Transduction of the IFN-gamma signal for HLA-DR expression in the promonocytic line THP-1 involves a late-acting PKC activity

Interferon gamma (IFN-gamma) is the most potent known lymphokine for activating macrophages and has been shown to induce expression of HLA-DR in THP-1 cells, a monocytic tumor cell line which expresses many of the properties of monocytes, in a dose- and time-dependent manner. Experiments were designed to examine, by FACS analysis and by measurement of messenger RNA levels, the molecular mechanism regulating the expression of HLA-DR molecules. The expression of HLA-DR molecules induced by IFN-gamma was blocked by the protein kinase C (PKC) inhibitors sphingosine, staurosporine, and H7. H7 when added up to 20 hr after the initial stimulation with IFN-gamma prevented the further expression of HLA-DR. The general kinase inhibitors H8, H9, and HA1004, all less potent PKC inhibitors than H7, did not block the IFN-gamma-induced expression of HLA-DR at the concentrations employed. W7, a calmodulin antagonist, but not a PKC inhibitor, was also unable to prevent the IFN-gamma-induced expression of HLA-DR. Treatment of THP-1 with phorbol 12-myristate 13-acetate (PMA), a direct activator of PKC, alone or with Ca2+ ionophore A23187, was unable to induce HLA-DR expression. However, pretreatment with PMA for 24 hr prior to IFN-gamma stimulation decreased the IFN-gamma-induced expression of HLA-DR without decreasing IFN-gamma receptor levels. These results suggest that PKC plays a significant role in the IFN-gamma-induced signal transduction pathway leading to the expression of HLA-DR in cells of the mononuclear phagocytic lineage, and that PKC activity is required throughout the course of events leading to the actual expression of HLA-DR.     

Pathogenicity of molecularly cloned bovine leukemia virus.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.     

The kinase inhibitor iso-H7 stimulates rat satellite cell differentiation through a non-protein kinase C pathway by increasing myogenin expression level

We analysed the signaling pathways involved in myogenic differentiation of primary cultures of rat satellite cells using substances targeting the protein kinase C (PKC) and the cAMP protein kinase (PKA) pathways. We have previously shown that iso-H7, which putatively inhibits both PKC and PKA, strongly stimulates satellite cell differentiation, as well as the PKA inhibitor HA 1004. In the study reported here, the effects of iso-H7 on satellite cell differentation were compared to those observed in the presence of agents which reduce PKC activity. It was shown that treatments with the highly specific PKC inhibitor GF109203X or with 12-O-tetradecanoylphorbol 13-acetate (TPA) which induced a partial PKC downregulation, did not significantly alter myogenic differentiation. Northern blot analyses showed that iso-H7 activated the expression of myogenin but not that of MyoD mRNA. Concurrently, iso-H7 increased myosin light-chain mRNA expression. In contrast, TPA had no effect on these syntheses. Taken together, these results showed that iso-H7 did not act intracellularly as a PKC inhibitor but rather as a PKA inhibitor as previously suggested. Our results are compatible with the hypothesis that a reduction in PKA activity controls satellite cell myogenesis through an increased myogenin mRNA expression.Abbreviations PKC protein kinase C - PKA cAMP-dependent protein kinase - CK creatine kinase - iso-H7 1-(5-isoquinolinesulfonyl)-3-methylpiperazine - H7 1-(5-isoquinolinesulfonyl)-2-methylpiperazine - HA 1004 N-(3-hydroxyethyl)-1-piperazine ethanesulfonate - TPA 12-O-tetradecanoyl phorbol 13-acetate - MLC myosin light chain - GAPDH glyceraldehyde 3-phosphate deshydrogenase     

Interleukin-2 regulates the activity of ornithine decarboxylase in a cloned murine T lymphocytic cell line: evidence for a protein kinase C-dependent pathway

