Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.

doi: 10.1359/jbmr.070805

Figure Lengend Snippet: FIG. 1. Expression of ASICs in the rat intervertbral disc. Sagittal sections of the intervertebral disc of embryonic (A and B) and mature rat (C–F) spines. Sections were treated with anti-ASIC3 antibody (B, D, and F) or counterstained with Alcian blue, eosin, and hematoxylin (A, C, and E). Note that nucleus pulposus cells expressed ASIC3 protein (B; arrow; magnification, ×20). ASIC3 expression was also evident in mature rat disc in the inner annulus zone (D; arrows). These cells are surrounded by an Alcian blue–positive matrix and display a round morphology (C, arrows), whereas the outer annulus fibrosus cells are surrounded by eosinophilic collagen rich matrix (C, arrowhead). ASIC3 expression was not detectable in the endplate chondrocytes of mature rat discs (F), which exhibit a round hypertrophic phenotype (E, arrows; magnification, ×20). (G) Expression of ASIC3 and ASIC2b in the intervertebral disc. mRNA was extracted from the nucleus pulposus (NP), the annulus fibrosus (AF), and the brain of adult rats and subjected to RT-PCR analysis. There was expression of ASIC3 and ASIC2b in both the discal tissues. Brain maximally expressed both ASIC isoforms. (H) Western blot analysis of ASIC3 and ASIC2b from adult rat discal tissues. Both nucleus pulposus and annulus fibrosus expressed ASIC3 and ASIC2b. (I) Immunofluorescent detection of ASIC proteins in discal cells. Cells were treated with antibodies to ASIC3 and ASIC2b. Both nucleus pulposus and annulus fibrosus cells expressed ASIC3 and ASIC2b (magnification, ×20). (J) Western blot analysis of ASIC proteins in cell lysates of discal cells. Western blots were performed using antibodies to ASIC3 (two separate antibodies for ASIC3) and ASIC2b. Both ASIC3 antibodies recognized a band at 60 kDa and an additional high molecular weight band at 90 kDa. (K) ASIC3 protein expression in the nuclear, cytosolic, and membrane fractions of disc cells. Fractionated protein extracts of the nucleus pulposus and annulus fibrosus cells were probed by Western blot for the expression of ASIC3. Note the prominent expression of this protein in the all the fractions. (L) Effects of deglycoslylation on ASIC3 protein molecular weight. Cell lysates were deglycosylated using PNGase F for 3 h at 37°C and probed for expression of ASIC3. The molecular weight of ASIC3 peptides were unchanged by treatment with the enzyme. (M) Immunoprecipitation (IP) analysis of ASIC3 and ASIC2b in disc cells. IP analysis was performed on tissue and cell lysates using anti-ASIC3 antibody. Immune complexes were separated by SDS-PAGE and probed with anti-ASIC2b antibody. Note the presence of ASIC2b in the ASIC3 immunoprecipitate of both the nucleus pulposus and annulus fibrosus tissue and nucleus pulposus cells.

Article Snippet: 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-ASIC3 antibody from two different manufacturers (Alpha Diagnostics and Alamone Laboratories) at a dilution of 1:500, ASIC2b, 1:500 (Alpha Diagnostics), pERK and ERK, 1:1000 (Cell Signaling), p75NTR, 1:1000 and TrkA, 1:1000 (Abcam), pTrkA; and 1:1000 (Cell Signaling).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, High Molecular Weight, Membrane, Molecular Weight, Immunoprecipitation, SDS Page

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.

doi: 10.1359/jbmr.070805

Figure Lengend Snippet: FIG. 2. Cloning of rat ASIC3 promoter and its activity in disc cells. (A) Cartoon showing map of successive PCR generated 5 deletion constructs of the rat ASIC3 promoter. The ASIC3 promoter contains three distinct domains: proximal, middle, and a distal activating domain. The ASIC3-D construct consists of a 2925-bp fragment containing 2831 bp of the upstream ASIC3 promoter sequence linked to 94 bp of exon 1 (i.e., −2831 to +94), whereas ASIC3-M and ASIC3-P constructs contain a 1571- (−1477 to +94) and a 1065-bp (−971 to + 94) fragement, respectively. (B) Agarose gel electrophoresis showing PCR amplicons corresponding to different size fragments of the rat ASIC3 promoter. PCR was performed on rat liver genomic DNA using a set of nested forward primers containing a MluI site and a common reverse primer with a XhoI site. The resulting DNA fragments were ligated into MluI and XhoI digested luciferase basic expression vector, pGL3. (C and D) Basal activities of ASIC3 promoter constructs relative to full-length construct ASIC3-D in nucleus pulposus (C) and annulus fibrosus cells (D). Both the cell types showed maximal luciferase activity for the ASIC3-P construct, whereas the longest construct, ASIC3-D, containing all three promoter domains, showed minimal activity. Values shown are mean ± SE of three independent experiments. *p < 0.05.

