novel MTOR-independent regulation. Noteworthy, while silencing of HIF1A affected hypoxic induction of BNIP3, it did not affect LC3-II levels, indicating hypoxia-induced autophagy is HIF1-independent. Importantly, there was no change in the number of LC3-positive autophagosomes in NP-specific Hif1a null mice. Finally, inhibition of autophagic flux did not affect the glycolytic metabolism of NP cells, suggesting a possible nonmetabolic role of autophagy. Taken together, our study for the first time shows that NP cells regulate autophagy in a noncanonical fashion independent of MTOR and HIF1A signaling

Tumor Necrosis Factor-α– and Interleukin-1β–Dependent Matrix Metalloproteinase-3 Expression in Nucleus Pulposus Cells Requires Cooperative Signaling via Syndecan 4 and Mitogen-Activated Protein Kinase–NF-κB Axis Implications in Inflammatory Disc Disease

Matrix metalloproteinase-3 (MMP-3) plays an important role in intervertebral disc degeneration, a ubiquitous condition closely linked to low back pain and disability. Elevated expression of syndecan 4, a cell surface heparan sulfate proteoglycan, actively controls disc matrix catabolism. However, the relationship between MMP-3 expression and syndecan 4 in the context of inflammatory disc disease has not been clearly defined. We investigated the mechanisms by which cytokines control MMP-3 expression in rat and human nucleus pulposus cells. Cytokine treatment increased MMP-3 expression and promoter activity. Stable silencing of syndecan 4 blocked cytokine-mediated MMP-3 expression; more important, syndecan 4 did not mediate its effects through NF-κB or mitogen-activated protein kinase (MAPK) pathways. However, treatment with MAPK and NF-κB inhibitors resulted in partial blocking of the inductive effect of cytokines on MMP-3 expression. Loss-of-function studies confirmed that NF-κB, p38α/β2/γ/δ, and extracellular signal–regulated kinase (ERK) 2, but not ERK1, contributed to cytokine-dependent induction of MMP3 promoter activity. Similarly, inhibitor treatments, lentiviral short hairpin-p65, and short hairpin-IκB kinase β significantly decreased cytokine-dependent up-regulation in MMP-3 expression. Finally, we show that transforming growth factor-β can block the up-regulation of MMP-3 induced by tumor necrosis factor (TNF)-α by counteracting the NF-κB pathway and syndecan 4 expression. Taken together, our results suggest that cooperative signaling through syndecan 4 and the TNF receptor 1–MAPK–NF-κB axis is required for TNF-α–dependent expression of MMP-3 in nucleus pulposus cells. Controlling these pathways may slow the progression of intervertebral disc degeneration and matrix catabolism