Abstract
Stimulation of the murine macrophage RAW 264.7 cell line with phorbol esters fails to promote nitric oxide synthesis as occurs in rat hepatocytes or peritoneal macrophages. Transfection of RAW 264.7 cells with plasmids harboring protein kinase C (PKC) -epsilon isotype but not with PKC-alpha, -beta1, -delta, or constitutively active -alpha and -beta1 isotypes resulted in the expression of nitric oxide synthase type II (iNOS), as reflected by the synthesis of nitric oxide measured in the culture medium of transfected cells. cotransfection of RAW 264.7 cells with the -1592 to +121-base pair promoter region of the murine iNOS gene and PKC isotypes specifically induced the transactivation of this promoter in the case of the plasmids containing the PKC-epsilon isotype. The mechanism by which PKC-epsilon induced iNOS expression involved the activation of nuclear factor binding to kappaB sites (NF-kappaB) as deduced by the suppressive effect of pyrrolidine dithiocarbamate on nitric oxide synthesis, an inhibitor of NF-kappaB activation, and by the activation of kappaB sites in cells transfected with a vector containing a kappaB motif linked to a chloramphenicol acetyltransferase reporter gene. These results suggest that PKC-epsilon can regulate a pathway that promotes iNOS expression in macrophages in response to phorbol ester activation.
Highlights
The knowledge of the mechanisms involved in the control of NO synthesis by different cell types is a subject of current interest because of the multiple physiological and pathological effects elicited by this molecule on different cell targets [1,2,3]
The different behavior observed between RAW 264.7 cells and peritoneal macrophages in the expression of iNOS in response to phorbol esters suggests the existence of cell-specific signaling pathways in these two cell types, either because of a distinct distribution of the protein kinase C (PKC) isotypes prevailing in each cell type or because of a divergent signaling mechanism downstream of the PKC network
Taking advantage of this inability of RAW 264.7 cells to display a significant PDBu-dependent NOxϪ synthesis, we studied whether a transient expression of PKC isotypes after transfection with plasmids harboring distinct PKC isoenzyme constructs might restore a PKCdependent induction of iNOS
Summary
The knowledge of the mechanisms involved in the control of NO synthesis by different cell types is a subject of current interest because of the multiple physiological and pathological effects elicited by this molecule on different cell targets [1,2,3]. The existence of a phorbol ester-mediated iNOS induction mechanism has been observed in a more reduced number of cell types, as for example rat peritoneal macrophages and hepatocytes [13, 14] In this regard, a role for PKC activation in the control of iNOS expression has been reported in the synergistic action of phorbol esters and IFN-␥ in murine peritoneal macrophages and in the cell line J774, as well as in astrocytes and microglia cells [15,16,17,18]. Priming with taxol of RAW 264.7 cells favors iNOS induction via a PKC-dependent mechanism, but treatment with phorbol esters alone has no effect on NO synthesis [19]. Our results show that expression of PKC-⑀ is sufficient to induce iNOS synthesis in RAW 264.7 cells through a mechanism that involves the transactivation of response elements located in the 1.8-kb region of the iNOS promoter
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