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Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies.

Smolders LA, Meij BP, Onis D, Riemers FM, Bergknut N, Wubbolts R, Grinwis GC, Houweling M, Groot Koerkamp MJ, van Leenen D, Holstege FC, Hazewinkel HA, Creemers LB, Penning LC, Tryfonidou MA - Arthritis Res. Ther. (2013)

Bottom Line: With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs.NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not.Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

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ABSTRACT

Introduction: Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods: Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results: Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions: Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

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Relative gene expression of relevant target genes. Relative gene expression of Brachyury, Cytokeratin 8, Axin2, Frizzled 1 (Fzd1), Low density lipoprotein receptor-related protein 5 (Lrp5), Wnt7b, Wnt inhibitory factor 1 (Wif1), Integrin linked kinase (Ilk), and Dickkopf homolog 3 (Dkk3) in the notochordal cell-rich (NC-rich), mixed, and chondrocyte-like cell rich (CLC-rich) NP from non-chondrodystrophic (NCD) and chondrodystrophic (CD) dogs (NCD, NC-rich NP was used as reference, set at 1). *Significant difference between NC-rich, mixed, and CLC-rich NP; §significant difference between NCD and CD dogs.
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Figure 2: Relative gene expression of relevant target genes. Relative gene expression of Brachyury, Cytokeratin 8, Axin2, Frizzled 1 (Fzd1), Low density lipoprotein receptor-related protein 5 (Lrp5), Wnt7b, Wnt inhibitory factor 1 (Wif1), Integrin linked kinase (Ilk), and Dickkopf homolog 3 (Dkk3) in the notochordal cell-rich (NC-rich), mixed, and chondrocyte-like cell rich (CLC-rich) NP from non-chondrodystrophic (NCD) and chondrodystrophic (CD) dogs (NCD, NC-rich NP was used as reference, set at 1). *Significant difference between NC-rich, mixed, and CLC-rich NP; §significant difference between NCD and CD dogs.

Mentions: qPCR analysis revealed no significant differences in the expression of the notochordal markers Brachyury and Cytokeratin 8 in the different histopathological stages in non-chondrodystrophic dogs, indicating that the expression of NC marker genes was preserved in all histopathological stages despite significant changes in IVD morphology (Figure 2 and Additional file 3). However, in chondrodystrophic dogs, Brachyury and Cytokeratin 8 gene expression was significantly downregulated in the CLC-rich group compared with the NC-rich and mixed groups; Brachyury and Cytokeratin 8 gene expression was significantly lower in CLC-rich NP from chondrodystrophic dogs than in CLC-rich NP from non-chondrodystrophic dogs. These results suggest that in chondrodystrophic dogs the transition from NC-rich to CLC-rich NP involves a significant downregulation in NC marker gene expression. These results were sustained by the microarray results, showing decreased gene expression in the CLC-rich group compared with the NC-rich group of notochordal markers Cytokeratin 8 and 19 [38,64,65].


Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies.

Smolders LA, Meij BP, Onis D, Riemers FM, Bergknut N, Wubbolts R, Grinwis GC, Houweling M, Groot Koerkamp MJ, van Leenen D, Holstege FC, Hazewinkel HA, Creemers LB, Penning LC, Tryfonidou MA - Arthritis Res. Ther. (2013)

Relative gene expression of relevant target genes. Relative gene expression of Brachyury, Cytokeratin 8, Axin2, Frizzled 1 (Fzd1), Low density lipoprotein receptor-related protein 5 (Lrp5), Wnt7b, Wnt inhibitory factor 1 (Wif1), Integrin linked kinase (Ilk), and Dickkopf homolog 3 (Dkk3) in the notochordal cell-rich (NC-rich), mixed, and chondrocyte-like cell rich (CLC-rich) NP from non-chondrodystrophic (NCD) and chondrodystrophic (CD) dogs (NCD, NC-rich NP was used as reference, set at 1). *Significant difference between NC-rich, mixed, and CLC-rich NP; §significant difference between NCD and CD dogs.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672710&req=5

Figure 2: Relative gene expression of relevant target genes. Relative gene expression of Brachyury, Cytokeratin 8, Axin2, Frizzled 1 (Fzd1), Low density lipoprotein receptor-related protein 5 (Lrp5), Wnt7b, Wnt inhibitory factor 1 (Wif1), Integrin linked kinase (Ilk), and Dickkopf homolog 3 (Dkk3) in the notochordal cell-rich (NC-rich), mixed, and chondrocyte-like cell rich (CLC-rich) NP from non-chondrodystrophic (NCD) and chondrodystrophic (CD) dogs (NCD, NC-rich NP was used as reference, set at 1). *Significant difference between NC-rich, mixed, and CLC-rich NP; §significant difference between NCD and CD dogs.
Mentions: qPCR analysis revealed no significant differences in the expression of the notochordal markers Brachyury and Cytokeratin 8 in the different histopathological stages in non-chondrodystrophic dogs, indicating that the expression of NC marker genes was preserved in all histopathological stages despite significant changes in IVD morphology (Figure 2 and Additional file 3). However, in chondrodystrophic dogs, Brachyury and Cytokeratin 8 gene expression was significantly downregulated in the CLC-rich group compared with the NC-rich and mixed groups; Brachyury and Cytokeratin 8 gene expression was significantly lower in CLC-rich NP from chondrodystrophic dogs than in CLC-rich NP from non-chondrodystrophic dogs. These results suggest that in chondrodystrophic dogs the transition from NC-rich to CLC-rich NP involves a significant downregulation in NC marker gene expression. These results were sustained by the microarray results, showing decreased gene expression in the CLC-rich group compared with the NC-rich group of notochordal markers Cytokeratin 8 and 19 [38,64,65].

Bottom Line: With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs.NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not.Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

View Article: PubMed Central - HTML - PubMed

ABSTRACT

Introduction: Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.

Methods: Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.

Results: Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.

Conclusions: Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.

Show MeSH
Related in: MedlinePlus