Article Snippet: 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-ASIC3 antibody from two different manufacturers (Alpha Diagnostics and Alamone Laboratories) at a dilution of 1:500, ASIC2b, 1:500 (Alpha Diagnostics), pERK and ERK, 1:1000 (Cell Signaling), p75NTR, 1:1000 and TrkA, 1:1000 (Abcam), pTrkA; and 1:1000 (Cell Signaling).

Techniques: Cloning, Activity Assay, Generated, Construct, Sequencing, Agarose Gel Electrophoresis, Luciferase, Expressing, Plasmid Preparation

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.

doi: 10.1359/jbmr.070805

Figure Lengend Snippet: FIG. 3. Evaluation of ASIC3 promoter re- sponsiveness to NGF treatment in nucleus pulposus, annulus fibrosus, and PC12 neuro- nal cells. Cells were transfected with full- length ASIC3 plasmid (ASIC3-D) and pRL- TK control vector and luciferase activity was measured 24 h after treatment with NGF (100 ng/ml). Note, in the nucleus pulposus (A) and annulus fibrosus cells (B), ASIC3 promoter activity did not change after NGF treatment, whereas in the PC12 cells (C), there was a 3- to 4-fold induction in promoter activity. NGF-mediated increase in ASIC3 promoter activity was suppressed to its basal level when PC12 cells were treated with anti- TrkA or anti-NGF antibody before addition of NGF. A similar experiment was per- formed using cells transfected with tyrosine hydroxylase (TH) promoter construct, a known NGF responsive gene. In both nucleus pulposus (D) and annulus fibrosus (E) cells, TH promoter activity did not change after NGF treatment. In contrast, in PC12 cells (F), a significant induction in ac- tivity was seen. The increase in TH promoter activity by NGF was suppressed by treatment with anti-TrkA or anti-NGF antibody. Val- ues shown are mean ± SD of three indepen- dent experiments. *p < 0.05.

Article Snippet: 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-ASIC3 antibody from two different manufacturers (Alpha Diagnostics and Alamone Laboratories) at a dilution of 1:500, ASIC2b, 1:500 (Alpha Diagnostics), pERK and ERK, 1:1000 (Cell Signaling), p75NTR, 1:1000 and TrkA, 1:1000 (Abcam), pTrkA; and 1:1000 (Cell Signaling).

Techniques: Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay, Construct

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.

doi: 10.1359/jbmr.070805

Figure Lengend Snippet: FIG. 5. Regulation of ASIC3 basal promoter activity by p75NTR in nucleus pulposus cells. (A) Effect of blocking p75NTR expression on ASIC3 promoter activity. Cells trans- fected with ASIC3-D plasmid were treated with anti-p75NTR an- tibody or isotype antibody (Ctr), and luciferase activity was mea- sured 24 h after the treatment. When p75NTR function was blocked, there was a significant suppression in ASIC3 basal pro- moter activity. (B) Effect of expression of DN-p75NTR on ASIC3 promoter activity. Nucleus pulposus cells were co-transfected with ecdysone-inducible DN-p75NTR and ecdysone receptor pVgRxR plasmids along with ASIC3-D reporter. The cells were treated with 1 g pronesterin-A for 24 h, and reporter activity was mea- sured. Pronesterin-A–induced expression of DN-p75NTR re- sulted in ∼50% suppression of ASIC3 promoter activity. Data shown are mean ± SD of three independent experiments per- formed in triplicate (n 3). *p < 0.05.

Article Snippet: 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-ASIC3 antibody from two different manufacturers (Alpha Diagnostics and Alamone Laboratories) at a dilution of 1:500, ASIC2b, 1:500 (Alpha Diagnostics), pERK and ERK, 1:1000 (Cell Signaling), p75NTR, 1:1000 and TrkA, 1:1000 (Abcam), pTrkA; and 1:1000 (Cell Signaling).

