PKC epsilon Signalling Activates ERK1/2, and Regulates Aggrecan, ADAMTS5, and miR377 Gene Expression in Human Nucleus Pulposus Cells: e82045

• Published in: PloS one 2013-11, Vol.8 (11)
• Main Authors: Tsirimonaki, Emmanouella, Fedonidis, Constantinos, Pneumaticos, Spiros G, Tragas, Adamantios A, Michalopoulos, Ioannis, Mangoura, Dimitra
• Format: Article
• Language: English
• Subjects: Cell culture
• ISSN: 1932-6203
• DOI: 10.1371/journal.pone.0082045

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 PKC epsilon -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 alpha , delta , epsilon , zeta , eta , iota , and mu were steadily detected, whereas beta , gamma , and [thetas] were not. Focusing on the signalling of PKC epsilon , an isoform that may confer protection against degeneration, we found that activation with the PKC epsilon -specific activator small peptide psi epsilon RACK 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, psi epsilon RACK induced upregulation of hsa-miR-377 expression, coupled to decreases in ADAMTS5 and cleaved aggrecan. Therefore, PKC epsilon activation in late passage NP cells may represent a molecular basis for aggrecan availability, as part of an PKC epsilon /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.