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Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Objective

Since nucleus pulposus cells reside under conditions of hypoxia, we determined if the expression of ANK, a pyrophosphate transporter, is regulated by the hypoxia-inducible factor (HIF) proteins.

Methods

Quantitative reverse transcription–polymerase chain reaction and Western blot analyses were used to measure ANK expression in nucleus pulposus cells from rats and humans. Transfections were performed to determine the effect of HIF-1/2 on ANK promoter activity.

Results

ANK was expressed in embryonic and mature rat discs. Oxygen-dependent changes in ANK expression in nucleus pulposus cells were minimal. However, silencing of HIF-1α and HIF-2α resulted in increased ANK expression and up-regulation of promoter activity. HIF-mediated suppression of ANK was validated by measuring promoter activity in HIF-1β–null embryonic fibroblasts. Under conditions of hypoxia, there was induction of promoter activity in the null cells as compared with the wild-type cells. Overexpression of HIF-1α and HIF-2α in nucleus pulposus cells resulted in a significant suppression of ANK promoter activity. Since the ANK promoter contains 2 hypoxia-responsive elements (HREs), we performed site-directed mutagenesis and measured promoter activity. We found that HIF-1 can bind to either of the HREs and can suppress promoter activity; in contrast, HIF-2 was required to bind to both HREs in order to suppress activity. Finally, analysis of human nucleus pulposus tissue showed that while ANK was expressed in normal tissue, there was increased expression of ANK along with alkaline phosphatase in the degenerated state.

Conclusion

Both HIF-1 and HIF-2 serve as negative regulators of ANK expression in the disc. We propose that baseline expression of ANK in the disc serves to prevent mineral formation under physiologic conditions.

Within the spine, the vertebrae are separated by a complex tissue, the intervertebral disc. At the periphery of the disc, a ligamentous tissue, the anulus fibrosus, encloses the proteoglycan-rich nucleus pulposus. Although details of the ontology of cells of the adult nucleus pulposus are obscure, it is known that they are derived from the notochord (1), an embryonic tissue with a limited blood supply. The superior and inferior boundaries of the intervertebral disc are formed by the cartilage end plates. A limited number of blood vessels infiltrate the end plates and the outer anulus fibrosus but do not enter the nucleus pulposus (2, 3). For this reason, there is considerable support for the view that the nucleus pulposus cells reside in a hypoxic environment (4–6). Surprisingly, while the disc contains both fibrous proteins and a hydrated extracellular matrix, calcified deposits are absent in the nucleus pulposus and the anulus fibrosus in healthy state.

Deposition of mineral salts is regulated by ANK, a multipass transmembrane channel that controls the transport of inorganic pyrophosphate (PPi), which is a powerful inhibitor of mineralization (7). Several genetic studies have identified multiple mutations in ANK that result in autosomal-dominant disorders that cause abnormal mineralization in the joints and bone (8–11). These disorders include familial chondrocalcinosis, which is characterized by excessive deposition of calcium pyrophosphate dihydrate (CPPD) crystals in affected joints, and craniometaphyseal dysplasia, Jackson type, whose phenotype includes hyperostosis of the craniofacial bones (8–11). In articular and growth plate cartilage, ANK expression is primarily localized to the superficial zone and the hypertrophic zone, respectively, suggesting that ANK expression is dependent on oxygen availability (12). Indeed, in a recent study, Zaka et al (13) demonstrated that the oxemic status of chondrocytes influences ANK expression and that its regulation was mediated by hypoxia-inducible factor 1α (HIF-1α).

The goal of the present investigation was 3-fold: to determine if ANK expression in cells of the intervertebral disc is dependent on the local oxygen tension, to determine the role of HIF in ANK regulation, and to investigate the relationship between disc degeneration and ANK expression. Using nucleus pulposus cells from the intervertebral discs of rats and humans and embryonic fibroblasts from HIF-1β–null mice, this study is the first to show that HIF-1 and HIF-2 regulate ANK gene expression in a unique manner. This finding lends further credence to the view that HIFs and ANK functionally adapt nucleus pulposus cells to the unique microenvironment of the disc.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Plasmids and reagents.

