RESUMO
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 ß, 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.
Assuntos
Proteínas ADAM/genética , Agrecanas/genética , Regulação da Expressão Gênica , Disco Intervertebral/metabolismo , MicroRNAs/genética , Proteína Quinase C-épsilon/metabolismo , Transdução de Sinais , Proteína ADAMTS5 , Sequência de Bases , Western Blotting , Diferenciação Celular , Primers do DNA , Ativação Enzimática , Humanos , Disco Intervertebral/citologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Despite advances in surgical technique and implant design, complications involving the extensor mechanism and patellofemoral joint after total knee arthroplasty (TKA) continue to be the most common cause of pain and the most commonly cited reason for revision surgery. Periprosthetic patellar fractures occur in 1.19% of all reported cases after TKA, with a clear correlation with resurfacing of the patella. In 88.32% of the cases reported the fracture is not associated with a traumatic event and it is identified at the follow-up examination during the first 2 years after knee replacement. Predisposing factors for fracture include lateral release, excessive bone removal, peg fixation and cementation, improper patellar tracking and prosthesis malpositioning. More than 50% of fractures are associated with a loose implant which complicates the fracture management. Non-operative treatment seems to offer acceptable functional results and pain relief, especially in cases of minimal displacement and stable implant fixation. However, when surgical reconstruction is undertaken, open reduction and internal fixation with tension band or cerclage wiring should not be the first choice of treatment as the rate of failure and subsequent non-union may be as high as 90%.