PKCβ Activation Research Articles

Functional role for protein kinase Cbeta as a regulator of stress-activated protein kinase activation and monocytic differentiation of myeloid leukemia cells.

Human myeloid leukemia cells respond to 12-O-tetradecanoylphorbol-13-acetate (TPA) and other activators of protein kinase C (PKC) with induction of monocytic differentiation. The present studies demonstrated that treatment of U-937 and HL-60 myeloid leukemia cells with TPA, phorbol-12,13-dibutyrate, or bryostatin 1 was associated with the induction of stress-activated protein kinase (SAPK). In contrast, TPA-resistant TUR and HL-525 cell variants deficient in PKCbeta failed to respond to activators of PKC with the induction of SAPK. A direct role for PKCbeta in TPA-induced SAPK activity in TUR and HL-525 cells that stably express PKCbeta was confirmed. We showed that TPA induced the association of PKCbeta with MEK kinase 1 (MEKK-1), an upstream effector of the SAPK/ERK kinase 1 (SEK1)-->SAPK cascade. The results also demonstrated that PKCbeta phosphorylated and activated MEKK-1 in vitro. The functional role of MEKK-1 in TPA-induced SAPK activity was further supported by the demonstration that the expression of a dominant negative MEKK-1 mutant abrogated this response. These findings indicate that PKCbeta activation is necessary for activation of the MEKK-1-->SEK1-->SAPK cascade in the TPA response of myeloid leukemia cells.

Promotion of HL-60 cell differentiation by 1,25'dihydroxyvitamin D 3 regulation of protein kinase C levels and activity

The hormone 1,25-dihydroxyvicamin D 3 [1,25-(OH) 2D 3] promotes differentiation of a number of cell types including HL-60 promyelocytic leukemia cells. It is now established that protein kinase Cβ (PKCβ) plays a critical role in HL-60 cell maturation to a monocyte/macrophage phenotype. In the present study, we investigated the importance of PKCβ levels and activation in 1,25-(OH) 2D 3-mediated differentiation of HL-60 cells. Cell differentiation promoted by 1,25-(OH) 2D 3 at 48 hr was 39 ± 3% (mean ± SEM) nitroblue tetrazolium (NB'T) positive and at 72 hr it was 35 ± 2% NBT positive and 70% CD14 positive. Thus, promotion of cell differentiation by 20 nM 1,25-(OH) 2D 3 treatment was maximal at 48–72 hr. When PKCβ levels and cell differentiation were assayed at 72 hr, treatment with 20 nM 1,25-(OH) 2D 3 for the initial 6 hr increased PKCβ levels by 175% but had little effect on cell differentiation (7 ± 2% NBT positive; 11% CD14 positive). The effect of ionomycin, a calcium ionophore, on PKCβ levels and cell differentiation also was examined. Alone, 5 μM ionomycin promoted few cells (3% CD14 positive) to differentiate. In contrast, cells treated with 5 μM ionomycin for 66 hr after a 6-hr pretreatment with 20 nM 1,25-(OH) 2D 3 resulted in 34 ± 5% NBT positive cells and 73% CD14 positive cells. Quantitatively, this induction of differentiation was identical to that observed in cultures continuously treated with 1,25-(OH) 2D 3 (35 ± 2% NBT positive; 70% CD14 positive). Therefore, ionomycin seemed to replace the requirement for the continuous presence of 1,25-(OH) 2D 3. Chelerythrine chloride (3 μM), a specific PKC inhibitor, blocked differentiation promoted by 1,25-(OH) 2D 3 alone (82 ± 2% inhibition) or in sequence with ionomycin (86 ± 3% inhibition). Taken together, our data show that the capacity of 1,25-(OH) 2D 3 to both increase PKCβ levels and activate PKC is utilized to promote HL-60 cell differentiation. These data further suggest that 1,25-(OH) 2D 3 has a genomic action to increase PKCβ levels and also a nongenomic action requiring its continuous presence to promote HL-60 cell differentiation.