Expression plasmid pCA-HIF-2α, with a triple mutation (P405A/P530A/N851A) (14), was provided by Dr. Celeste Simon (University of Pennsylvania, Philadelphia, PA), and expression plasmids p(HA)HIF-1α(401Δ603) and pARNT were provided by Dr. Eric Huang (National Cancer Research Institute, Bethesda, MD). HIF-2α small interfering RNA (siRNA) and control siRNA duplexes were purchased from Dharmacon (On-Target Plus SMARTpool). An siHIF-1α (PBS/pU6-HIF-1α) plasmid developed by Dr. Connie Cepko (15) was obtained from Addgene (plasmid 21103). The construction of wild-type and mutant ANK reporter plasmids has been described previously (13). The wild-type reporter contains a 388-bp proximal ANK promoter from –684 bp to –296 bp with respect to the translation initiation into the pGL4.10 luciferase reporter vector. As an internal transfection control, pRL-TK (Promega) containing Renilla reniformis luciferase genes was used. For Western blotting, rabbit polyclonal antibodies directed against 2 different peptide sequences in mouse ANK were used: Ab36 (encompassing amino acids 36–49: RGIAAVKEDAVEML) and Ab218 (encompassing amino acids 218–232: DIIPDRSGPELGGDA).

Isolation of nucleus pulposus cells.

Rat nucleus pulposus cells were isolated using a previously described explant culture method (16). Nucleus pulposus cells migrated out of the explant after 1 week. When confluent, the cells were lifted using a trypsin (0.25%)/EDTA (1 mM) solution and were subcultured in 10-cm dishes. These cells expressed high levels of aggrecan and type II collagen and were used for the studies described below.

Collection and grading of human tissues.

Lumbar disc tissues were collected as surgical waste from patients undergoing elective surgical procedures on the spine (average age 54 years [range 38–82 years]). In accordance with the Institutional Review Board guidelines of Thomas Jefferson University, informed consent for sample collection was obtained from each patient. Assessment of the disease state was performed using the modified Thompson grading system (17).

Cell culture under conditions of hypoxia.

Nucleus pulposus cells, and mouse embryonic fibroblasts from HIF-1β wild-type (+/+) and null (–/–) mice (kindly provided by Dr. Celeste Simon, University of Pennsylvania) were maintained in Dulbecco's modified Eagle's medium and 10% fetal bovine serum (FBS) supplemented with antibiotics. Cells were cultured for 24–48 hours in an Invivo2 Hypoxia Workstation (Ruskinn) with a mixture of 1% O2, 5% CO2, and 93% N2.

Immunohistologic studies.

Freshly isolated spines or whole embryos were immediately fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) and then embedded in paraffin. Transverse and coronal sections measuring 6–8μ in thickness were deparaffinized in xylene, rehydrated through graded ethanol, and stained with Alcian blue and with hematoxylin and eosin (H&E). For localization of ANK, sections were incubated overnight at 4°C with the peptide-directed rabbit polyclonal anti-ANK218 antibody in 2% bovine serum albumin in PBS at a dilution of 1:200. After thoroughly washing the sections, the bound primary antibody was incubated for 45 minutes at room temperature with Alexa Fluor 488–conjugated anti-rabbit secondary antibody (Invitrogen) at a dilution of 1:100. Sections were mounted in mounting medium containing DAPI and visualized using a fluorescence microscope (Olympus).

Western blotting.

