Search

cAMP signals are received and transmitted by multiple isoforms of cAMP-dependent protein kinases, typically determined by their specific regulatory subunits. In the brain the major regulatory isoform RIIbeta and the RII-anchor protein, AKAP150 (rat) or 75 (bovine), are differentially expressed. Cortical neurons express RIIbeta and AKAP75; conversely, granule cerebellar cells express predominantly RIalpha and RIIalpha. Cortical neurons accumulate PKA catalytic subunit and phosphorylated cAMP responsive element binding protein very efficiently into nuclei upon cAMP induction, whereas granule cerebellar cells fail to do so. Down-regulation of RIIbeta synthesis by antisense oligonucleotides inhibited cAMP-induced nuclear signaling in cortical neurons. Expression in cerebellar granule cells of RIIbeta and AKAP75 genes by microinjection of specific expression vectors, markedly stimulated cAMP-induced transcription of the lacZ gene driven by a cAMP-responsive element promoter. These data indicate that the composition of PKA in cortical and granule cells underlies the differential ability of these cells to transmit cAMP signals to the nucleus.

The primary cilium is a microtubule‐based sensory organelle that dynamically links signalling pathways to cell differentiation, growth, and development. Genetic defects of primary cilia are responsible for genetic disorders known as ciliopathies. Orofacial digital type I syndrome (OFDI) is an X‐linked congenital ciliopathy caused by mutations in the OFD1 gene and characterized by malformations of the face, oral cavity, digits and, in the majority of cases, polycystic kidney disease. OFD1 plays a key role in cilium biogenesis. However, the impact of signalling pathways and the role of the ubiquitin‐proteasome system (UPS) in the control of OFD1 stability remain unknown. Here, we identify a novel complex assembled at centrosomes by TBC1D31, including the E3 ubiquitin ligase praja2, protein kinase A (PKA), and OFD1. We show that TBC1D31 is essential for ciliogenesis. Mechanistically, upon G‐protein‐coupled receptor (GPCR)‐cAMP stimulation, PKA phosphorylates OFD1 at ser735, thus promoting OFD1 proteolysis through the praja2‐UPS circuitry. This pathway is essential for ciliogenesis. In addition, a non‐phosphorylatable OFD1 mutant dramatically affects cilium morphology and dynamics. Consistent with a role of the TBC1D31/praja2/OFD1 axis in ciliogenesis, alteration of this molecular network impairs ciliogenesis in vivo in Medaka fish, resulting in developmental defects. Our findings reveal a multifunctional transduction unit at the centrosome that links GPCR signalling to ubiquitylation and proteolysis of the ciliopathy protein OFD1, with important implications on cilium biology and development. Derangement of this control mechanism may underpin human genetic disorders., OFD1 (oro‐facial digital type I syndrome protein) resides in a centrosomal complex that links GPCR signalling to ubiquitylation and degradation of OFD1, controlling cilium morphology and dynamics and vertebrate development.

The endoplasmic reticulum stress and the unfolded protein response are triggered following an imbalance between protein load and protein folding. Until recently, two possible outcomes of the unfolded protein response have been considered: life or death. We sought to substantiate a third alternative, dedifferentiation, mesenchymal shift, and activation of the antioxidant response by using typical endocrine cells, i.e. thyroid cells. The thyroid is a unique system both of endoplasmic reticulum stress (a single protein, thyroglobulin represents the majority of proteins synthesized in the endoplasmic reticulum by the thyrocyte) and of polarized epithelium (the single layer of thyrocytes delimiting the follicle). Following endoplasmic reticulum stress, in thyroid cells the folding of thyroglobulin was disrupted. The mRNAs of unfolded protein response were induced or spliced (X-box binding protein-1). Differentiation was inhibited: mRNA levels of thyroid specific genes, and of thyroid transcription factors were dramatically downregulated, at least in part, transcriptionally. The dedifferentiating response was accompanied by an upregulation of mRNAs of antioxidant genes. Moreover, cadherin-1, and the thyroid (and kidney)-specific cadherin-16 mRNAs were downregulated, vimentin, and SNAI1 mRNAs were upregulated. In addition, loss of cortical actin and stress fibers formation were observed. Together, these data indicate that ER stress in thyroid cells induces dedifferentiation, loss of epithelial organization, shift towards a mesenchymal phenotype, and activation of the antioxidant response, highlighting, at the same time, a new and wide strategy to achieve survival following ER stress, and, as a sort of the other side of the coin, a possible new molecular mechanism of decline/loss of function leading to a deficit of thyroid hormones formation.

