The role of protein kinase C in the development of the complications of diabetes.

Diabetes mellitus produces a state of chronic hyperglycemia which in turn leads to the development of severe complications including retinopathy, nephropathy, neuropathy, and atherosclerosis. Many different mechanisms have been put forward to attempt to explain how glucose elevations can damage these various organ systems. Protein kinase C activation is one of the sequelae of hyperglycemia and is thought to play a role in the development of diabetic complications. There are multiple mechanisms for its activation in the diabetic state and multiple downstream effects attributable to that activation. The role of protein kinase C activation in the development of the above-mentioned complications of diabetes is discussed in this chapter. In addition, the potential use of isoform-specific inhibitors of protein kinase C for the treatment of diabetic complications is proposed.

Protein kinase C decreases the hepatocyte growth factor-induced activation of Erk1/Erk2 MAP kinases.

HGF and phorbol ester induce the scattering of HepG2 cells. Recently, we have reported that the motility and morphological responses that accompany this process require the activation of Erk1/Erk2 MAP kinases, and phosphatidylinositol 3-kinase contributes to the activation of Erk1/Erk2 in HGF-induced cells. The cell scattering-associated appearance of a high-M(r) (>300 kDa) protein pair has also been observed, and has been proven to be a sensitive marker of the intensity of Erk1/Erk2 activation. Our present study demonstrates that in HGF-induced cells protein kinase C and phosphatidylinositol 3-kinase regulate oppositely the expression of these cell scattering-associated proteins. While in phorbol ester-treated cells the sustained activation of protein kinase C is essential for this expression, in HGF-induced cells the inhibition of protein kinase C with bisindolylmaleimide I stimulates the expression. Protein kinase C reduces the HGF-induced phosphorylation of Erk1/Erk2, and in this way it can limit the intensity of Erk1/Erk2-dependent gene-expression

Amelioration of accelerated diabetic mesangial expansion by treatment with a PKC beta inhibitor in diabetic db/db mice, a rodent model for type 2 diabetes.

Activation of protein kinase C (PKC) is implicated as an important mechanism by which diabetes causes vascular complications. We have recently shown that a PKC beta inhibitor ameliorates not only early diabetes-induced glomerular dysfunction such as glomerular hyperfiltration and albuminuria, but also overexpression of glomerular mRNA for transforming growth factor beta1 (TGF-beta1) and extracellular matrix (ECM) proteins in streptozotocin-induced diabetic rats, a model for type 1 diabetes. In this study, we examined the long-term effects of a PKC beta inhibitor on glomerular histology as well as on biochemical and functional abnormalities in glomeruli of db/db mice, a model for type 2 diabetes. Administration of a PKC beta inhibitor reduced urinary albumin excretion rates and inhibited glomerular PKC activation in diabetic db/db mice. Administration of a PKC beta inhibitor also prevented the mesangial expansion observed in diabetic db/db mice, possibly through attenuation of glomerular expression of TGF-beta and ECM proteins such as fibronectin and type IV collagen. These findings provide the first in vivo evidence that the long-term inhibition of PKC activation in the renal glomeruli can ameliorate glomerular pathologies in diabetic state, and thus suggest that a PKC beta inhibitor might be an useful therapeutic strategy for the treatment of diabetic nephropathy.

Enzymatic rationale and preclinical support for a potent protein kinase C beta inhibitor in cancer therapy.

The macrocyclic bisindolylmaleimide, LY333531, selectively inhibits protein kinase C beta 1 and beta 2 isoforms with an approximate IC50 of 5 nanomolar. The efficacy of LY333531 administered alone and in combination with cytotoxic cancer therapies in models of non-small cell lung carcinoma and brain tumors was determined in vivo. In the Lewis lung carcinoma, administration of LY333531 enhanced the activity of paclitaxel and fractionated radiation and, to a lesser degree, carboplatin and gemcitabine. In the human T98G glioblastoma multiforme xenograft, the addition of LY333531 to treatment with carmustine (BCNU) resulted in enhanced tumor response in a nodule grown subcutaneously and increased life-span in animals bearing an intracranial tumor from 37 days in the control animals to 64 days in the BCNU treated animals, and to 104 days in the LY333531 plus BCNU treated animals with 4 out of 5 animals being long-term survivors.

Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis.