The role of protein kinase C (PKC) in the regulation of ornithine decarboxylase (ODC) activity during interleukin-2 (IL-2)-dependent cell growth was investigated. A large biphasic increase in the activity of ODC was observed after treatment of IL-2-deprived CTLL-2 cells with recombinant human IL-2 (rec IL-2). The PKC activators phorbol 12-myristate 13-acetate (PMA) and 4 beta-phorbol 12,13-didecanoate (4 beta-PDD), but not the inactive analog 4 alpha-PDD, induced ODC activity in exponentially growing cultures. Unlike IL-2, however, phorbol esters were poor inducers of IL-2-depleted cultures. H-7, a potent inhibitor of PKC and cyclic nucleotide-dependent protein kinases (CN-PK), suppressed the IL-2-induced ODC activity, while HA1004, a more potent inhibitor of CN-PK than of PKC, had opposite effects depending on its concentration. The results suggest that activation of PKC is involved in but is not the sole mechanism for the induction of ODC by rec IL-2. At concentrations which suppressed the induction of ODC activity by IL-2, H-7 inhibited DNA synthesis and HA1004 did not.     

Regulation of mitogen-stimulated human T-cell proliferation, interleukin-2 production, and interleukin-2 receptor expression by protein kinase C inhibitor, H-7

Recently published reports suggest that the activation of protein kinase C (PKC) plays an important role in the activation pathway of many cell types. In this study, we examined the role of PKC in human T-cell proliferation, IL-2 production, and IL-2R expression, when cultured with the mitogen PHA, the PKC inhibitor H-7, and H-7 control HA1004. H-7 inhibited the PHA-stimulated [3H]thymidine uptake, IL-2 production, and IL-2R expression in a dose-related manner. Further, we found H-7 inhibited T-cell proliferation, IL-2 production, IL-2 mRNA from PHA plus PMA-stimulated cultures. We also found that H-7 inhibited the early-stage activation of PHA-stimulated cells. The presence of exogenous purified human IL-2 or rIL-4 partly reversed the immunosuppression caused by H-7. In contrast, HA1004 had no effect on cell proliferation, IL-2 production, or IL-2R expression. Our results demonstrate that PKC activation is one major pathway through which T-cells become activated.     

Inhibition of human natural killer cell activity by the protein kinase C inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpiperazine is an early but post-binding event

Recent evidence has demonstrated a protein kinase C (PKC)-dependent step in cytotoxic T lymphocyte activation. Here, we examined the influence of PKC in the lytic response of human NK cells to K562, an NK-sensitive tumor target cell. We used the known protein kinase inhibitors 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and HA1004. H-7 caused a dose-related inhibition of NK cell-mediated cytolysis (CMC) when the inhibitor was present throughout the course of the 3-h chromium release assay. The 50% inhibitory concentration for H-7 was 7 microM. In contrast, HA1004, which exerts a greater inhibitory effect on cyclic nucleotide-dependent protein kinases than PKC, had no effect on NK-CMC. The suppression of NK-CMC by H-7 was not due to inhibition of binding of the effector cells to target cells and could be reversed by the addition of PMA. H-7 was most effective in abrogating NK-CMC when added to the assay within the first 30 min and treatment of the effector and target cells with H-7 resulted in no loss of NK-CMC. Because nearly 50% of the normal NK lytic activity had taken place by 30 min, this suggested that H-7 inhibited an early event. H-7 exerted a dose-related suppression of antibody-dependent cell-mediated cytotoxicity (ADCC) suggesting that NK-CMC and ADCC share the utilization of PKC, however, HA1004 did not inhibit ADCC. Treating NK cells with IL-2 or IFN-beta did not overcome the inhibition of NK-CMC by H-7. In this study, we have thus demonstrated the presence of a PKC-dependent step in NK-CMC and ADCC.     

Role of protein kinase C in IFN-mediated Ly-6E antigen induction.

