Makarand V Risbud

Thomas Jefferson University, Philadelphia, Pennsylvania, United States

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Publications (98)392.52 Total impact

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    ABSTRACT: Intervertebral disc degeneration is the leading cause of chronic back pain. Recent studies show that raised level of SDC4, a cell-surface heparan sulfate (HS) proteoglycan, plays a role in pathogenesis of disc degeneration. However, in nucleus pulposus (NP) cells of the healthy intervertebral disc, the mechanisms that control expression of SDC4 and its physiological function are unknown. Hypoxia induced SDC4 mRNA and protein expression by ∼2.4- and 4.4-fold (P<0.05), respectively, in NP cells. While the activity of the SDC4 promoter containing hypoxia response element (HRE) was induced 2-fold (P<0.05), the HRE mutation decreased the activity by 40% in hypoxia. Transfections with plasmids coding prolyl-4-hydroxylase domain protein 2 (PHD2) and ShPHD2 show that hypoxic expression of SDC4 mRNA and protein is regulated by PHD2 through controlling hypoxia-inducible factor 1α (HIF-1α) levels. Although overexpression of HIF-1α significantly increased SDC4 protein levels, stable suppression of HIF-1α and HIF-1β decreased SDC4 expression by 50% in human NP cells. Finally, suppression of SDC4 expression, as well as HS function, resulted in an ∼2-fold increase in sex-determining region Y (SRY)-box 9 (Sox9) mRNA, and protein (P<0.05) and simultaneous increase in Sox9 transcriptional activity and target gene expression. Taken together, our findings suggest that in healthy discs, SDC4, through its HS side chains, contributes to maintenance of the hypoxic tissue niche by controlling baseline expression of Sox9.-Fujita, N., Hirose, Y., Tran, C. M., Chiba, K., Miyamoto, T., Toyama, Y., Shapiro, I. M., Risbud, M. V. HIF-1-PHD2 axis controls expression of syndecan 4 in nucleus pulposus cells.
    The FASEB Journal 02/2014; · 5.70 Impact Factor
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    ABSTRACT: The objective of the study was to examine the regulation of CCN2 by inflammatory cytokines, IL-1β and TNF-α and to determine if CCN2 modulates IL-1β-dependent catabolic gene expression in nucleus pulposus (NP) cells. IL-1β 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-1β in combination with CCN2 suppressed the inductive effect of IL-1β on catabolic genes, including MMP-3, ADAMTS-5, syndecan 4 (SDC4) and prolyl hydroxylase 3 (PHD3). 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-1β was independent of modulation of NF-κB signaling. Using disintegrins, echistatin (ECH) and VLO4, peptide inhibitors to αvβ3 and α5β1 integrins, we showed that CCN2 binding to both integrins was required for the inhibition of IL-1β-induced catabolic gene expression. Noteworthy, 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.
    Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
  • Makarand V Risbud, Irving M Shapiro
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    ABSTRACT: Degeneration of the intervertebral discs (IVDs) is a major contributor to back, neck and radicular pain. IVD degeneration is characterized by increases in levels of the proinflammatory cytokines TNF, IL-1α, IL-1β, IL-6 and IL-17 secreted by the IVD cells; these cytokines promote extracellular matrix degradation, chemokine production and changes in IVD cell phenotype. The resulting imbalance in catabolic and anabolic responses leads to the degeneration of IVD tissues, as well as disc herniation and radicular pain. The release of chemokines from degenerating discs promotes the infiltration and activation of immune cells, further amplifying the inflammatory cascade. Leukocyte migration into the IVD is accompanied by the appearance of microvasculature tissue and nerve fibres. Furthermore, neurogenic factors, generated by both disc and immune cells, induce expression of pain-associated cation channels in the dorsal root ganglion. Depolarization of these ion channels is likely to promote discogenic and radicular pain, and reinforce the cytokine-mediated degenerative cascade. Taken together, an enhanced understanding of the contribution of cytokines and immune cells to these catabolic, angiogenic and nociceptive processes could provide new targets for the treatment of symptomatic disc disease. In this Review, the role of key inflammatory cytokines during each of the individual phases of degenerative disc disease, as well as the outcomes of major clinical studies aimed at blocking cytokine function, are discussed.
