Nucleus Pulposus Cell Metabolism Research Articles

Activation of Nuclear Factor Erythroid 2-Related Factor 2 Antagonizes the Reactive Oxygen Species Modulator 1-Induced Oxidative Stress in Nucleus Pulposus Cells

This study investigates the role of Reactive Oxygen Species Modulator 1 (ROMO1) in oxidative stress within nucleus pulposus (NP) cells and its potential regulation by Nuclear factor erythroid 2-related factor 2 (Nrf2). Intervertebral disc samples from patients were collected, and ROMO1, Nrf2, collagen I/II levels were analyzed to establish their potential connection. Human NP cells were cultured and exposed to H2O2 to induce oxidative stress. To elucidate ROMO1’s impact on NP cell metabolism, NP cells were transfected with ROMO1. Concurrently, Nrf2 activators and inhibitors were used to modulate Nrf2 expression during culturing. Oxidative stress was assessed through CAT and SOD1 gene expression analysis and measurement of cellular reactive oxygen species (ROS) production. NP cell status was determined by evaluating cell viability and collagen I/II expression. Results indicated elevated ROMO1 expression in severe intervertebral disc degeneration (IDD) and after H2O2 treatment. ROMO1 overexpression increased ROS production, suppressed CAT, SOD, and collagen II expression, while elevating collagen I and negatively affecting cell viability. However, Nrf2 activation effectively suppressed ROMO1 expression and protected NP cells from oxidative stress induced by H2O2 or ROMO1. In conclusion, ROMO1 exacerbates oxidative stress and contributes to NP cell degeneration, a process mitigated by Nrf2 activation.

Medical Prospect of Melatonin in the Intervertebral Disc Degeneration through Inhibiting M1-Type Macrophage Polarization via SIRT1/Notch Signaling Pathway.

The study aims to explore the medical prospect of melatonin (MLT) and the underlying therapeutic mechanism of MLT-mediated macrophage (Mφ) polarization on the function of nucleus pulposus (NP) in intervertebral disc degeneration (IDD). RAW 264.7 Mφs were induced by lipopolysaccharide (LPS) to simulate Mφ polarization and the inflammatory reaction of Mφs with or without MLT were detected. Conditioned medium (CM) collected from these activated Mφs with or without MLT treatment were further used to incubate NP cells. The oxidative stress, inflammation and extracellular matrix (ECM) metabolism in NP cells were determined. Then, the changes in SIRT1/Notch signaling were detected. The agonist (SRT1720) and inhibitor (EX527) of SIRT1 were used to further explore the association among MLT. The interaction between SIRT1 and NICD was detected by immunoprecipitation (IP). Finally, puncture-induced rat IDD models were established and IDD degrees were clarified by X-ray, MRI, H&E staining and immunofluorescence (IF). The results of flow cytometry and inflammation detection indicated that LPS could induce M1-type Mφ polarization with pro-inflammatory properties. MLT significantly inhibited the aforementioned process and inhibited M1-type Mφ polarization, accompanied by the alleviation of inflammation. Compared with those without MLT, the levels of oxidative stress, pro-inflammatory cytokines and ECM catabolism in NP cells exposed to CM with MLT were markedly downregulated in a dose-dependent manner. The inhibition of SIRT1 and the enhancement of Notch were observed in activated Mφs and they can be reversed after MLT treatment. This prediction was further confirmed by using the SRT1720 and EX527 to activate or inhibit the signaling. The interaction between SIRT1 and NICD was verified by IP. In vivo study, the results of MRI, Pfirrmann grade scores and H&E staining demonstrated the degree of disc degeneration was significantly lower in the MLT-treated groups when compared with the IDD control group. The IF data showed M1-type Mφ polarization decreased after MLT treatment. MLT could inhibit M1-type Mφ polarization and ameliorate the NP cell injury caused by inflammation in vitro and vivo, which is of great significance for the remission of IDD. The SIRT1/Notch signaling pathway is a promising target for MLT to mediate Mφ polarization.

Rosuvastatin suppresses TNF-α-induced matrix catabolism, pyroptosis and senescence via the HMGB1/NF-κB signaling pathway in nucleus pulposus cells.

