Modulation of nitric oxide-induced apoptotic death of HL-60 cells by protein kinase C and protein kinase A through mitogen-activated protein kinases and CPP32-like protease pathways.

To define the signaling pathways during NO-induced apoptotic events and their possible modulation by two protein kinase systems, we explored the involvement of three structurally related mitogen-activated protein kinase subfamilies. Exposure of HL-60 cells to sodium nitroprusside (SNP) strongly activated p38 kinase, but did not activate c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). In addition, SNP-induced apoptosis was markedly blocked by the selective p38 kinase inhibitor (SB203580) but not by MEK1 kinase inhibitor (PD098059), indicating that p38 kinase serves as a mediator of NO-induced apoptosis. In contrast, treatment of cells with phorbol 12-myristate 13-acetate (PMA) strongly activated not only JNK but also ERK, while not affecting p38 kinase. However, although SNP by itself weakly activated CPP32-like protease, SNP in combination with PMA markedly increased the extent of CPP32-like protease activation. Interestingly, N6,O2-dibutylyl cAMP (DB-cAMP) significantly blocked SNP- or SNP plus PMA-induced activation of CPP32-like protease and the resulting induction of apoptosis. DB-cAMP also blocked PMA-induced JNK activation. Collectively, these findings demonstrate the presence of specific up- or down-modulatory mechanisms of cell death pathway by NO in which (1) p38 kinase serves as a mediator of NO-induced apoptosis, (2) PKC acts at the point and/or upstream of JNK and provides signals to potentiate NO-induced CPP32-like protease activation, and (3) PKA lies upstream of either JNK or CPP32-like protease to protect NO- or NO plus PMA-induced apoptotic cell death in HL-60 cells.     

Functional role of extracellular signal-regulated kinase activation and c-Jun induction in phorbol ester-induced promoter activation of human 12(S)-lipoxygenase gene

The functional role of mitogen-activated protein kinase (MAPK) signaling and c-Jun induction in phorbol 12-myristate 13-acetate (PMA)-induced human 12(S)-lipoxygenase gene expression was studied in human epidermoid carcinoma A431 cells. Among the family of MAPK, PMA only increased the activity of extracellular signal-regulated kinase (ERK). Treatment of cells with PD98059, which is an inhibitor of mitogen-activated protein kinase kinase (MEK), decreased the PMA-induced expression of 12(S)-lipoxygenase. Transfection of cells with Ras, Raf and ERK2 dominant negative mutants inhibited the PMA-induced promoter activation of the 12(S)-lipoxygenase gene in all cases. PMA-induced expression of c-Jun was inhibited by pretreatment with PD98059. Following treatment with PMA, the interaction between c-Jun and simian virus 40 promoter factor 1 (Sp1) in cells increased with time. Enhancement of binding between the c-Jun-Sp1 complex and the Sp1 oligonucleotide was observed in cells treated with PMA, suggesting the possible interaction of c-Jun-Sp1 with GC-rich binding sites in the gene promoter. These results indicate that PMA treatment induced ERK activation mainly through the Raf-MEK-ERK signaling pathway following induction of c-Jun expression, and the formation of the c-Jun-Sp1 complex. Finally, PMA activated the promoter activity of the 12(S)-lipoxygenase gene in cells overexpressing protein kinase C (PKC)delta but not PKCalpha, indicating that PKCdelta played the functional role in mediating the gene activation of 12(S)-lipoxygenase induced by PMA.     

Mixed lineage kinase 3 inhibits phorbol myristoyl acetate-induced DNA synthesis but not osteopontin expression in rat mesangial cells

