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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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Morphologies and growth rate of human NP cells in culture.(A) Phase contrast micrographs (black-and-white panels), and immunocytochemical decoration of mitotic spindles (microtubules visualized with β-tubulin-rhodamine and chromosomes with Hoechst 33258, upper row middle panel) and of the actin-binding protein tropomyosin (visualized with fluorescein, last two panels) show gains in numbers and a stable morphology throughout patients samples and passaging, even after freeze-thawing cycles (examples are given for samples NP6, 9, 11, 13, and 14; arrows and arrowheads are described in the text). NP or AC cells grew without contact inhibition, while NP cells could form cellular hives in early passages (triple arrow in NP14 P6). Large NP cells, accounting for ~1%, were observed in all samples (arrowheads in NP7 P5 and in NP14 P6); bar=10 μm in phase contrast photographs. (B) Cell number (logarithmic scale, y-axis ) versus days in culture (linear scale, x-axis): NP cells had impressive growth rates in early passages with similar curves amongst patient samples (data shown are the average of all samples); arrows indicate times of cell culture propagation through passaging.
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pone-0082045-g001: Morphologies and growth rate of human NP cells in culture.(A) Phase contrast micrographs (black-and-white panels), and immunocytochemical decoration of mitotic spindles (microtubules visualized with β-tubulin-rhodamine and chromosomes with Hoechst 33258, upper row middle panel) and of the actin-binding protein tropomyosin (visualized with fluorescein, last two panels) show gains in numbers and a stable morphology throughout patients samples and passaging, even after freeze-thawing cycles (examples are given for samples NP6, 9, 11, 13, and 14; arrows and arrowheads are described in the text). NP or AC cells grew without contact inhibition, while NP cells could form cellular hives in early passages (triple arrow in NP14 P6). Large NP cells, accounting for ~1%, were observed in all samples (arrowheads in NP7 P5 and in NP14 P6); bar=10 μm in phase contrast photographs. (B) Cell number (logarithmic scale, y-axis ) versus days in culture (linear scale, x-axis): NP cells had impressive growth rates in early passages with similar curves amongst patient samples (data shown are the average of all samples); arrows indicate times of cell culture propagation through passaging.

Mentions: In order to attain sufficient NP cell numbers to study mechanisms of differentiation, we reasoned that NP cells should be allowed to first partially de-differentiate and thus gain in proliferation capacity. In pilot experiments we established the conditions, described in Methods, which permitted both cell cultures from every patient to be established and propagation of these cultures for up to 30 passages. When freshly plated, cells exhibited spherical shapes as they underwent cell division (Figure 1A, NP6 Passage 0 (P0), culture day 3 (C3), arrowhead, and NP8 P0, C2), before spreading onto the substrate (double arrows, NP6 P0, C3). Over the next days, cells acquired a polygonal body with several processes radiating away from the cell body, thus assuming an irregular stellate appearance (Figure 1A, NP6, P0, C12); such NP cell morphology with several lamellipodia with filopodia has been previously reported for human (e.g., 16,63) or bovine NP cell cultures, while in cell cultures established from explants [11] or through mechanical dissociation (filters) [17] lesser lamellipodia, often without filopodia, were observed at least during early passages. Cells grew exponentially during the first few passages (Figure 1B), and proliferation rate remained high up to passages 10-12, even after propagation (repeated passages and cycles of freeze-and-thaw) that covered cell maintenance periods (culture plus storage) of over two years in total. Cell morphology was also very similar over different patient samples e.g., Figure 1A, NP6 P0 at C12, and NP9 P7, NP13 P17, and NP11 P28 (all at C10). It is noteworthy that also up to passage 10-12, about 1-2% of the cell population consisted of large body cells with intricate process outgrowth patterns (Figure 1A, arrowheads in NP7 P5 and NP14 P6). Beyond that, cell proliferation slowed down, yet cells kept their stellate appearance and responded to plating on chitosan with a rounded morphology (data not shown). In general, cells grew without contact inhibition and we often observed cellular hives (Figure 1A, triple arrow) at least up to P10. Contact inhibition was also absent in cultures of human articular chondrocytes (AC), which were used as reference of a chondrocytic phenotype; these cells, however, grew without hive formation (Figure 1A, AC P5).


