Protein kinase C delta and eta differently regulate the expression of loricrin and Jun family proteins in human keratinocytes.

Barrier function of the epidermis is maintained by precise expression of keratinocyte-specific structural proteins to form the cornified cell envelope (CE). Loricrin, a major component of the CE, is expressed at the late stage of keratinocyte differentiation. In this study, we reveal the isoform-specific function of protein kinase C (PKC) in the regulation of loricrin expression. Both PKCdelta and PKCeta have been recognized as differentiation-promoting isoforms. However, loricrin expression was inversely controlled by PKCdelta and PKCeta in cultured keratinocytes and 3D skin culture; i.e. loricrin expression was decreased by PKCdelta and increased by PKCeta. To clarify the mechanisms that PKCdelta and PKCeta oppositely regulate the loricrin expression, we examined the expression of activator protein-1 (AP-1) family proteins, which modulate the transcription of loricrin and are downstream molecules of PKC. PKCdelta decreased c-Jun expression, whereas PKCeta increased JunD, which are positive regulators of loricrin transcription. These findings suggest that inverse effects of PKCdelta and PKCeta on loricrin expression attributes to the expression of c-Jun and JunD.

Toward global standardization of conducting fair investigations of allegations of research misconduct.

In the United States, through nation-wide discussions, the procedures for handling allegations of research misconduct are now well established. Procedures are geared toward carefully treating both complainants and respondents fairly in accordance with the US framework. Other countries, which have their own cultural and legal framework, also need fair and legally compatible procedures for conducting investigations of allegations of research misconduct. Given the rapid growth of international collaboration in research, it is desirable to have a global standard, or common ground, for misconduct investigations. Institutions need clear guidance on important subjects such as what information should be included in the investigation reports, how the investigation committee should be organized once research misconduct allegation has been received, how to conduct the investigation, how the data and information obtained should be taken as evidence for vs. against misconduct, and what policies the investigation committee should follow. We explore these issues from the viewpoint of members of committees investigating accusations of research misconduct (hereafter referred to as "investigation committees") as well as persons overseeing the committees in Japan. We hope to engender productive discussions among experts in misconduct investigations, leading to a formulation of international standards for such investigation.

PKCalpha mediates TGFbeta-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 beta (TGFbeta) shares the pathway with high Ca2+. On exposure of the cells to TGFbeta1, 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 alpha (PKCalpha) was shown to phosphorylate 10Thr of S100C/A11, which is a critical event for the signal transduction. The TGFbeta1-induced growth inhibition was almost completely mitigated when PKCalpha activity was blocked or when S100C/A11 was functionally sequestered. These results indicate that, in addition to the well-characterized Smad-mediated pathway, the PKCalpha-S100C/A11-mediated pathway is involved in and essential for the growth inhibition of normal human keratinocytes cells by TGFbeta1.

Discovery of novel motilin antagonists: Conversion of tetrapeptide leads to orally available peptidomimetics.

We successfully discovered peptidomimetic motilin antagonists (17c and 17d) through the improvement of physicochemical properties of a tetrapeptide antagonist (2). Furthermore, with oral administration and based on motilin antagonistic activity, both compounds suppressed motilin-induced colonic and gastric motility in conscious dogs.

Physiological essay on Gulliver's Travels: a correction after three centuries.

Gulliver's Travels by Jonathan Swift, published in 1726, was analyzed from the viewpoint of scaling in comparative physiology. According to the original text, the foods of 1724 Lilliputians, tiny human creatures, are needed for Gulliver, but the author found that those of 42 Lilliputians and of 1/42 Brobdingnagians (gigantic human creatures) are enough to support the energy of Gulliver. The author further estimated their heartbeats, respiration rates, life spans and blood pressure. These calculations were made by the use of three equations, i.e., body mass index (BMI = W/H2) and quarter-power laws (E∝W3/4 and T∝W1/4), where W, H, E, and T denote body weight, height, energy and time, respectively. Their blood pressures were estimated with reference to that of the giraffe and barosaurus, a long-neck dinosaur. Based on the above findings, the food requirement of Gulliver in the original text should be corrected after almost three centuries.

