Genetic modification of human embryonic stem cells with adenoviral vectors: differences of infectability between lines and correlation of infectability with expression of the coxsackie and adenovirus receptor.

Adenovirus is an efficient vector for expression of transgenes in dividing and nondividing cells. However, very few studies of human embryonic stem cells (hESCs) have utilized adenoviral vectors. We examine here the ability of adenovirus to infect naive hESCs and the differentiated derivatives of multiple hESC lines. We found a striking variation in adenovirus infection rates between lines. The variability in infection rates was positively correlated with the expression of the coxsackievirus and adenovirus receptor, but not that of alpha(nu)-integrin. Adenoviral infection did not interfere with the expression of pluripotency markers, even after passaging. In addition, infection did not affect differentiation of hESC-derived neural precursors in vitro. We also found that green fluorescent protein expression mediated by adenovirus can be a useful marker for tracking hESC in xenografts. We conclude that adenovirus is a practical vector for genetic modification of naive hESC from most, but not all lines, but may be more generally useful for gene transfer into differentiated derivatives of hESC lines.

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.

Coordinated phosphorylation of insulin receptor substrate-1 by glycogen synthase kinase-3 and protein kinase C betaII in the diabetic fat tissue.

Serine/threonine phosphorylation of insulin receptor substrate-1 (IRS-1) is an important negative modulator of insulin signaling. Previously, we showed that glycogen synthase kinase-3 (GSK-3) phosphorylates IRS-1 at Ser(332). However, the fact that GSK-3 requires prephosphorylation of its substrates suggested that Ser(336) on IRS-1 was the "priming" site phosphorylated by an as yet unknown protein kinase. Here, we sought to identify this "priming kinase" and to examine the phosphorylation of IRS-1 at Ser(336) and Ser(332) in physiologically relevant animal models. Of several stimulators, only the PKC activator phorbol ester PMA enhanced IRS-1 phosphorylation at Ser(336). Treatment with selective PKC inhibitors prevented this PMA effect and suggested that a conventional PKC was the priming kinase. Overexpression of PKCalpha or PKCbetaII isoforms in cells enhanced IRS-1 phosphorylation at Ser(336) and Ser(332), and in vitro kinase assays verified that these two kinases directly phosphorylated IRS-1 at Ser(336). The expression level and activation state of PKCbetaII, but not PKCalpha, were remarkably elevated in the fat tissues of diabetic ob/ob mice and in high-fat diet-fed mice compared with that from lean animals. Elevated levels of PKCbetaII were also associated with enhanced phosphorylation of IRS-1 at Ser(336/332) and elevated activity of GSK-3beta. Finally, adenoviral mediated expression of PKCbetaII in adipocytes enhancedphosphorylation of IRS-1 at Ser(336). Taken together, our results suggest that IRS-1 is sequentially phosphorylated by PKCbetaII and GSK-3 at Ser(336) and Ser(332). Furthermore, these data provide evidence for the physiological relevance of these phosphorylation events in the pathogenesis of insulin resistance in fat tissue.

Novel insights into wound healing sequence of events.

Effective wound healing leads to restoration of tissue integrity and occurs through a highly organized multistage process involving various cell types. Currently, methods for wound healing assessment lack a structured system for analysis of quantitative parameters. We have established a unique quantitative assessment strategy of wound healing stages based on histological criteria. Distinctive immunohistochemical parameters including re-epithelialization, epidermal differentiation, cell migration, proliferation, inflammatory response as well as dermal closure, matrix distribution, and skin remodelling were identified and followed during the timeline of wound healing progression. Assessment was based on various defined characteristics and each stage-specific parameter was independently quantified for complete wound closure. This analysis allowed a follow-up of wound healing dynamics and identified the contribution of critical and specific parameters to wound healing physiology and pathology. In this review we demonstrate our assessment strategy of crucial wound healing events and introduce a unique quantification system for each of the processes involved in wound repair. We believe that our unique method can be utilized as a diagnostic platform for standardizing assessment of wound healing progression as well as a screening tool for potential therapies.

Immediate tight sealing of skin incisions using an innovative temperature-controlled laser soldering device: in vivo study in porcine skin.