Keloids are benign skin tumors that develop in individuals who have a positive family history of keloid disorders. Keloids are characterized by a deregulated wound-healing process, atypical fibroblasts with extreme deposition of extracellular matrix components, particularly collagen, increased cell proliferation and associated failure of apoptosis. Recently ingenol-mebutate has been used as a novel agent with anti-proliferative activity on human keloids as an alternative treatment option in patients, once conventional therapies have failed. We hypothesized that microRNAs (miR/miRNA) may be involved in the balance between lesion formation and repair. A comprehensive understanding of the molecular mechanism underlying the Ingenol-mebutate response in keloid fibroblast following Ingenol-mebutate exposure has been established previously. Therefore, the present study analyzed changes in miRNAs and apoptotic gene regulation in Ingenol-mebutate treated keloid fibroblast, by reverse transcription-quantitative polymerase chain reaction and a DNA fragmentation assay. The range of upregulated miRNAs and downregulated genes encoding cell death appeared to be associated with the degree of the morphological alterations in Ingenol-mebutate treated keloids. In particular, the upregulation of miR-34a was detected in keloid fibroblasts during and following Ingenol-mebutate exposure. Keloid fibroblasts that overexpressed miR-34a showed differential expression of genes involved in the apoptotic signaling pathway such as p53. In conclusion, the Ingenol-mebutate treatment used here was effective in reducing keloid fibroblast growth in cell culture experiments and the expression of particular miRNAs modulated the pro-apoptotic gene expression following Ingenol-mebutate treatment.

AIMS/HYPOTHESIS: Beta cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (decline of glucose-stimulated insulin secretion, downregulation of specific gene expression). Apoptosis and dysfunction are caused, at least in part, by lipoglucotoxicity. The mechanisms implicated are oxidative stress, increase in the hexosamine biosynthetic pathway (HBP) flux and endoplasmic reticulum (ER) stress. Oxidative stress plays a role in glucotoxicity-induced beta cell dedifferentiation, while glucotoxicity-induced ER stress has been mostly linked to beta cell apoptosis. We sought to clarify whether ER stress caused by increased HBP flux participates in a dedifferentiating response of beta cells, in the absence of relevant apoptosis. METHODS: We used INS-1E cells and murine islets. We analysed the unfolded protein response and the expression profile of beta cells by real-time RT-PCR and western blot. The signal transmission pathway elicited by ER stress was investigated by real-time RT-PCR and immunofluorescence. RESULTS: Glucosamine and high glucose induced ER stress, but did not decrease cell viability in INS-1E cells. ER stress caused dedifferentiation of beta cells, as shown by downregulation of beta cell markers and of the transcription factor, pancreatic and duodenal homeobox 1. Glucose-stimulated insulin secretion was inhibited. These effects were prevented by the chemical chaperone, 4-phenyl butyric acid. The extracellular signal-regulated kinase (ERK) signal transmission pathway was implicated, since its inhibition prevented the effects induced by glucosamine and high glucose. CONCLUSIONS/INTERPRETATION: Glucotoxic ER stress dedifferentiates beta cells, in the absence of apoptosis, through a transcriptional response. These effects are mediated by the activation of ERK1/2.

In this study, selenocystine, a nutritionally available selenoamino acid, was identified for the first time as a novel agent with anti proliferative activity on human keloids. The 20 μM concentration after 48. h treatment used here was the most effective to reduce keloid fibroblast growth. We analyzed the gene expression profile of selenocystine treatment response in keloid fibroblasts by the microarray system to characterize the effects of selenocystine on human keloids. The major alterations in keloid fibroblasts following selenocystine exposure included up-regulation of the genes encoding cell death and transcription factors. Prominent down-regulation of genes involved in development, cell adhesion and cytoskeleton, as well as extra cellular matrix genes, usually strongly up-regulated in keloids, resulted following selenocystine exposure. The range of the down-regulated genes and the degree of the decreased expression appeared to be correlated with the degree of the morphological alterations in selenocystine treated keloids. © 2011 Elsevier Inc.