Mixed lineage kinase 3 (MLK 3) (also called SPRK or PTK-1) is a recently described member of the family of the mixed lineage kinase subfamily of Ser/Thr protein kinases that interacts with mitogen-activated protein kinase pathways. In order to test the biological relevance and potential interaction of MLK 3 with protein kinase C-mediated signaling pathways, human MLK 3 was stably expressed in rat glomerular mesangial cells using a retroviral vector (LXSN) and the effects of phorbol myristoyl acetate (PMA) on DNA synthesis and osteopontin mRNA expression were examined. In control (vector-transfected) mesangial cells PMA increased [³H]-thymidine incorporation in a concentration-dependent manner. In mesangial cells stably expressing MLK 3, the PMA-induced increase in [³H]-thymidine incorporation was significantly reduced (> 50%). However, the PMA-induced increase in osteopontin mRNA was not affected by MLK 3 expression. To determine the mechanisms of these effects, activation of ERK2, JNK1 and p38 in response to PMA was examined in both vector and MLK 3 transfected cells. ERK2 activation was increased several fold by PMA in control cells but was attenuated significantly in MLK 3 expressing cells, suggesting that MLK 3 expression in mesangial cells can negatively regulate the ERK pathway. PMA had no significant effect on JNK and P38 activation, in either vector- or MLK 3-expressing cells. PD98059, a MEK inhibitor blocked PMA-induced DNA synthesis without affecting osteopontin expression. These results suggest that while protein kinase C activation increases cellular proliferation and osteopontin mRNA expression, over-expression of MLK 3 affects only the PKC-induced DNA synthesis, probably through inhibition of ERK. These results also indicate a novel mechanism of growth regulation by a member of the mixed-lineage kinase family that might have significant therapeutic implications in proliferative glomerulonephritis.     

The protein kinase inhibitor SB203580 uncouples PMA-induced differentiation of HL-60 cells from phosphorylation of Hsp27

HL-60 cells are an attractive model for studies of human myeloid cell differentiation. Among the well-examined parameters correlated to differentiation of HL-60 cells are the expression and phosphorylation of the small heat shock protein Hsp27. Here we demonstrate that PMA treatment of HL-60 cells stimulates different MAP kinase cascades, leading to significant activation of ERK2 and p38 reactivating kinase (p38RK). Using the protein kinase inhibitor SB 203580, we specifically inhibited p38RK and, thereby, activation of its target MAP kinase-activated protein kinase 2(MAPKAP kinase 2), which is the major enzyme responsible for small Hsp phosphorylation. As a result, PMA-induced Hsp27 phosphorylation is inhibited in SB 203580-treated HL-60 cells indicating that p38RK and MAPKAP kinase 2 are components of the PMA-induced signal transduction pathway leading to Hsp27 phosphorylation. We further demonstrate that, although PMA-induced phosphorylation is inhibited, SB 203580-treated HL-60 cells are still able to differentiate to the macrophage-like phenotype as judged by decrease in cell proliferation, induction of expression of the cell surface antigen CD11b and changes in cell morphology. These results indicate that, although correlated, Hsp27 phosphorylation is not required for HL-60 cell differentiation. However, the results do not exclude that increased Hsp27 expression is involved in HL-60 cell differentiation.     

IL-1R-associated kinase-1 mediates protein kinase Cδ-induced IL-1β production in monocytes

The role of IL-1R-associated kinase (IRAK)1 and its interaction with protein kinase C (PKC)δ in monocytes to regulate IL-1β production has not been reported so far. The present study thus investigates such mechanisms in the THP1 cell line and human monocytes. PMA treatment to THP1 cells induced CD11b, TLR2, TLR4, CD36, IRAK1, IRAK3, and IRAK4 expression, IRAK1 kinase activity, PKCδ and JNK phosphorylation, AP-1 and NF-κB activation, and secretory IL-1β production. Moreover, PMA-induced IL-1β production was significantly reduced in the presence of TLR2, TLR4, and CD11b Abs. Rottlerin, a PKCδ-specific inhibitor, significantly reduced PMA-induced IL-1β production as well as CD11b, TLR2 expression, and IRAK1-JNK activation. In PKCδ wild-type overexpressing THP1 cells, IRAK1 kinase activity and IL-1β production were significantly augmented, whereas recombinant inactive PKCδ and PKCδ small interfering RNA significantly inhibited basal and PMA-induced IRAK1 activation and IL-1β production. Endogenous PKCδ-IRAK1 interaction was observed in quiescent cells, and this interaction was regulated by PMA. IRAK1/4 inhibitors, their small interfering RNAs, and JNK inhibitor also attenuated PMA-induced IL-1β production. NF-κB activation inhibitor and SN50 peptide inhibitor, however, failed to affect PMA-induced IL-1β production. A similar role of IRAK1 in IL-1β production and its regulation by PKCδ was evident in the primary human monocytes, thus signifying the importance of our finding. To our knowledge, the results obtained demonstrate for the first time that IRAK1 and PKCδ functionally interact to regulate IL-1β production in monocytic cells. A novel mechanism of IL-1β production that involves TLR2, CD11b, and the PKCδ/IRAK1/JNK/AP-1 axis is thus being proposed.     