Cells were placed on ice immediately following treatment and washed with ice-cold Hanks' balanced salt solution. All of the wash buffers and the final resuspension buffer included 1× protease inhibitor cocktail (Roche), NaF (5 mM), and Na3VO4 (200 μM). Total cell proteins were resolved by electrophoresis on 8–12% sodium dodecyl sulfate–polyacrylamide gels and transferred by electroblotting to polyvinylidene difluoride membranes (Bio-Rad). The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C in 3% nonfat dry milk in TBST with the anti-ANK (1:1,500 dilution) or anti–β-tubulin (1:3,000 dilution; Developmental Studies Hybridoma Bank) antibody. Immunolabeling was detected using ECL Reagent (Amersham Biosciences). Blot intensity was determined by densitometric analysis using Kodak 1D 3.6 software.

Real-time reverse transcription–polymerase chain reaction (RT-PCR) analysis.

Total RNA was extracted from nucleus pulposus cells using RNeasy Mini Columns (Qiagen). Before elution from the column, RNA was treated with RNase-free DNase I (Qiagen). For human samples, total RNA was isolated from 100–300 mg of nucleus pulposus tissue. Tissue was homogenized in TRIzol (Invitrogen) on ice using an Omni TH Homogenizer (Omni International). Following TRIzol extraction, RNA was passed through the RNeasy Mini Columns. The purified, DNA-free RNA was converted to complementary DNA (cDNA) using SuperScript III reverse transcriptase (Invitrogen). Template cDNA and gene-specific primers (for rat ANK, 5′-ATGGGCTGGCGTATTCTCTGATGA-3′ [forward] and for human ANK, 5′-CAACAAACTGGTGAGCACGAGCAA-3′ [forward]) were added to Fast SYBR Green master mix (Applied Biosystems), and messenger RNA (mRNA) expression was quantified using a 7900HT Fast Real-Time PCR system (Applied Biosystems). Both 18S and GAPDH were used to normalize the mRNA expression. Melting curves were analyzed to verify the specificity of the RT-PCR reaction and the absence of primer–dimer formation. Each sample was analyzed in duplicate and included a template-free control. All of the primers we used were synthesized by Integrated DNA Technologies.

Immunofluorescence microscopy.

Cells were plated in 96-well flat-bottomed plates (4 × 103/well) and cultured for 24 hours under conditions of hypoxia (1% O2). After incubation, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS for 10 minutes, blocked with PBS containing 5% FBS, and incubated overnight at 4°C with antibodies against ANK (1:200 dilution). As a negative control, cells were reacted with isotype IgG under similar conditions. After washing, the cells were incubated with Alexa Fluor 488–conjugated anti-rabbit secondary antibody (Invitrogen), at a dilution of 1:50, for 45 minutes at room temperature. Cells were imaged using a laser scanning confocal microscope (Olympus FluoView).

Transfections and dual-luciferase assay.

One day before transfection, cells were transferred to 24-well plates at a density of 4 × 104 cells/well. To investigate the effects of HIF-1/2 overexpression on ANK promoter activity, cells were cotransfected with 100–300 ng of pCA-HIF-1α or pCA-HIF-2α or with backbone vectors pcDNA3.1 and 100 ng of pARNT, with 300 ng of ANK reporter and 300 ng of pRL-TK plasmid added to all cotransfections. For silencing experiments, we used 100–300 ng of siHIF-1α or siHIF-2α or respective control siRNA with 400 ng of ANK reporter with 300 ng pRL-TK plasmid. Lipofectamine 2000 (Invitrogen) was used as a transfection reagent. For each transfection, plasmids were premixed with the transfection reagent.

Twenty-four hours after transfection, the cells were transferred to a hypoxia work station (1% O2) or were maintained under conditions of normoxia (21% O2) for 24–48 hours. Cells were harvested, and a Dual-Luciferase Reporter Assay system (Promega) was used for sequential measurements of firefly and Renilla luciferase activities. Quantification of luciferase activities and calculation of relative ratios were performed using a luminometer (TD-20/20; Turner Designs). At least 3 independent transfections were performed, and all analyses were carried out in triplicate.

Statistical analysis.

