Role of Prostaglandin I2 in the Gene Expression Induced by  Mechanical Stress in Spinal Ligament Cells Derived from Patients with  Ossification of the Posterior Longitudinal Ligament

Abstract Background: The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remains to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy associated gene expression was correlated with the expression of osteogenic differentiation genes. Methods: Expression of autophagy associated genes was detected in 37 samples from 21 OPLL patients and 16 non-OPLL patients. The correlation of autophagy associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson’s correlation. The expression of autophagy associated genes of ligament fibroblasts was assessed by reverse transcription-quantitative Polymerase Chain Reaction (RT-qPCR), western blotting and immunofluorescence. The incidence of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts. Results: In clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) (r=0.8233, P<0.001), alkaline phosphatase, biomineralization associated (ALP) (r=0.7821, P<0.001) and collagen type 1 (COL 1) (r=0.6078, P=0.001). Consistently, the upregulation of autophagy associated genes in ligament fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The incidence of autophagy was also increased in ligament fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP and COL 1 by 63.2% (P<0.01), 52% (P<0.01) and 53.2% (P<0.01) in ligament fibroblasts from patients with OPLL, respectively. Conclusions: Beclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts, and promoted the development of OPLL.

Download Full-text

Alleviation of murine osteoarthritis by deletion of the focal adhesion mechanosensitive adapter, Hic-5

Scientific Reports ◽

10.1038/s41598-019-52301-7 ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Aya Miyauchi ◽

Joo-ri Kim-Kaneyama ◽

Xiao-Feng Lei ◽

Song Ho Chang ◽

Taku Saito ◽

...

Keyword(s):

Gene Expression ◽

Articular Cartilage ◽

Mechanical Stress ◽

Focal Adhesion ◽

Catabolic Gene ◽

Catabolic Genes ◽

A Disintegrin And Metalloproteinase ◽

Factor Α

Abstract Excessive mechanical stress is a major cause of knee osteoarthritis. However, the mechanism by which the mechanical stress begets osteoarthritis development remains elusive. Hydrogen peroxide-inducible clone-5 (Hic-5; TGFβ1i1), a TGF-β inducible focal adhesion adaptor, has previously been reported as a mediator of mechanotransduction. In this study, we analyzed the in vivo function of Hic-5 in development of osteoarthritis, and found that mice lacking Hic-5 showed a significant reduction in development of osteoarthritis in the knee. Furthermore, we found reduced expression of catabolic genes, such as metalloproteinase-13 and a disintegrin and metalloproteinase with thrombospondin type 1 motif 5 in osteoarthritic lesions in mice lacking Hic-5. During osteoarthritis development, Hic-5 is detected in chondrocytes of articular cartilage. To investigate the role of Hic-5 in chondrocytes, we isolated chondrocytes from articular cartilage of wild type and Hic-5-deficient mice. In these primary cultured chondrocytes, Hic-5 deficiency resulted in suppression of catabolic gene expression induced by osteoarthritis-related cytokines such as tumor necrosis factor α and interleukin 1β. Furthermore, Hic-5 deficiency in chondrocytes suppressed catabolic gene expression induced by mechanical stress. Revealing the regulation of chondrocyte catabolism by Hic-5 contributes to understanding the pathophysiology of osteoarthritis induced by mechanical stress.

Download Full-text

Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-020-02017-6 ◽

2020 ◽

Vol 15 (1) ◽

Author(s):

Yuehua Yang ◽

Zunwen Lin ◽

Jiangwei Chen ◽

Sheng Ding ◽

Weiwei Mao ◽

...