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)

Morphologies and growth rate of human NP cells in culture.(A) Phase contrast micrographs (black-and-white panels), and immunocytochemical decoration of mitotic spindles (microtubules visualized with β-tubulin-rhodamine and chromosomes with Hoechst 33258, upper row middle panel) and of the actin-binding protein tropomyosin (visualized with fluorescein, last two panels) show gains in numbers and a stable morphology throughout patients samples and passaging, even after freeze-thawing cycles (examples are given for samples NP6, 9, 11, 13, and 14; arrows and arrowheads are described in the text). NP or AC cells grew without contact inhibition, while NP cells could form cellular hives in early passages (triple arrow in NP14 P6). Large NP cells, accounting for ~1%, were observed in all samples (arrowheads in NP7 P5 and in NP14 P6); bar=10 μm in phase contrast photographs. (B) Cell number (logarithmic scale, y-axis ) versus days in culture (linear scale, x-axis): NP cells had impressive growth rates in early passages with similar curves amongst patient samples (data shown are the average of all samples); arrows indicate times of cell culture propagation through passaging.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0082045-g001: Morphologies and growth rate of human NP cells in culture.(A) Phase contrast micrographs (black-and-white panels), and immunocytochemical decoration of mitotic spindles (microtubules visualized with β-tubulin-rhodamine and chromosomes with Hoechst 33258, upper row middle panel) and of the actin-binding protein tropomyosin (visualized with fluorescein, last two panels) show gains in numbers and a stable morphology throughout patients samples and passaging, even after freeze-thawing cycles (examples are given for samples NP6, 9, 11, 13, and 14; arrows and arrowheads are described in the text). NP or AC cells grew without contact inhibition, while NP cells could form cellular hives in early passages (triple arrow in NP14 P6). Large NP cells, accounting for ~1%, were observed in all samples (arrowheads in NP7 P5 and in NP14 P6); bar=10 μm in phase contrast photographs. (B) Cell number (logarithmic scale, y-axis ) versus days in culture (linear scale, x-axis): NP cells had impressive growth rates in early passages with similar curves amongst patient samples (data shown are the average of all samples); arrows indicate times of cell culture propagation through passaging.
Mentions: In order to attain sufficient NP cell numbers to study mechanisms of differentiation, we reasoned that NP cells should be allowed to first partially de-differentiate and thus gain in proliferation capacity. In pilot experiments we established the conditions, described in Methods, which permitted both cell cultures from every patient to be established and propagation of these cultures for up to 30 passages. When freshly plated, cells exhibited spherical shapes as they underwent cell division (Figure 1A, NP6 Passage 0 (P0), culture day 3 (C3), arrowhead, and NP8 P0, C2), before spreading onto the substrate (double arrows, NP6 P0, C3). Over the next days, cells acquired a polygonal body with several processes radiating away from the cell body, thus assuming an irregular stellate appearance (Figure 1A, NP6, P0, C12); such NP cell morphology with several lamellipodia with filopodia has been previously reported for human (e.g., 16,63) or bovine NP cell cultures, while in cell cultures established from explants [11] or through mechanical dissociation (filters) [17] lesser lamellipodia, often without filopodia, were observed at least during early passages. Cells grew exponentially during the first few passages (Figure 1B), and proliferation rate remained high up to passages 10-12, even after propagation (repeated passages and cycles of freeze-and-thaw) that covered cell maintenance periods (culture plus storage) of over two years in total. Cell morphology was also very similar over different patient samples e.g., Figure 1A, NP6 P0 at C12, and NP9 P7, NP13 P17, and NP11 P28 (all at C10). It is noteworthy that also up to passage 10-12, about 1-2% of the cell population consisted of large body cells with intricate process outgrowth patterns (Figure 1A, arrowheads in NP7 P5 and NP14 P6). Beyond that, cell proliferation slowed down, yet cells kept their stellate appearance and responded to plating on chitosan with a rounded morphology (data not shown). In general, cells grew without contact inhibition and we often observed cellular hives (Figure 1A, triple arrow) at least up to P10. Contact inhibition was also absent in cultures of human articular chondrocytes (AC), which were used as reference of a chondrocytic phenotype; these cells, however, grew without hive formation (Figure 1A, AC P5).

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