Techniques: Activity Assay, Blocking Assay, Expressing, Plasmid Preparation, Luciferase, Transfection

Journal: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research

Article Title: Expression of acid-sensing ion channel 3 (ASIC3) in nucleus pulposus cells of the intervertebral disc is regulated by p75NTR and ERK signaling.

doi: 10.1359/jbmr.070805

Figure Lengend Snippet: FIG. 6. Regulation of ASIC3 gene promoter by MEK/ERK signaling. (A) Influence of NGF treatment on ERK activity in nucleus pulposus cells. Cells were treated with NGF (100 ng/ml) for increasing time periods (0–180 min), and cell lysates were analyzed by Western blot. NGF treatment induced pERK1/2 expression within 5 min, and levels remained higher than basal levels for 1 h, although there was a decline after 10 min of exposure. (B) p75NTR-mediated activation of ERK in response to NGF treatment. Cells were co-transfected with ELK1-TAD plasmid (50 ng) or empty backbone vector (Gal4DBD; 50 ng) along with DN-p75NTR, and ELK1 transactivation was measured. NGF treatment significantly increased ELK1-TAD activity; the increase in activity was suppressed by pronesterin A–inducible DN-p75NTR. Backbone vector Gal4DBD showed minimal activity in untreated control cells and cells treated with NGF. (C) Effect of ERK inhibition on ASIC3 promoter activity. Cells were co-transfected with ASIC3-D reporter plasmid along with DN-MEK1 or empty backbone vector (pMCL), and reporter activity was measured. There was ∼30% suppression of basal ASIC3 promoter activity with 50 ng DN-MEK plasmid, which was further suppressed at 100 ng. Further increases in DN-MEK plasmid concentration (up to 400 ng) suppressed basal reporter activity by almost 80%. (C) Effect of overexpression of MEK1 on ASIC3 promoter activity. Cells were co-transfected with CA-MEK1 expression vector or empty vector pMCL along with ASIC3-D reporter, and luciferase activity was measured. A significant induction in ASIC3 promoter activity was observed with 50 ng of MEK1 plasmid. When the dose of MEK1 plasmid was 400 ng, there was a 2.25-fold increase in ASIC3 reporter activity. Values shown are representative of three independent experiments, performed in triplicate; mean ± SD. *p < 0.05.

Article Snippet: 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-ASIC3 antibody from two different manufacturers (Alpha Diagnostics and Alamone Laboratories) at a dilution of 1:500, ASIC2b, 1:500 (Alpha Diagnostics), pERK and ERK, 1:1000 (Cell Signaling), p75NTR, 1:1000 and TrkA, 1:1000 (Abcam), pTrkA; and 1:1000 (Cell Signaling).

Techniques: Activity Assay, Western Blot, Expressing, Activation Assay, Transfection, Plasmid Preparation, Control, Inhibition, Concentration Assay, Over Expression, Luciferase

Journal: Scientific Reports

Article Title: Vacuolar-ATPase-mediated muscle acidification caused muscular mechanical nociceptive hypersensitivity after chronic stress in rats, which involved extracellular matrix proteoglycan and ASIC3

doi: 10.1038/s41598-023-39633-1

Figure Lengend Snippet: Changes in RCS-induced decrease of MMWT by antagonists to ASIC3 and TRPs ( A ), in mRNAs expression of ASIC3 and TRPV1 ( B ) and in protein of ASIC3 in DRGs after RCS ( C , D ). ( A ) The presentation methods are the same as those in Fig. . The arrow shows the period at which APETx2 and RR were injected (3–5 d after RCS). Two-way ANOVA with repeated measures (see Supplementary Table ) followed by Bonferroni’s post hoc test. ## : p < 0.01 compared to the PBS group (main effect of the “drug”), $$, $$$: p < 0.01, 0.001 compared to “pre RCS”, *, **: p < 0.05, 0.01 compared to “pre Inj” in each drug group, &&&: p < 0.001 compared to PBS at the time point, APETx2 (APET) 0.73 μM and 2.2 μM reversed the mechanical withdrawal threshold decreased after RCS but RR neither 1.5 μM nor 15 μM did. ( B ) Vertical axis: ASIC3 mRNA and TRPV1 mRNA relative to GAPDH mRNA. DRGs were removed from rats 1 week after RCS. *: p < 0.05. Note that only ASIC3 mRNA level increased significantly after RCS. ( C ) Western blot analysis of ASIC3 protein expression in DRGs of RCS and control (CTR) rats. Blots from two rats for each group were represented. GAPDH was used as an internal control. ( D ) ASIC3 protein levels in DRGs quantified from the Western blot (n = 6 for each group). Data were shown as relative values to GAPDH. * : p < 0.05. (See Supplementary Table for statistics summary for ( B ) and ( D )).

Article Snippet: The upper piece of membrane was probed with anti-ASIC3 antibody raised in rabbit (1:200, GeneTex, Irvine, CA, USA) and goat anti-rabbit IgG conjugated with HRP (1:2000, Sigma Aldrich), and the lower piece with mouse anti-GAPDH antibody (1:10,000, GeneTex) and goat anti-mouse IgG conjugated with HRP (1:1000, Thermo Fisher Scientific Inc.) to use as loading control.

Techniques: Expressing, Injection, Western Blot