Ciliary parathyroid hormone signaling activates transforming growth factor-β to maintain intervertebral disc homeostasis during aging

© 2018 The Author(s). 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

Mechanical overloading-induced miR-325-3p reduction promoted chondrocyte senescence and exacerbated facet joint degeneration

Abstract Objective Lumbar facet joint (LFJ) degeneration is one of the main causes of low back pain (LBP). Mechanical stress leads to the exacerbation of LFJ degeneration, but the underlying mechanism remains unknown. This study was intended to investigate the mechanism of LFJ degeneration induced by mechanical stress. Methods Here, mice primary chondrocytes were used to screen for key microRNAs induced by mechanical overloading. SA-β-gal staining, qRT-PCR, western blot, and histochemical staining were applied to detect chondrocyte senescence in vitro and in vivo. We also used a dual-luciferase report assay to examine the targeting relationship of miRNA-325-3p (miR-325-3p) and Trp53. By using NSC-207895, a p53 activator, we investigated whether miR-325-3p down-regulated trp53 expression to reduce chondrocyte senescence. A mice bipedal standing model was performed to induce LFJ osteoarthritis. Adeno-associated virus (AAV) was intraarticularly injected to evaluate the effect of miR-325-3p on facet joint degeneration. Results We observed chondrocyte senescence both in human LFJ osteoarthritis tissues and mice LFJ after bipedally standing for 10 weeks. Mechanical overloading could promote chondrocyte senescence and senescence-associated secretory phenotype (SASP) expression. MicroRNA-array analysis identified that miR-325-3p was obviously decreased after mechanical overloading, which was further validated by fluorescence in situ hybridization (FISH) in vivo. Dual-luciferase report assay showed that miR-325-3p directly targeted Trp53 to down-regulated its expression. MiR-325-3p rescued chondrocyte senescence in vitro, however, NSC-207895 reduced this effect by activating the p53/p21 pathway. Intraarticular injection of AAV expressing miR-325-3p decreased chondrocyte senescence and alleviated LFJ degeneration in vivo. Conclusion Our findings suggested that mechanical overloading could reduce the expression of miR-325-3p, which in turn activated the p53/p21 pathway to promote chondrocyte senescence and deteriorated LFJ degeneration, which may provide a promising therapeutic strategy for LFJ degeneration

Effect of miR-21 on capillary network formation of HUVECs.

<p>(A) After transfection with miR-21 mimics, miR-21 mimic negative control, antagomir-21 or antagomir-21 negative control, HUVECs were exposed to OGD and then trypsinized and seeded onto Matrigel. Cells were incubated and allowed to form networks for 12 h, then stained with calcein-AM (Green) for 15 min. Bar = 200 μm. (B) Comparison of the number of branch nodes in the treatment groups relative to respective negative control groups at 12 h. Values represent means±S.D; n = 3; *p<0.05.</p

The Angiogenic Effect of microRNA-21 Targeting TIMP3 through the Regulation of MMP2 and MMP9

<div><p>microRNAs are a novel set of small, non-protein-coding nucleotide RNAs that negatively regulate the expression of target mRNAs. miRNA-21 is a microRNA that is highly enriched in endothelial cells. miRNA-21 has been shown to be a potential pro-angiogenic factor in some biological systems. Our previous study showed that the expression of miRNA-21 was up-regulated after spinal cord injury. However, the effect of miRNA-21 on angiogenesis in the spinal cord was unclear. In this study, to understand the role of miRNA-21 on injured endothelial cells exclusively, an oxygen and glucose deprivation model of endothelial cells was constructed, and the up-regulation of miRNA-21 was discovered in this model. An increased level of miRNA-21 by mimics promoted the survival, migration and tube formation of endothelial cells, which simultaneously inhibited tissue inhibitor of metalloproteinase-3 (TIMP3) expression and promoted matrix metalloproteinase-2 (MMP2) and matrix metalloproteinase-9 (MMP9) expression and secretion. A decreased level of miRNA-21 by antagomir exerted an opposite effect. As is well known, survival, migration and tube formation of endothelial cells are necessary prerequisites for angiogenesis after injury. TIMP3 was validated as a direct target of miRNA-21 by dual-luciferase reporter assay. Silencing with small interfering RNA against TIMP3 promoted tube formation and increased MMP2 and MMP9 expression at the protein level. <i>In vivo</i>, we found that decreased levels of miRNA-21 inhibited angiogenesis after spinal cord injury in rats using synchrotron radiation micro-computed tomography. In summary, these findings suggest that miRNA-21 has a protective effect on angiogenesis by reducing cell death and promoting cell survival, migration and tube formation via partially targeting the TIMP3 by potentially regulating MMP2 and MMP9. TIMP3 is a functional target gene. Identifying the role of miRNA-21 in the protection of angiogenesis might offer a novel therapeutic target for secondary spinal cord injury, in which angiogenesis is indispensable.</p></div

Effect of miR-21 on TIMP3, MMP2 and MMP9 expression.

<p>HUVECs were transfected with miR-21 mimics, miR-21 mimic negative control, antagomir-21 or antagomir-21 negative control and exposed to OGD for 4 h and then recovered for 24h. (A-C) qRT-PCR reveals the different mRNA expression levels of TIMP3, MMP2 and MMP9 between the treatment groups and respective negative groups. Values represent means±S.D; n = 3; *p<0.05. (D) Western blot bands show the protein expression of TIMP3, MMP2 and MMP9. (E-G) Comparison of TIMP3, MMP2 and MMP9 protein expression between the treatment groups and respective negative groups. Values represent means±S.D; n = 3; *p<0.05.</p

3D vascular morphologic changes and quantitative analysis after injury <i>in vivo</i>.

<p>(A) 3D vascular morphology changes after SCI in rats treated with antagomir-21 or antagomir-21 negative control. (B) Effect of antagomir-21 on the density of bifurcation points (DBP). (C) Effect of antagomir-21 on the sum of vascular volume (SVV). These results revealed that the down-regulation of miR-21 was adverse to remodeling of the vasculature 3,7 or 14 days after SCI in rats (Bar = 500 μm, Values represent means±S.D; n = 3; *p<0.05).</p

Regulation of miR-21 expression in HUVECs.

<p>(A) miR-21 expression of HUVECs exposed to 4 h OGD and recovered for 24 h increased significantly compared with cells without OGD. (B) Evaluation of transfection efficiency by qRT-PCR. After exposure to 4 h OGD and recovery for 24 h, miR-21 mimics increased the expression of miR-21 compared with miR-21 mimic negative control; antagomir-21 decreased the expression of miR-21 compared with antagomir-21 negative control. Values represent means±S.D; n = 3; ^#p<0.01.</p