    Nature Reviews Rheumatology 10/2013; · 9.75 Impact Factor
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    ABSTRACT: Despite many advances in our understanding of the molecular basis of disc degeneration, there remains a paucity of preclinical models which can be used to study the biochemical and molecular events that drive disc degeneration, and the effects of potential therapeutic interventions. The goal of this study is to characterize global gene expression changes in a disc organ culture system that mimics early nontraumatic disc degeneration. To mimic a degenerative insult, rat intervertebral discs were cultured in the presence of TNF-a, IL-1ß and serum-limiting conditions. Gene expression analysis was performed using a microarray to identify differential gene expression between experimental and control groups. Differential pattern of gene expression was confirmed using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) or Western blot. Treatment resulted in significant changes in expression of more than 1,000 genes affecting many aspects of cell function including cellular movement, the cell cycle, cellular development, and cell death and proliferation. Many of the most highly upregulated and downregulated genes have known functions in disc degeneration and extracellular matrix hemostasis. Construction of gene networks based on known cellular pathways and expression data from our analysis demonstrated that the network associated with cell death, cell cycle regulation and DNA replication and repair was most heavily affected in this model of disc degeneration. This rat organ culture model uses cytokine exposure to induce wide gene expression changes with the most affected genes having known reported functions in disc degeneration. We propose that this model is a valuable tool to study the etiology of disc degeneration and evaluate potential therapeutic treatments.
    Arthritis research & therapy 09/2013; 15(5):R121. · 4.27 Impact Factor
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    ABSTRACT: Study Design: Laboratory study.Objective: The aims of this study were 1) To confirm that Substance P (SP) is expressed by nucleus pulposus (NP) and annulus fibrosus (AF) cells; 2) To determine the effect of SP on expression of inflammatory mediators in human disc cells and the effect of inflammatory mediators on expression of SP; and 3) To characterize the relative expression of SP receptor isoforms in disc tissue and describe whether exposure to SP changes receptor expression.Summary of Background Data: Substance P, classically described as a neurotransmitter, acts as an inflammatory regulator in other tissue types but its role within the intervertebral disc has not been characterized.Methods: Human AF and NP cells from 7 individuals were expanded in monolayer and maintained in alginate bead culture. Cells were treated with SP or IL-1β/TNF-α. After treatment, the cells were recovered, RNA was isolated and transcribed into cDNA. Quantitative RT-PCR was performed to evaluate expression of inflammatory mediators, SP and its receptors.Results: Disc cells treated with SP demonstrated significant upregulation of IL-1β, IL-6 and IL-8 in NP and AF cells while significant upregulation of RANTES and TNF occurred only in the AF. SP was expressed by AF and NP cells at low levels; expression did not change significantly with SP treatment but was significantly upregulated after treatment with IL-1β/TNF-α. Both SP receptor isoforms were expressed by NP and AF cells.Conclusion: SP upregulates inflammatory mediators in disc cells. SP and SP receptors were expressed in both NP and AF and expression did not change after treatment with SP but increased after treatment with IL-1β/TNF-α. SP likely acts in an autocrine or paracrine manner in IVD cells and may be involved in "crosstalk" between disc cells and neurons, providing a potential mechanism for transmission of painful discogenic stimuli.
    Spine 07/2013; · 2.16 Impact Factor
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    ABSTRACT: STUDY DESIGN.: Laboratory study. OBJECTIVE.: To evaluate expression of chemokine regulated and normal T cell expressed and secreted (RANTES)/C-C motif ligand 5 (CCL5) and interleukins in intervertebral discs (IVDs) specimens from patients with discogram-proven painful degeneration. SUMMARY OF BACKGROUND DATA.: Discogenic back pain results in tremendous costs related to treatment and lost productivity. The relationship between inflammation, degeneration (IVD), and cytokine upregulation is well established, but other mediators of the inflammatory cascade are not well characterized. METHODS.: Painful IVDs were taken from 18 patients undergoing surgery for discogenic pain with positive preoperative discogram. Painless control tissue was taken at autopsy from patients without back pain/spinal pathology or spinal levels with negative discograms resected for deformity.Quantitative real time polymerase chain reaction (qRT-PCR) was performed to evaluate RANTES, IL-1β, IL-6, and IL-8 expression in painful and control discs. RANTES and interleukin expression were analyzed on the basis of Pfirrmann grade.Disc cells were cultured in alginate beads using 2 groups: an untreated group and a group treated with 10 ng/mL IL-1β, 10 ng/mL TNF-α, and 1% fetal bovine serum to induce a degenerative phenotype. RESULTS.: Nine painless IVD specimens and 7 painful IVD specimens were collected. RANTES expression demonstrated a 3.60-fold increase in painful discs versus painless discs, a significant difference (P = 0.049). IL-1β expression demonstrated significantly higher expression in painful discs (P = 0.03). RANTES expression data demonstrated significant upregulation with increasing Pfirrmann grade (P = 0.045). RANTES expression correlated significantly with IL-1β expression (ρ = 0.67, P < 0.0001). RANTES expression increased more than 200-fold in the alginate culture model in cells treated with IL-1β/TNF-α, 1% fetal bovine serum (P < 0.001). CONCLUSION.: RANTES and IL-1β expression was significantly elevated in painful IVDs after careful selection of painless versus painful IVD tissue. RANTES expression was found to correlate significantly with expression of IL-1β. RANTES was upregulated by IL-1β/TNF-α/1% fetal bovine serum an in vitro treatment to induce a degenerative phenotype.