All measurements were performed in triplicate, data are presented as the mean ± SEM. Differences between groups were analyzed by Student's t-test. P values less than 0.05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

Sagittal sections of discs from neonatal (Figures 1A and B) and skeletally mature (Figures 1C and D) rats were stained with an antibody to ANK (Figures 1A and C) or were counterstained with H&E and Alcian blue (Figures 1B and D). ANK was expressed by cells of the nucleus pulposus, anulus fibrosus, and cartilaginous end plate (Figures 1A and C). In all cases, staining was localized to the membrane and cytosol (Figures 1A and C). Expression of ANK in disc tissues and cultured cells was examined by Western blot analysis. As shown in Figure 1E, nucleus pulposus and anulus fibrosus tissues (top) expressed a prominent 54.4-kd ANK band; moreover, nucleus pulposus and anulus fibrosus cells (bottom) showed robust expression of ANK.

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Figure 1. A–D, Sagittal sections of intervertebral discs from an embryonic rat (A and B) and a mature rat (C and D). Sections were treated with ANK antibody (A and C) or were counterstained with hematoxylin and eosin (B) or Alcian blue (D). ANK was expressed in the nucleus pulposus (NP) from the adult and neonate rats. Anulus fibrosus and end plate cartilage (EP) are indicated (original magnification × 20). E, Western blot of ANK expression in rat disc tissues (top) and cultured cells (bottom) from the nucleus pulposus and anulus fibrosus (AF). GAPDH and β-tubulin were included as controls. F, Real-time reverse transcription–polymerase chain reaction analysis of ANK expression by nucleus pulposus cells under conditions of hypoxia (1% O2) at the indicated times. There was no significant change in ANK mRNA expression under conditions of hypoxia. NS = not significant. G, Western blot analysis of ANK expression in nucleus pulposus cells under conditions of hypoxia (Hx), as determined using 2 different ANK antibodies, Ab218 and Ab36. A prominent ANK band was detected at 54.4 kd. β-tubulin was included as control. Nx = normoxia. H, Densitometric analysis of ANK expression on multiple Western blots. There was no significant change in ANK expression under conditions of hypoxia at the indicated times. I, Immunofluorescence analysis of nucleus pulposus cells cultured under conditions of hypoxia or normoxia. There was a similar level of ANK expression under both conditions (original magnification × 20). Values in F and H are the mean and SEM of 3 independent experiments.

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We examined the expression of ANK in the nucleus pulposus in relationship to its oxemic status. When nucleus pulposus cells were cultured under conditions of hypoxia, there was no induction of ANK mRNA (Figure 1F). We then examined the effect of hypoxia on ANK protein expression in nucleus pulposus cells by Western blotting (Figures 1G and H) and immunofluorescence microscopy (Figure 1I). Two peptide-directed antibodies against ANK (Ab218 and Ab36) were used for Western blotting. Again, there was no significant difference between normoxic and hypoxic expression of ANK protein (Figures 1G–I).

To investigate whether HIF-1 and HIF-2 played a role in the regulation of ANK expression in nucleus pulposus cells, we evaluated ANK expression in HIF-silenced nucleus pulposus cells under conditions of hypoxia (Figures 2A–C). As expected, HIF-1α– and HIF-2α–silenced cells evidenced a decrease in protein and mRNA expression of HIF-1α (Figures 2A and C) and HIF-2α (Figure 2B), as compared with cells transfected with control siRNA. In addition, we confirmed that suppression of HIF-1α lowered its target gene expression. When HIF-1α was silenced and examined under conditions of normoxia and hypoxia, there was significant suppression of enolase 1 promoter activity, a known HIF-1α target gene in nucleus pulposus cells (results not shown). Moreover, under conditions of hypoxia, silencing of either HIF-1α (Figures 2A and C) or HIF-2α (Figure 2B) resulted in increased expression of ANK protein (Figures 2A, B, and D) and ANK mRNA (Figure 2C) as compared with cells transfected with control siRNA.

