Protein kinase C-eta (PKC-η) is required for the expression of the inducible nitric oxide synthase (NOS II) in human monocytic cells: a correlation in transcription between PKC-η and NOS II in inflammatory arthritides

Pham, Tram Ngoc Quynh (2003) Protein kinase C-eta (PKC-η) is required for the expression of the inducible nitric oxide synthase (NOS II) in human monocytic cells: a correlation in transcription between PKC-η and NOS II in inflammatory arthritides. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Nitric oxide (NO), produced by the inducible nitric oxide synthase (NOS II), is important in host defence against invading pathogens including bacteria, fungi and certain viruses. Its induction by LPS and pro-inflammatory cytokines (e.g., TNF-α, IFN-γ, and IL-1β) is well established in murine monocytic cells. However, the same cannot be said for human monocytic cells. This work explored the possibility that the failure of human cells to express NOS II following LPS stimulation might be due to a lack of key signalling molecule(s) essential to the pathway. -- Since studies with PKC inhibitors have implicated protein kinase C (PKC) in NOS II regulation in murine monocytic cells, a complete investigation comparing PKC isotype expression between human and murine monocytic cells was needed to address whether a lack of one of the isotypes may be responsible for the failure of human monocytic cells to express NOS II. We found that human monocytic cells lacked PKC-η (eta), while the murine counterparts lacked PKC-β1 (beta 1 ). Subsequently, it was determined that human monocytic cells transfected with PKC-η could induce NOS II expression and produced NO production following LPS stimulation. This provides direct evidence that PKC-η may be involved in NOS II regulation. We have now established expression correlation between PKC-η and NOS II in monocyte-derived macrophages (MDM) from patients with inflammatory diseases (lA) (rheumatoid arthritis [RA] and spondyloarthropathies [SpA]). Severely- affected lA patients (with > 5 swollen joints) showed highly elevated plasma NO (237.8 ± 34.41 μM, n=10) relative to healthy (131.1 ± 18.9 μM, n=9), osteoarthritis (126.9 ± 40.9 μM, n=13), or mildly affected lA individuals (with< 5 swollen joints) (131.1 ± 18.9 μM, n=10). Analysis of transcriptional expression of NOS II revealed that only in lA patients with elevated plasma NO was NOS II present in MDM, and that PKC-η was always expressed in the NOS It-reactive cases. MDM in individuals with normal circulating NO were negative for both PKC-η and NOS II. This was true in 15 out of 16 (94%) cases tested implicating for the first time a requirement of PKC-η for the development of a NOS It-positive phenotype in human monocytic cells. -- In our study, TNF-α did not seem to be involved in NOS II regulation as lA patients n=7), who responded well clinically to infliximab (anti-TNF-α monoclonal antibody), showed elevated NO and expressed both NOS II and PKC-η in MDM. In contrast, lA patients responding well to anakinra (IL-1 receptor antagonist) had normal plasma NO, did not express NOS II but were positive for PKC-η in MDM. We propose that in severely affected lA, there may be three stages in the development of NOS II positive phenotype in monocytes with IL-1 somehow being involved in the regulation of NOS II expression. -- Two synthetic (JPK-101 and JPK-109) and one natural (JPK-113) compounds from Trypterigyum plant, whose extract is used as traditional Chinese medication for the treatment for RA, were investigated for their effects on the induction of NOS II as well as production of TNF-α, NO, and matrix metalloprotease (MMP). We found that the compounds could inhibit production of NO (via suppression of NOS II) and TNF-α, but not MMP. These findings support their well known, but poorly explained, anti-inflammatory effects.

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