    Spine 05/2013; 38(11):873-80. · 2.16 Impact Factor
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    ABSTRACT: We investigated TNF-α and IL-1β regulation of ADAMTS-4 expression in nucleus pulposus (NP) cells and its role in aggrecan degradation. Real-time quantitative RT-PCR, Western blotting, and transient transfections with rat NP cells and lentiviral silencing with human NP cells were performed to determine the roles of MAPK and NF-κB in cytokine-mediated ADAMTS-4 expression and function. ADAMTS4 expression and promoter activity increased in NP cells after TNF-α and IL-1β treatment. Treatment of cells with MAPK and NF-κB inhibitors abolished the inductive effect of the cytokines on ADAMTS4 mRNA and protein expression. Although ERK1, p38α, p38β2, and p38γ were involved in induction, ERK2 and p38δ played no role in TNF-α-dependent promoter activity. The inductive effect of p65 on ADAMTS4 promoter was confirmed through gain and loss-of-function studies. Cotransfection of p50 completely blocked p65-mediated induction. Lentiviral transduction with shRNA plasmids shp65, shp52, shIKK-α, and shIKK-β significantly decreased TNF-α-dependent increase in ADAMTS-4 and -5 levels and aggrecan degradation. Silencing of either ADAMTS-4 or -5 resulted in reduction in TNF-α-dependent aggrecan degradation in NP cells. By controlling activation of MAPK and NF-κB signaling, TNF-α and IL-1β modulate expression of ADAMTS-4 in NP cells. To our knowledge, this is the first study to show nonredundant contribution of both ADAMTS-4 and ADAMTS-5 to aggrecan degradation in human NP cells in vitro.
    American Journal Of Pathology 04/2013; · 4.52 Impact Factor
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    ABSTRACT: The objective of the study was to investigate how inflammatory cytokines, IL-1β and TNF-α control Notch signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective Notch receptors (Notch1, 2), ligand (Jagged 2) and target genes (Hes1, Hey1 and Hey2) was observed in NP cells following cytokine treatment. A concomitant increase in Notch signaling as evidenced by induction in activity of target gene Hes1 and Hey1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2 kb Notch2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on Notch2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on Notch2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting the promoter studies, lentiviral delivery of sh-p65, and sh-IKKβ significantly decreased cytokine dependent change in Notch2 expression. Interestingly, MAPK signaling showed an isoform specific control of Notch2 promoter, p38α/β2/δ, ERK1 and ERK2 contributed to cytokine dependent induction while p38γ played no role. Analysis of human NP tissues showed that Notch 1, 2 and Hey2 expression correlated with each other. Moreover, expression of Notch2 and IL-1β as well as the number of cells immunopositive for Notch2 significantly increased in histologically degenerate discs compared to non-degenerate discs. Taken together these results explain the observed dysregulated expression of Notch genes in degenerative disc disease. Thus controlling IL-1β and TNF-αactivities during disc disease may restore Notch signaling and nucleus pulposus cell function.
    Journal of Biological Chemistry 04/2013; · 4.65 Impact Factor
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    Cassie M Tran, Irving M Shapiro, Makarand V Risbud
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    ABSTRACT: Connective tissue growth factor (CCN2/CTGF) plays an important role in extracellular matrix synthesis, especially in skeletal tissues such as cartilage, bone, and the intervertebral disc. As a result there is a growing interest in examining the function and regulation of this important molecule in the disc. This review discusses the regulation of CCN2 by TGF-β and hypoxia, two critical determinants that characterize the disc microenvironment, and discusses known functions of CCN2 in the disc. The almost ubiquitous regulation of CCN2 by TGF-β, including that seen in the disc, emphasizes the importance of the TGF-β-CCN2 relationship, especially in terms of extracellular matrix synthesis. Likewise, the unique cross-talk between CCN2 and HIF-1 in the disc highlights the tissue and niche specific mode of regulation. Taken together the current literature supports an anabolic role for CCN2 in the disc and its involvement in the maintenance of tissue homeostasis during both health and disease. Further studies of CCN2 in this tissue may reveal valuable targets for the biological therapy of disc degeneration.
