title: PKCε signalling activates ERK1/2, and regulates aggrecan, ADAMTS5, and miR377 gene expression in human nucleus pulposus cells.
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.
authors: Biomedical Research Foundation of the Academy of Athens, Athens, Greece.
authors: Emmanouella
authors: E
authors: Tsirimonaki
authors: Constantinos
authors: C
authors: Fedonidis
authors: Spiros G
authors: SG
authors: Pneumaticos
authors: Adamantios A
authors: AA
authors: Tragas
authors: Ioannis
authors: I
authors: Michalopoulos
authors: Dimitra
authors: D
authors: Mangoura
mesh_terms: D051722:ADAM Proteins / Q000235:genetics*; D000071116:ADAMTS5 Protein; D053674:Aggrecans / Q000235:genetics*; D001483:Base Sequence; D015153:Blotting, Western; D002454:Cell Differentiation; D017931:DNA Primers; D004789:Enzyme Activation; D005786:Gene Expression Regulation; D006801:Humans; D007403:Intervertebral Disc / Q000166:cytology / Q000378:metabolism*; D035683:MicroRNAs / Q000235:genetics*; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D053674:Aggrecans; D017931:DNA Primers; C534063:MIRN377 microRNA, human; D035683:MicroRNAs; D051744:Protein Kinase C-epsilon; D051722:ADAM Proteins; D000071116:ADAMTS5 Protein; C121532:ADAMTS5 protein, human