The YAC T cell lymphoma normally does not express Ly-6E mRNA or Ly-6E surface molecules but can be induced to do so on incubation with either IFN-gamma or IFN-alpha/beta. This system afforded a model to assess the possible role of protein kinase C (PKC) in IFN-mediated Ly-6E induction. First, we used various pharmacologic agents known to interfere with the function of PKC or other kinases. The PKC inhibitors H-7 and phloretin were found to block Ly-6E induction by IFN-gamma or IFN-alpha/beta both at the mRNA and protein levels. In contrast, inhibitors of cyclic nucleotide-dependent kinases (HA1004), of myosin L chain kinase (ML-9, A-3) or of calmodulin (R24157, W-7) failed to suppress this induction. Next, we investigated the effects of the PKC activators PMA and mezerein (MEZ) on Ly-6E expression. Although neither PMA nor MEZ by themselves could induce Ly-6E in YAC cells, both agents enhanced by up to fivefold the induction of Ly-6 mRNA and Ly-6E surface expression triggered by IFN-gamma. However, the induction of Ly-6E expression caused by IFN-alpha/beta was only marginally increased by cotreatment of YAC cells with PMA or MEZ. Altogether, these observations demonstrate that PKC or a related kinase is involved in the transduction mechanisms that lead to Ly-6E induction. However, activation of PKC is not sufficient for this induction and requires other unidentified signal(s) provided by IFN. Our data also indicate that IFN-gamma and IFN-alpha/beta induce Ly-6E through overlapping but distinct intracellular pathways with different sensitivities to PKC activators.     

Regulation of Bovine Leukemia Virus tax and pol mRNA Levels by Interleukin-2 and -10

Recently, particular cytokines have been identified to affect progression of a variety of diseases and retrovirus infections. Previously, we demonstrated that interleukin-2 (IL-2), IL-12, and gamma interferon increased in peripheral blood mononuclear cells (PBMCs) from animals with early disease and decreased in PBMCs from animals with late disease stages of bovine leukemia virus (BLV) infection. In contrast, IL-10 increased with disease progression. To examine the effects of these cytokines on BLV expression, BLV tax and pol mRNA and p24 protein were quantified by competitive PCR and immunoblotting, respectively. IL-10 inhibited BLV tax and pol mRNA levels in BLV-infected PBMCs; however, the inhibitory effect of IL-10 was prevented in PBMCs depleted of monocytes and/or macrophages (monocyte/macrophages). To determine whether these factors were secreted or monocyte/macrophage associated, monocyte/macrophage-depleted PBMCs were cultured with isolated monocyte/macrophages in transwells where contact between monocyte/macrophages and nonadherent PBMCs was blocked. BLV tax and pol mRNA levels increased in transwell cultures similar to cultures containing nonseparated cells, and IL-10 addition inhibited the increase of BLV tax and pol mRNA. These results suggest that monocyte/macrophages secrete soluble factor(s) that increases BLV mRNA levels and that secretion of these soluble factor(s) could be inhibited by IL-10. In contrast, IL-2 increased BLV tax and pol mRNA and p24 protein production. Thus, IL-10 production by BLV-infected animals with late stage disease may serve to control BLV mRNA levels, while IL-2 may increase BLV mRNA in the early disease stage. To determine a correlation between cell proliferation and BLV expression, the effect of IL-2 and IL-10 on PBMC proliferation was tested. As anticipated, IL-2 stimulated while IL-10 suppressed antigen-specific PBMC proliferation. The present study, combined with our previous findings, suggests that increased IL-10 production in late disease stages suppresses BLV mRNA levels, while IL-2-activated immune responses stimulate BLV expression by BLV-infected B cells.     

Involvement of protein kinase C in the regulation of ornithine decarboxylase mRNA by phorbol esters in rat hepatoma cells

The tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulates a rapid increase in ornithine decarboxylase (EC 4.1.1.17; ODC) activity in target cells. Here we demonstrate that this process involves a rapid accumulation of ODC mRNA, which is maximal 3 h after treatment (three- to eightfold greater than control cells) and decays to control levels within 18 h. Stimulation of ODC mRNA by TPA is blocked by phorbol dibutyrate down-regulation of protein kinase C (PKC). ODC mRNA was also induced by the PKC activators, phospholipase C and 1-oleoyl-2-acetyl-rac-glycerol, and blocked by kinase inhibitors (trifluoroperazine, H7, and palmitoyl-L-carnitine), consistent with a requirement for PKC activation in the induction mechanism. However, the non-PKC-specific protein kinase inhibitor HA1004 also suppressed expression of ODC mRNA in response to TPA, under conditions where it did not inhibit PKC, suggesting that additional kinases may be involved in the intracellular signalling process. The stability of the ODC mRNA (control value = 6.2 +/- 1.6 h) is not significantly changed by either TPA (5.7 +/- 0.8 h) or by cycloheximide (6.0 h). These results are inconsistent with any contribution from altered mRNA half-life towards the accumulation of ODC mRNA following treatment with phorbol ester tumor promoters.     