The growth of any solid tumor depends on angiogenesis. Among the known angiogenic factors, vascular endothelial growth factor (VEGF) has been shown to play a pivotal role in tumor angiogenesis. However, to date, the signal transduction pathway initiated by VEGF is still not fully understood. It has been suggested that protein kinase C (PKC) plays an important role in the VEGF-induced signal transduction pathway in vitro, although the role of PKC in tumor angiogenesis in vivo still remains to be elucidated. By delivering the VEGF gene within the self-contained tetracycline-regulated retroviral vector (Retro-Tet) into hepatocellular carcinoma (HCC) cells, we manipulated VEGF expression by providing tetracycline in the drinking water to assess the tumor kinetics mediated exclusively by VEGF. In this study, we combined this Retro-tet system and LY333531, an inhibitor of the PKC-beta isoform, to elucidate the role of PKC-beta in tumor development and angiogenesis. Using a syngenic xenograft model, tumor augmentation induced by VEGF overexpression in HCC was markedly suppressed by oral administration of the PKC-beta inhibitor, with an accompanying reduction of neovascularization and p44/42 mitogen-activated protein kinase activation. This inhibitory effect was achieved even after the tumor was fully established. Immunohistochemical analysis revealed that apoptosis increased markedly in the tumor upon PKC-beta inhibitor treatment, whereas tumor cell proliferation itself did not change. Furthermore, with orthotopical transplantation, PKC-beta inhibition suppressed HCC tumor development in the liver. These results suggest that PKC-beta lies on the signal transduction pathway by which VEGF augments development and angiogenesis not only at the initial stage but also after the tumor is fully established.

Overexpression of protein kinase C-alpha in MCF-7 breast cancer cells results in differential regulation and expression of alphavbeta3 and alphavbeta5.

MCF-7 breast cancer cells stably transfected with protein kinase C-alpha (MCF-7-PKC-alpha cells) show anchorage-independent growth and exhibit increased tumorigenicity in nude mice. Since integrins are involved in tumor growth and metastatic spread, we investigated whether integrin expression is differentially regulated in MCF-7-PKC-alpha cells. Fluorescence-activated cell sorting revealed that alphavbeta3 is highly expressed on MCF-7-PKC-alpha cells, but is undetectable on MCF-7V cells (MCF-7 cells transfected with vector only). In contrast, MCF-7-PKC-alpha cells have reduced expression of alphavbeta5. Blocking experiments with antibodies to alphavbeta3 and alphavbeta5 revealed that these receptors are used by MCF-7-PKC-alpha cells to adhere primarily to vitronectin and osteopontin. Consistent with heterodimer expression, MCF-7-PKC-alpha cells express increased beta3 and decreased beta5 on their surface. Surface expression of alphav on MCF-7-PKC-alpha cells is unchanged. Western blotting, Northern analysis, and nuclear run-on assays indicated that post-translational mechanisms increase the surface expression of beta3 on MCF-7-PKC-alpha cells. In contrast, reduced beta5 transcription diminishes beta5 surface expression on MCF-7-PKC-alpha cells. These results indicate that overexpression of PKC-alpha in MCF-7 cells alters beta5 and beta3 expression by transcriptional and post-translational mechanisms, respectively, resulting in altered heterodimer expression. These findings suggest that the increased metastatic capacity of tumor cells with elevated protein kinase C levels may result, in part, from modulation of integrin expression.

Effects of protein kinase C inhibitors on thromboxane production by thrombin-stimulated platelets.

The purpose of these studies was to identify a possible role for protein kinase C in thromboxane production. The effects of four putative protein kinase C inhibitors were studied with platelet stimulation by thrombin (0.5-150 nM), Thrombin Quick I (1.5-500 nM) or a thrombin receptor (protease activated receptor-1) agonist peptide (TRAP) (5-120 microM). Thromboxane production was increased by the bisindolylmaleimide derivative, 2-[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3-yl)-maleimi de (GF 109203X), unchanged by the inhibitors 12-(2-cyanoethyl)-6,7, 12,13-tetrahydro-13-methyl-5-oxo-5H-indolo (2,3-a) pyrrolo (3, 4-c)-carbazole (Gö 6976) and 5,21:12,17-dimetheno-18H-dibenzo[i, o]pyrrolo[3,4-l][1,8]diazacyclohexadecine-18,20(19H)-dione, 8-[(dimethylamino)methyl]-6,7,8,9,10,11-hexahydro-, monomethanesulfonate (379196), the latter of which is protein kinase C beta-selective, and decreased by 1-[6-[(3-acetyl-2,4, 6-trihydroxy-5-methylphenyl)methyl]-5,7-dihydroxy-2, 2-dimethyl-2H-1-benzopyran-8-yl]-3-phenyl-2-propen-1-one (rottlerin), an inhibitor selective for protein kinase C delta. These results indicate complex regulation of thromboxane synthesis in human platelets including a probable role for protein kinase C delta. The results taken together further suggest that GF 109203X may suppress negative feedback resulting from an unidentified kinase and that the classical protein kinase C isoforms alpha and beta do not have a significant role in regulating thromboxane production by platelets.

