Results are expressed as percentages of variation compared with the control

n respond to transcriptional activity that follows activation of signalling pathways, as part of 20142041 transcriptional program regulation, and thus co-regulate major biological pathways with potential clinical uses. Aside the human nucleus pulposus cells where expression of PKC isoforms has not been addressed, many studies have dealt with the role of individual PKCs in all basic cellular processes of chondrocytes, including its action as a major signalling effector of activated vitamin D receptors, yet there is no clear consensus on specific mechanisms. The difficulty in delineating the roles of individual PKCs primarily lies with the fact that isoform-specific activators and inhibitors have only very recently been developed, while long term effects of PKC genetic manipulation usually include interregulation amongst the isoforms. Thus, in studies used in culture models activities of PKC and are reduced during nitric oxide-induced apoptosis in chondrocytes; their role in dedifferentiation, however, appears as necessary or indifferent. In human articular chondrocytes cultures, PKC is 1235481-90-9 required for cytokine-mediated expression of aggrecanases, a hallmark of osteoarthritis and of metalloproteinases, an irreversible mechanism of cartilage collagenolysis during inflammatory joint diseases. Similarly, PKC is shown as necessary for chondrocytic survival, but, when activated by basic fibroblast growth factor, stimulates MMP activity. The novel PKC has been detected in all tested chondrocytic lineages across species, yet its specific roles have to be addressed. PKC was not found implicated in reactive oxygen speciesinduced chondrocyte cell death, while its activation was reduced by bFGF, a “catabolic” agonist in human articular chondrocytes. Further pointing out a protective role, PKC has not been detected in screens that identified genes upregulated in diseases of chondrocytes, like for example PKCI, and or . Moreover, PKC does not appear to participate in crosstalks between major biological pathways in chondrocytes, like PKC and . The study for the specific actions of PKC has become possible with the development of a rationally designed peptide, namely RACK, an PKC allosteric activator derived from a sequence that regulates autoinhibitory interactions. RACK, conjugated to TAT peptide, has been administered in culture and in vivo with equal success in producing PKC specific biological effects. With this background, we undertook these studies to investigate the mechanistic details of PKC activation and study its potential function as a chondrocytic phenotype regulator in human nucleus pulposus cells. Materials and Methods Materials Culture media and supplements were from Life Technologies and fetal bovine serum from Biowest. We used the following antibodies: polyclonal antibodies to tropomyocin from Sigma, to phosphorylated species of PKC and myristoylated alanine-rich C-kinase substrate , to PKC, ERK1/2, and to activator protein 1 from Santa Cruz, to cAMP responsive element 11336787 binding protein 1 from Millipore; mouse monoclonal antibodies to MARCKS and phosho-ERK1/2 from Santa Cruz, to p120GAP from Upstate Biotechnology, to neo-epitope ARG on aggrecan, clone BC-3 from ABCAM, and to -tubulin from Sigma; and secondary antibodies conjugated to horseradish peroxidase or fluorochromes from Santa Cruz. The PKC-selective activator peptide, RACK was synthesized and conjugated to a peptide derived from the trans-activating transcriptional activator 2 PKC/ERK Signal