Intervertebral disc degeneration is mainly caused by irregular matrix metabolism in nucleus pulposus cells and involves inflammatory factors such as TNF-α. Rosuvastatin, which is widely used in the clinic to reduce cholesterol levels, exerts anti-inflammatory effects, but whether rosuvastatin participates in IDD remains unclear. The current study aims to investigate the regulatory effect of rosuvastatin on IDD and the potential mechanism. In vitro experiments demonstrate that rosuvastatin promotes matrix anabolism and suppresses catabolism in response to TNF-α stimulation. In addition, rosuvastatin inhibits cell pyroptosis and senescence induced by TNF-α. These results demonstrate the therapeutic effect of rosuvastatin on IDD. We further find that HMGB1, a gene closely related to cholesterol metabolism and the inflammatory response, is upregulated in response to TNF-α stimulation. HMGB1 inhibition or knockdown successfully alleviates TNF-α-induced ECM degradation, senescence and pyroptosis. Subsequently, we find that HMGB1 is regulated by rosuvastatin and that its overexpression abrogates the protective effect of rosuvastatin. We then verify that the NF-κB pathway is the underlying pathway regulated by rosuvastatin and HMGB1. In vivo experiments also reveal that rosuvastatin inhibits IDD progression by alleviating pyroptosis and senescence and downregulating HMGB1 and p65. This study might provide new insight into therapeutic strategies for IDD.

BACH1 promotes intervertebral disc degeneration by regulating HMOX1/GPX4 to mediate oxidative stress, ferroptosis, and lipid metabolism in nucleus pulposus cells.

Intervertebral disc degeneration (IDD) is a primary health problem worldwide that involves oxidative stress, ferroptosis, and lipid metabolism. However, the underlying mechanism remains unclear. We investigated whether the transcription factor BTB and CNC homology 1 (BACH1) affected IDD progression by regulating HMOX1/GPX4-mediated ferroptosis and lipid metabolism in nucleus pulposus cells (NPCs). A rat IDD model was created to detect BACH1 expression in intervertebral disc tissues. Next, rat NPCs were isolated and treated with tert-butyl hydroperoxide (TBHP). BACH1, HMOX1, and GPX4 were knocked down, and oxidative stress and ferroptosis-related marker levels were examined. The binding of BACH1 to HMOX1 and of BACH1 to GPX4 was verified using chromatin immunoprecipitation (ChIP). Finally, untargeted lipid metabolism analysis was performed. An IDD model was successfully created, and BACH1 activity was found to be enhanced in the rat IDD tissues. BACH1 inhibited TBHP-induced oxidative stress and oxidative stress-induced ferroptosis in NPCs. Simultaneously, ChIP verified that BACH1 protein bound to HMOX1 and targeted the HMOX1 transcription inhibition to affect oxidative stress in NPCs. ChIP also verified that BACH1 bound to GPX4 and targeted the GPX4 inhibition to affect ferroptosis in NPCs. Finally, BACH1 inhibition in vivo improved IDD and affected lipid metabolism. The transcription factor BACH1 promoted IDD by regulating HMOX1/GPX4 to mediate oxidative stress, ferroptosis, and lipid metabolism in NPCs.

Nutrient metabolism of the nucleus pulposus: A literature review

Cells take in, consume, and synthesize nutrients for numerous physiological functions. This includes not only energy production but also macromolecule biosynthesis, which will further influence cellular signaling, redox homeostasis, and cell fate commitment. Therefore, alteration in cellular nutrient metabolism is associated with pathological conditions.Intervertebral discs, particularly the nucleus pulposus (NP), are avascular and exhibit unique metabolic preferences. Clinical and preclinical studies have indicated a correlation between intervertebral degeneration (IDD) and systemic metabolic diseases such as diabetes, obesity, and dyslipidemia. However, a lack of understanding of the nutrient metabolism of NP cells is masking the underlying mechanism. Indeed, although previous studies indicated that glucose metabolism is essential for NP cells, the downstream metabolic pathways remain unknown, and the potential role of other nutrients, like amino acids and lipids, is understudied.In this literature review, we summarize the current understanding of nutrient metabolism in NP cells and discuss other potential metabolic pathways by referring to a human NP transcriptomic dataset deposited to the Gene Expression Omnibus, which can provide us hints for future studies of nutrient metabolism in NP cells and novel therapies for IDD.