    Matrix biology: journal of the International Society for Matrix Biology 04/2013; · 3.56 Impact Factor
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    ABSTRACT: The objective of the study was to investigate if HIF-1α and CCN2 form a regulatory network in hypoxic nucleus pulposus (NP) cells. A decrease in CCN2 expression and proximal promoter activity was observed in NP cells after hypoxic culture. Analysis of both human and mouse CCN2 promoters using the JASPAR core database revealed the presence of putative hypoxia response elements (HREs). Transfection experiments showed that both promoter activities and CCN2 expression decreases in hypoxia in a HIF-1α-dependent fashion. Interestingly, deletion analysis and mutation of the HREs individually or in combination resulted in no change in promoter activity in response to hypoxia or in response to HIF-1α, suggesting an indirect mode of regulation. Notably, silencing of endogenous CCN2 increased HIF-1α levels and its target gene expression, suggesting a role for CCN2 in controlling basal HIF-1α levels. On the other hand, treatment of cells with rCCN2 resulted in a decrease in the ability of HIF-1α transactivating domain to recruit co-activators and diminished target gene expression. Lastly, knockdown of CCN2 in NP cells results in a significant decrease in GAG synthesis and expression of aggrecan and collagen II. Immunohistochemical staining of intervertebral discs of CCN2 null embryos shows a decrease in aggrecan. These findings reveal a negative feedback loop between CCN2 and HIF-1α in NP cells and demonstrate a role for CCN2 in maintaining matrix homeostasis in this tissue.
    Journal of Biological Chemistry 03/2013; · 4.65 Impact Factor
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    ABSTRACT: OBJECTIVE.: To investigate TNF-α and IL-1β regulation of CCL3 expression in nucleus pulposus (NP) cells and in macrophage migration. METHODS.: qRT-PCR and immunohistochemistry were used to measure CCL3 expression in NP cells. Transfections were used to determine the role of NF-κB, C/EBP-β and MAPK on cytokine mediated CCL3 promoter activity. Effect of NP-conditioned medium on macrophage migration was measured using a transwell system. RESULTS. AN INCREASE: in CCL3 expression and promoter activity was observed in NP cells after TNF-α or IL-1β treatment. Treatment of cells with NF-κB and MAPK inhibitors abolished the effect of the cytokines on CCL3 expression. The inductive effect of p65 and C/EBP-β on CCL3 promoter was confirmed through gain- and loss-of-function studies. Noteworthy, co-transfection of p50 completely blocked cytokine and p65 dependent induction. In contrast, c-Rel and RelB had little effect on promoter activity. Lentiviral transduction with Sh-p65 and Sh-Ikkβ significantly decreased TNF-α dependent increase in CCL3 expression. Analysis of degenerate human NP tissues showed that CCL3, but not CCL4 expression correlated positively with the grade of tissue degeneration. Importantly, treatment of macrophages with conditioned medium of NP cells treated with TNF-α or IL-1β?promoted their migration; pretreatment of macrophages with antagonist to CCR1, primary receptor for CCL3 and CCL4, blocked cytokine mediated migration. CONCLUSIONS.: By controlling the activation of MAPK, NF-κB and C/EBPβ signaling, TNF-α and IL-1β modulate the expression of CCL3 in NP cells. The CCL3-CCR1 axis may play an important role in promoting macrophage infiltration in degenerate, herniated discs. © 2012 American College of Rheumatology.
    Arthritis & Rheumatology 12/2012; · 7.48 Impact Factor
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    ABSTRACT: Despite the high prevalence of intervertebral disc disease, little is known about changes in intervertebral disc cells and their regenerative potential with ageing and intervertebral disc degeneration. Here we identify populations of progenitor cells that are Tie2 positive (Tie2(+)) and disialoganglioside 2 positive (GD2(+)), in the nucleus pulposus from mice and humans. These cells form spheroid colonies that express type II collagen and aggrecan. They are clonally multipotent and differentiated into mesenchymal lineages and induced reorganization of nucleus pulposus tissue when transplanted into non-obese diabetic/severe combined immunodeficient mice. The frequency of Tie2(+) cells in tissues from patients decreases markedly with age and degeneration of the intervertebral disc, suggesting exhaustion of their capacity for regeneration. However, progenitor cells (Tie2(+)GD2(+)) can be induced from their precursor cells (Tie2(+)GD2(-)) under simple culture conditions. Moreover, angiopoietin-1, a ligand of Tie2, is crucial for the survival of nucleus pulposus cells. Our results offer insights for regenerative therapy and a new diagnostic standard.
