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. 2018 Jul 18;6:21.
doi: 10.1038/s41413-018-0022-y. eCollection 2018.

Ciliary Parathyroid Hormone Signaling Activates Transforming Growth Factor-β to Maintain Intervertebral Disc Homeostasis During Aging

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Free PMC article

Ciliary Parathyroid Hormone Signaling Activates Transforming Growth Factor-β to Maintain Intervertebral Disc Homeostasis During Aging

Liwei Zheng et al. Bone Res. .
Free PMC article

Abstract

Degenerative disc disease (DDD) is associated with intervertebral disc degeneration of spinal instability. Here, we report that the cilia of nucleus pulposus (NP) cells mediate mechanotransduction to maintain anabolic activity in the discs. We found that mechanical stress promotes transport of parathyroid hormone 1 receptor (PTH1R) to the cilia and enhances parathyroid hormone (PTH) signaling in NP cells. PTH induces transcription of integrin αvβ6 to activate the transforming growth factor (TGF)-β-connective tissue growth factor (CCN2)-matrix proteins signaling cascade. Intermittent injection of PTH (iPTH) effectively attenuates disc degeneration of aged mice by direct signaling through NP cells, specifically improving intervertebral disc height and volume by increasing levels of TGF-β activity, CCN2, and aggrecan. PTH1R is expressed in both mouse and human NP cells. Importantly, knockout PTH1R or cilia in the NP cells results in significant disc degeneration and blunts the effect of PTH on attenuation of aged discs. Thus, mechanical stress-induced transport of PTH1R to the cilia enhances PTH signaling, which helps maintain intervertebral disc homeostasis, particularly during aging, indicating therapeutic potential of iPTH for DDD.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
IVD volume and TGF-β activity decrease during aging. a 3D propagation phase contrast micro-tomography (PPCT) images of IVDs of 2-month and 18-month-old mice. Scale bars, 500 μm. b Quantitative analysis of IVD height and volume. c 3D upper and lower surface PPCT images showing thickness distribution among five areas of IVDs in 2-month and 18-month old mice and (d) quantitative analysis of (c). Scale bar, color code indicating degree of thickness from blue (100 μm) to red (800 μm). e A 3D PPCT image of IVD of lumbar 3rd and 4th was randomly selected to draw this schematic diagram to define the five regions with labeling on the IVD including C: central region, R: right region, A: anterior region, L: left region, P: posterior region based on the anatomical characters. f, g Safranin-O staining images of IVD tissue sections showing nucleus pulposus (NP) area (f) and quantitative analysis of the cell numbers in NP area and IVD histological scores of 2-month and 18-month old mice (g). Scale bars, 100 μm. h Immunostaining images of IVD sections showing expression of aggrecan (ACAN), CCN2, and pSmad2/3 positive cells in the NP area. Scale bars, 200 μm. i Quantitative analysis of ACAN-positive and CCN2-positive areas as percentage of total IVD area and pSmad2/3-positive cells in NP area (Ar). Scale bars, 50 μm. j Western blot analysis showing pSmad2 levels in NP tissues of 2-month and 18-month old mice. All data shown as mean ± s.d. *P < 0.05, **P < 0.01, n = 8 per group. Statistical significance was determined by Student's t-test
Fig. 2
Fig. 2
PTH directly induces cAMP production and phosphorylation of CREB in NP cells. a Immunostaining images of mouse IVD sections showing PTH1R (brown) in NP cells with IgG antibody as negative control (NC). Scale bar, 100 μm. b Western blot analysis showing PTH1R expression in NP and AF cells with HEK293 cells as negative control (NC) and UMR-106 osteoblast-like cells as positive control (PC). c Lineage mapping of PTH1R expression in NP cells of notochordal origin (top yellow) using NotoCre; ROSA26-GFP mice. Scale bar, 20 μm (top) and 50 μm (bottom). NP cells stained positively for notochord origin (green) and presence of PTH1R receptor (red). Cells of notochordal origin (green) and PTH1R positive cells (red) stained abundantly with in NP area of disc tissue co-localization (yellow). d ELISA analysis of cellular cAMP levels in NP cells with PTH treatment. e Immunostaining of IVD sections showing pCREB-positive cells in NP area at different time points with PTH treatment. Scale bars, 100 μm. f Percentage of pCREB-positive cells versus total NP cells with PTH treatment. g Western blot analysis of pCREB levels in NP cells with PTH treatment. h Western blot analysis of PTH1R expression in human NP specimens at different ages. i qRT-PCR of PTH1R mRNA levels in NP tissues from young and older patients shown as fold changes. All data shown as mean ± s.d. *P < 0.05, **P < 0.01. n = 8 per group. Statistical significance was determined by Student's t-test