Specific PFKFB3 Inhibitor Memorably Ameliorates Intervertebral Disc Degeneration via Inhibiting NF-κB and MAPK Signaling Pathway and Reprogramming of Energy Metabolism of Nucleus Pulposus Cells.

Intervertebral disc (IVD) degeneration (IVDD) is a characteristic of the dominating pathological processes of nucleus pulposus (NP) cell senescence, abnormal synthesis and irregular distribution of extracellular matrix (ECM), and tumor necrosis factor-α (TNF-α) induced inflammation. Nowadays, IVD acid environment variation which accelerates the pathological processes mentioned above arouses researchers' attention. KAN0438757 (KAN) is an effective inhibitor of selective metabolic kinase phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) that has both energy metabolism reprogramming and anti-inflammatory effects. Therefore, a potential therapeutic benefit of KAN lies in its ability to inhibit the development of IVDD. This study examined in vitro KAN toxicity in NP primary cells (NPPs). Moreover, KAN influenced tumor necrosis factor-α (TNF-α) induced ECM anabolism and catabolism; the inflammatory signaling pathway activation and the energy metabolism phenotype were also examined in NPPs. Furthermore, KAN's therapeutic effect was investigated in vivo using the rat tail disc puncture model. Phenotypically speaking, the KAN treatment partially rescued the ECM degradation and glycolysis energy metabolism phenotypes of NPPs induced by TNF-α. In terms of mechanism, KAN inhibited the activation of MAPK and NF-κB inflammatory signaling pathways induced by TNF-α and reprogramed the energy metabolism. For the therapeutic aspect, the rat tail disc puncture model demonstrated that KAN has a significant ameliorated effect on the progression of IVDD. To sum up, our research successfully authenticated the potential therapeutic effect of KAN on IVDD and declaimed its mechanisms of both novel energy metabolism reprogramming and conventional anti-inflammation effect.

Exosomes-derived miR-125-5p from cartilage endplate stem cells regulates autophagy and ECM metabolism in nucleus pulposus by targeting SUV38H1

The study aimed to explore the effects of normal CESC-derived exosomes (N-CESC-exo) on autophagy, apoptosis and extracellular matrix (ECM) metabolism of nucleus pulposus cells (NPCs) and their underlying molecular mechanisms in vivo and in vitro.Tert-buty l hydroperoxide (TBHP) was used to induce CESCs and NPCs degeneration models in vitro. Flow cytometry and TUNEL staining were used to assess apoptosis. Proteins expression were detected by Western blotting. qRT-PCR was applied to detect miR-125-5p and SUV39H1 expression. The miRNA differences were analyzed by bioinformatics. Dual-luciferase reporter assay was applied to detect the target relationship. The degeneration of intervertebral disc tissue was observed by hematoxylin-eosin (H&E) staining. The disc damage was assessed with Safranin-O and Fast Green staining. LC3B expression was detected by immunofluorescence.We observed that NPCs could ingest N-CESC-exo. N-CESC-exo reduced degenerated nucleus pulposus cells (TBHP-NPC) apoptosis, bax, MMP13, and p62 expression, while increased bcl2, ACAN, LC3-II/I expression, and the fluorescence intensity of GFP-LC3. Bioinformatics analysis confirmed that miR-125-5p was low expression, while SUV39H1 was overexpressed in IDD. Further, the dual-luciferase reporter assays confirmed the targeting relationship between miR-125-5p and SUV39H1.CESC-exomiR−125−5p inhibited TBHP-NPCs apoptosis by inhibiting SUV39H1, bax, MMP13, and p62 expression, while increased bcl2, ACAN, LC3-II/I expression, and the fluorescence intensity of GFP-LC3, thereby alleviating rat IDD.

Role of autophagy in intervertebral disc degeneration.