Phosphatidylinositol 3-kinase contributes to Erk1/Erk2 MAP kinase activation associated with hepatocyte growth factor-induced cell scattering.

MAP kinase cascade-dependent responses were investigated during scattering of HepG2 human hepatoma cells stimulated by HGF or phorbol ester. Inhibition of phosphatidylinositol 3-kinase with LY294002 prevented completely the dissociation of cells. Inhibition of MAP kinase kinase (MEK) with PD98059 prevented the development of characteristic morphological changes associated with cell migration. EGF, which failed to induce cell scattering, caused a short-term increase in the phosphorylation of Erk1/Erk2 MAP kinases. On the contrary, HGF or phorbol ester stimulated the phosphorylation of MAP kinases for a long time. Experiments performed with LY294002 indicated that phosphatidylinositol 3-kinase contributed to the HGF-stimulated phosphorylation of Erk1/Erk2. This finding was confirmed by the demonstration that the MAP kinase cascade-dependent expression of a high-Mr (>300 kDa) protein pair appearing in the course of cell scattering was inhibited by LY294002 in HGF-induced cells but was not inhibited in phorbol ester-treated cells.

Targeted overexpression of protein kinase C beta2 isoform in myocardium causes cardiomyopathy.

Increased cardiovascular mortality occurs in diabetic patients with or without coronary artery disease and is attributed to the presence of diabetic cardiomyopathy. One potential mechanism is hyperglycemia that has been reported to activate protein kinase C (PKC), preferentially the beta isoform, which has been associated with the development of micro- and macrovascular pathologies in diabetes mellitus. To establish that the activation of the PKCbeta isoform can cause cardiac dysfunctions, we have established lines of transgenic mice with the specific overexpression of PKCbeta2 isoform in the myocardium. These mice overexpressed the PKCbeta2 isoform transgene by 2- to 10-fold as measured by mRNA, and proteins exhibited left ventricular hypertrophy, cardiac myocyte necrosis, multifocal fibrosis, and decreased left ventricular performance without vascular lesions. The severity of the phenotypes exhibited gene dose-dependence. Up-regulation of mRNAs for fetal type myosin heavy chain, atrial natriuretic factor, c-fos, transforming growth factor, and collagens was also observed. Moreover, treatment with a PKCbeta-specific inhibitor resulted in functional and histological improvement. These findings have firmly established that the activation of the PKCbeta2 isoform can cause specific cardiac cellular and functional changes leading to cardiomyopathy of diabetic or nondiabetic etiology.

Transcriptional regulation of insulin receptor substrate 1 by protein kinase C.

Insulin receptor substrate-1 (IRS-1) is involved in insulin signal transduction distal to receptor occupation. Targeted disruption of IRS-1 leads to insulin resistance and hyperglycemia in mice, which suggests that altered IRS-1 expression could contribute to the insulin resistance seen in non-insulin-dependent diabetes mellitus. In vitro studies using phorbol esters have implicated the protein kinase C (PKC) pathway as being involved in the pathogenesis of insulin resistance. Using the MCF-7 breast cancer cell, a role for PKC in regulating IRS-1 expression was examined. In an MCF-7 cell line (MCF-7-PKC-alpha) that exhibits multiple alterations in PKC isoform expression, IRS-1 content was reduced to negligible levels relative to parental MCF-7 cells. This decrease in IRS-1 content was associated with a 30-fold reduction in IRS-1 transcription. In parental MCF-7 cells, PKC inhibitors (GF109203X (bisindolylmaleimide I) and staurosporine) reduced IRS-1 content. Chronic exposure to 12-O-tetradecanoylphorbol-13-acetate (TPA; >8 h) reduced IRS-1 content and down-regulated the novel PKC-delta isoform. Bryostatin 1 inhibited TPA-induced depletion of both IRS-1 and PKC-delta expression in MCF-7 cells. Associated with TPA-induced reduction in IRS-1 content was a reduction in IRS-1 transcription. These data demonstrate that PKC can modulate IRS-1 content and suggest a potential role for PKC-delta in positively regulating IRS-1 expression.

