Toshio Kuroki

Induction of differentiation in normal human keratinocytes by adenovirus-mediated introduction of the eta and delta isoforms of protein kinase C.

ABSTRACT Protein kinase C (PKC) plays a crucial role(s) in regulation of growth and differentiation of cells. In the present study, we examined possible roles of the α, δ, η, and ζ isoforms of PKC in squamous differentiation by overexpressing these genes in normal human keratinocytes. Because of the difficulty of introducing foreign genes into keratinocytes, we used an adenovirus vector system, Ax, which allows expression of these genes at a high level in almost all the cells infected for at least 72 h. Increased kinase activity was demonstrated in the cells overexpressing the α, δ, and η isoforms. Overexpression of the η isoform inhibited the growth of keratinocytes of humans and mice in a dose (multiplicity of infection [MOI])-dependent manner, leading to G1 arrest. The η-overexpressing cells became enlarged and flattened, showing squamous cell phenotypes. Expression and activity of transglutaminase 1, a key enzyme of squamous cell differentiation, were induced in the η-overexpressing cells in dose (MOI)- and time-dependent manners. The inhibition of growth and the induction of transglutaminase 1 activity were found only in the cells that express the η isoform endogenously, i.e., in human and mouse keratinocytes but not in human and mouse fibroblasts or COS1 cells. A dominant-negative η isoform counteracted the induction of transglutaminase 1 by differentiation inducers such as a phorbol ester, 1α,25-dihydroxyvitamin D3, and a high concentration of Ca2+. Among the isoforms examined, the δ isoform also inhibited the growth of keratinocytes and induced transglutaminase 1, but the α and ζ isoforms did not. These findings indicate that the η and δ isoforms of PKC are involved crucially in squamous cell differentiation.

Role of Smad4 (DPC4) inactivation in human cancer

The tumor suppressor gene Smad4 (DPC4) at chromosome 18q21.1 belongs to the Smad family, which mediates the TGFbeta signaling pathway suppressing epithelial cell growth. This review summarizes the mutational events of the Smad4 gene in human cancer. The Smad4 gene is genetically responsible for familial juvenile polyposis, an autosomal dominant disease characterized by predisposition to gastrointestinal polyps and cancer. In this syndrome, polyps are formed by inactivation of the Smad4 gene through germline mutation and loss of the unaffected wild-type allele. In pancreatic and colorectal cancer, inactivation of the Smad4 gene through homozygous deletion or intragenic mutation occurs frequently in association with malignant progression. However, mutation of this gene is seen only occasionally in the rest of human cancers. The majority of Smad4 gene mutations in human cancer are missense, nonsense, and frameshift mutations at the mad homology 2 region (MH2), which interfere with the homo-oligomer formation of Smad4 protein and the hetero-oligomer formation between Smad4 and Smad2 proteins, resulting in disruption of TGFbeta signaling. Supporting evidence for the above observation was provided by genetically manipulated mice carrying either a heterozygote of the Smad4 gene or a compound heterozygote of the Smad4 and APC genes, which develop either gastrointestinal polyps/cancer mimicking familial juvenile polyposis or progressed colorectal cancer, respectively.

Activation of Protein Kinase Cζ Induces Serine Phosphorylation of VAMP2 in the GLUT4 Compartment and Increases Glucose Transport in Skeletal Muscle

ABSTRACT Insulin stimulates glucose uptake into skeletal muscle tissue mainly through the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The precise mechanism involved in this process is presently unknown. In the cascade of events leading to insulin-induced glucose transport, insulin activates specific protein kinase C (PKC) isoforms. In this study we investigated the roles of PKCζ in insulin-stimulated glucose uptake and GLUT4 translocation in primary cultures of rat skeletal muscle. We found that insulin initially caused PKCζ to associate specifically with the GLUT4 compartments and that PKCζ together with the GLUT4 compartments were then translocated to the plasma membrane as a complex. PKCζ and GLUT4 recycled independently of one another. To further establish the importance of PKCζ in glucose transport, we used adenovirus constructs containing wild-type or kinase-inactive, dominant-negative PKCζ (DNPKCζ) cDNA to overexpress this isoform in skeletal muscle myotube cultures. We found that overexpression of PKCζ was associated with a marked increase in the activity of this isoform. The overexpressed, active PKCζ coprecipitated with the GLUT4 compartments. Moreover, overexpression of PKCζ caused GLUT4 translocation to the plasma membrane and increased glucose uptake in the absence of insulin. Finally, either insulin or overexpression of PKCζ induced serine phosphorylation of the GLUT4-compartment-associated vesicle-associated membrane protein 2. Furthermore, DNPKCζ disrupted the GLUT4 compartment integrity and abrogated insulin-induced GLUT4 translocation and glucose uptake. These results demonstrate that PKCζ regulates insulin-stimulated GLUT4 translocation and glucose transport through the unique colocalization of this isoform with the GLUT4 compartments.