Intervertebral disc degeneration (IDD) is a leading contributor to low back pain. The intervertebral disc (IVD) is composed of three tissue types: the central gelatinous nucleus pulposus (NP) tissue, the surrounding annulus fibrosus (AF) tissue, and the inferior and superior cartilage endplates. The IVD microenvironment is hypoxic, acidic, hyperosmotic, and low in nutrients because it is mostly avascular. The cellular processes that underlie IDD initiation and progression are still poorly understood. Specifically, a lack of understanding regarding NP cell metabolism and physiology hinders the development of effective therapeutics to treat IDD patients. Autophagy is a vital intracellular degradation process that removes damaged organelles, misfolded proteins, and intracellular pathogens and recycles the degraded components for cellular energy and function. NP cells have adapted to survive within their harsh tissue microenvironment using processes that are largely unknown, and we postulate autophagy is one of these undiscovered mechanisms. In this review, we describe unique features of the IVD tissue, review how physiological stressors impact autophagy in NP cells in vitro, survey the current understanding of autophagy regulation in the IVD, and assess the relationship between autophagy and IDD. Published studies confirm autophagy markers are present in IVD tissue, and IVD cells can regulate autophagy in response to cellular stressors in vitro. However, data are still lacking to determine the exact mechanisms regulating autophagy in IVD cells. More in-depth research is needed to establish whether autophagy is necessary to maintain IVD cell health and validate autophagy as a relevant therapeutic target for treating IDD.

Influence of Angiopoietin Treatment with Hypoxia and Normoxia on Human Intervertebral Disc Progenitor Cell’s Proliferation, Metabolic Activity, and Phenotype

Increasing evidence implicates intervertebral disc (IVD) degeneration as a major contributor to low back pain. In addition to a series of pathogenic processes, degenerated IVDs become vascularized in contrast to healthy IVDs. In this context, angiopoietin (Ang) plays a crucial role and is involved in cytokine recruitment, and anabolic and catabolic reactions within the extracellular matrix (ECM). Over the last decade, a progenitor cell population has been described in the nucleus pulposus (NP) of the IVD to be positive for the Tie2 marker (also known as Ang-1 receptor). In this study, we investigated the influence of Ang-1 and Ang-2 on human NP cell (Tie2+, Tie2− or mixed) populations isolated from trauma patients during 7 days in normoxia (21% O2) or hypoxia (≤5% O2). At the end of the process, the proliferation and metabolic activity of the NP cells were analyzed. Additionally, the relative gene expression of NP-related markers was evaluated. NP cells showed a higher proliferation depending on the Ang treatment. Moreover, the study revealed higher NP cell metabolism when cultured in hypoxia. Additionally, the relative gene expression followed, with an increase linked to the oxygen level and Ang concentration. Our study comparing different NP cell populations may be the start of new approaches for the treatment of IVD degeneration.

LDHA-Mediated Glycolytic Metabolism in Nucleus Pulposus Cells Is a Potential Therapeutic Target for Intervertebral Disc Degeneration.

The intervertebral disc degeneration (IDD) is considered to be an initiator of a series of spinal diseases, among which changes in the nucleus pulposus (NP) are the most significant. NP cells reside in a microenvironment with a lack of blood vessels, hypoxia, and low glucose within the intervertebral disc. Due to the strong activity of HIF-1α, glycolysis is the main pathway for energy metabolism in NP cells. Our previous study found that higher SIRT1 expression is beneficial to delay the degeneration of NP cells. In order to find the downstream genes by which SIRT1 acts on NP cells, we used iTRAQ sequencing to detect the differences between degenerated NP cells overexpressing SIRT1 and a control group (human NP cells were derived from surgery) and found that the expression of LDHA changed in the same direction with SIRT1. This suggests that SIRT1 may delay the degeneration of NP cells by regulating glycolysis. We then used a Seahorse XFe24 analyzer to measure the bioenergetic parameters of NP cells and obtained three findings: (a) glycolysis is the main energy metabolism pathway in NP cells, (b) there is a large difference in ATP production between senescent cells and young cells, and (c) SIRT1 can regulate the production of ATP from glycolysis by regulating LDHA. We also found that SIRT1 in NP cells has a positive regulatory effect on c-Myc which is an upstream gene of LDHA. Through observing IDD-related indicators such as apoptosis, proliferation, senescence, and extracellular matrix, we found that SIRT1 can delay degeneration, and interference with c-Myc and LDHA, respectively, weakens the protective effect of SIRT1. Interfering with LDHA alone can also inhibit glycolysis and accelerate degeneration. Overall, we found that the inhibition of glycolysis in Np cells significantly affects their normal physiological functions and determined that LDHA is a potential therapeutic target for the treatment of IDD.

High-dose TGF-β1 degrades human nucleus pulposus cells via ALK1-Smad1/5/8 activation.

