Claude Backendorf

Human Papillomavirus Deregulates the Response of a Cellular Network Comprising of Chemotactic and Proinflammatory Genes

Despite the presence of intracellular pathogen recognition receptors that allow infected cells to attract the immune system, undifferentiated keratinocytes (KCs) are the main targets for latent infection with high-risk human papilloma viruses (hrHPVs). HPV infections are transient but on average last for more than one year suggesting that HPV has developed means to evade host immunity. To understand how HPV persists, we studied the innate immune response of undifferentiated human KCs harboring episomal copies of HPV16 and 18 by genome-wide expression profiling. Our data showed that the expression of the different virus-sensing receptors was not affected by the presence of HPV. Poly(I:C) stimulation of the viral RNA receptors TLR3, PKR, MDA5 and RIG-I, the latter of which indirectly senses viral DNA through non-self RNA polymerase III transcripts, showed dampening in downstream signalling of these receptors by HPVs. Many of the genes downregulated in HPV-positive KCs involved components of the antigen presenting pathway, the inflammasome, the production of antivirals, pro-inflammatory and chemotactic cytokines, and components downstream of activated pathogen receptors. Notably, gene and/or protein interaction analysis revealed the downregulation of a network of genes that was strongly interconnected by IL-1β, a crucial cytokine to activate adaptive immunity. In summary, our comprehensive expression profiling approach revealed that HPV16 and 18 coordinate a broad deregulation of the keratinocytes inflammatory response, and contributes to the understanding of virus persistence.

Interdependent transcription control elements regulate the expression of the SPRR2A gene during keratinocyte terminal differentiation.

Expression of the SPRR2A gene, a member of the small proline-rich family of cornified cell envelope precursor proteins, is strictly linked to keratinocyte terminal differentiation both in vivo and in vitro. In this study, we explored the molecular mechanisms underlying this regulation in transiently transfected primary keratinocytes induced to differentiate in vitro. Deletion mapping and site-directed mutagenesis of SPRR2A promoter-chloramphenicol acetyltransferase constructs indicate that four transcription control elements are essential and sufficient for promoter activity. These elements were further characterized by electrophoretic mobility shift and identified as (i) an inverted octamer doublet, bound by the POU domain factor Oct-11 (Skn-1a/i, Epoc-1), (ii) an interferon-stimulated response element recognized by interferon regulatory factors 1 and 2, (iii) an Ets binding site partially overlapping the interferon-stimulated response element, and (iv) a TG box recognized by the Sp1 family of zinc finger transcription factors. Destruction of a single terminal differentiation element is sufficient to completely abolish transcription from the SPRR2A promoter, indicating that these transcription control elements function in concert in an interdependent manner. Apparently, integration of signals transmitted by the above-mentioned transcription factors is necessary and sufficient to promote gene expression during keratinocyte terminal differentiation.

Human Papillomavirus (HPV) Upregulates the Cellular Deubiquitinase UCHL1 to Suppress the Keratinocyte's Innate Immune Response

Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPVs capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1) in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3) K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.

Folic Acid-Modified Mesoporous Silica Nanoparticles for Cellular and Nuclear Targeted Drug Delivery

Site-specific stimuli responsive nanomaterials are an important breakthrough for the improvement of modern therapies in nanomedicine. Mesoporous silica nanoparticles are good candidate for the development of targeted delivery system as their surface can be easily modified with functional groups in order to achieve controlled and specific release. We designed a drug delivery system based on mesoporous silica nanoparticles modified with folic acid as a specific targeting moiety. The functionalization forms a nanovalve system in which the surface is modified with an aliphatic chain. This stalk tethers a cyclodextrin with the specific role to prevent undesired release of the cargo. To avoid any movement of the cyclodextrin the folic acid is placed at the end of the chain. The release kinetics were investigated with UV/VIS spectroscopy and cellular uptake was extensively studied using flow cytometry. Through this study we demonstrated the biocompatibility of folic acid modified MSNs and the effective release of an encapsulated anticancer drug using TUNEL and Western Blot assays. Chapter 3: Folic Acid Modified Mesoporous Silica Nanoparticles for Cellular and Nuclear Targeted Drug Delivery

Enhanced DDB2 Expression Protects Mice from Carcinogenic Effects of Chronic UV-B Irradiation