UV irradiation increases ROS production via PKCdelta 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 earth's 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 PKCdelta and PKCalpha in photoaging versus cellular senescence. While chronological aging is accompanied by overexpression and activation of PKCalpha, UV irradiation and ROS production are associated with photoaging accompanied by PKCdelta downregulation and nuclear translocation.

New Classification of Research Misconduct from the Viewpoint of Truth, Trust, and Risk.

Fabrication, Falsification and Plagiarism (FFP) and Questionable Research Practice (QRP) have been used worldwide in the classification of research misconduct. However, FFP comprises two distinct categories of misconduct: FF is extreme research misconduct that betrays truth, while P undermines trust of science community. Irreproducibility and inadequate practice of research also betray trust. Research misconduct has the potential to cause serious risk of safety in daily life. The proposed classification system is outlined as follows: Class I misconduct: Betrayal of the truth: (1) Fabrication and (2) Falsification. Class II misconduct: Betrayal of trust: (1) Plagiarism of text ; Irreproducibility; and (3) Inadequate research practice. Class III misconduct: Risk to safety of health and industrial products: (1) Risk to safety of health and (2) Risk to safety of industrial products. The proposed classification reflects deeper values of truth, trust, and risk more directly than the previous classification and elucidates issues about nature and significance of misconduct.

Repeating probability of authors with retracted scientific publications.

Both the scientific community and the general public have expressed concern over scientific misconduct. The number of retracted articles has increased dramatically over the past 20 years and now comprises about .02% of the 2 million articles published each year. Retraction of publications available in large public databases can be analyzed as an objective measure for scientific misconduct and errors. In this project, we analyzed retractions of scientific publications using the Web of Science (WoS) and PubMed databases. We found that a power law is applicable to distributions of retracting authors and retracted publications with exponents of about -.6 and -3.0, respectively. Application of a power-law model for retracted publications implies that retraction is not a random event. Analysis of the retraction distributions suggests that a small fraction (1-2%) of retracting authors with ≧5 retractions are responsible for around 10% of retraction. The probabilities for their repeating retraction are calculated using a statistical model: 3-5% likelihood of repeat retraction for authors with a single retraction at five years after the latest retraction and 26-37% for authors with five retractions at five years after the latest retraction. By focusing on those with repeated retractions, this analysis could contribute to identification of measures to reduce such repetition of retractions.

Protein kinase Cdelta amplifies ceramide formation via mitochondrial signaling in prostate cancer cells.

We studied the role of protein kinase C isoform PKCdelta in ceramide (Cer) formation, as well as in the mitochondrial apoptosis pathway induced by anticancer drugs in prostate cancer (PC) cells. Etoposide and paclitaxel induced Cer formation and apoptosis in PKCdelta-positive LNCaP and DU145 cells but not in PKCdelta-negative LN-TPA or PC-3 cells. In contrast, these drugs induced mitotic cell cycle arrest in all PC cell lines. Treatment with Rottlerin, a specific PKCdelta inhibitor, significantly inhibited drug-induced Cer formation and apoptosis in LNCaP cells, as did overexpression of dominant negative-type PKCdelta. Overexpression of wild-type PKCdelta had an opposite effect in PC-3 cells. Notably, etoposide induced biphasic Cer formation in LNCaP cells. The early and transient Cer increase resulted from de novo Cer synthesis, while the late and sustained Cer accumulation was derived from sphingomyelin hydrolysis by neutral sphingomyelinase (nSMase). Cer, in turn, induced mitochondrial translocation of PKCdelta and stimulated the activity of this kinase, promoting cytochrome c release and caspase-9 activation. Furthermore, the specific caspase-9 inhibitor LEHD-fmk significantly inhibited etoposide-induced nSMase activation, Cer accumulation, and PKCdelta mitochondrial translocation. These results indicate that PKCdelta plays a crucial role in activating anticancer drug-induced apoptosis signaling by amplifying the Cer-mediated mitochondrial amplification loop.