Transforming growth factor β1 (TGF-β1) can promote the proliferation and differentiation of intervertebral disc cells and participates in its repair process. However, whether TGF-β1 engages in the process of disc degeneration has not yet been fully elucidated. The present study aimed to investigate the function of high-dose TGF-β1 on the metabolism of nucleus pulposus cells (NPCs). TGF-β1 levels in human degenerative intervertebral disc tissues and tumor necrosis factor (TNF)-α-induced degenerative NPCs were analyzed. Furthermore, NPCs were treated with TGF-β1 and inhibitors of TGF-β1 receptors [ALK tyrosine kinase receptor (ALK) 1 and ALK5] to determine the effect of the receptors in the mediation of NPC degeneration. The NPC state was determined by the components of secretory collagen I/II, tissue inhibitor of metalloproteinase-3 (TIMP-3) and matrix metalloproteinase (MMP)-13. The mRNA expression of Smad1/2/3/5/8, the downstream gene of TGF-β1 mediated by ALK, was also measured. Results showed that TGF-β1 and ALK1 were positively associated with the degree of degeneration of NP or NPCs in vitro, but negatively associated with ALK5. Furthermore, high-doses of TGF-β1 suppressed collagen II, but enhanced collagen I, TIMP-3, MMP-13, ALK1/5 and Smad1/2/3/5/8 expression. ALK5 inhibition induced the suppression of Smad2/3 and aggravated high-dose TGF-β1-induced NPC degeneration, as shown by the reduction in collagen II and increase in collagen I, TIMP-3 and MMP-13. By contrast, ALK1 inhibition resulted in Smad1/5/8 suppression and alleviated high-dose TGF-β1-induced NPC degeneration. Taken together, it was concluded that high-doses of TGF-β1 contributed to the degeneration of NPCs via the upregulation of ALK1 and Smad1/5/8.

BRD4 inhibition regulates MAPK, NF-κB signals, and autophagy to suppress MMP-13 expression in diabetic intervertebral disc degeneration.

Diabetes mellitus may lead to intervertebral disc degeneration (IVDD). Matrix metalloproteinase-13 (MMP-13) is one of the major catabolic factors in extracellular matrix (ECM) metabolism of nucleus pulposus cells (NPCs) and contributes to diabetic IVDD. Bromodomain-containing protein 4 (BRD4) is a member of the bromodomain and extraterminal protein family and is implicated in chronic inflammation. Here, we report that the expression of BRD4 and MMP-13 was elevated in diabetic nucleus pulposus tissues as well as in advanced glycation end products (AGEs)-treated NPCs; also, the regulatory effect of BRD4 on MMP-13 was studied. We found that MMP-13 was regulated by MAPK and NF-κB signaling as well as autophagy in AGEs-treated NPCs. Next, we explored the role of BRD4 in regulation of MAPK, NF-κB signaling, and autophagy. The results showed that BRD4 is the upstream regulator of all of these 3 factors, and inhibition of BRD4 may suppress MAPK and NF-κB signaling while activating autophagy in AGEs-treated NPCs. Finally, we demonstrated that BRD4 inhibition may suppress MMP-13 expression in diabetic NPCs in vitro as well as in vivo; meanwhile, it may preserve ECM in diabetic rats. Our study demonstrates that inhibition of BRD4 may suppress MAPK and NF-κB signaling and activate autophagy to suppress MMP-13 expression in diabetic IVDD, and diabetic IVDD may be compromised by BRD4 inhibitors.-Wang, J., Hu, J., Chen, X., Huang, C., Lin, J., Shao, Z., Gu, M., Wu, Y., Tian, N., Gao, W., Zhou, Y., Wang, X., Zhang, X. BRD4 inhibition regulates MAPK, NF-κB signals, and autophagy to suppress MMP-13 expression in diabetic intervertebral disc degeneration.