Thrombin causes increased monocytic-cell adhesion to endothelial cells through a protein kinase C-dependent pathway.

The coagulation protein thrombin has been shown to stimulate multiple endothelial-cell (EC) functions, including production of platelet-derived growth factor and of platelet-activating factor (PAF), and neutrophil adhesion. We have found that thrombin causes increased binding of monocytic cells (U937 cells and normal human monocytes) to cultured EC of various species. Maximum adhesion of monocytes to pig aortic EC occurred 6 h after thrombin treatment and remained elevated through 24 h. Stimulation of adherence by bovine alpha-thrombin was half-maximal at 15 units/ml, and reached a plateau at 50 units/ml. Catalytically inactive thrombin (phenylmethanesulphonyl fluoride-treated) had no effect on monocyte adhesion to EC. Heparin, but not the endotoxin antagonist polymyxin B, suppressed the stimulation of adhesion by thrombin without altering basal adhesion. Two lines of evidence suggested that protein kinase C (PKC) was involved in the intracellular signalling to increase monocyte adhesion to EC. First the PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated monocytic-cell adhesion to EC at a dose consistent with stimulation of PKC (half-maximal response at 1-3 nM) and with a time course similar to that for thrombin stimulation (maximal by 4 h). Diacylglycerol, a physiological activator of PKC, also stimulated U937-cell adhesion to EC. Secondly, H7, a PKC inhibitor, completely blocked stimulation of monocyte adhesion to EC by thrombin or PMA. The structural analogue of H7, HA1004, which preferentially inhibits cyclic-AMP- and cyclic-GMP-dependent protein kinases, had no effect on stimulated monocyte adhesion. The PKC inhibitor also blocked the stimulation of monocyte adhesion to EC by interleukin-1 and endotoxin, but did not alter the basal level of monocyte binding to unstimulated EC. Thrombin stimulation of monocyte adhesion differed from the reported stimulation of neutrophil adhesion by thrombin in that the latter process reached a maximum in minutes rather than hours. In addition, neither PAF itself nor agents known to stimulate PAF production by EC, such as arachidonate and the Ca2+ ionophore A23187, had any effect on monocyte adhesion. These results demonstrate a PKC-dependent cytokine-like action of the coagulation protein thrombin in modulating monocytic-cell adhesion to EC, a phenomenon of potential importance in many pathological and physiological processes.     

Activation of protein kinases A and C increase lymphocyte penetration through endothelial monolayers

A brief incubation of lymphocytes with either PMA, stimulating protein kinase C, or with dibutyryl-cAMP, leading to protein kinase A activation, led to increased lymphocyte penetration through intact endothelial monolayers in vitro. The PMA-induced penetration could be dose-dependently down-regulated with a protein kinase C inhibitor, H7. Similarly HA 1004, being mainly a protein kinase A inhibitor, decreased the dibutyryl-cAMP induced penetration. Treatment of lymphocytes with PMA and cAMP did not alter the expression of CD44 homing receptors on lymphocytes. Stimulation of lymphocytes with dibutyryl-cGMP or calcium ionophore had no effect on lymphocyte penetration. These results suggest that activation of both protein kinases A and C is important in the lymphocyte binding to endothelium.     

H7, a protein kinase C inhibitor, increases the glutathione content of neuroblastoma cells.

It is shown that the intracellular glutathione (GSH) concentration of neuroblastoma-2a cells in culture increases with a maximum at 24 h after starting treatment with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), an inhibitor of protein kinase C (PKC). Other inhibitors of this and other protein kinases, e.g. sphingosine, staurosporine, and HA 1004, at the concentrations tested, had a less marked or negligible effect on intracellular GSH concentration. 12-O-Tetradecanoylphorbol-13-acetate (TPA) was also tested and showed no significant effect 24 h after addition.     