Fig. 3
Fig. 3
iPTH attenuates disc degeneration by activation of TGF-β. a 3D PPCT images of IVDs with iPTH (1–34) injection of 40 μg·kg-1 daily or vehicle of 18-month old mice, 5 days per week for 8 weeks. Scale bars, 500 μm. b Quantitative analysis of mouse IVD height and IVD volume. c,d 3D PPCT images and quantitative analysis showing thickness distribution of five regions of IVD treated with iPTH or vehicle. Color code indicating degree of thickness from blue (100 μm) to red (800 μm). e, f MRI scan of mouse lumbar spine showing signal intensity of the discs (yellow arrow) of 18-month old mice treated with iPTH or vehicle in comparison with MRI scan of 2-month old mice (e) and quantitative measurements of the disc signal intensity (f). Scale bars, 1 mm. g, h Safranin-O staining images of IVD sections showing NP area of 18-month old mice with iPTH or vehicle (g) and quantitative analysis of cell numbers in NP area and IVD histological scores (h). Scale bars, 100 μm. i, j Immunostaining images of IVD sections showing expression of ACAN, CCN2, and pSmad2/3 positive cells in NP area. Scale bars, 200 μm. k Quantitative analysis of the ACAN-positive, CCN2-positive areas and the number of pSmad2/3 positive cells as a percentage of total IVD area (Ar). l, m ELISA analysis of total and active TGF-β levels from NP tissue in 18-month old mice treated with iPTH or vehicle. n Western blot analysis showing pSmad2 levels on in NP tissues from 18-month-old mice injected with PTH or vehicle in comparison with that of NP tissues from 2-month old. All data are reported as the mean ± s.d. *P < 0.05, **P < 0.01. n = 8 per group. Statistical significance was determined by one-way ANOVA and Student's t-test
Fig. 4
Fig. 4
PTH induces integrin αvβ6 expression to activate latent TGF-β. a Immunostaining images showing various types of integrin expressions in IVD tissue from 18-month-ld mice injected with PTH or vehicle and quantitative analysis (b). Scale bar, 50 μm. c qRT-PCR analysis of the mRNA levels of various integrin in NP tissue from 18-month-old mice injected with PTH or vehicle. Results reported as fold change. d Western blot analysis of integrin β6 expression in NP cells of 18-month-old mice at different time points post PTH injection (PTH1-34, 100 nmol·L-1). e, f Chromatin immunoprecipitation assay with four different potential pCREB binding sites (primers 1, 2, 3 and 4) in the β6 integrin promoter. g pCREB, Integrin αVβ6, pSmad2/3, or Safranin-O staining of IVD sections from an IVD ex vivo compression model of 30-month-old rat with treatment of either vehicle or PTH (PTH1-34, 100 nmol·L-1). Scale bar, 20 μm. h Quantitative analysis of the percentage of pCREB, pSmad2/3 positive cells and the Integrin αVβ6 positive areas as a percentage of total IVD area (Ar) of (g). All data are reported as the mean ± s.d. *P < 0.05. n = 8 per group. Statistical significance was determined by one-way ANOVA and Student's t-test
Fig. 5
Fig. 5
Conditional knockout of PTH1R reduces spinal flexibility. a Immunostaining images showing no PTH1R expression in NP tissue of PTH1R-deficient mice (PTH1R−/−) relative to their wild type littermates (PTH1R+/+). IVDs (top); NP (bottom). Scale bar, 50 μm. b 3D PPCT images of IVD thickness distribution in PTH1R−/− mice at different ages compared to PTH1R+/+ mice. Scale bar, color code indicating the degree of thickness from blue (100 μm) to red (800 μm). c Quantitative analysis of IVD volume in PTH1R−/− mice at different ages compared to PTH1R+/+ mice. d, e Images of the 3D finite element analysis model for testing spine flexibility in PTH1R-deficient mice at 6-month and 12-month-old mice. d The upper surface of L3 and bottom surface of L4 were fixed with rigid bars to mimic the loading of IVD for flexibility measurement. e For each model, the torque loading was applied to simulate motion in four different directions; dorsiflexion, anteflexion, left, and right lateral flexion measurement. f Quantitative analysis of spine flexibility in PTH1R-deficient mice at 6-month and 12-month of age. *P < 0.05, **P < 0.01. n = 8 per group. Statistical significance was determined by one-way ANOVA and Student's t-test. All data are reported as the mean ± s.d.