BACKGROUND: A feedback temperature-controlled laser soldering system (TCLS) was used for bonding skin incisions on the backs of pigs. The study was aimed: 1) to characterize the optimal soldering parameters, and 2) to compare the immediate and long-term wound healing outcomes with other wound closure modalities. MATERIALS AND METHODS: A TCLS was used to bond the approximated wound margins of skin incisions on porcine backs. The reparative outcomes were evaluated macroscopically, microscopically, and immunohistochemically. RESULTS: The optimal soldering temperature was found to be 65 degrees C and the operating time was significantly shorter than with suturing. The immediate tight sealing of the wound by the TCLS contributed to rapid, high quality wound healing in comparison to Dermabond or Histoacryl cyanoacrylate glues or standard suturing. CONCLUSIONS: TCLS of incisions in porcine skin has numerous advantages, including rapid procedure and high quality reparative outcomes, over the common standard wound closure procedures. Further studies with a variety of skin lesions are needed before advocating this technique for clinical use.

Selective cyclooxygenase-2 inhibitors stimulate glucose transport in L6 myotubes in a protein kinase Cdelta-dependent manner.

Selective inhibitors of cyclooxygenase-2 (prostaglandin-endoperoxide synthase-2; COX-2) augment the rate of hexose uptake in myotubes by recruiting glucose transporter-4 (GLUT-4) to the plasma membrane in an insulin- and AMPKalpha-independent manner [Alpert E, Gruzman A, Lardi-Studler B, Cohen G, Reich R, Sasson S. Cyclooxygenase-2 (PTGS2) inhibitors augment the rate of hexose transport in L6 myotubes in an insulin- and AMPKalpha-independent manner. Diabetologia 2006;49:562-70]. We aimed at elucidating the molecular interactions that mediate this effect of COX-2 inhibitors in L6 myotubes. The effects of the inhibitors niflumic acid, nimesulide and rofecoxib on activities and phosphorylation state of key proteins in the insulin transduction pathway were determined. These inhibitors did not induce specific tyrosine phosphorylation in IRS-1, could not assemble a functional IRS-PI3K-PKB/Akt complex and did not activate GSK3alpha/beta, JNK1/2, ERK1/2, p38-MAPK or c-Cbl by site-specific phosphorylation(s). Yet, like insulin, they activated mTOR and induced downstream threonine phosphorylation in p70S6K and 4EBP1. However, rapamycin, which inhibits mTOR enzymatic activity, did not interfere with COX-2 inhibitor-induced stimulation of hexose uptake in myotube. Thus, mTOR activation was not required for COX-2 inhibitor-dependent augmentation of hexose transport in myotubes. Because PKCdelta has also been shown to activate mTOR, we asked whether COX-2 inhibitors activate mTOR by a prior activation of PKCdelta. Indeed, all three inhibitors induced tyrosine phosphorylation in PKCdelta and stimulated its kinase activity. Moreover, pharmacological inhibition of PKCdelta or the expression of a dominant-negative form of PKCdelta in myotubes completely abolished COX-2 inhibitor-dependent stimulation of hexose uptake. This study shows that selective COX-2 inhibitors activate a unique PKCdelta-dependent pathway to increase GLUT-4 abundance in the plasma membrane of myotubes and augment the rate of hexose transport.

Nek7 kinase is enriched at the centrosome, and is required for proper spindle assembly and mitotic progression.

Members of the NIMA-related kinases (NRK) family are recently emerging as central regulators of various aspects of the cell cycle. However, the cellular roles of the mammalian NRK, Nek7, remain obscure. We show here that the endogenous Nek7 protein is enriched at the centrosome in a microtubule-independent manner. Overexpression of wt or kinase-defective Nek7 resulted in cells of rounder appearance, and higher proportions of multinuclear and apoptotic cells. Down-regulation of Nek7 using a small interfering RNA approach resulted in a significant increase in mitotic cells presenting multipolar spindle phenotype. These results suggest a role for Nek7 in regulating proper spindle assembly and mitotic progression.

The role of protein kinase C delta activation and STAT3 Ser727 phosphorylation in insulin-induced keratinocyte proliferation.

Activation of the STAT family of transcription factors is regulated by cytokines and growth factors. STAT tyrosine and serine phosphorylation are linked to the transcriptional activation and function of STAT. We have previously described a unique pathway inducing keratinocyte proliferation, which is mediated by insulin stimulation and depends on protein kinase C delta (PKCdelta). In this study, we assessed STAT3 activation downstream of this pathway and characterized the role of PKCdelta activation in STAT3 tyrosine and serine phosphorylation and keratinocyte proliferation. Following insulin stimulation, STAT3 interacted with PKCdelta but not with any other PKC isoform expressed in skin. Activated forms of PKCdelta and STAT3 were essential for insulin-induced PKCdelta-STAT3 activation in keratinocyte proliferation. Abrogation of PKCdelta activity inhibited insulin-induced STAT3 phosphorylation, PKCdelta-STAT3 association and nuclear translocation. In addition, overexpression of STAT3 tyrosine mutant eliminated insulin-induced PKCdelta activation and keratinocyte proliferation. Finally, overexpression of a STAT3 serine mutant abrogated insulin-induced STAT3 serine phosphorylation and STAT3-induced keratinocyte proliferation, whereas STAT3 tyrosine phosphorylation was induced and nuclear localization remained intact. This study indicates that PKCdelta activation is a primary regulator of STAT3 serine phosphorylation and that PKCdelta is essential in directing insulin-induced signaling in keratinocyte proliferation.