Keyword(s):

Gene Expression ◽

Osteogenic Differentiation ◽

Western Blotting ◽

Molecular Mechanisms ◽

Quantitative Polymerase Chain Reaction ◽

Pearson Correlation ◽

Posterior Longitudinal Ligament ◽

Interfering Rna ◽

Collagen Type 1 ◽

Spinal Ligament

Abstract Background The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remain to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy-associated gene expression was correlated with the expression of osteogenic differentiation genes. Methods Expression of autophagy-associated genes was detected in 37 samples from 21 OPLL patients and 16 non-OPLL patients. The correlation of autophagy-associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson’s correlation. The expression of autophagy-associated genes of ligament fibroblasts was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunofluorescence. The incidence of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts. Results In clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) (r = 0.8233, P < 0.001), alkaline phosphatase, biomineralization associated (ALP) (r = 0.7821, P < 0.001), and collagen type 1 (COL 1) (r = 0.6078, P = 0.001). Consistently, the upregulation of autophagy-associated genes in ligament fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The incidence of autophagy was also increased in ligament fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP, and COL 1 by 63.2% (P < 0.01), 52% (P < 0.01), and 53.2% (P < 0.01) in ligament fibroblasts from patients with OPLL, respectively. Conclusions Beclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts and promoted the development of OPLL.

Download Full-text

Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament

10.21203/rs.3.rs-22864/v1 ◽

2020 ◽

Author(s):

yuehua yang ◽

zunwen Lin ◽

Jiangwei Chen ◽

Sheng Ding ◽

Weiwei Mao ◽

...

Keyword(s):

Gene Expression ◽

Osteogenic Differentiation ◽

Western Blotting ◽

Molecular Mechanisms ◽

Quantitative Polymerase Chain Reaction ◽

Pearson Correlation ◽

Posterior Longitudinal Ligament ◽

Interfering Rna ◽

Collagen Type 1 ◽

Spinal Ligament

Abstract Background The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remains to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy associated gene expression was correlated with the expression of osteogenic differentiation genes. Methods Expression of autophagy associated genes was detected in 21 samples from patients with OPLL patients and 16 non-OPLL patients. The correlation of autophagy associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson’s correlation. The expression of autophagy associated genes of fibroblasts was assessed by reverse transcription-quantitative Polymerase Chain Reaction (RT-qPCR), western blotting and immunofluorescence. The rate of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts. Results In clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) (r = 0.8233, P < 0.001), alkaline phosphatase, biomineralization associated (ALP) (r = 0.7821, P < 0.001) and collagen type 1 (COL I) (r = 0.6078, P = 0.001). Consistently, the upregulation of autophagy associated genes in fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The rate of autophagy was also increased in fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP and COL I by 63.2% (P < 0.01), 52% (P < 0.01) and 53.2% (P < 0.01) in ligament fibroblasts from patients with OPLL, respectively. Conclusions Beclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts, and promoted the development of OPLL.

Download Full-text

Role of small nuclear RNAs in eukaryotic gene expression

Essays in Biochemistry ◽

10.1042/bse0540079 ◽

2013 ◽

Vol 54 ◽

pp. 79-90 ◽

Cited By ~ 34

Author(s):

Saba Valadkhan ◽

Lalith S. Gunawardane

Keyword(s):

Gene Expression ◽

Protein Interactions ◽

Rna Stability ◽

Splice Sites ◽

Branch Site ◽

Small Nuclear Rnas ◽

Eukaryotic Gene Expression ◽

Eukaryotic Gene ◽

Rna Biogenesis

Eukaryotic cells contain small, highly abundant, nuclear-localized non-coding RNAs [snRNAs (small nuclear RNAs)] which play important roles in splicing of introns from primary genomic transcripts. Through a combination of RNA–RNA and RNA–protein interactions, two of the snRNPs, U1 and U2, recognize the splice sites and the branch site of introns. A complex remodelling of RNA–RNA and protein-based interactions follows, resulting in the assembly of catalytically competent spliceosomes, in which the snRNAs and their bound proteins play central roles. This process involves formation of extensive base-pairing interactions between U2 and U6, U6 and the 5′ splice site, and U5 and the exonic sequences immediately adjacent to the 5′ and 3′ splice sites. Thus RNA–RNA interactions involving U2, U5 and U6 help position the reacting groups of the first and second steps of splicing. In addition, U6 is also thought to participate in formation of the spliceosomal active site. Furthermore, emerging evidence suggests additional roles for snRNAs in regulation of various aspects of RNA biogenesis, from transcription to polyadenylation and RNA stability. These snRNP-mediated regulatory roles probably serve to ensure the co-ordination of the different processes involved in biogenesis of RNAs and point to the central importance of snRNAs in eukaryotic gene expression.

Download Full-text