Mutant epidermal growth factor receptor undergoes less protein degradation due to diminished binding to c-Cbl.

Gefitinib (Iressa) sensitivity in non-small cell lung cancer (NSCLC) is associated with activating mutations in epidermal growth factor receptor (EGFR). It was reported that autophosphorylation of the mutant EGFR is prolonged compared with wild-type EGFR. To explore the mechanism of sustained autophosphorylation, the mutant and wild-type EGFR degradation activities were examined in NSCLC cell lines. EGFR degradation activity was measured by 125I-EGF. The degradation rate of EGFR was lower in the PC-9 NSCLC cell line, which expressed 15-bp deletion mutant EGFR, compared with that in the PC-14 NSCLC (wild-type EGFR). To clarify the mechanism, the stable transfected cell lines, 293_pEGFR and 293_pdelta15, expressing wild-type and mutant EGFR, respectively, were used. In 293_pdelta15, EGFR degradation and binding of c-Cbl ubiquitin ligase to this receptor were reduced compared with 293_pEGFR. Based on these results, we conclude that the mutant EGFR underwent less protein degradation due to diminished binding to c-Cbl.

Localization of phospholipase D1 to caveolin-enriched membrane via palmitoylation: implications for epidermal growth factor signaling.

Phospholipase D (PLD) has been suggested to mediate epidermal growth factor (EGF) signaling. However, the molecular mechanism of EGF-induced PLD activation has not yet been elucidated. We investigated the importance of the phosphorylation and compartmentalization of PLD1 in EGF signaling. EGF treatment of COS-7 cells transiently expressing PLD1 stimulated PLD1 activity and induced PLD1 phosphorylation. The EGF-induced phosphorylation of threonine147 was completely blocked and the activity of PLD1 attenuated by point mutations (S2A/T147A/S561A) of PLD1 phosphorylation sites. The expression of a dominant negative PKCalpha mutant by adenovirus-mediated gene transfer greatly inhibited the phosphorylation and activation of PLD1 induced by EGF in PLD1-transfected COS-7 cells. EGF-induced PLD1 phosphorylation occurred primarily in the caveolin-enriched membrane (CEM) fraction, and the kinetics of PLD1 phosphorylation in the CEM were strongly correlated with PLD1 phosphorylation in the total membrane. Interestingly, EGF-induced PLD1 phosphorylation and activation and the coimmunoprecipitation of PLD1 with caveolin-1 and the EGF receptor in the CEM were significantly attenuated in the palmitoylation-deficient C240S/C241S mutant, which did not localize to the CEM. Immunocytochemical analysis revealed that wild-type PLD1 colocalized with caveolin-1 and the EGF receptor and that phosphorylated PLD1 was localized exclusively in the plasma membrane, although some PLD1 was also detected in vesicular structures. Transfection of wild-type PLD1 but not of C240S/C241S mutant increased EGF-induced raf-1 translocation to the CEM and ERK phosphorylation. This study shows, for the first time, that EGF-induced PLD1 phosphorylation and activation occur in the CEM and that the correct localization of PLD1 to the CEM via palmitoylation is critical for EGF signaling.

Enhancement of sensitivity to tumor necrosis factor alpha in non-small cell lung cancer cells with acquired resistance to gefitinib.