UV-damaged DNA-binding protein (UV-DDB) is essential for global genome repair (GGR) of UV-induced cyclobutane pyrimidine dimers (CPD). Unlike human cells, rodent epidermal cells are deficient in GGR of CPDs and express a subunit of UV-DDB, DDB2, at a low level. In this study, we generated mice (K14-DDB2) ectopically expressing mouse DDB2 at elevated levels. Enhanced expression of DDB2 both delayed the onset of squamous cell carcinoma and decreased the number of tumors per mouse in chronically UV-B light-exposed hairless mice. Enhanced expression of DDB2 improved repair of both CPDs and pyrimidine(6-4)pyrimidone photoproducts (6-4PP) in dermal fibroblasts. However, GGR of CPDs in K14-DDB2 mice did not reach the level of efficiency of human cells, suggesting that another repair protein may become rate limiting when DDB2 is abundantly present. To complement these studies, we generated mice in which the DDB2 gene was disrupted. DDB2-/- and DDB2+/- mice were found to be hypersensitive to UV-induced skin carcinogenesis. On the cellular level, we detected a delay in the repair of 6-4PPs in DDB2-/- dermal fibroblasts. Neither the absence nor the enhanced expression of DDB2 affected the levels of UV-induced apoptosis in epidermal keratinocytes or cultured dermal fibroblasts. Our results show an important role for DDB2 in the protection against UV-induced cancer and indicate that this protection is most likely mediated by accelerating the repair of photolesions.

AP-1 and Ets Transcription Factors Regulate the Expression of the Human SPRR1A Keratinocyte Terminal Differentiation Marker

The 173-base pair proximal promoter ofSPRR1A is necessary and sufficient for regulated expression in primary keratinocytes induced to differentiate either by increasing extracellular calcium or by 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. Whereas calcium-induced expression depends both on an AP-1 and an Ets binding site in this region, responsiveness to TPA resides mainly (but not exclusively) on the Ets element, indicating that Ets factors are important targets for protein kinase C signaling during keratinocyte terminal differentiation. This conclusion is further substantiated by the finding that expression of ESE-1, an Ets transcription factor involved in SPRR regulation, is also induced by TPA, with kinetics similar to SPRR1A. The strict AP-1 requirement inSPRR1A for calcium-induced differentiation is not found forSPRR2A, despite the presence of an identical AP-1 consensus binding site in this gene. Binding site swapping indicates that both the nucleotides flanking the TGAGTCA core sequence and the global promoter context are essential in determining the contribution of AP-1 factors in gene expression during keratinocyte terminal differentiation. In the distal SPRR1A promoter region, a complex arrangement of positive and negative regulatory elements, which are only conditionally needed for promoter activity, are likely involved in gene-specific fine-tuning of the expression of this member of the SPRR gene family.

Cellular functions of 14-3-3|[zeta]| in apoptosis and cell adhesion emphasize its oncogenic character

14-3-3 proteins are relevant to cancer biology as they are key regulators of major cellular processes such as proliferation, differentiation, senescence and apoptosis. So far, the sigma isoform (14-3-3σ) has most directly been implicated in carcinogenesis and was recognized as a tumour-suppressor gene. The other six members of the mammalian 14-3-3 gene family likely behave as oncogenes, although direct evidence supporting this view is largely circumstantial. In this report, we show that knockdown of 14-3-3ζ induces at least two isoform-specific phenotypes that are consistent with a potential oncogenic activity during tumorigenesis. Firstly, downregulation of 14-3-3ζ sensitized cells to stress-induced apoptosis and JNK/p38 signalling and secondly, it enforced cell–cell contacts and expression of adhesion proteins. Apparently, the zeta isoform restrains both cell adhesion and the cellular propensity for apoptosis, two activities that are also restrained during carcinogenesis. The assumption that 14-3-3ζ has oncogenic properties was substantiated with a web-based meta-analysis (Oncomine), revealing that 14-3-3ζ is overexpressed in various types of carcinomas. As the highly conserved human 14-3-3 gene family encodes proteins with either tumour-promoting or tumour-suppressing activities, we infer that the cellular balance between the various 14-3-3 isoforms is crucial for the proper functioning of cells.