PKCα mediates TGFβ-induced growth inhibition of human keratinocytes via phosphorylation of S100C/A11

Growth regulation of epithelial cells is of major concern because most human cancers arise from them. We demonstrated previously a novel signal pathway involving S100C/A11 for high Ca2+-induced growth inhibition of normal human keratinocytes (Sakaguchi, M., M. Miyazaki, M. Takaishi, Y. Sakaguchi, E. Makino, N. Kataoka, H. Yamada, M. Namba, and N.H. Huh. 2003. J. Cell Biol. 163:825–835). This paper addresses a question whether transforming growth factor β (TGFβ) shares the pathway with high Ca2+. On exposure of the cells to TGFβ1, S100C/A11 was phosphorylated, bound to nucleolin, and transferred to the nucleus, resulting in induction of p21WAF1/CIP1 and p15INK4B through activation of Sp1. Protein kinase C α (PKCα) was shown to phosphorylate 10Thr of S100C/A11, which is a critical event for the signal transduction. The TGFβ1-induced growth inhibition was almost completely mitigated when PKCα activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCα–S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFβ1.

PKCη associates with cyclin E/cdk2/p21 complex, phosphorylates p21 and inhibits cdk2 kinase in keratinocytes

PKC is activated on the cell membrane by phospholipids, thereby transducing signals to intracellular pathways. We provide here another function of PKC, namely, regulating cell cycle by interaction with the cyclin E/cdk2/p21 complex. Among the 10 isoforms of PKC, PKCη is predominantly expressed in squamous cell epithelia and induces terminal differentiation of keratinocytes. PKCη that is endogenously expressed or overexpressed was found to associate with the cyclin E/cdk2/p21 complex in keratinocytes of mice and humans. Requirement of a possible adaptor protein to the binding was suggested by the reconstitution of PKCη and the cyclin E/cdk2/p21 complex which were prepared from human keratinocytes or Sf9 insect cells. Colocalization of PKCη with cdk2 and cyclin E was observed in the cytoplasm, particularly in the perinuclear region. p21 was phosphorylated in the complex in a PKC-activator dependent manner. Association of PKCη with cdk2 resulted in marked inhibition of cdk2-kinase activity when measured by phosphorylation of Rb. Dominant negative PKCη associated with the cyclin E/cdk2/p21 complex, but caused a little inhibition of cdk2 kinase activity. Among the known regulatory mechanisms of cdk2 activity, dephosphorylation of Thr160 was demonstrated.

UV irradiation increases ROS production via PKCδ signaling in primary murine fibroblasts

Ultraviolet (UV) irradiation is a major environmental factor responsible for a high incidence of premature skin aging, referred to as photoaging, as well as skin cancer and melanoma. UVA irradiation represents 90% of the solar UV light reaching the earths surface, and yet the mechanisms by which it exerts its biological effects are not clear. UVA penetrates into the skin tissue, reaching the basal layers of the active dividing cells and, therefore, the contribution of UVA to skin damage may be significant. The majority of UVA energy is absorbed by unidentified photosensitizers in the cells which are postulated to generate reactive oxygen species (ROS). It has been believed that both chronological aging and photoaging share the same molecular features and, as such, it is very common to utilize UV irradiation for induction of skin aging. To determine the involvement of protein kinase isoforms in chronological aging and photoaging, we utilized in vitro aging model systems of primary murine fibroblasts and primary fibroblasts isolated from PKC null mice. We show for the first time distinct involvement of PKC isoforms PKCδ and PKCα in photoaging versus cellular senescence. While chronological aging is accompanied by overexpression and activation of PKCα, UV irradiation and ROS production are associated with photoaging accompanied by PKCδ downregulation and nuclear translocation. J. Cell. Biochem. 105: 194–207, 2008.