Protein kinase Calpha is a major mediator of the stimulatory effect of phorbol ester on phospholipase D-mediated hydrolysis of phosphatidylethanolamine.

Stimulation of phospholipase D (PLD)-mediated hydrolysis of phosphatidylcholine (PtdCho) by phorbol 12-myristate 13-acetate (PMA) has been shown to be mediated by the alpha- and betaI-isoforms of protein kinase C (PKC). To determine the role of various PKC isozymes in the regulation of PLD-mediated phosphatidylethanolamine (PtdEtn) hydrolysis, MCF-7 human breast carcinoma cells overexpressing the alpha- and theta-isoforms, and R6 rat fibroblasts overexpressing the alpha-, betaI-, and epsilon-isoforms were used. In the vector control MCF-7 cells, which contain low levels of PKC-alpha, PMA (100 nM) had only small effects on the hydrolysis of PtdEtn (1.1-1.35-fold) and PtdCho (1.15-1.6-fold). Stable expression of PKC-alpha in MCF-7 cells, which was accompanied by increased levels of the betaI- and theta-isoforms as well, greatly enhanced both PMA-induced PLD-mediated formation of phosphatidylethanol (approximately 5-fold) and the hydrolysis of PtdEtn (2.5-2.9-fold) and PtdCho (5.5-7.2-fold). The effects of PMA on the hydrolysis of PtdEtn (and PtdCho) in MCF-7/PKC-alpha cells were significantly inhibited by 0.5-3 microM concentrations of Gö 6976, a selective inhibitor of the conventional PKC subfamily. Stable expression of PKC-alpha in R6 fibroblasts enhanced, at a shorter (10 min) incubation time, the effects of PMA on the hydrolysis of both PtdEtn and, to a lesser extent, PtdCho. In contrast, stable expression of PKC-betaI in R6 fibroblasts, which originally did not contain this enzyme, enhanced the effects of PMA only on PtdCho, but not PtdEtn, hydrolysis. Overexpression of either PKC-theta in MCF-7 cells or PKC-epsilon in R6 and NIH 3T3 fibroblasts had no detectable effects on PMA-induced hydrolysis of PtdEtn. Collectively, the results suggest that PKC-alpha has a major role in the mediation of phorbol ester action on PtdEtn hydrolysis, while PtdCho hydrolysis may be regulated by both the alpha and betaI isoforms.

Phorbol ester-stimulated phosphorylation of PU.1: association with leukemic cell growth inhibition.

PU.1, a member of the ets transcription factor family, has been previously shown to be necessary for tetradecanoylphorbol-13 acetate (TPA)-induced U937 leukemic cell maturation. We examined the effects of TPA on PU.1 content and PU.1 DNA binding activity in U937 cells. Unstimulated cells expressed PU.1 mRNA transcripts and TPA did not increase these levels. However, TPA treatment induced phosphorylation of PU.1. Gel-shift analysis using a labeled PU.1 oligomer showed that TPA induced a unique PU.1 binding activity. This binding activity was phosphorylation-dependent, as indicated by the ability of phosphatase treatment to abolish its detection. The PU.1 binding activity was generated at TPA-13 concentrations stimulating growth arrest and was blocked by the PKC inhibitor GF109203X, which antagonized TPA-induced growth inhibition. Bryostatin 1, another protein kinase C activator, induced only a modest degree of U937 growth inhibition and antagonized TPA-stimulated growth arrest. Bryostatin 1 was unable to induce this TPA-generated PU.1 binding activity. High bryostatin 1 concentrations inhibited generation of this TPA-induced band shift. These data suggest that TPA-induced growth inhibition is associated with phosphorylation of PU.1 and generation of a unique PU.1 binding activity.