ROS Quenching Potential of the Epidermal Cornified Cell Envelope

The cornified cell envelope (CE) is a specialized structure assembled beneath the plasma membrane of keratinocytes in the outermost layers of the epidermis. It is essential for the physical and permeability properties of the barrier function of the skin. Our skin is continuously exposed to atmospheric oxygen and threatened by reactive oxygen species (ROS). Here, we identify the CE as a first line of antioxidant defense and show that the small proline-rich (SPRR) family of CE precursor proteins have a major role in ROS detoxification. Cysteine residues within these proteins are responsible for ROS quenching, resulting in inter- and intramolecular S-S bond formation, both in isolated proteins and purified CEs. The related keratinocyte proline-rich protein is also oxidized on several cysteine residues within the CE. Differences in antioxidant potential between various SPRR family members are likely determined by structural differences rather than by the amount of cysteine residues per protein. Loricrin, a major component of the CE with a higher cysteine content than SPRRs, is a weak ROS quencher and oxidized on a single cysteine residue within the CE. It is inferred that SPRR proteins provide the outermost layer of our skin with a highly adaptive and protective antioxidant shield.

Construction and properties of an Epstein-Barr-virus-derived cDNA expression vector for human cells

A cDNA expression vector containing the element oriP and the sequence encoding the Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) as well as the hygromycin B-resistance dominant marker gene has been constructed. Its characteristics have been compared to a similar vector lacking the EBV sequences. (a) The EBV+ vector is maintained as an episome with a copy number of approx. 50 per cell, whereas the number of the integrated EBV- copies is in general smaller than 10, when simian virus 40-transformed xeroderma pigmentosum fibroblasts (XP20S-SV) constitute the recipient cell line. (b) The presence of the EBV sequences in the vector resulted in a five- to ten-fold higher transfection efficiency with the Ca.phosphate precipitation technique. (c) cDNA inserts in the EBV+ vector are shown to be efficiently and properly expressed in the recipient cell. (d) If transfection is performed with a mixture of EBV+ vectors with different inserts, transfectants are shown to harbour different plasmids within one cell. (e) The ratio between these plasmids in one cell can be shifted in favour of a vector with a particular insert, when selection for this insert is performed. (f) Reconstruction experiments indicated that isolation of a low-abundance sequence from a mixture of vectors is at least 100-fold more efficient with the EBV+ system, than with the EBV- system. (g) Rescue of the episomal vector from transfected cells can be readily achieved.

Regulation of keratinocyte proliferation and differentiation by all-trans-retinoic acid, 9-cis-retinoic acid and 1,25-dihydroxy vitamin D3

We studied the effect of all-trans retinoic acid, (all-trans-RA), 9-cis-retinoic acid (9-cis-RA) and 1,25-dihydroxy vitamin D3 (1,25(OH)2D3) on proliferation and differentiation of human keratinocytes cultured in a submerged culture system for up to 5 weeks and evaluated changes in cell morphology and in the expression of proliferation- and terminal differentiation-related genes on both the mRNA and the protein levels. Under control culture conditions, the expression of small proline-rich proteins (SPRR1 and SPRR2), involucrin, Ki67 and c-jun, reached a maximum after 2 weeks in culture (1 week postoconfluence) and then decreased as the tissue architecture of the cultures deteriorated. Upon simulataneous treatment withboth retinoids and 1,25(OH)2D3 a culture was generated that remained stable for 4 weeks with at least eight living cell layers. Furthermore, this culture showed a pattern of SPRR2 and involucrin expression which closely resembled that of native epidermis, a maintained, Ki67 expression and a strongly induced c-jun expression. Treatment with 1,25(OH)2D3 alone inhibited cell proliferation, and stimulated cell differentiation resulting in acceleration, of the differentiated phenotype and was accompanied by inhibition of c-jun and Ki67 expression and also, surprisingly, by inhibition, of SPRR1, SPRR2 and involucrin expression. In contrast, treatment with all-trans-RA and/or 9-cis-RA induced a more proliferative phenotype with a prolonged lifespan as compared to control cultures. SPRR1 was weakly repressed, SPRR2 was strongly repressed, a delayed induction of involucrin occurred, and c-jun and Ki67 expression were mainteined. These results show that modulation of the composition of the medium by the addition of various, vitamins results in changes in the balance between keratinocyte proliferation and differentiation which correspond to changes in the expression of proliferation and differentiation markers and prolongation, of the culture lifespan.