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Figure 2. Hypoxia-inducible factor (HIF) regulation of ANK expression. Cells were transfected with HIF-1α small interfering RNA (siHIF-1α) (A, C, and D), HIF-2α siRNA (B), or with their respective control siRNA and cultured under conditions of hypoxia. A and B, Immunofluorescence staining for HIF-1α and ANK (A) and for HIF-2α and ANK (B) in HIF-silenced cells under conditions of hypoxia. Note the increase in ANK staining in HIF-silenced cells as compared with the controls. As expected, the silenced cells show decreased staining for the respective HIFs (original magnification × 20). C, Expression of mRNA for HIF-1α and ANK in HIF-1α–silenced cells. Silencing of HIF-1α resulted in increased ANK mRNA expression as compared with control. Values are the mean and SEM of 3 independent experiments. = P < 0.05. D, Western blot analysis of ANK in siHIF-1α–transfected cells. Higher expression of ANK protein is evident in HIF-1α–silenced cells as compared with control cells. β-tubulin was included as control.

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We further evaluated the relationship between ANK transcription and oxygen tension by measuring the activity of the ANK proximal promoter under normoxic and hypoxic conditions. Figure 3A shows that in nucleus pulposus cells, ANK promoter activity was similar under both hypoxic and normoxic conditions. In addition, to confirm the role of HIF-1 and HIF-2 in the transcriptional regulation of ANK expression, promoter activity was measured by performing loss-of-function studies. When HIF-1α expression was silenced using siRNA, ANK promoter activity was induced under both normoxic and hypoxic conditions (Figure 3B). However, induction of activity was more pronounced under conditions of hypoxia.

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Figure 3. Effect of oxygen tension and hypoxia-inducible factor (HIF) on ANK promoter activity in nucleus pulposus cells. A, Cells were transfected with ANK reporter constructs, and luciferase reporter activity was measured under conditions of normoxia (21% O2) or hypoxia (1% O2). There was no evidence of hypoxic changes in ANK promoter activity. B and C, Effect of transfection with HIF-1α small interfering RNA (siHIF-1α) (B) or HIF-2α siRNA (siHIF-2α) (C) or with their respective control siRNA. Cells were transfected and cultured under conditions of normoxia (Nx) or hypoxia (Hx). Silencing of HIF-1α or HIF-2α resulted in a significant induction of ANK promoter activity. Induction was more pronounced under conditions of hypoxia. D, Effect of hypoxia on ANK promoter activity in HIF-1β wild-type (WT) and knockout (KO; null) mouse embryonic fibroblasts. In contrast to wild-type cells, hypoxia induced ANK promoter activity in null cells. Values are the mean and SEM of 3 independent experiments. = P < 0.05. NS = not significant.

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To confirm the role of HIF-2 in the regulation of ANK, we cotransfected nucleus pulposus cells with HIF-2α siRNA. As shown in Figure 3C, HIF-2α silencing resulted in the induction of promoter activity independently of the oxemic status. Again, hypoxic silencing of HIF-2α caused a profound induction of promoter activity. The role of HIF-1 and HIF-2 in the regulation of ANK promoter activity was further examined using wild-type and null mouse embryonic fibroblasts derived from HIF-1β homozygous null (–/–) and wild-type (+/+) mice. Wild-type mouse embryonic fibroblasts did not show a significant change in ANK promoter activity under conditions of hypoxia (Figure 3D). In contrast, null cells exhibited a significant induction of ANK promoter activity under hypoxic conditions (Figure 3D).

As further validation of whether the ANK promoter activity was responsive to HIF signaling, we cotransfected nucleus pulposus cells with plasmids encoding pCA-HIF-1α or pCA-HIF-2α, both of which are insensitive to oxidative degradation (Figures 4A and B). Transfection with pCA-HIF-1α as well as with pCA-HIF-2α significantly suppressed the ANK promoter activity (Figures 4A and B). The inhibitory effect of pCA-HIF-1α on ANK promoter activity was such that the level of inhibition remained constant when the concentration of plasmid was decreased from 300 ng to 100 ng (Figure 4A). For pCA-HIF-2α, while suppression of promoter activity was evident at 100 ng, the effect was further enhanced when the concentration of plasmid was increased from to 200 ng (Figure 4B).