Protein kinase C isoform expression during the differentiation of 3T3-L1 preadipocytes: loss of protein kinase C-alpha isoform correlates with loss of phorbol 12-myristate 13-acetate activation of nuclear factor kappaB and acquisition of the adipocyte phenotype.

The regulated expression of protein kinase C (PKC) isoforms was examined during the differentiation program of 3T3-L1 preadipocytes. In a parallel analysis, differentiation was blocked by treatment of the cells with tumor necrosis factor-alpha (TNF) to determine differentiation-specific changes in isoform expression from growth or treatment-induced effects. This analysis revealed that the expression of the conventional PKC-alpha isoform was reduced by 85% as cells attained the adipocyte phenotype. PKC-beta expression was measurable only during the early stages of the differentiation process and was not detectable in fully differentiated cells. An upregulation of PKC-theta, a novel PKC isoform, occurred during the latter stage of differentiation. Expression of PKC-zeta an atypical PKC isoform suggested to participate in TNF signal transduction, occurred throughout the time course with similar levels of expression in both preadipocytes and adipocytes. Nuclear run-on analysis demonstrated an approximately 85% reduction in the transcription of the PKC-alpha gene during differentiation. The reduced expression of this isoform corresponded with the decreased ability to activate nuclear factor kapppaB (NF-kappaB) in response to phorbol 12-myristate 13-acetate (PMA) treatment in the adipocytes. These data suggest that PMA responsiveness in 3T3-L1 adipocytes is markedly diminished.

Effect of ionizing radiation on AP-1 binding activity and basic fibroblast growth factor gene expression in drug-sensitive human breast carcinoma MCF-7 and multidrug-resistant MCF-7/ADR cells.

We studied the effect of ionizing radiation on the activation of the AP-1 transcription factors and the regulation of basic fibroblast growth factor (bFGF) gene expression in drug-sensitive human breast carcinoma (MCF-7) cells and its drug-resistant variant (MCF-7/ADR) cells. Northern blot and gel mobility shift assays showed that 135 cGy of ionizing radiation induced c-jun and c-fos gene expression, AP-1 binding activity, as well as bFGF gene expression in MCF-7/ADR cells. In MCF-7 cells, however, we observed little/no induction of bFGF gene expression and AP-1 binding activity after the stress. Nevertheless, MCF-7 cells transfected with plasmids containing c-jun gene contain high levels of bFGF protein. H-7 (60 micrograms/ml), a potent protein kinase C (PKC) inhibitor, inhibited the stress-induced AP-1 binding activity and bFGF gene expression in MCF-7/ADR cells. Corroborating this observation, overexpression of PKC alpha induced bFGF gene expression in MCF-7 cells. Taken together, these results suggest that stress-induced bFGF gene expression is mediated through the activation of PKC and AP-1 transcription factors. Differences in the levels of PKC activity and AP-1 binding factors may be responsible for differential expression of bFGF among breast cancer cell lines. Although there are large differences in response to ionizing radiation between MCF-7 and MCF-7/ADR cell lines, we observed no significant differences in radiocytotoxicity between them.

Phorbol ester treatment of U937 cells with altered protein kinase C content and distribution induces cell death rather than differentiation.

Overexpression of protein kinase C (PKC)-zeta, an atypical PKC isoform, in U937 cells stimulates certain parameters of phenotypic maturation and increases expression of endogenous alpha and beta PKC isoforms. In response to 12-O-tetradecanoylphorbol-13-acetate (TPA), parental U937 cells displayed growth arrest and differentiated into a monocyte/macrophage-like cell line, while PKC-zeta cells underwent death. The ability of GF109203X to inhibit TPA-induced death of PKC-zeta cells suggested that activation of a conventional isoform was necessary to induce apoptosis. While exhibiting unique morphological changes, parameters indicative of a further degree of differentiation were not observed in TPA-treated PKC-zeta cells. TPA-induced down-regulation of PKC activity was similar in both cells. While modest quantitative differences in individual isoform down-regulation existed, intracellular localization of isoforms prior to activation differed significantly between U937 and PKC-zeta cells. Expression of gadd45 was induced by TPA in PKC-zeta but not parental cells and occurred as a primary response to TPA and prior to the onset of cell death. These data suggest that the decision of a cell to undergo death or differentiation in response to phorbol esters may, in part, be modulated by alterations within the PKC signal transduction pathway.