Tumor cells that have acquired resistance to gefitinib through continuous drug administration may complicate future treatment. To investigate the mechanisms of acquired resistance, we established PC-9/ZD2001, a non-small-cell lung cancer cell line resistant to gefitinib, by continuous exposure of the parental cell line PC-9 to gefitinib. After 6 months of culture in gefitinib-free conditions, PC-9/ZD2001 cells reacquired sensitivity to gefitinib and were established as a revertant cell line, PC-9/ZD2001R. PC-9/ZD2001 cells showed collateral sensitivity to several anticancer drugs (vinorelbine, paclitaxel, camptothecin, and 5-fluorouracil) and to tumor necrosis factor alpha (TNF-alpha). Compared with PC-9 cells, PC-9/ZD2001 cells were 67-fold more sensitive to TNF-alpha and PC-9/ZD2001R cells were 1.3-fold more sensitive. Therefore, collateral sensitivity to TNF-alpha was correlated with gefitinib resistance. PC-9/ZD2001 cells expressed a lower level of epidermal growth factor receptor (EGFR) than did PC-9 cells; this down-regulation was partially reversed in PC-9/ZD2001R cells. TNF-alpha-induced autophosphorylation of EGFR (cross-talk signaling) was detected in all three cell lines. However, TNF-alpha-induced Akt phosphorylation and IkappaB degradation were observed much less often in PC-9/ZD2001 cells than in PC-9 cells or PC-9/ZD2001R cells. Expression of the inhibitor of apoptosis proteins c-IAP1 and c-IAP2 was induced by TNF-alpha in PC-9 and PC-9/ZD2001R cells but not in PC-9/ZD2001 cells. This weak effect of EGFR on Akt pathway might contribute to the TNF-alpha sensitivity of PC-9/ZD2001 cells. These results suggest that therapy with TNF-alpha would be effective in some cases of non-small-cell lung cancer that have acquired resistance to gefitinib.

Disruption of protein kinase Ceta results in impairment of wound healing and enhancement of tumor formation in mouse skin carcinogenesis.

We have generated a mouse strain lacking protein kinase C (PKC) eta to evaluate its significance in epithelial organization and tumor formation. The PKCeta-deficient mice exhibited increased susceptibility to tumor formation in two-stage skin carcinogenesis by single application of 7,12-dimethylbenz(a)anthracene (DMBA) for tumor initiation and repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) for tumor promotion. The tumor formation was not enhanced by DMBA or TPA treatment alone, suggesting that PKCeta suppresses tumor promotion. Epidermal hyperplasia induced by topical TPA treatment was prolonged in the mutant mice. The enhanced tumor formation may be closely associated with the prolonged hyperplasia induced by topical TPA treatment. In the mutant mice, after inflicting injury by punch biopsy, wound healing on the dorsal skin, particularly reepithelialization, was significantly delayed and impaired in structure. Impairment of epithelial regeneration in wound healing indicates a possibility that PKCeta plays a role in maintenance of epithelial architecture. Homeostasis in epithelial tissues mediated by PKCeta is important for tumor formation in vivo. We propose that PKCeta is involved in tumor formation modulated by regulation of proliferation and remodeling of epithelial cells in vivo.

High incidence of protein-truncating mutations of the p53 gene in liver metastases of colorectal carcinomas.

To clarify the significance of p53 mutations in liver metastasis of colorectal carcinogenesis, the characteristics of p53 mutations from 51 liver metastases and 76 primary invasive carcinomas without liver metastasis (Dukes' A, B and C) were compared. The frequency of tumors with p53 mutations was 61% (31 out of 51) in the liver metastases, and 51% (39 out of 76) in the primary carcinomas without liver metastasis. Approximately 90% of the informative cases having p53 mutation showed 17pLOH. Mutations detected within exons 4-10 of the p53 gene included missense, nonsense, frameshift, inframe deletion, and inframe insertion mutations. Out of the tumors with p53 mutations, we found that the percentage of tumors with protein-truncating mutations (nonsense and frameshift mutations) was extremely higher in liver metastases (16 out of 31, 52%) than in primary carcinomas without liver metastasis (5 out of 39, 13%) (P=0.0005). The present results suggest that protein-truncating mutations of the p53 gene are more relevant than missense mutations as one of the prognostic factors in liver metastasis of colorectal carcinomas.