MicroRNA-145 overexpression attenuates apoptosis and increases matrix synthesis in nucleus pulposus cells

AimsLower back pain is often associated with intervertebral disc degeneration (IDD), which results from a decrease in nucleus pulposus (NP) cells and an imbalance between the degradation and synthesis of extracellular matrix (ECM) components. Multiple microRNAs play crucial roles in the modulation of NP cell apoptosis and matrix degradation. miR-145 is an important microRNA related to degenerative diseases such as osteoarthritis. Here, the effect of miR-145 in IDD was elucidated. The aim of this study was to explore the role and mechanism of miR-145 in the apoptosis of NP cells and in matrix metabolism in NP cells. Materials and methodsReal-time PCR, western blotting and flow cytometry analysis were used to observe the effect of miR-145 on NP cell apoptosis in the absence or presence of oxidative stress. Cell transfection, loss-of-function experiments using an ADAM17 inhibitor or lentiviral shADAM17, immunofluorescence, real-time PCR and western blotting were performed to demonstrate the role and mechanism of miR-145 in NP cell matrix metabolism. Key findingsmiR-145 attenuated NP cell apoptosis in the absence and presence of oxidative stress. Moreover, miR-145 overexpression increased and miR-145 suppression decreased matrix synthesis. ADAM17, which is expressed in degenerative discs, is the target of miR-145. ADAM17 gene suppression with lentiviral shRNA or an inhibitor enhanced matrix synthesis in NP cells. In addition, siADAM17 reversed the matrix degradation induced by miR-145 inhibition. SignificancemiR-145 suppresses apoptosis and promotes ECM synthesis in NP cells. miR-145 is thus a potential therapeutic microRNA for IDD.

Generation of Proinflammatory Mediator of Intervertebral Disc Cells by Nicotine Stimulation

Study Design: Experimental investigation in vitro. Objectives: To evaluate the relationship between the degeneration of intervertebral disc cells, and low back pain induced by degeneration of intervertebral disc cells and increases in use of proinflammatory mediators via nicotine stimulation. Summary of Literature Review: Smoking is a leading cause of degeneration of intervertebral disc cells and low back pain. According to the existing literature, nicotine, one of the main ingredients in cigarettes, causes the degeneration of intervertebral disk cells including decrease of glycoprotein through generation of carboxy-hemoglobin, vasoconstriction, and disability of fibrinolysis and changes of metabolism of nucleus pulposus cells. Materials and Methods: Annulus fibrosus of intervertebral disc and knee joint cartilage were collected from pigs; these cells were acquired by gradual enzyme decomposition. Using Trypan blue, concentration and survival rate of cells were examined; cells were inserted on alginate beads for tertiary cultivation. Nicotine was then applied at 0, 50, 100, 200 and 300 nM, respectively, and the samples were cultivated for three, six and nine days, respectively. After collecting culture fluid, it was measured for interleukin(IL)-1β, IL-6 and IL-8 with the ELISA Test. DNA of cells used for cultivation was quantitated and the amount of the resulting proinflammatory mediator was normalized. The results were then compared with the result of same study on cartilage of porcine knee joints. Results: For changes of the inflammatory mediator based on the concentration of nicotine, in nicotine stimulation with low concentration of 50 nM and the control group, there was no significant change, while transient increases of inflammatory mediator showed in nicotine stimulation with concentrations of 100, 200 nM, respectively. There was not a significant increase of IL-1β observed in all nicotine stimulation groups; these were the same results in porcine cartilage study. The level of IL-6 in 200, 300 nM nicotine concentration showed significant increases, respectively. The level of IL-8 in high dose nicotine stimulation groups also showed significant increases of DNA on the sixth day. And in porcine cartilage study group, significant changes were observed in 200, 300 nM, but the absolute value was lower than that of annulus fibrous cells group. Conclusion: Inflammatory mediators such as IL-6 and IL-8 increased as the result of tertiary cultivation of annulus fibrosus cells of porcine intervertebral disk and nicotine stimulation. It is believed that the cells of the disc annulus are more sensitive than articular chondrocytes to nicotine stimulation. This may be the focus of future long-term studies effects of nicotine other inflammatory cytokines

The expression and role of non‐canonical (PKC) signaling in nucleus pulposus cell metabolism

