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PKCε signalling activates ERK1/2, and regulates aggrecan, ADAMTS5, and miR377 gene expression in human nucleus pulposus cells.

Tsirimonaki E, Fedonidis C, Pneumaticos SG, Tragas AA, Michalopoulos I, Mangoura D - PLoS ONE (2013)

Bottom Line: Focusing on the signalling of PKCε, an isoform that may confer protection against degeneration, we found that activation with the PKCε-specific activator small peptide ψεRACK led sequentially to a prolonged activation of ERK1/2, increased abundance of the early gene products ATF, CREB1, and Fos with concurrent silencing of transcription for Ki67, and increases in mRNA expression for aggrecan.More importantly, ψεRACK induced upregulation of hsa-miR-377 expression, coupled to decreases in ADAMTS5 and cleaved aggrecan.Moreover, this pathway should be considered as a target for understanding the molecular mechanism of IVD degeneration and for therapeutic restoration of degenerated disks.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Foundation of the Academy of Athens, Athens, Greece.

ABSTRACT
The protein kinase C (PKC) signaling, a major regulator of chondrocytic differentiation, has been also implicated in pathological extracellular matrix remodeling, and here we investigate the mechanism of PKCε-dependent regulation of the chondrocytic phenotype in human nucleus pulposus (NP) cells derived from herniated disks. NP cells from each donor were successfully propagated for 25+ culture passages, with remarkable tolerance to repeated freeze-and-thaw cycles throughout long-term culturing. More specifically, after an initial downregulation of COL2A1, a stable chondrocytic phenotype was attested by the levels of mRNA expression for aggrecan, biglycan, fibromodulin, and lumican, while higher expression of SOX-trio and Patched-1 witnessed further differentiation potential. NP cells in culture also exhibited a stable molecular profile of PKC isoforms: throughout patient samples and passages, mRNAs for PKC α, δ, ε, ζ, η, ι, and µ were steadily detected, whereas β, γ, and θ were not. Focusing on the signalling of PKCε, an isoform that may confer protection against degeneration, we found that activation with the PKCε-specific activator small peptide ψεRACK led sequentially to a prolonged activation of ERK1/2, increased abundance of the early gene products ATF, CREB1, and Fos with concurrent silencing of transcription for Ki67, and increases in mRNA expression for aggrecan. More importantly, ψεRACK induced upregulation of hsa-miR-377 expression, coupled to decreases in ADAMTS5 and cleaved aggrecan. Therefore, PKCε activation in late passage NP cells may represent a molecular basis for aggrecan availability, as part of an PKCε/ERK/CREB/AP-1-dependent transcriptional program that includes upregulation of both chondrogenic genes and microRNAs. Moreover, this pathway should be considered as a target for understanding the molecular mechanism of IVD degeneration and for therapeutic restoration of degenerated disks.

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PKC isoform expression in human NP cells of early or late passage.Representative images of RT-PCR results for PKC isoform profiling demonstrate an indistinguishable profile amongst various NP samples and cell passages (NP11, P0; NP10, P23; NP1, at P3 and P13), and AC cells (AC, P17) in culture, with the exception of the ζ isoform, which appears at a slightly different molecular size (402 versus 321 - arrowheads versus arrows). The MPNST 90-8 cell line which expresses all the PKC isoforms but βI, served as a positive control. For each sample, the expression of each gene was examined using identical amounts of cDNA template, as for the housekeeping GAPDH (shown at the bottom row). The ladder used was the GeneRuler 100 bp (NEB), where the intense band corresponds to the 500 bp size.
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pone-0082045-g004: PKC isoform expression in human NP cells of early or late passage.Representative images of RT-PCR results for PKC isoform profiling demonstrate an indistinguishable profile amongst various NP samples and cell passages (NP11, P0; NP10, P23; NP1, at P3 and P13), and AC cells (AC, P17) in culture, with the exception of the ζ isoform, which appears at a slightly different molecular size (402 versus 321 - arrowheads versus arrows). The MPNST 90-8 cell line which expresses all the PKC isoforms but βI, served as a positive control. For each sample, the expression of each gene was examined using identical amounts of cDNA template, as for the housekeeping GAPDH (shown at the bottom row). The ladder used was the GeneRuler 100 bp (NEB), where the intense band corresponds to the 500 bp size.

Mentions: PKCs have important role in the proliferation, differentiation, or apoptosis of chondrocytes, yet, their molecular profile has not been previously addressed in human NP cells. Thus, we used semi-quantitative PCR to detect all known PKC isoforms in human NP-derived cultures, using as reference AC cells as well as the 90-8 cell line that expresses all PKCs, but βI (Figure 4). Throughout patient samples, mRNAs for PKC α, δ, ε, ζ, η, ι, and µ were steadily detected, whereas β, γ, and θ were not. This pattern was stable and was retained in all culture passages, either early (NP11, P0; NP1, P3) or late (NP10, P23; NP1, P13). An intriguing observation was that, upon subculturing, the ζ isoform consistently appeared at a slightly different molecular size (402 versus 321, arrowheads versus arrow), possibly due to alternative splicing, and this eventually became the prominent transcript. AC cells presented with an almost identical molecular PKC phenotype to that of NP cells, expressing the same isoforms at comparable ratios.