Inhibition of the MEK/ERK signaling pathway by the novel antimetastatic agent NAMI-A down regulates c-myc gene expression and endothelial cell proliferation.

Imidazolium trans-imidazoledimethyl sulfoxide-tetrachlororuthenate (NAMI-A) is a novel ruthenium-containing experimental antimetastatic agent. Compelling evidence ascribes a pivotal role to endothelial cells in the orchestration of tumor angiogenesis and metastatic growth, suggesting antiangiogenic therapy as an attractive approach for anticancer treatment. In this context, activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signaling pathway has been found fundamental in transducing extracellular stimuli that modulate a number of cellular process including cell proliferation, migration and invasion. Here we show that exposure of the transformed endothelial cell line ECV304 to NAMI-A significantly inhibited DNA synthesis, as well as the expression of the proliferating cell nuclear antigene (PCNA). These responses were associated with a marked down-regulation of ERK phosphorylation in serum-cultured cells. In addition, NAMI-A markedly reduced serum stimulated- and completely suppressed phorbol 12-myristate 13-acetate (PMA)-triggered MAPK/ERK kinase activity. NAMI-A was also able to inhibit the phosphorylation of MEK, the upstream activator of ERK, and, similar to both the protein kinase C (PKC) inhibitor GF109203X and the MAPK/ERK (MEK) inhibitor PD98059, it completely counteracted PMA-induced ERK phosphorylation. Finally, NAMI-A and PD98059 down regulated c-myc gene expression to the same extent in serum-cultured cells and dose-dependently counteracted, and ultimately abolished, the increase in c-myc gene expression elicited by PMA in serum-free cells. These results suggest that inhibition of MEK/ERK signaling by NAMI-A may have an important role in modulating c-myc gene expression and ECV304 proliferation.     

Induction of ANGPTL4 expression in human airway smooth muscle cells by PMA through activation of PKC and MAPK pathways

In this study, we demonstrate that protein kinase C (PKC) activators, including phorbol-12-myristate-13-acetate (PMA), 1,2-dioctanoyl-sn-glycerol (DOG), and platelet-derived growth factor α are potent inducers of angiopoietin-like protein 4 (ANGPTL4) expression in several normal lung cell types and carcinoma cell lines. In human airway smooth muscle (HASM) cells induction of ANGPTL4 expression is observed as early as 2 h after the addition of PMA. PMA also increases the level of ANGPTL4 protein released in the medium. PKC inhibitors Ro31-8820 and Gö6983 greatly inhibit the induction of ANGPTL4 mRNA by PMA suggesting that this up-regulation involves activation of PKC. Knockdown of several PKCs by corresponding siRNAs suggest a role for PKCα. PMA does not activate MAPK p38 and p38 inhibitors have little effect on the induction of ANGPTL4 indicating that p38 is not involved in the regulation of ANGPTL4 by PMA. In contrast, treatment of HASM by PMA induces phosphorylation and activation of Ra, MEK1/2, ERK1/2, JNK, Elk-1, and c-Jun. The Ras inhibitor manumycin A, the MEK1/2 inhibitor U0126, and the JNK inhibitor SP600125, greatly reduce the increase in ANGPTL4 expression by PMA. Knockdown of MEK1/2 and JNK1/2 expression by corresponding siRNAs inhibits the induction of ANGPTL4. Our observations suggest that the induction of ANGPTL4 by PMA in HASM involves the activation of PKC, ERK, and JNK pathways. This induction may play a role in tissue remodeling during lung injury and be implicated in several lung pathologies.     