Canonical Wnt/β-catenin (hereafter Wnt) signaling regulates the proliferation and differentiation of various cell types. However, the role of non-canonical signaling including protein kinase C (PKC) signaling has not been investigated in intervertebral disc (IVD) cells. The aim of this study was to elucidate whether the activation of PKC signaling act to modulate Wnt signaling in IVD cells. We performed several reporter assays, real-time reverse transcription polymerase chain reaction (RT-PCR), immunohistochemical and immunofluorescence analyses, and western blot analyses using rat nucleus pulposus (NP) cells. We also examined the cell proliferation and cell cycle distribution under phorbol 12-myristate 13-acetate (PMA) stimulation, a known activator of PKC signaling. We found that NP cells exhibited decreased β-catenin mRNA and protein levels upon stimulation with PMA. PMA treatment promoted proliferation and cell cycle progression in a time- and dose-dependent manner. In addition, activation of the PKC signaling also regulated the expression of aggrecan. Finally, activation by PMA induced the expression of several PKC isoforms in NP cells. It is concluded that activation of PKC signaling might lead to an increase in matrix synthesis and cell proliferation, thereby inhibiting IVD degeneration. Crosstalk in these signaling pathways plays an important role in the regulation of IVD homeostasis.

Nucleus pulposus cells express HIF-1α under normoxic culture conditions: A metabolic adaptation to the intervertebral disc microenvironment

Nucleus pulposus (NP) cells of the intervertebral disc reside in an environment that has a limited vascular supply and generate energy through anaerobic glycolysis. The goal of the present study was to examine the expression and regulation of HIF-1alpha, a transcription factor that regulates oxidative metabolism in nucleus pulposus cells. Nucleus pulposus cells were isolated from rat, human, and sheep disc and maintained at either 21% or 2% oxygen for various time periods. Cells were also treated with desferrioxamine (Dfx), a compound that mimics the effects of hypoxia (Hx). Expression and function of HIF-1alpha were assessed by immunofluorescence microscopy, Western blot analysis, gel shift assays, and luciferase reporter assays. In normoxia (Nx), rat, sheep, and human nucleus pulposus cells consistently expressed the HIF-1alpha subunit. Unlike other skeletal cells, when maintained under low oxygen tension, the nucleus pulposus cells exhibited a minimal induction in HIF-1alpha protein levels. Electromobility shift assays confirmed the functional binding of normoxic HIF-1alpha protein to its putative DNA binding motif. A dual luciferase reporter assay showed increased HIF-1alpha transcriptional activity under hypoxia compared to normoxic level, although this induction was small when compared to HeLa and other cell types. These results indicate that normoxic stabilization of HIF-1alpha is a metabolic adaptation of nucleus pulposus cells to a unique oxygen-limited microenvironment. The study confirmed that HIF-1alpha can be used as a phenotypic marker of nucleus pulposus cells.

Segmental Variation in the In Vitro Cell Metabolism of Nucleus Pulposus Cells Isolated From a Series of Bovine Caudal Intervertebral Discs

This study focuses on the association between cell metabolism and molecular matrix composition of nucleus pulposus (NP) tissue with spine level in sequential bovine caudal intervertebral discs. To explore the hypothesis that the molecular composition of NP tissue and corresponding cell metabolism varies with caudal spine. A secondary hypothesis is tested that potential cellular differences are maintained after monolayer culture. In articular cartilage, cell metabolism and molecular matrix composition are influenced by loading history. This may also be a feature of intervertebral discs in series. NP cells (nonpooled or level pooled) were isolated from four sequential bovine caudal intervertebral discs (levels 3-4, 4-5, 5-6, and 6-7) and cultured in alginate beads immediately or following monolayer culture. Levels of 3H-TdR (proliferation) and 35SO4 (GAG synthesis) incorporation were determined from 14 animals. In a separate set of 6 animals, total content of water, DNA, collagen (and type), GAG (and type) were also determined. The rate of 3H-TdR and 35SO4 incorporation in freshly isolated NP cells increased nonlinearly from level 3-4 to 6-7 (P < 0.05). Monolayer cultured cells retained level-specific differences for 35SO4 and 3H-TdR incorporation similar to that of freshly isolated cells. GAG content and chondroitin sulfate proportion decreased distally (P < 0.05); however, total collagen and Type I proportion increased distally (P < 0.05). No significant differences in water or DNA content could be determined. The results support the hypothesis that level-specific differences in NP cell metabolism and molecular composition are dependent on spine level potentially reflecting subtle mechanical differences between levels. Retention of level-specific differences in monolayer may suggest a certain level of cell "programming." This may be important for cellular strategies to repairspecific sites of degeneration.