PKC-delta-dependent activation of oxidative stress in adipocytes of obese and insulin-resistant mice: role for NADPH oxidase.

Oxidative stress is thought to be one of the causative factors contributing to insulin resistance and type 2 diabetes. Previously, we showed that reactive oxygen species (ROS) production is significantly increased in adipocytes from high-fat diet-induced obese and insulin-resistant mice (HF). ROS production was also associated with the increased activity of PKC-delta. In the present studies, we hypothesized that PKC-delta contributes to ROS generation and determined their intracellular source. NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI) reduced ROS levels by 50% in HF adipocytes, and inhibitors of NO synthase (L-NAME, 1 mM), xanthine oxidase (allopurinol, 100 microM), AGE formation (aminoguanidine, 10 microM), or the mitochondrial uncoupler (FCCP, 10 microM) had no effect. Rottlerin, a selective PKC-delta inhibitor, suppressed ROS levels by approximately 50%. However, neither GO-6976 nor LY-333531, effective inhibitors toward conventional PKC or PKC-beta, respectively, significantly altered ROS levels in HF adipocytes. Subsequently, adenoviral-mediated expression of wild-type PKC-delta or its dominant negative mutant (DN-PKC-delta) in HF adipocytes resulted in either a twofold increase in ROS levels or their suppression by 20%, respectively. In addition, both ROS levels and PKC-delta activity were sharply reduced by glucose depletion. Taken together, these results suggest that PKC-delta is responsible for elevated intracellular ROS production in HF adipocytes, and this is mediated by high glucose and NADPH oxidase.

Differential regulation of alpha6beta4 integrin by PKC isoforms in murine skin keratinocytes.

In mammalian epidermis, alpha6beta4 integrin is expressed exclusively on the basal layer localized to the hemidesmosomes, where it interacts extracellularly with the laminin-5 ligand. During differentiation, loss of alpha6beta4 is associated with keratinocyte detachment from the basement membrane and upward migration. The protein kinase C (PKC) family of isoforms participates in regulation of integrin function and is linked to skin differentiation. Exposure of primary murine keratinocytes to PKC activators specifically downregulates alpha6beta4 expression. Utilizing recombinant adenoviruses, we selectively overexpressed skin PKC isoforms in primary keratinocytes. PKCdelta and PKCzeta induced downregulation of alpha6beta4 protein expression, leading to reduced keratinocyte attachment to laminin-5 and enhanced gradual detachment from the underlying matrix. In contrast, PKCalpha upregulated alpha6beta4 protein expression, leading to increased keratinocyte attachment to laminin-5 and to the underlying matrix. Altogether, these results suggest distinct roles for specific PKC isoforms in alpha6beta4 functional regulation during the early stages of skin differentiation.

PESI--an intelligent system for prediction of enzyme-substrate interactions based on experimental constraints.

We present a system for predicting protein-protein modifications, and demonstrate its usefulness in the field of signal transduction research. Signal transduction is one of the most important areas of investigation in biological research. One of the major mechanisms frequently employed by cells to regulate signal transduction processes involves protein phosphorylation by various kinases. As many as 1,000 protein kinases and 500 protein phosphatases in the human genome are thought to be involved in phosphorylation processes which regulate all aspects of cell function. The complexity of such interactions stems from the enormous number of factors and interactions, which makes the identification of putative substrates for any given enzyme by straightforward experimentation increasingly difficult. We present here a data mining algorithm, based on the similarity between the modifier proteins and between the modified proteins, and on experimental constraints. The application presented here (PESI) focuses on substrate phosphorylation by various enzymes. This algorithm reduces the number of substrate candidates for experimental study by about two orders of magnitude. Moreover, this algorithm has already yielded predictions for previously unknown substrates of the enzymes PKCdelta and PKCeta, which we have confirmed experimentally.