MCF-7 breast cancer cells transfected with protein kinase C-alpha exhibit altered expression of other protein kinase C isoforms and display a more aggressive neoplastic phenotype.

Increased protein kinase C (PKC) activity in malignant breast tissue and positive correlations between PKC activity and expression of a more aggressive phenotype in breast cancer cell lines suggest a role for this signal transduction pathway in the pathogenesis and/or progression of breast cancer. To examine the role of PKC in the progression of breast cancer, human MCF-7 breast cancer cells were transfected with PKC-alpha, and a group of heterogenous cells stably overexpressing PKC-alpha were isolated (MCF-7-PKC-alpha). MCF-7-PKC-alpha cells expressed fivefold higher levels of PKC-alpha as compared to parental or vector-transfected MCF-7 cells. MCF-7-PKC-alpha cells also displayed a substantial increase in endogenous expression of PKC-beta and decreases in expression of the novel delta- and eta-PKC isoforms. MCF-7-PKC-alpha cells displayed an enhanced proliferative rate, anchorage-independent growth, dramatic morphologic alterations including loss of an epithelioid appearance, and increased tumorigenicity in nude mice. MCF-7-PKC-alpha cells exhibited a significant reduction in estrogen receptor expression and decreases in estrogen-dependent gene expression. These findings suggest that the PKC pathway may modulate progression of breast cancer to a more aggressive neoplastic process.

Persistent enhancement of sustained calcium-dependent glutamate release by phorbol esters: role of calmodulin-independent serine/threonine phosphorylation and actin disassembly.

The phorbol ester 4 beta-phorbol 12,13-dibutyrate increases the final extent of Ca(2+)-dependent glutamate release during the continuous depolarization of the synaptosomal plasma membrane. Based on this finding, we suggested that the sustained activation of protein kinase C has a positive influence on the efficiency of synaptic vesicle recycling in the presence of saturating concentrations of Ca2+. Previous work from our laboratory demonstrated that this 4 beta-phorbol 12,13-dibutyrate-dependent enhancement of synaptic vesicle recycling persists following the removal of 4 beta-phorbol 12,13-dibutyrate, requires localized Ca2+ entry through voltage-regulated channels, and is insensitive to the protein kinase inhibitor staurosporine. In the present study, we examined the possibility that the facilitation of glutamate release may be propagated through interactions between the protein kinase C- and multifunctional Ca2+/calmodulin-dependent protein kinase pathways. However, our data argue strongly against the involvement of such a mechanism in the persistent enhancement of sustained glutamate release. We observed that 4 beta-phorbol 12,13-dibutyrate did not increase the availability of cytosolic free calmodulin or the level of autonomous Ca2+/calmodulin-dependent protein kinase activity. In addition, we determined the effects of various serine/threonine kinase and phosphatase inhibitors on the phorbol ester-dependent enhancement of sustained glutamate release and found that protein kinase C increased the extent, but not the duration, of Ca(2+)-dependent glutamate release through a kinase-independent mechanism. Given our finding that the actin-depolymerizing agent cytochalasin D totally occluded the eb1ect of 4 beta-phorbol 12,13-dibutyrate on release, we postulate that protein kinase C signals may be transduced through direct interactions between protein kinase C isoforms and cytoskeletal protein kinase C binding proteins.

Overexpression of protein kinase C-zeta stimulates leukemic cell differentiation.

A function for protein kinase C-zeta (PKC-zeta), a member of the phorbol ester nonresponsive atypical protein kinase C subfamily, in modulating differentiation was examined in the leukemic U937 cell. Transfected U937 cells stably overexpressing PKC-zeta displayed a longer doubling time, lower saturation density at confluency, and an increase in adherence to plastic as compared to control cells. PKC-zeta cells expressed a more differentiated phenotype as assessed by changes in morphology, surface antigen expression, and lysosomal enzyme activities and were distinct from parental U937 cells stimulated to differentiate by exposure to phorbol esters. In contrast to parental U937 cells, PKC-zeta cells constitutively expressed mRNA transcripts for c-jun and a low mobility AP-1 binding activity. Thus, PKC-zeta overexpression stimulates a type of phenotypic differentiation that differs significantly from maturation occurring upon activation of other PKC subfamilies induced by phorbol ester treatment. Increased expression of the c-jun protooncogene and an increase in AP-1 binding activity in PKC-zeta cells provides a potential mechanism for explaining the altered differentiation status of this cell.