    Nature Communications 12/2012; 3:1264. · 10.02 Impact Factor
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    ABSTRACT: Recent studies suggest a differential role of PHD isoforms in controlling hypoxia inducible factor (HIF)-α degradation and activity in nucleus pulposus (NP) cells. However, the regulation and function of PHDs under inflammatory conditions that characterize disc disease is not yet known. Here, we show that in NP cells, TNF-α and IL-1β induce PHD3 expression through NF-κB. Lentiviral delivery of Sh-P65 and Sh-IKKβ confirms that cytokine-mediated PHD3 expression is NF-κB dependent. Noteworthy, although both cytokines induce HIF activity, use of Sh-HIF-1α and PHD3 promoter/enhancer constructs harboring well characterized hypoxia response element (HRE) show lack of HIF involvement in cytokine mediated PHD3 expression. Loss of function studies clearly indicate that PHD3 serves as a co-activator of NF-κB signaling activity in NP cells; PHD3 interacts with, and co-localizes with, p65. We observed that when PHD3 is silenced there is a significant decrease in TNF-α-induced expression of catabolic markers that include ADAMTS5, syndecan4, MMP13 and COX2, at the same time there is restoration of aggrecan and collagen type II expression. Noteworthy, hydroxylase function of PHDs is not required for mediating cytokine-dependent gene expression. These findings show that by enhancing the activity of inflammatory cytokines PHD3 may serve a critical role in degenerative disc disease.
    Journal of Biological Chemistry 09/2012; · 4.65 Impact Factor
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    ABSTRACT: To study the regulation of expression of β-1,3-glucuronosyltransferase 1 (GlcAT-1), an important regulator of glycosaminoglycan (GAG) synthesis, by Smad3 in nucleus pulposus (NP) cells. GlcAT-1 expression was examined in rat NP and anulus fibrosus (AF) cells treated with transforming growth factor β (TGFβ). The effects of Smad signaling and Smad suppression on GlcAT-1 were examined in rat NP cells. GlcAT-1 expression was analyzed in the discs of Smad3-null mice and in degenerated human NP tissue. TGFβ increased the expression of GlcAT-1 in rat NP but not rat AF cells. Suppression of GlcAT-1 promoter activity was evident with dominant-negative ALK-5 (DN-ALK-5). Cotransfection with Smad3 strongly induced promoter activity independent of TGFβ. Bioinformatics analysis indicated the presence of several Smad binding sites in the promoter; deletion analysis showed that the region between -274 and -123 bp was required for Smad3 response. DN-Smad3, Smad 3 small interfering RNA, and Smad7 strongly suppressed basal as well as TGFβ-induced promoter activity. Induction of promoter activity by Smad3 was significantly blocked by DN-Smad3; Smad7 had a very small effect. Lentiviral transduction of NP cells with short hairpin RNA Smad3 resulted in a decrease in GlcAT-1 expression and accumulation of GAG. Compared to wild-type mice, significantly lower expression of GlcAT-1 was seen in the discs of Smad3-null mice. Analysis of degenerated human NP tissue specimens showed no positive correlation between GlcAT-1 and TGFβ expression. Moreover, isolated cells from degenerated human tissue showed a lack of induction of GlcAT-1 expression following TGFβ treatment, suggesting an altered response. Our findings demonstrate that in healthy NP cells, the TGFβ-Smad3 axis serves as a regulator of GlcAT-1 expression. However, an altered responsiveness to TGFβ during disc degeneration may compromise GAG synthesis.