Cellular pathways involved in the ex vivo expression of bovine leukemia virus.

Bovine leukemia virus (BLV) is the etiologic agent of enzootic bovine leukosis. The virus adopts a strategy based on the lack of viral expression in vivo; only very rare BLV-infected B lymphocytes express viral information. When the cells are isolated from animals in persistent lymphocytosis and cultivated ex vivo, a tremendous increase in viral expression occurs. To gain insight into this mechanism, we employed a general approach using chemicals that interfere specifically with cellular pathways involved in signal transduction from the cell membrane to the nucleus. Our data demonstrate that BLV expression is not correlated with the activity of protein kinase A (PKA) and is even inhibited by cyclic AMP (cAMP). The cAMP/PKA pathway is thus apparently not involved in ex vivo viral expression. In contrast, PKC appears to play a key role in this process. Phorbol myristate acetate can directly activate viral expression in B cells (in the absence of T cells). Furthermore, calphostin C, a highly specific inhibitor of PKC, partly decreases ex vivo BLV expression. Our data further demonstrate that calmodulin and calcineurin, a calmodulin-dependent phosphatase, play a key role in the induction of viral expression. The involvement of this calmodulin-dependent pathway could explain the induction of expression that cannot be assigned to PKC. Furthermore, it appears that the activation of viral expression requires a calmodulin but not a PKA-dependent pathway. These data highlight major differences between transient transfection and ex vivo experiments. Finally, despite their homologies, BLV and human T-cell leukemia virus appear to use different signal transduction pathways to induce viral expression.     

Induction of gene expression by IL-1 in NIH 3T3 cells. Possible requirement of protein kinase C activity and independence from arachidonic acid metabolism

NIH 3T3 fibroblasts were transfected with the chloramphenicol-acetyltransferase (CAT) gene under the control of the SV40 early promoter, which can be stimulated by IL-1. CAT activity in cell lysates and PGE2 release in the supernatants were measured in control and stimulated cell cultures in parallel. Human IL-1 beta (180 pM) and human rTNF-alpha (3 nM) significantly stimulated both CAT activity and PGE2 release. The combined incubation of the two cytokines resulted in a synergistic effect on PGE2 release. The addition of AA (30 microM) greatly stimulated PGE2 release without affecting CAT activity. Similarly, drugs interfering with AA metabolism were without effect on CAT activity although profoundly reducing PGE2 release. Forskolin (0.1 microM) did not modify either parameter. The glucocorticoid fluocinolone (20 nM) was able to decrease both parameters. Protein kinase inhibitors H7 (5-50 microM) and sphingosine (50 microM) inhibited only IL-1-induced CAT activity, whereas H8 (5-50 microM) and HA1004 (50 microM) were ineffective on both parameters. PMA (0.5 microM) and R59 022, a diacylglycerol kinase inhibitor (10 microM), did not modify either control or IL-1-induced CAT activity. IL-1-stimulated PGE2 release was potentiated by PMA, although this effect was not inhibited by H7. The data suggest that: 1) in NIH 3T3 cells the activation of AA metabolism by IL-1 is not involved in IL-1-induced gene expression; 2) protein kinase C activity is required but not sufficient for IL-1-induced gene expression; and 3) PMA may stimulate AA metabolism by a mechanism in part independent of protein kinase activity.     

Differential effect of molecular size HA in mouse chondrocytes stimulated with PMA

Background

Hyaluronan (HA) fragments elicit the expression of inflammatory mediators through a mechanism involving the CD44 receptor. This study investigated the effects of HA at different molecular weights on PMA-induced inflammation in mouse chondrocytes.

Methods

mRNA and related protein levels were measured for CD44, PKCδ, PKC?, TNF-α, IL-1β, MMP-13, and iNOS in chondrocytes, untreated or PMA treated, with and without the addition of HA. The level of NF-kB activation was also assayed.