Keloids are benign skin tumors and are the effect of a dysregulated wound-healing process in genetically predisposed patients. They are characterized by formation of excess scar tissue beyond the boundaries of the wound. Keloids are often confused with hypertrophic scars because of an apparent lack of morphologic differences. The molecular distinction between scars and keloid is still controversial and, until today, there is no appropriate treatment yet for keloid disease. In this study, we have found, for the first time, p53 mutations in both hypertrophic scar and keloids fibroblasts from cultured cells to various extents. Since p53 plays a central role in the DNA damage response by inducing cell cycle arrest and/or apoptotic cell death, we also set up time course experiments making cell cultures at different times to investigate the phenomenon of apoptosis and its involvement in the process of pathological scarring in both hypertrophic scars and keloids. The extent of apoptosis in this study was investigated by DNA fragmentation and MTT assays, propidium iodide staining, p53 expression, and subcellular distribution. Moreover, the correlation of apoptosis and ROS levels in keloid and hypertrophic scars fibroblasts was assessed. Understanding the molecular mechanisms that determine the regulation of apoptosis during wound healing might allow us to therapeutically modulate these pathways so that apoptotic cell death is reactivated in dysregulated and hypertrophic cells. © Springer Science+Business Media, LLC. 2009.

The antioxidant enzyme Cu,Zn superoxide dismutase has so far been considered costitutively expressed and exclusively localized into cytosol. In this paper we investigated Cu,Zn superoxide dismutase export in neuroblastoma SK-N-BE cells by flow cytometry analysis, confocal immunofluorescence analysis and enzyme-linked immunosorbed assay. Immunofluorescence analysis shows that the enzyme is exported by microvesicular granules; moreover the treatment of cells with brefeldin A and with 2-deoxy-D-glucose and sodium azide strongly decreases the amount of CuZn superoxide dismutase detected in the medium. Therefore the involvement of ATP-dependent mechanisms, likely including BFA-sensitive intracytoplasmic vesicles in Cu,Zn SOD export from SK-N-BE cells, has to be hypothesized. Microvesicular-mediated Cu,Zn SOD export in neurons could represent a relevant phenomenon able to influence cell excitability that is affected by reactive oxygen species.

Reactive oxygen species (ROS) behave as second messengers in signal transduction for a series of receptor/ligand interactions. A major regulatory role is played by hydrogen peroxide (H2O2), more stable and able to freely diffuse through cell membranes. Copper-zinc superoxide dismutase (CuZn-SOD)-1 is a cytosolic enzyme involved in scavenging oxygen radicals to H2O2 and molecular oxygen, thus representing a major cytosolic source of peroxides. Previous studies suggested that superoxide anion and H2O2 generation are involved in T cell receptor (TCR)-dependent signaling. Here, we describe that antigen-dependent activation of human T lymphocytes significantly increased extracellular SOD-1 levels in lymphocyte cultures. This effect was accompanied by the synthesis of SOD-1-specific mRNA and by the induction of microvesicle SOD-1 secretion. It is of note that SOD-1 increased its concentration specifically in T cell population, while no significant changes were observed in the "non-T" cell counterpart. Moreover, confocal microscopy showed that antigen-dependent activation was able to modify SOD-1 intracellular localization in T cells. Indeed, was observed a clear SOD-1 recruitment by TCR clusters. The ROS scavenger N-acetylcysteine (NAC) inhibited this phenomenon. Further studies are needed to define whether SOD-1-dependent superoxide/peroxide balance is relevant for regulation of T cell activation, as well as in the functional cross talk between immune effectors. © 2013 Elsevier B.V.