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Figure 4. Effect of pCA-HIF-1α (A) or pCA-HIF-2α (B) expression on ANK promoter activity in nucleus pulposus cells. Note the significant suppression of ANK reporter activity even when cells were transfected with 100 ng of hypoxia-inducible factor (HIF) plasmid. Values are the mean and SEM of 3 independent experiments. = P < 0.05.

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The interaction of HIFs with hypoxia-responsive elements (HREs) in the ANK promoter was examined using reporters containing a mutation (underlined bases) in either HRE-1 (CGCGTGCC to CGTATGCC) or HRE-2 (CGCGTGCT to CGTATGCT) (Figure 5A). Regardless of the oxemic tension, a mutation in HRE-1 resulted in decreased basal activity as compared with the wild-type reporter, whereas a mutation in HRE-2 had no effect on basal activity (Figure 5B). We examined the effect of HIF silencing on the activity of HRE mutants under conditions of hypoxia. The results clearly showed that HIF-1α silencing led to increased activation of both HRE-1 and HRE-2 mutant reporters (Figure 5C). In contrast, the activity of both HRE mutant reporters was unresponsive to HIF-2α silencing (Figure 5D). Moreover, overexpression of pCA-HIF-1α resulted in suppression of the activity of both the HRE-1 and HRE-2 mutant reporters (Figure 5E). In contrast, pCA-HIF-2α failed to suppress the activity of either of the mutant reporters (Figure 5F).

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Figure 5. Interaction of hypoxia-inducible factors (HIFs) with hypoxia-responsive elements (HREs) in the ANK promoter. A, Schematic of the different ANK reporter constructs used in the transfections. Arrow indicates transcription start site. WT = wild-type; Mut1 = HRE-1 mutant; Mut2 = HRE-2 mutant; Luc = luciferase. B, Relative luciferase activity in cells transfected with wild-type, mutant 1, and mutant 2 ANK reporter constructs, as measured under conditions of normoxia (Nx) and hypoxia (Hx). C and D, Effect of silencing of HIF-1α with small interfering RNA (siHIF-1α) or HIF-2α siRNA (siHIF-2α) or with their respective control siRNA on mutant ANK reporters. Silencing with siHIF-1α induced the activity of both mutant 1 and mutant 2, as compared with cells transfected with control siRNA (C). Silencing with siHIF-2α had no effect on the activity of either mutant (D). E and F, Effect of pCA-HIF-1α (E) or pCA-HIF-2α (F) coexpression on mutant ANK reporter activity. HIF-1α suppressed the activity of both mutants, whereas neither mutant was responsive to HIF-2α. Values are the mean and SEM of 3 independent experiments. = P < 0.05. NS = not significant.

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We evaluated the expression of ANK and tissue-nonspecific alkaline phosphatase (TNAP) in degenerated human disc tissues graded according to a modified Thompson grading system (Figure 6A). Real-time RT-PCR analysis showed a trend toward an increase in the expression of mRNA for ANK (Figure 6B) as well as mRNA for TNAP (Figure 6C) in degenerated tissues as compared with normal control tissues. We extracted proteins from the same tissue samples and confirmed their integrity by gel electrophoresis. All tissue extracts showed discrete protein bands with minimal sample deterioration (Figure 6C). Western blot analysis was performed on equal amounts of these tissue proteins using an anti-ANK antibody. As indicated in Figure 6D, there was a discrete ANK protein band (54.4 kd) in all the samples, with a lower level of expression evident in the normal control tissue. Inspection of the combined data set suggested a trend toward increased ANK expression with degeneration. Not surprisingly, patient-to-patient variation within the same grade was also evident.