Divergent dimerization properties of mutant beta 1 thyroid hormone receptors are associated with different dominant negative activities.

Syndromes of resistance to thyroid hormones are caused by mutations in the T3-binding domain of the c-erbA beta thyroid hormone receptor gene. The S receptor (deletion of THR332) is a potent dominant negative protein cloned from a kindred with generalized resistance to thyroid hormones. The G-H receptor (ARG311HIS) has compromised dominant negative function and was found in both normal individuals and in a patient with severe pituitary resistance to thyroid hormones. We have investigated the mechanism responsible for the difference in receptor phenotypes by analyzing the binding of S and G-H receptors to thyroid hormone response elements with electrophoretic mobility shift analysis. Wild-type human c-erbA beta 1 (WT), S, and G-H receptors were synthesized in reticulocyte lysate, reacted with a thyroid hormone response element consisting of a direct repeat with 4 base pairs (DR+4; AGGTCA CAGG AGGTCA), and the products analyzed by gel shift. G-H receptor homodimerization was greatly impaired; G-H formed predominantly monomeric complex compared with monomeric and homodimeric WT complexes. The G-H receptor was able to form heterodimeric complexes with cellular thyroid hormone receptor auxiliary protein (TRAP) factors including the human retinoid X receptor-alpha. When TRAP was limiting, the levels of G-H heterodimeric complex were 2- to 3-fold reduced compared with WT receptor. In contrast to the WT and G-H receptors, the S receptor formed almost exclusively homodimeric complex with DR+4; the approximate ratio of S:WT:G-H homodimeric complexes at equivalent concentrations of receptors was 60:20:1. A measurable increase (1.2- to 2.6-fold) in heterodimeric complex formation was observed with the S receptor relative to WT when TRAP was at limiting concentration. As reported previously by others, thyroid hormone significantly reduced the WT homodimeric complex with DR+4. There was no effect on the S homodimeric complex. Finally, the WT, S, and G-H receptors formed different complexes with the element consisting of an inverted repeat with 5 base pairs (IR+5; AGGTCA ACAGT TGACCT) and the IR element (AGGTCA TGACCT), which were differently regulated by thyroid hormone. The S receptor bound as a homodimer with IR+5, whereas the WT receptor bound as a homodimer only with thyroid hormone. No homodimeric complex formed with IR+5 and the G-H receptor. Qualitatively similar results were observed with the IR element. We conclude that the ARG311HIS mutation severely perturbs the homodimerization and, to a much less degree, heterodimerization functions of the c-erbA beta 1 receptor. Furthermore, the THR332 deletion mutation augments homodimerization of the c-erbA beta 1 receptor. These results indicate that different mutations in the c-erbA beta 1 thyroid hormone receptor have divergently affected dimerization activities which seem to influence the level of dominant negative activity in man.

12-Deoxyphorbol-13-O-phenylacetate-20-acetate is not protein kinase C-beta isozyme-selective in vivo.

The phorbol ester, 12-deoxyphorbol-13-O-phenylacetate-20-acetate (DOPPA) has been shown to activate specifically the protein kinase C (PKC)-beta 1 isozyme in vitro (1). We have investigated the potential of DOPPA as a PKC-beta 1/2 isozyme-specific agonist in intact cells, employing U937 cells, which express beta 1/2, epsilon and zeta PKC and in Swiss 3T3 cells which lack PKC-beta 1/2 but express alpha, delta, epsilon and zeta PKC. Immunoblot analysis with isozyme-specific antibodies indicated that DOPPA can mediate the subcellular redistribution and down-modulation of all endogenous PKC isozymes (except PKC-zeta) in both U937 and Swiss 3T3 cells. Prolonged treatment (> 6 h) of cultures in down-modulation studies is complicated by the metabolism of DOPPA to 12-deoxyphorbol-13-phenylacetate (DOPP), a compound which activates all PKC isozymes tested in vitro (Ryves, W. J., et al. (1991) FEBS Lett., 288, 5-9). Nevertheless, because DOPPA induced rapid and dose-dependent phosphorylation of p80 in cells which do not express PCK-beta, p80 phosphorylation in Swiss 3T3 cells indicates that DOPPA can activate a non-beta PKC in vivo. The data suggest that DOPPA cannot be used as a PKC-beta-selective agonist in intact cell studies.