The η isoform of protein kinase C inhibits UV-induced activation of caspase-3 in normal human keratinocytes

Abstract Protein kinase C (PKC) fulfills a central role in the decision of cell fate in keratinocytes. Both PKCδ and PKCη induce growth inhibition and differentiation of normal human keratinocytes (NHK). Here we show that PKCδ and PKCη play opposite roles in UVB-induced apoptosis in NHK. PKCδ enhanced UVB-induced caspase-3 activity, while overexpression of PKCη reduced it. In keeping with these observations, the dominant negative mutant of PKCδ significantly inhibited the activation of caspase-3, whereas dominant negative PKCη increased it in a dose (MOI)-dependent manner. Unlike PKCδ, cleavage and translocation to mitochondria of PKCη were not observed, resulting in no detection of cytochorome c release. Furthermore, UV-induced activation of p38 MAP kinase, which suppressed the caspase-3 activity in NHK, was blocked by dominant negative PKCη. These findings suggest that PKCη negatively regulates UV-induced apoptosis through its localization, resistance to cleavage, and the p38 MAPK pathway.

Cholesterol sulfate, an activator of protein kinase C mediating squamous cell differentiation : a review

Activity of protein kinase C (PKC) depends on the interaction with polar head-groups of two membrane lipids, i.e., phosphatidylserine and diacylglycerol. We demonstrated that cholesterol metabolism is directly involved in activation of the eta isoform of protein kinase C (PKCeta), which is predominantly expressed in epithelial tissues in close association with epithelial differentiation. We found that PKCeta was activated by cholesterol sulfate (CS), a metabolite of cholesterol formed during squamous cell differentiation. In the presence of CS, phorbol ester only weakly enhanced the activity of PKCeta. CS also activated PKCeta, PKCdelta and PKCepsilon in a dose-dependent manner, when assayed using purified recombinant materials. However, when partially purified materials were used from overexpressing normal human keratinocytes, only PKCeta was activated by CS among the isoforms examined. All the existing lines of evidence, mainly supplied from our laboratory, suggest that CS is involved in a signal transduction of squamous cell differentiation and thereby modifying squamous cell carcinogenesis.

Enhancement of anchorage-independent growth of human pancreatic carcinoma MIA PaCa-2 cells by signaling from protein kinase C to mitogen-activated protein kinase

We found that 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) promoted anchorage‐independent growth but did not affect anchorage‐dependent growth of MIA PaCa‐2 human pancreatic carcinoma cells. TPA markedly activated mitogen‐activated protein kinase (MAPK)/extracellular signal–regulated kinase in an anchorage‐independent manner. Two protein kinase C (PKC) isoforms, conventional PKC (cPKC) and novel PKC (nPKC), but not apical PKC, translocated from the cytosolic to the particulate fraction upon TPA treatment. To identify the PKC isoforms involved in the regulation of anchorage‐independent growth, four PKC isoforms (α, δ, ε, and ζ) were forced to be expressed in MIA PaCa‐2 cells with an adenovirus vector. Overexpression of nPKCδ or nPKCε activated MAPK and promoted anchorage‐independent growth. Overexpression of cPKCα alone did not influence anchorage‐independent growth but lowered the concentration of TPA that was required to enhance such growth. Expression of constitutively active MAPK kinase‐1 (MEK1) also promoted anchorage‐independent growth. Furthermore, PKC inhibitors or an MEK inhibitor completely suppressed both TPA‐induced activation of MAPK and promotion of anchorage‐independent growth, but a cPKC‐selective inhibitor partially suppressed TPA‐induced promotion of the growth. Based on these results, we suggest that MAPK activation, mediated by certain isoforms of PKC, plays a part in oncogenic growth of MIA PaCa‐2 cells. In summary, our data indicated that specific inhibitors of the cPKC and nPKC signaling pathway might be selective anti‐oncogenic growth agents for some types of human pancreatic cancer.

Eighth Japanese‐German Workshop on Molecular and Cellular Aspects of Carcinogenesis1

Initiated in 1987, the biennial Japanese-German Workshops of the Essen series on “Molecular and Cellular Aspects of Carcinogenesis” are organized traditionally at the Institute of Cell Biology (Cancer Research) [IFZ], University of Essen Medical School and West German Cancer Center Essen, Germany. This 8th Workshop was generously supported by the International Committee for the Cancer Research Program of the Ministry of Education, Culture, Sports, Science & Technology and the International Cooperation Program for 2nd-Term Comprehensive 10-Year Strategy for Cancer Control of the Ministry of Health, Labor & Welfare on the Japanese side, and by the Federal Ministry of Education and Research (BMBF), the Deutsche Forschungsgemeinschaft (DFG) and the Alfried Krupp von Bohlen und Halbach-Stiftung, Essen, in Germany. Additional support from numerous other sponsors in Germany and Japan is gratefully acknowledged. The names and affiliations of Workshop participants are listed at the end of this report.