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Figure 6. Expression of ANKH in nucleus pulposus samples from patients with degenerative disc disease. A, Representative magnetic resonance images (MRIs) showing grades II–V degenerative disc disease. MRIs were graded according to a modified Thompson grading system (17). B and C, Real-time reverse transcription–polymerase chain reaction analysis of ANKH (B) and tissue-nonspecific alkaline phosphatase (TNAP) (C) mRNA expression in nucleus pulposus tissue samples from multiple patients. Samples from 3 different patients were used for each tissue grade. With increased severity of disc degeneration, there is a relative increase in the expression of ANKH and TNAP mRNA. Mean expression in normal control tissues (Co) was set at 1.0. D, Protein extracts from human degenerated nucleus pulposus tissues were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and staining with Coomassie blue. The distinct bands in each sample are verification of the quality of the protein samples. E, Western blot analysis of ANKH expression in human degenerated nucleus pulposus tissue samples. ANKH expression is increased in samples of all grades of degenerated tissues as compared with control.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

The goal of this investigation was to examine the expression of ANK in the intervertebral disc and to test the hypothesis that oxygen tension regulated ANK expression; in addition, we assessed whether ANK expression was dependent on HIF activity. We examined the expression and localization of ANK in the intervertebral disc of animals at 2 different stages of skeletal maturity. Our results indicated that there was significant expression of ANK in the nucleus pulposus, with mature animals exhibiting a higher level of expression than neonates.

In light of a previous report from our laboratories, demonstrating that ANK expression is sensitive to oxygen tension (13), and in recognition of the fact that in the adult disc, the nucleus pulposus is completely devoid of vasculature and the oxygen tension is low, the observed high levels of ANK expression were unanticipated. We were not surprised, however, to observe that, analogous to ANK expression in the growth plate, ANK expression was high in the calcified zone of the end plate, a region rich in vascular supply. Our observations suggested the existence of intrinsic differences in ANK regulation between the nucleus pulposus and the growth plate and led us to explore in more detail the effect of oxygen on ANK expression in the nucleus pulposus.

Consistent with the findings of our previous studies of constitutively high expression of HIF in the nucleus pulposus in a manner that was independent of oxygen tension, we observed that ANK expression was also not responsive to PO2. Since the levels of HIF-1 and, to some extent, HIF-2 in nucleus pulposus cells are refractory to changes in oxygen tension (6, 16, 18–20), it is difficult to show that ANK is regulated by HIF. For this reason, we used gain and loss of function studies with the promoter sequences of the murine ANK gene containing 2 conserved HRE motifs: a proximal motif at –354 bp (HRE-2) and a distal motif at –485 bp (HRE-1).

When we silenced the expression of HIF-1α or HIF-2α in the cells, we found that ANK expression was induced under conditions of hypoxia at both the mRNA and protein levels, suggesting that ANK expression is regulated in an HIF-dependent manner. Furthermore, forced expression of HIF-1α or HIF-2α caused suppression in ANK reporter activity in nucleus pulposus cells. This finding was further confirmed by site-directed mutagenesis studies. Interaction of HIF-1 with wild-type HRE-1 or HRE-2 suppressed promoter activity; however, mutation of either HRE-1 or HRE-2 abolished the responsiveness of the ANK promoter to changes in the HIF-2 levels, indicating that the binding of HIF-2 to both HREs is likely required for suppression of ANK promoter activity.

Our observation that a mutation in HRE-1 resulted in decreased basal promoter activity suggests that the binding of HIF to this motif is likely required for recruitment of a regulatory factor that maintains baseline promoter activity. Importantly, these experiments highlighted differences between 2 HIF isoforms; compared with HIF-2, HIF-1 robustly influences ANK promoter activity, even under conditions of normoxia. It is interesting that despite similar levels of HIF proteins under conditions of normoxia and hypoxia, silencing of both HIF isoforms under conditions of hypoxia resulted in significantly higher induction of ANK reporter, suggesting that hypoxia and HIF may exert opposing effects on ANK transcription. In support of this notion, we found that, unlike wild-type cells, hypoxic conditions induced ANK promoter activity in HIF-1β–null cells that were effectively null with respect to HIF-1α and HIF-2α activity. Based on these complementary findings, we postulate that in nucleus pulposus cells, hypoxia may also regulate ANK promoter activity in an HIF-independent manner.