Differential effects of tumor necrosis factor-alpha on protein kinase C isoforms alpha and delta mediate inhibition of insulin receptor signaling.

Tumor necrosis factor-alpha (TNF-alpha) is a multifunctional cytokine that interferes with insulin signaling, but the molecular mechanisms of this effect are unclear. Because certain protein kinase C (PKC) isoforms are activated by insulin, we examined the role of PKC in TNF-alpha inhibition of insulin signaling in primary cultures of mouse skeletal muscle. TNF-alpha, given 5 min before insulin, inhibited insulin-induced tyrosine phosphorylation of insulin receptor (IR), IR substrate (IRS)-1, insulin-induced association of IRS-1 with the p85 subunit of phosphatidylinositol 3-kinase (PI3-K), and insulin-induced glucose uptake. Insulin and TNF-alpha each caused tyrosine phosphorylation and activation of PKCs delta and alpha, but when TNF-alpha preceded insulin, the effects were less than that produced by each substance alone. Insulin induced PKCdelta specifically to coprecipitate with IR, an effect blocked by TNF-alpha. Both PKCalpha and -delta are constitutively associated with IRS-1. Whereas insulin decreased coprecipitation of IRS-1 with PKCalpha, it increased coprecipitation of IRS-1 with PKCdelta. TNF-alpha blocked the effects of insulin on association of both PKCs with IRS-1. To further investigate the involvement of PKCs in inhibitory actions of TNF-alpha on insulin signaling, we overexpressed specific PKC isoforms in mature myotubes. PKCalpha overexpression inhibited basal and insulin-induced IR autophosphorylation, whereas PKCdelta overexpression increased IR autophosphorylation and abrogated the inhibitory effect of TNF-alpha on IR autophosphorylation and signaling to PI3-K. Blockade of PKCalpha antagonized the inhibitory effects of TNF-alpha on both insulin-induced IR tyrosine phosphorylation and IR signaling to PI3-K. We suggest that the effects of TNF-alpha on IR tyrosine phosphorylation are mediated via alteration of insulin-induced activation and association of PKCdelta and -alpha with upstream signaling molecules.

Transcriptional and post-transcriptional regulation of the PKC delta gene by etoposide in L1210 murine leukemia cells: implication of PKC delta autoregulation.

Protein kinase C delta (PKC delta) plays an important role in the regulation of apoptosis in response to diverse anticancer agents. PKC delta is cleaved irreversibly to a catalytically active fragment in response to apoptotic stimuli; however, little information is available about the regulation of PKC delta gene expression. In this study, we found that the amount of steady-state PKC delta mRNA and protein was increased by etoposide in mouse L1210 leukemia cells. The transcriptional rate of the PKC delta gene and the stability of PKC delta mRNA were increased by treatment with etoposide, resulting in the accumulation of PKC delta protein. Rottlerin inhibited etoposide-induced PKC delta gene expression significantly, while Go6976, LY294002 and PD98059 had no effect. Further, both stable and adenovirus-mediated expression of a dominant negative PKC delta(KR) abrogated etoposide-induced PKC delta expression. Etoposide-stimulated PKC delta transcription but not PKC delta mRNA stability was blocked completely by pretreatment with rottlerin. Our data reveal a novel mechanism whereby PKC delta gene is regulated at the transcriptional and post-transcriptional level in the L1210 leukemia cell line.

Requirement of Ca(2+) and PKCdelta for Janus kinase 2 activation by angiotensin II: involvement of PYK2.

