Limits...
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.

Show MeSH

Related in: MedlinePlus

Caveolin-1 expression in the course of early intervertebral disc degeneration. (A) Caveolin-1 gene expression and (B) proportion of the NP surface area that stained for Caveolin-1, and mean gray value for Caveolin-1 protein expression in the notochordal cell (NC)-rich, mixed, and chondrocyte-like cell (CLC)-rich nucleus pulposus (NP) from non-chondrodystrophic (NCD) and chondrodystrophic (CD) dogs. *Significant difference between degeneration stages; §significant difference between NCD and CD dogs. The proportion of the NP surface area that stained for caveolin-1 was not divided into NCD and CD dogs because no significant differences were found between breed types. (C) Typical examples of NP samples stained for Caveolin-1, showing the NC-rich NP, mixed cell population NP with NCs and CLCs, and the CLC-rich NP. In the NC-rich and mixed groups, membranous (arrow) and cytoplasmic (arrowhead) staining can be observed. Note that Caveolin-1 staining is not observed in CLCs. (D) Immunofluorescent staining of the NC-rich NP for the proteins Caveolin-1 (green) and β-catenin (red), and for DNA (blue). Region of interest (ROI) lines drawn across cell bodies were used to generate profile intensity plots (right) for the signal intensity of the Caveolin-1 (green), β-catenin (red), and Topro-3 (blue). The signal intensity peaks for Caveolin-1 correspond with the signal peaks of β-catenin at the cell membrane (located at the same distance of the ROI line), indicating colocalization of these proteins. Also, the central β-catenin signal peaks correspond with the Topro-3 signal peaks, indicating nuclear localization of β-catenin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3672710&req=5

Figure 3: Caveolin-1 expression in the course of early intervertebral disc degeneration. (A) Caveolin-1 gene expression and (B) proportion of the NP surface area that stained for Caveolin-1, and mean gray value for Caveolin-1 protein expression in the notochordal cell (NC)-rich, mixed, and chondrocyte-like cell (CLC)-rich nucleus pulposus (NP) from non-chondrodystrophic (NCD) and chondrodystrophic (CD) dogs. *Significant difference between degeneration stages; §significant difference between NCD and CD dogs. The proportion of the NP surface area that stained for caveolin-1 was not divided into NCD and CD dogs because no significant differences were found between breed types. (C) Typical examples of NP samples stained for Caveolin-1, showing the NC-rich NP, mixed cell population NP with NCs and CLCs, and the CLC-rich NP. In the NC-rich and mixed groups, membranous (arrow) and cytoplasmic (arrowhead) staining can be observed. Note that Caveolin-1 staining is not observed in CLCs. (D) Immunofluorescent staining of the NC-rich NP for the proteins Caveolin-1 (green) and β-catenin (red), and for DNA (blue). Region of interest (ROI) lines drawn across cell bodies were used to generate profile intensity plots (right) for the signal intensity of the Caveolin-1 (green), β-catenin (red), and Topro-3 (blue). The signal intensity peaks for Caveolin-1 correspond with the signal peaks of β-catenin at the cell membrane (located at the same distance of the ROI line), indicating colocalization of these proteins. Also, the central β-catenin signal peaks correspond with the Topro-3 signal peaks, indicating nuclear localization of β-catenin.

Mentions: In chondrodystrophic dogs, the gene expression of Caveolin-1 was significantly downregulated in the CLC-rich compared with the NC-rich and mixed groups (Figure 3); no significant changes were found in non-chondrodystrophic dogs. The gene expression of Caveolin-1 in the CLC-rich NP was significantly lower in chondrodystrophic dogs than in non-chondrodystrophic dogs.


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)

Caveolin-1 expression in the course of early intervertebral disc degeneration. (A) Caveolin-1 gene expression and (B) proportion of the NP surface area that stained for Caveolin-1, and mean gray value for Caveolin-1 protein expression in the notochordal cell (NC)-rich, mixed, and chondrocyte-like cell (CLC)-rich nucleus pulposus (NP) from non-chondrodystrophic (NCD) and chondrodystrophic (CD) dogs. *Significant difference between degeneration stages; §significant difference between NCD and CD dogs. The proportion of the NP surface area that stained for caveolin-1 was not divided into NCD and CD dogs because no significant differences were found between breed types. (C) Typical examples of NP samples stained for Caveolin-1, showing the NC-rich NP, mixed cell population NP with NCs and CLCs, and the CLC-rich NP. In the NC-rich and mixed groups, membranous (arrow) and cytoplasmic (arrowhead) staining can be observed. Note that Caveolin-1 staining is not observed in CLCs. (D) Immunofluorescent staining of the NC-rich NP for the proteins Caveolin-1 (green) and β-catenin (red), and for DNA (blue). Region of interest (ROI) lines drawn across cell bodies were used to generate profile intensity plots (right) for the signal intensity of the Caveolin-1 (green), β-catenin (red), and Topro-3 (blue). The signal intensity peaks for Caveolin-1 correspond with the signal peaks of β-catenin at the cell membrane (located at the same distance of the ROI line), indicating colocalization of these proteins. Also, the central β-catenin signal peaks correspond with the Topro-3 signal peaks, indicating nuclear localization of β-catenin.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Caveolin-1 expression in the course of early intervertebral disc degeneration. (A) Caveolin-1 gene expression and (B) proportion of the NP surface area that stained for Caveolin-1, and mean gray value for Caveolin-1 protein expression in the notochordal cell (NC)-rich, mixed, and chondrocyte-like cell (CLC)-rich nucleus pulposus (NP) from non-chondrodystrophic (NCD) and chondrodystrophic (CD) dogs. *Significant difference between degeneration stages; §significant difference between NCD and CD dogs. The proportion of the NP surface area that stained for caveolin-1 was not divided into NCD and CD dogs because no significant differences were found between breed types. (C) Typical examples of NP samples stained for Caveolin-1, showing the NC-rich NP, mixed cell population NP with NCs and CLCs, and the CLC-rich NP. In the NC-rich and mixed groups, membranous (arrow) and cytoplasmic (arrowhead) staining can be observed. Note that Caveolin-1 staining is not observed in CLCs. (D) Immunofluorescent staining of the NC-rich NP for the proteins Caveolin-1 (green) and β-catenin (red), and for DNA (blue). Region of interest (ROI) lines drawn across cell bodies were used to generate profile intensity plots (right) for the signal intensity of the Caveolin-1 (green), β-catenin (red), and Topro-3 (blue). The signal intensity peaks for Caveolin-1 correspond with the signal peaks of β-catenin at the cell membrane (located at the same distance of the ROI line), indicating colocalization of these proteins. Also, the central β-catenin signal peaks correspond with the Topro-3 signal peaks, indicating nuclear localization of β-catenin.
Mentions: In chondrodystrophic dogs, the gene expression of Caveolin-1 was significantly downregulated in the CLC-rich compared with the NC-rich and mixed groups (Figure 3); no significant changes were found in non-chondrodystrophic dogs. The gene expression of Caveolin-1 in the CLC-rich NP was significantly lower in chondrodystrophic dogs than in non-chondrodystrophic dogs.

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