    Arthritis & Rheumatology 06/2012; 64(10):3324-33. · 7.48 Impact Factor
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    ABSTRACT: Adaptive response to hypoxia in nucleus pulposus cells of the intervertebral disc is regulated by the hypoxia-inducible factors, HIF-1α and HIF-2α. Moreover, oxygen-dependent turnover of HIF-1α in these cells is controlled by the prolyl-4-hydroxylase domain (PHD) family of proteins. Whether HIF homologues control expression of PHDs and whether PHDs control hypoxia-inducible factor (HIF) turnover and/or activity under hypoxia is not known. Here, we show that in nucleus pulposus cells, hypoxia robustly induces PHD3 expression and, to a lesser extent, of PHD2 and PHD1. Reporter analysis shows that the hypoxic induction of the PHD2 promoter is HIF-1α dependent, whereas PHD3 promoter/enhancer activity is dependent on both HIF-1α and HIF-2α. Lentiviral delivery of HIF-1α, ShHIF-1α, and ShHIF-1β confirmed these observations. Noteworthy, HIF-1α maintains basal expression of PHD1 in hypoxia at the posttranscriptional level. Finally, loss of function studies using lentiviral transduction of ShPHDs clearly shows that even at 1% O(2), PHD2 selectively degrades HIF-1α. In contrast, in hypoxia, PHD3 enhances HIF-1α transcriptional activity without affecting protein levels. To correlate these observations with disc disease, a condition characterized by tissue vascularization, we analyzed human tissues. Increased PHD1 mRNA expression but decreased PHD2 and PHD3 expression is observed in degenerate tissues. Interestingly, the hypoxic responsiveness of all the PHDs is maintained in isolated nucleus pulposus cells regardless of the disease state. We propose that PHD2 and PHD3 can be used as a biomarker of tissue oxygenation in the disc and that, as such, it may have important clinical implications.
    Journal of Biological Chemistry 03/2012; 287(20):16975-86. · 4.65 Impact Factor
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    ABSTRACT: The objective of our study was to examine the regulation of hypoxic expression of heat shock protein 70 (Hsp70) in nucleus pulposus cells and to determine if Hsp70 promoted hypoxia-inducible factor (HIF)-1α degradation. Rat nucleus pulposus cells were maintained in culture in either 21% or 1% oxygen. To determine the regulation of Hsp70 expression by tonicity enhancer binding protein (TonEBP) and HIF-1/2, loss-of-function and gain-of-function experiments and mutational analysis of the Hsp70 promoter were performed. Hypoxia increased Hsp70 expression in nucleus pulposus cells. Noteworthy, hypoxia increased TonEBP transactivation and mutation of TonE motifs blocked hypoxic induction of the Hsp70 promoter. In contrast, mutation of hypoxia response element (HRE) motifs coupled with loss-of-function experiments suggested that HIF-1 and HIF-2 suppressed Hsp70 promoter activity and transcription. Interestingly, HIF-α interferes with TonEBP function and suppresses the inductive effect of TonEBP on the Hsp70 promoter. In terms of Hsp70 function, when treated with Hsp70 transcriptional inhibitor, KNK437, there was an increase in HIF-1α protein stability and transcriptional activity. Likewise, when Hsp70 was overexpressed, the stability of HIF-1α and its transcriptional activity decreased. Hsp70 interacted with HIF-1α under hypoxic conditions and evidenced increased binding when treated with MG132, a proteasomal inhibitor. These results suggest that Hsp70 may promote HIF-1α degradation through the proteasomal pathway in nucleus pulposus cells. In hypoxic and hyperosmolar nucleus pulposus cells, Hsp70, TonEBP, and HIFs form a regulatory loop. We propose that the positive regulation by TonEBP and negative regulation of Hsp70 by HIF-1 and HIF-2 may serve to maintain Hsp70 levels in these cells, whereas Hsp70 may function in controlling HIF-1α homeostasis.
    Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 02/2012; 27(5):1106-17. · 6.04 Impact Factor

Publication Stats

2k Citations
392.52 Total Impact Points

Institutions

  • 2002–2013
    • Thomas Jefferson University
      • Department of Orthopaedic Surgery
      Philadelphia, Pennsylvania, United States
    • Sree Chitra Tirunal Institute for Medical Sciences and Technology
      Tiruvananantapuram, Kerala, India
  • 2012
    • Thomas Jefferson University Hospitals
      Philadelphia, Pennsylvania, United States
    • Tokai University
      Hiratuka, Kanagawa, Japan
  • 2004
    • Charité Universitätsmedizin Berlin
      Berlín, Berlin, Germany
    • National University of Singapore
      Tumasik, Singapore
  • 1999–2003
    • National Centre For Cell Science, Pune
      Poona, Mahārāshtra, India
  • 2001
    • Humboldt-Universität zu Berlin
      • Department of Psychology
      Berlín, Berlin, Germany
  • 2000
    • Indian Institute of Technology Bombay
      Mumbai, Mahārāshtra, India