A Role for the MEK/MAPK Pathway in PMA-Induced Cell Cycle Arrest: Modulation of Megakaryocytic Differentiation of K562 Cells

In vitromegakaryocytic differentiation of the pluripotent K562 human leukemia cell line is induced by PMA. Treatment of K562 cells with PMA results in growth arrest, polyploidy, morphological changes, and increased cell–cell and cell–substrate adhesion. These PMA-induced changes in K562 cells are preceded by a rapid rise in the activity of MEK (MAP kinase/extracellular regulated kinases) that leads to a sustained activation of ERK2 (extracellular regulated kinase; MAPK). Blockade of MEK1 activation by PD098059, a recently described specific MEK inhibitor [D. T. Dudleyet al.(1995).Proc. Natl. Acad. Sci. USA92, 7686–7689], reverses both the growth arrest and the morphological changes of K562 cells induced by PMA treatment. These changes are not associated with a disruption of PMA-induced down-regulation of BCR-ABL kinase or early integrin signaling events but are associated with a block of the cell-surface expression of the gpIIb/IIIa (CD41) integrin, a cell marker of megakaryocytic differentiation. These results demonstrate that the PMA-induced signaling cascade initiated by protein kinase C activation requires the activity of the MEK/ERK signaling complex to regulate cell cycle arrest, thus regulating the program that leads to the cell-surface expression of markers associated with megakaryocytic differentiation.     

Inhibition of apolipoprotein AI gene expression by tumor necrosis factor alpha: roles for MEK/ERK and JNK signaling

Plasma high-density lipoprotein and apolipoprotein AI (apoAI) levels are suppressed by tumor necrosis factor alpha. To determine the molecular mechanisms responsible for the effect of TNF alpha on the apoAI promoter activity, HepG2 cells were exposed to both genetic and pharmacological modulators of TNF alpha-mediated signaling in the presence or absence of TNF alpha. Exogenous ERK1 and ERK2 expression suppressed basal apoAI promoter activity; however, only ERK2 enhanced the ability of TNF alpha to suppress apoAI promoter activity. Exogenous expression of all three MEK isoforms (MEK1, MEK2A, and MEK2E) suppressed basal apoAI promoter activity and further aggravated TNF alpha-related apoAI promoter activity inhibition. Treatment with SB202190 (p38 MAP kinase inhibitor) alone significantly increased apoAI promoter activity; however, in the presence of TNF alpha, apoAI promoter activity was suppressed to an extent similar to that in cells not treated with SB202190. ApoAI promoter activity increased in cells treated with the specific JNK inhibitor SP600125, but unlike SB202190 treatment, the level of TNF alpha-related apoAI promoter inhibition was reduced by 50%. Similarly, the level of TNF alpha-related apoAI promoter inhibition was reduced in cells transfected with JNK1 siRNA. Finally, treatment of cells with the NF-kappaB inhibitors BAY and SN-50 or overexpression of NF-kappaB subunits p50 and p65 had no effect on the ability of TNF alpha to repress apoAI promoter activity. These results suggest that TNF alpha suppresses apoAI promoter activity through both the MEK/ERK and JNK pathways but is not mediated by either p38 MAP kinase activity or NF-kappaB activation.     

Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-kappaB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IkappaBalpha phosphorylation, IkappaBalpha degradation, and the formation of a NF-kappaB-specific DNA-protein complex. The PMA-mediated increase in kappaB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.     

Role of ras-dependent ERK activation in phorbol ester-induced endothelial cell barrier dysfunction

The treatment of endothelial cell monolayers with phorbol 12-myristate 13-acetate (PMA), a direct protein kinase C (PKC) activator, leads to disruption of endothelial cell monolayer integrity and intercellular gap formation. Selective inhibition of PKC (with bisindolylmaleimide) and extracellular signal-regulated kinases (ERKs; with PD-98059, olomoucine, or ERK antisense oligonucleotides) significantly attenuated PMA-induced reductions in transmonolayer electrical resistance consistent with PKC- and ERK-mediated endothelial cell barrier regulation. An inhibitor of the dual-specificity ERK kinase (MEK), PD-98059, completely abolished PMA-induced ERK activation. PMA also produced significant time-dependent increases in the activity of Raf-1, a Ser/Thr kinase known to activate MEK ( approximately 6-fold increase over basal level). Similarly, PMA increased the activity of Ras, which binds and activates Raf-1 ( approximately 80% increase over basal level). The Ras inhibitor farnesyltransferase inhibitor III (100 microM for 3 h) completely abolished PMA-induced Raf-1 activation. Taken together, these data suggest that the sequential activation of Ras, Raf-1, and MEK are involved in PKC-dependent endothelial cell barrier regulation.     