In vascular smooth muscle cells, angiotensin II (AngII) stimulates association of its G protein-coupled AngII type 1 (AT(1)) receptor with Janus kinase 2 (JAK2), resulting in the activation of signal transducer and activator of transcription proteins. Although the association and activation of subsequent signal transducer and activator of transcription proteins appear to prerequire JAK2 activation, the signaling mechanism by which the AT(1) receptor activates JAK2 remains uncertain. Here, we have examined the signaling mechanism required for JAK2 activation by AngII in vascular smooth muscle cells. We found that AngII, through the AT(1) receptor, rapidly stimulated JAK2 phosphorylation at Tyr(1007/1008), the critical sites for the kinase activation. By using selective agonists and inhibitors, we demonstrated that PLC and its derived signaling molecules, phosphatidylinositol triphosphate/Ca(2+) and diacylglycerol/PKC, were essential for AngII-induced JAK2 phosphorylation. The PKC isoform required for JAK2 activation appears to be PKCdelta since a selective PKCdelta but not PKCalpha/beta inhibitor and dominant-negative PKCdelta overexpression inhibited JAK2 activation. We further examined a link between JAK2 and a Ca(2+)/PKC-sensitive tyrosine kinase, PYK2. We found that PYK2 activation by AngII requires PKCdelta, and that PYK2 associates with JAK2 constitutively. Moreover, transfection of two distinct PYK2 dominant-negative mutants markedly inhibited AngII-induced JAK2 activation. From these data we conclude that AT(1)-derived signaling molecules, specifically Ca(2+) and PKCdelta, participate in AngII-induced JAK2 activation through PYK2. These data provide a new mechanistic insight by which the hormone AngII exerts its cytokine-like actions in mediating vascular remodeling.

Protein kinase C alpha associates with phospholipase D1 and enhances basal phospholipase D activity in a protein phosphorylation-independent manner in human melanoma cells.

It is well known that phospholipase D plays a crucial part in the signal transduction of many types of cells, and is activated by protein kinase C alpha when cells are stimulated. To elucidate the role of phospholipase D in melanoma, the expression of phospholipase D1 and protein kinase C alpha in primary and metastatic lesions of acral lentiginous melanoma and superficial spreading melanoma was investigated using immunohistologic techniques. In addition, the mechanism of regulation of phospholipase D1 by protein kinase C alpha was examined in a human melanoma cell line HM3KO using an adenovirus-mediated gene transfer technique. Both phospholipase D1 and protein kinase C alpha were strongly expressed in primary and metastatic lesions of superficial spreading melanoma. Conversely, in acral lentiginous melanoma lesions, the expression of these two proteins increased dramatically with tumor progression; the expression of both phospholipase D1 and protein kinase C alpha was almost negative in the radial growth phase of primary acral lentiginous melanoma lesions, and increased synchronously in a progression-related manner in advanced acral lentiginous melanoma lesions, including vertical growth phase and metastatic lesions. Immunoprecipitation study showed that phospholipase D1 and protein kinase C alpha are associated physiologically in resting melanoma cells. Further immunoprecipitation study using HM3KO cells after adenovirus-mediated simultaneous overexpression of phospholipase D1 and protein kinase C alpha, or phospholipase D1 and the kinase-negative mutant of protein kinase C alpha revealed that both protein kinase C alpha and the kinase-negative mutant of protein kinase C alpha are associated with phospholipase D1 in melanoma cells in the absence of an external signal. Overexpression of protein kinase C alpha or the kinase-negative mutant of protein kinase C alpha in melanoma cells by the adenovirus vectors resulted in the enhancement of basal phospholipase D activity in a viral concentration-dependent manner. Furthermore, enhanced basal phospholipase D activity increased the in vitro invasive potential of HM3KO cells. These results suggest that upregulation of phospholipase D1 and protein kinase C alpha plays a part in the progression of acral lentiginous melanoma from the radial growth phase to the vertical growth phase. The present results also suggest that protein kinase C alpha associates with phospholipase D1 and enhances basal phospholipase D activity in a protein phosphorylation-independent manner in melanoma cells, which contributes to the cell's high invasive potential.