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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.

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.

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Immunofluorescence of Caveolin-1 in primary notochordal cells in monolayer culture. Immunofluorescence images of the notochordal cell clusters on days 0, 2, 4, 6, 8, and 10 in culture. Scale bar: 50 μm. Nuclear staining (Topro-3) and Caveolin-1 staining are depicted in blue and green, respectively. Caveolin-1 protein was located in intracellular membranes, as suggested by the inhomogeneous appearance of the immunolabeled membrane-embedded marker, and in the notochordal cell membrane.
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Figure 4: Immunofluorescence of Caveolin-1 in primary notochordal cells in monolayer culture. Immunofluorescence images of the notochordal cell clusters on days 0, 2, 4, 6, 8, and 10 in culture. Scale bar: 50 μm. Nuclear staining (Topro-3) and Caveolin-1 staining are depicted in blue and green, respectively. Caveolin-1 protein was located in intracellular membranes, as suggested by the inhomogeneous appearance of the immunolabeled membrane-embedded marker, and in the notochordal cell membrane.

Mentions: The relative gene expression of Caveolin-1 in primary NCs on day 0 in culture was comparable to that in NC-rich NP tissue ex vivo, but thereafter increased significantly on days 2, 4, and 6 and remained stable on days 8 and 10 (see Additional files 7, 9 and 10). Caveolin-1 protein was located in intracellular membranes, as suggested by the inhomogeneous appearance of the immunolabeled membrane-embedded marker, and in the NC cell membrane (Figure 4). The expression of Caveolin-1 protein per cell was significantly higher on day 4 in culture than on days 0 and 2, but levels decreased thereafter on days 6, 8 and 10.


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)

Immunofluorescence of Caveolin-1 in primary notochordal cells in monolayer culture. Immunofluorescence images of the notochordal cell clusters on days 0, 2, 4, 6, 8, and 10 in culture. Scale bar: 50 μm. Nuclear staining (Topro-3) and Caveolin-1 staining are depicted in blue and green, respectively. Caveolin-1 protein was located in intracellular membranes, as suggested by the inhomogeneous appearance of the immunolabeled membrane-embedded marker, and in the notochordal cell membrane.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Immunofluorescence of Caveolin-1 in primary notochordal cells in monolayer culture. Immunofluorescence images of the notochordal cell clusters on days 0, 2, 4, 6, 8, and 10 in culture. Scale bar: 50 μm. Nuclear staining (Topro-3) and Caveolin-1 staining are depicted in blue and green, respectively. Caveolin-1 protein was located in intracellular membranes, as suggested by the inhomogeneous appearance of the immunolabeled membrane-embedded marker, and in the notochordal cell membrane.
Mentions: The relative gene expression of Caveolin-1 in primary NCs on day 0 in culture was comparable to that in NC-rich NP tissue ex vivo, but thereafter increased significantly on days 2, 4, and 6 and remained stable on days 8 and 10 (see Additional files 7, 9 and 10). Caveolin-1 protein was located in intracellular membranes, as suggested by the inhomogeneous appearance of the immunolabeled membrane-embedded marker, and in the NC cell membrane (Figure 4). The expression of Caveolin-1 protein per cell was significantly higher on day 4 in culture than on days 0 and 2, but levels decreased thereafter on days 6, 8 and 10.

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