Significantly, the findings of this study lend further strength to the recent observations of Zaka et al (13) that ANK is a HIF-1 target gene. In contrast to the observed low levels of ANK expression in hypoxic regions of the growth plate, however, our studies of ANK in the nucleus pulposus demonstrated robust ANK expression despite the hypoxic nature of the tissue. Under normal physiologic conditions, local control of mineralization in the nucleus pulposus is necessary to prevent ectopic mineralization of the disc (21). Although the precise function of ANK in the intervertebral disc is unknown, we suggest that, in part, control of mineralization is facilitated by the ability of ANK to transport the mineralization inhibitor PPi (22, 23). It is therefore likely that the factors that limit the expression of ANK under conditions of hypoxia in the growth plate do not apply to the expression of ANK in the nucleus pulposus.

Zaka et al (13) proposed that the hypoxic repression of ANK expression in the growth plate may be mediated by recruitment of a secondary transcription factor that may interfere with the binding of HIF-1 to the responsive HRE in the proximal promoter of ANK. Further studies are currently under way in our laboratory to determine the precise nature of HIF-mediated regulation of ANK expression in the nucleus pulposus. Nevertheless, we conclude that in the hypoxic intervertebral disc, the expression of HIF-1 and HIF-2 serves to maintain basal ANK expression. We further hypothesize that the expression of ANK in nucleus pulposus cells represents an adaptational response to its unique microenvironmental niche and metabolic status within the intervertebral disc (18, 19).

Calcification of intervertebral discs is associated with disease and with aging (24, 25). Indeed, studies by Gruber et al (26) and Melrose et al (27) indicate that calcified deposits are evident in human as well as animal discs. Since ANK is known to be involved in the local control of mineralization in tissues such as bone and in the calcified zone of the growth plate, and since ANK protein expression is elevated in the articular cartilage of patients with osteoarthritis and patients with CPPD crystal deposition disease (28, 29), we chose to evaluate the levels of ANK expression in degenerated human nucleus pulposus. While the numbers of control tissues were very limited (due to practical difficulties in acquiring normal human discs), measurement of the expression of both mRNA and protein suggested that as the nucleus became degenerated, there was a rise in expression of the ANK.

In addition to the transcriptional regulation of ANK expression by HIF proteins, ANK expression has also been shown to respond to a variety of other stimuli, including transforming growth factor β (TGFβ) (30). Indeed, we have recently demonstrated that ANK expression is up-regulated in response to treatment with TGFβ in nucleus pulposus cells (Skubutyte R, et al: unpublished observations). It is therefore not unreasonable to assume that in the degenerated disc, dysregulation of HIF activity, as well as elevated levels of factors such as TGFβ, may contribute to the increased ANK levels (30).

From a functional perspective, we conclude that basal expression of ANK is required to prevent dystrophic mineral formation in the normal intervertebral disc. In the degenerated disc, however, high levels of ANK expression, raised levels of extracellular PPi in conjunction with PPi-generating ectoenzymes, such as plasma cell membrane glycoprotein 1 and cartilage intermediate-layer protein, and increased elaboration of TNAP could promote hydroxyapatite deposition (29, 31). Future studies are aimed toward investigating this hypothesis.

AUTHOR CONTRIBUTIONS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES

All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Risbud had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study conception and design. Skubutyte, Markova, Freeman, Anderson, Dion, Williams, Shapiro, Risbud.

Acquisition of data. Skubutyte, Markova, Freeman, Anderson, Dion, Williams, Shapiro, Risbud.

Analysis and interpretation of data. Skubutyte, Markova, Freeman, Anderson, Dion, Williams, Shapiro, Risbud.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. AUTHOR CONTRIBUTIONS
  7. REFERENCES