Conditions perturbing the homeostasis of the endoplasmic reticulum (ER) cause accumulation of unfolded proteins and trigger ER stress. In PC Cl3 thyroid cells, thapsigargin and tunicamycin interfered with the folding of thyroglobulin, causing accumulation of this very large secretory glycoprotein in the ER. Consequently, mRNAs encoding BiP and XBP-1 were induced and spliced, respectively. In the absence of apoptosis, differentiation of PC Cl3 cells was inhibited. mRNA and protein levels of the thyroid-specific genes encoding thyroglobulin, thyroperoxidase and the sodium/iodide symporter and of the genes encoding the thyroid transcription factors TTF-1, TTF-2 and Pax-8 were dramatically downregulated. These effects were, at least in part, transcriptional. Moreover, they were selective and temporally distinct from the general and transient PERK-dependent translational inhibition. Thyroid dedifferentiation was accompanied by changes in the organization of the polarized epithelial monolayer. Downregulation of the mRNA encoding E-cadherin, and upregulation of the mRNAs encoding vimentin, α-smooth muscle actin, α(1)(I) collagen and SNAI1/SIP1, together with formation of actin stress fibers and loss of trans-epithelial resistance were found, confirming an epithelial-mesenchymal transition (EMT). The thyroid-specific and epithelial dedifferentiation by thapsigargin or tunicamycin were completely prevented by the PP2 inhibitor of Src-family kinases and by stable expression of a dominant-negative Src. Together, these data indicate that ER stress induces dedifferentiation and an EMT-like phenotype in thyroid cells through a Src-mediated signaling pathway.

The p63 gene belongs to the p53 gene family and encodes for sequence-specific transcription factors. p63 has been characterized primarily in the context of epidermis where is implicated in the establishment of keratinocyte cell fate and in maintenance of epithelial self-renewal. ΔNp63 isoform has been showed to be involved in several kinds of human tumors of epidermal origin, even nonmalignant, for the neoplastic and proliferative potential. Here, we report the differential expression and the cellular localization of the ΔNp63 isoform in fibroblasts isolated from human keloids and hypertrophic scars compared to normal skin. Differently from hypertrophic scar, our results show that ΔNp63 has a nuclear localization and is overexpressed only in keloid fibroblasts, suggesting an essential role of ΔNp63 in vivo in human keloids. Consistent with our results, we hypothesize that ΔNp63 overexpression may be oncogenic because of its ability to block the activity of p53 since p53 is underexpressed in fibroblasts from keloids. © Mary Ann Liebert, Inc.

cAMP-dependent protein kinase (PKA) is composed of two genetically distinct catalytic (C) and regulatory (R) subunits. There are two different classes of PKA, designated as type I and type II, which contain distinct R subunits (RI or RII, respectively) but share a common C subunit. Enhanced expression of type I PKA has been correlated with cell proliferation and neoplastic transformation. Detection of the different PKA subunits is usually performed by photoaffinity labeling with 8-N3-32P-cAMP or by radioimmunolabeling techniques. Both techniques are time consuming and require a high number of cells and the use of radioactive reagents. Using the MCF-10A normal human mammary cell line infected with a recombinant retroviral vector containing the human RI alpha gene (MCF-10A RI alpha), we have developed a flow-cytometric assay to detect the intracellular content of RI alpha protein in human cells. MCF-10A and MCF-10A RI alpha cells were fixed in 1.5% paraformaldehyde at 37 degrees C for 15 min and permeabilized by methanol and acetone (1:1) at -20 degrees C for 5 min before staining with a specific IgG2a MoAb followed by a FITC-conjugate rabbit-anti mouse IgG. This procedure was also successfully utilized to recognize RI alpha protein content in human peripheral blood lymphocytes. Flow-cytometric detection of the RI alpha subunit in human cells is feasible and allows the study of the role of type I PKA in cell growth and neoplastic transformation.

The presence of a specific SOD surface membrane receptor in rat hepatocyte cells was investigated using ligand blot analysis of the SOD receptor with 125I-SOD, and binding studies with iodinated and fluorescinated SOD. A specific SOD receptor with a molecular weight of about 320 kDa was identified. We suggest that the modulatory role of SOD on HMG-CoA reductase is exerted through this specific receptor. © 1994.