PKCε signalling activates ERK1/2, and regulates aggrecan, ADAMTS5, and miR377 gene expression in human nucleus pulposus cells.

Tsirimonaki E, Fedonidis C, Pneumaticos SG, Tragas AA, Michalopoulos I, Mangoura D - PLoS ONE (2013)

PKC isoform expression in human NP cells of early or late passage.Representative images of RT-PCR results for PKC isoform profiling demonstrate an indistinguishable profile amongst various NP samples and cell passages (NP11, P0; NP10, P23; NP1, at P3 and P13), and AC cells (AC, P17) in culture, with the exception of the ζ isoform, which appears at a slightly different molecular size (402 versus 321 - arrowheads versus arrows). The MPNST 90-8 cell line which expresses all the PKC isoforms but βI, served as a positive control. For each sample, the expression of each gene was examined using identical amounts of cDNA template, as for the housekeeping GAPDH (shown at the bottom row). The ladder used was the GeneRuler 100 bp (NEB), where the intense band corresponds to the 500 bp size.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3842981&req=5

pone-0082045-g004: PKC isoform expression in human NP cells of early or late passage.Representative images of RT-PCR results for PKC isoform profiling demonstrate an indistinguishable profile amongst various NP samples and cell passages (NP11, P0; NP10, P23; NP1, at P3 and P13), and AC cells (AC, P17) in culture, with the exception of the ζ isoform, which appears at a slightly different molecular size (402 versus 321 - arrowheads versus arrows). The MPNST 90-8 cell line which expresses all the PKC isoforms but βI, served as a positive control. For each sample, the expression of each gene was examined using identical amounts of cDNA template, as for the housekeeping GAPDH (shown at the bottom row). The ladder used was the GeneRuler 100 bp (NEB), where the intense band corresponds to the 500 bp size.
Mentions: PKCs have important role in the proliferation, differentiation, or apoptosis of chondrocytes, yet, their molecular profile has not been previously addressed in human NP cells. Thus, we used semi-quantitative PCR to detect all known PKC isoforms in human NP-derived cultures, using as reference AC cells as well as the 90-8 cell line that expresses all PKCs, but βI (Figure 4). Throughout patient samples, mRNAs for PKC α, δ, ε, ζ, η, ι, and µ were steadily detected, whereas β, γ, and θ were not. This pattern was stable and was retained in all culture passages, either early (NP11, P0; NP1, P3) or late (NP10, P23; NP1, P13). An intriguing observation was that, upon subculturing, the ζ isoform consistently appeared at a slightly different molecular size (402 versus 321, arrowheads versus arrow), possibly due to alternative splicing, and this eventually became the prominent transcript. AC cells presented with an almost identical molecular PKC phenotype to that of NP cells, expressing the same isoforms at comparable ratios.

Bottom Line: Focusing on the signalling of PKCε, an isoform that may confer protection against degeneration, we found that activation with the PKCε-specific activator small peptide ψεRACK led sequentially to a prolonged activation of ERK1/2, increased abundance of the early gene products ATF, CREB1, and Fos with concurrent silencing of transcription for Ki67, and increases in mRNA expression for aggrecan.More importantly, ψεRACK induced upregulation of hsa-miR-377 expression, coupled to decreases in ADAMTS5 and cleaved aggrecan.Moreover, this pathway should be considered as a target for understanding the molecular mechanism of IVD degeneration and for therapeutic restoration of degenerated disks.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Research Foundation of the Academy of Athens, Athens, Greece.

ABSTRACT
The protein kinase C (PKC) signaling, a major regulator of chondrocytic differentiation, has been also implicated in pathological extracellular matrix remodeling, and here we investigate the mechanism of PKCε-dependent regulation of the chondrocytic phenotype in human nucleus pulposus (NP) cells derived from herniated disks. NP cells from each donor were successfully propagated for 25+ culture passages, with remarkable tolerance to repeated freeze-and-thaw cycles throughout long-term culturing. More specifically, after an initial downregulation of COL2A1, a stable chondrocytic phenotype was attested by the levels of mRNA expression for aggrecan, biglycan, fibromodulin, and lumican, while higher expression of SOX-trio and Patched-1 witnessed further differentiation potential. NP cells in culture also exhibited a stable molecular profile of PKC isoforms: throughout patient samples and passages, mRNAs for PKC α, δ, ε, ζ, η, ι, and µ were steadily detected, whereas β, γ, and θ were not. Focusing on the signalling of PKCε, an isoform that may confer protection against degeneration, we found that activation with the PKCε-specific activator small peptide ψεRACK led sequentially to a prolonged activation of ERK1/2, increased abundance of the early gene products ATF, CREB1, and Fos with concurrent silencing of transcription for Ki67, and increases in mRNA expression for aggrecan. More importantly, ψεRACK induced upregulation of hsa-miR-377 expression, coupled to decreases in ADAMTS5 and cleaved aggrecan. Therefore, PKCε activation in late passage NP cells may represent a molecular basis for aggrecan availability, as part of an PKCε/ERK/CREB/AP-1-dependent transcriptional program that includes upregulation of both chondrogenic genes and microRNAs. Moreover, this pathway should be considered as a target for understanding the molecular mechanism of IVD degeneration and for therapeutic restoration of degenerated disks.

Show MeSH
Related in: MedlinePlus