PMA-induced activation of the p42/44ERK- and p38RK-MAP kinase cascades in HL-60 cells is PKC dependent but not essential for differentiation to the macrophage-like phenotype

The signaling mechanisms leading to phorbol ester myristate (PMA)-induced differentiation of HL-60 cells to the macrophagelike phenotype were investigated by using different protein kinase inhibitors. The protein kinase C inhibitor Ro 31-8220 specifically blocks PMA-induced differentiation, activation of the p42/44^ERK- and p38^RK-MAP kinase cascades and Hsp27-phosphorylation in HL-60 cells. Because Ro 31-8220 does not inhibit activation of the MAP kinase cascades by protein kinase C (PKC)-independent signals such as epidermal growth factor (EGF), heat shock, or anisomycin in these cells, only PMA-induced activation of the MAP kinases can be downstream of PKC. The MEK1 inhibitor PD 098059 and the p38^RK inhibitor SB 203580 also were used to analyze whether the PMA-induced PKC-dependent activation of MAP kinases is involved in the differentiation process. Under certain conditions, PD 098059 can completely block the PMA-induced activation of the p42^ERK as monitored by imunoprecipitation kinase assay by using the substrate myelin basic protein. SB 203580 specifically inhibits activation of p38^RK as judged by MAPKAP kinase 2 activity against the substrate Hsp27 and also blocks Hsp27 phosphorylation in the cells. In contrast, neither PD 098059 nor SB 203580 nor both inhibitors together prevent PMA-induced differentiation of the HL-60 cells to the macrophagelike phenotype. The results suggest the existence of a diversification of PMA-induced signaling in HL-60 cells downstream of PKC, leading to activation of MAP kinases that are not essential for differentiation and to phosphorylation of other, so far unidentified, targets responsible for differentiation. J. Cell. Physiol. 173:310–318, 1997. © 1997 Wiley-Liss, Inc.     

Hypertonicity-induced aquaporin-1 (AQP1) expression is mediated by the activation of MAPK pathways and hypertonicity-responsive element in the AQP1 gene

Aquaporin-1 (AQP1) is a water channel that is induced by hypertonicity. The present study was undertaken to clarify the osmoregulation mechanism of AQP1 in renal medullary cells. In cultured mouse medullary (mIMCD-3) cells, AQP1 expression was significantly induced by hypertonic treatment with impermeable solutes, whereas urea had no effect on AQP1 expression. This result indicates the requirement of a hypertonic gradient. Hypertonicity activated ERK, p38 kinase, and JNK in mIMCD-3 cells. Furthermore, all three MAPKs were phosphorylated by the upstream activation of MEK1/2, MKK3/6, and MKK4, respectively. The treatments with MEK inhibitor U0126, p38 kinase inhibitor SB203580, and JNK inhibitor SP600125 significantly attenuated hypertonicity-induced AQP1 expression in mIMCD-3 cells. In addition, hypertonicity-induced AQP1 expression was significantly reduced by both the dominant-negative mutants of JNK1- and JNK2-expressing mIMCD-3 cells. NaCl-inducible activity of AQP1 promoter, which contains a hypertonicity response element, was attenuated in the presence of U0126, SB203580, and SP600125 in a dose-dependent manner and was also significantly reduced by the dominant-negative mutants of JNK1 and JNK2. These data demonstrate that the activation of ERK, p38 kinase, and JNK pathways and the hypertonicity response element in the AQP1 promoter are involved in hypertonicity-induced AQP1 expression in mIMCD-3 cells.