Results

CD44, PKCδ, and PKC? mRNA expression resulted higher than controls in chondrocytes treated with PMA. PMA also induced NF-kB up-regulation and increased TNF-α, IL-1β, MMP-13, and iNOS expression. HA treatment produced different effects: low MW HA up-regulated CD44 expression, increased PKCδ and PKC? levels, and enhanced inflammation in untreated chondrocytes; while in PMA-treated cells it increased CD44, PKCδ, PKC?, NF-kB, TNF-α, IL-1β, MMP-13, and iNOS expression and enhanced the effects of PMA; medium MW HA did not exert action; high MW HA had no effect on untreated chondrocytes; however, it reduced PKCδ, PKC?, NF-kB activation and inflammation in PMA-stimulated cells. Specific CD44 blocking antibody was utilised to confirm CD44 as the target of HA modulation.

General Significance

These data suggest that HA via CD44 may modulate inflammation via its different molecular mass.     

Transactivation of hsp70-1/2 in geldanamycin-treated human non-small cell lung cancer H460 cells: involvement of intracellular calcium and protein kinase C

Geldanamycin is an antitumor drug that binds HSP90 and induces a wide range of heat shock proteins, including HSP70s. In this study we report that the induction of HSP70s is dose-dependent in geldanamycin-treated human non-small cell lung cancer H460 cells. Analysis of the induction of HSP70s specific isoform using LC-ESI-MS/MS analysis and Northern blotting showed that HSP70-1/2 are the major inducible forms under geldanamycin treatment. Transactivation of hsp70-1/2 was determined by electrophoretic mobility-shift assay using heat shock element (HSE) as a probe. The signaling pathway mediators involved in hsp70-1/2 transactivation were screened by the kinase inhibitor scanning technique. Pretreatment with serine/threonine protein kinase inhibitors H7 or H8 blocked geldanamycin-induced HSP70-1/2, whereas protein kinase A inhibitor HA1004, protein kinase G inhibitor KT5823, and myosin light chain kinase inhibitor ML-7 had no effect. Furthermore, the protein kinase C (PKC)-specific inhibitor Ro-31-8425 and the Ca2+-dependent PKC inhibitor G?-6976 diminished geldanamycin-induced HSP70-1/2, suggesting an involvement of the PKC in the process. In addition, geldanamycin treatment causes a transient increase of intracellular Ca2+. Chelating intracellular Ca2+ with BAPTA-AM or depletion of intracellular Ca2+ store with A23187 or thapsigargin significantly decreased geldanamycin-transactivated HSP70-1/2 expression. Taken together, our results demonstrate that geldanamycin-induced specific HSP70-1/2 isoforms expression in H460 cells through signaling pathway mediated by Ca2+ and PKC.     

Up-regulation of tissue inhibitor of metalloproteinases-3 gene expression by TGF-β in articular chondrocytes is mediated by serine/threonine and tyrosine kinases

The balance between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) regulates extracellular matrix turn-over in normal animal development, cancer cell metastasis, atherosclerotic plaque rupture and erosion of arthritic cartilage. Transforming growth factor beta (TGF-β), an inducer of matrix synthesis, potently enhances mRNA and protein of a recently characterized MMP inhibitor, TIMP-3, in bovine articular chondrocytes. We examined the implication of protein kinases in the TGF-β-mediated induction of TIMP-3 expression by utilizing activators and inhibitors of these enzymes. Protein kinase A activators, dibutyryl cyclic AMP, or forskolin had little or no effect, respectively, while phorbol 12-myristate 13-acetate (PMA), a PKC activator, increased TIMP-3 gene expression. H7, a serine/threonine protein kinase inhibitor, markedly reduced the response of TIMP-3 gene to TGF-β. Furthermore, two protein tyrosine kinase inhibitors, genistein and herbimycin A, inhibited TGF-β induction of TIMP-3. H7 and genistein also suppressed TGF-β-induced TIMP-3 protein expression. These results suggest that TGF-β signaling for TIMP-3 gene induction involves H7-sensitive serine/threonine kinase as well as herbimycin A- and genistein-sensitive protein tyrosine kinases. J. Cell. Biochem. 70:517–527, 1998. © 1998 Wiley-Liss, Inc.