Fig. 6
Fig. 6
Conditional knockout of PTH1R accelerates disc degeneration. a Schematic representation of the unstable spine model generated by resection of the third and fourth lumbar spinous processes along with the supraspinous and interspinous ligaments from second to fifth lumbar vertebra. b 3D PPCT images of IVD thickness distribution in PTH1R-deficient mice at different time points post-surgery compared with those of PTH1R+/+ mice. c Quantitative analysis of IVD volume of (b). d 3D PPCT images showing IVD thickness distribution in 12-month-old PTH1R-deficient mice or PTH1R+/+ mice treated with iPTH or vehicle. e Quantitative analysis of IVD volume in (d). f Western blot analysis of pSmad2/3, CCN2 and Acan in NP tissue from PTH1R-deficient mice treated with iPTH or vehicle. g qRT-PCR analysis of the mRNA expression levels of CCN2 and Acan in NP tissue from PTH1R-deficient mice or PTH1R+/+ mice treated with iPTH or vehicle. Results reported as fold change. *P < 0.05, **P < 0.01. n = 8 per group. Statistical significance was determined by one-way ANOVA and Student's t-test. All data are reported as the mean ± s.d.
Fig. 7
Fig. 7
PTH stimulates transport of PTH1R to primary cilia of NP cells. a Immunostaining for acetylated α-tubulin (green) and DAPI (blue) showing the length of primary cilia of NP cells from PTH1R-deficient mice or PTH1R+/+ mice. b Quantitative measurements of primary cilia length of (a). c Immunostaining for acetylated α-tubulin or PTH1R showing that PTH-stimulated transport of PTH1R to primary cilia of NP cells. d Quantitative analysis of PTH1R intensity in cilia of (c). e Immunostaining for DAPI, pCREB, or acetylated α-tubulin showing that PTH-stimulated phosphorylation of CREB at primary cilia of NP cells. f Quantitative analysis of pCREB intensity in cilia of (e). g, h Co-immunoprecipitation of cell lysates from NP cells treated with PTH or vehicle using antibody against pCREB and blotted with PTH1R (g) or using antibody against PTH1R and blotted with pCREB (h) showing the interaction between PTH1R and pCREB in the acetylated α-tubulin extracts. *P < 0.05. n = 8 per group. Statistical significance was determined by one-way ANOVA and Student's t-test. All data are reported as the mean ± s.d.
Fig. 8
Fig. 8
Shear stress enhances transport of PTH1R to primary cilia. a Immunostaining for acetylated α-tubulin or PTH1R showing that shear stress stimulated transport of PTH1R to primary cilia of both mouse and human NP cells. b Quantitative analysis of PTH1R intensity in mouse (left) and human (right) cilia of (a). c Immunostaining for acetylated α-tubulin or PTH1R showing that PTH-stimulated transport of PTH1R to primary cilia of NP cells in the presence of compression loading in an IVD ex vivo compression model of 3-month-old and 30-month-old rat. d Immunostaining of the mouse and human NP cells treated with pallidin siRNA or control siRNA for acetylated α-tubulin or PTH1R showing that PTH-stimulated transport of PTH1R to primary cilia of NP cells was inhibited. Scale bar, 10 μm. e Quantitative analysis of PTH1R intensity in mouse (left) and human (right) cilia of (d). f Western blot analysis of PTH-induced pCREB levels in NP cells treated with pallidin siRNA or control siRNA. n = 8 per group
Fig. 9
Fig. 9
Primary cilia regulates PTH signaling in NP cells for disc anabolic activity. a 3D PPCT and immunostaining images showing that PTH effect on IVDs diminished in IFT88−/− mice. PPCT Scale bar, 500 μm. Safranin-O Scale bars, 100 μm. b Quantitative analysis of IVD volume and (c) IVD histological scores of (a). d Western blot analysis of pCREB in NP cells isolated from IFT88−/− or IFT88+/+ mice injected with iPTH or vehicle with or without shear stress. e Immunostaining for pSmad2/3 (brown), CCN2 and ACAN (red) with DAPI (blue) in 2-month IFT88−/− or IFT88+/+ mice treated with PTH or vehicle. pSmad2/3 and CCN2 Scale bar, 50 μm; ACAN Scale bar, 100 μm. f, g and h Quantitative analysis of percentages of pSmad2/3 positive cells in NP area and CCN2 and ACAN-positive area of total IVD area. *P < 0.05. n = 8, per group. Statistical significance was determined by one-way ANOVA and Student's t-test. All data are reported as the mean ± s.d.

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