Nuclear staining and relative distance for quantifying epidermal differentiation in biomarker expression profiling

<p>Abstract</p> <p>Background</p> <p>The epidermal physiology results from a complex regulated homeostasis of keratinocyte proliferation, differentiation and death and is tightly regulated by a specific protein expression during cellular maturation. Cellular <it>in silico </it>models are considered a promising and inevitable tool for the understanding of this complex system. Hence, we need to incorporate the information of the differentiation dependent protein expression in cell based systems biological models of tissue homeostasis. Such methods require measuring tissue differentiation quantitatively while correlating it with biomarker expression intensities.</p> <p>Results</p> <p>Differentiation of a keratinocyte is characterized by its continuously changing morphology concomitant with its movement from the basal layer to the surface, leading to a decreased average nuclei density throughout the tissue. Based thereon, we designed and evaluated three different mathematical measures (nuclei based, distance based, and joint approach) for quantifying differentiation in epidermal keratinocytes. We integrated them with an immunofluorescent staining and image analysis method for tissue sections, automatically quantifying epidermal differentiation and measuring the corresponding expression of biomarkers. When studying five well-known differentiation related biomarkers in an epidermal neck sample only the resulting biomarker profiles incorporating the relative distance information of cells to the tissue borders (distance based and joint approach) provided a high-resolution view on the whole process of keratinocyte differentiation. By contrast, the inverse nuclei density approach led to an increased resolution at early but heavily decreased resolution at late differentiation. This effect results from the heavy non-linear decay of DAPI intensity per area, probably caused by cytoplasmic growth and chromatin decondensation. In the joint approach this effect could be compensated again by incorporating distance information.</p> <p>Conclusion</p> <p>We suppose that key mechanisms regulating tissue homeostasis probably depend more on distance information rather than on nuclei reorganization. Concluding, the distance approach appears well suited for comprehensively observing keratinocyte differentiation.</p

The E5 protein of the human papillomavirus type 16 down-regulates HLA-I surface expression in calnexin-expressing but not in calnexin-deficient cells

The human papillomavirus type 16 E5 protein (HPV16 E5) down-regulates surface expression of HLA-I molecules. The molecular mechanisms underlying this effect are so far unknown. Here we show that HPV16 E5 down-regulates HLA-I surface expression in calnexin-containing but not in calnexin-deficient cells. Immunoprecipitation experiments reveal that calnexin and HPV16E5 can be co-precipitated and that this association depends on the presence of a wild-type first hydrophobic region of E5. When an E5 mutant (M1) in which the first putative transmembrane helix had been disrupted was used for the transfections calnexin-E5 co-precipitation was strongly impaired. In addition, we show that the M1 mutant is only able to marginally down-regulate HLA-I surface expression compared to the wild-type protein. Besides, we demonstrate that E5 forms a ternary complex with calnexin and the heavy chain of HLA-I, which is mediated by the first hydrophobic region of the E5 protein. On the basis of our results we conclude that formation of this complex is responsible for retention of HLA-I molecules in the ER of the cells

Mechano-transduction in periodontal ligament cells identifies activated states of MAP-kinases p42/44 and p38-stress kinase as a mechanism for MMP-13 expression

<p>Abstract</p> <p>Background</p> <p>Mechano-transduction in periodontal ligament (PDL) cells is crucial for physiological and orthodontic tooth movement-associated periodontal remodelling. On the mechanistic level, molecules involved in this mechano-transduction process in PDL cells are not yet completely elucidated.</p> <p>Results</p> <p>In the present study we show by western blot (WB) analysis and/or indirect immunofluorescence (IIF) that mechanical strain modulates the amount of the matrix metalloproteinase MMP-13, and induces non-coherent modulation in the amount and activity of signal transducing molecules, such as FAK, MAP-kinases p42/44, and p38 stress kinase, suggesting their mechanistic role in mechano-transduction. Increase in the amount of FAK occurs concomitant with increased levels of the focal contact integrin subunits β3 and β1, as indicated by WB or optionally by IIF. By employing specific inhibitors, we further identified p42/44 and p38 in their activated, i.e. phosphorylated state responsible for the expression of MMP-13. This finding may point to the obedience in the expression of this MMP as extracellular matrix (ECM) remodelling executioner from the activation state of mechano-transducing molecules. mRNA analysis by pathway-specific RT-profiler arrays revealed up- and/or down-regulation of genes assigning to MAP-kinase signalling and cell cycle, ECM and integrins and growth factors. Up-regulated genes include for example focal contact integrin subunit α3, MMP-12, MAP-kinases and associated kinases, and the transcription factor c-fos, the latter as constituent of the AP1-complex addressing the MMP-13 promotor. Among others, genes down-regulated are those of COL-1 and COL-14, suggesting that strain-dependent mechano-transduction may transiently perturbate ECM homeostasis.</p> <p>Conclusions</p> <p>Strain-dependent mechano-/signal-transduction in PDL cells involves abundance and activity of FAK, MAP-kinases p42/44, and p38 stress kinase in conjunction with the amount of MMP-13, and integrin subunits β1 and β3. Identifying the activated state of p42/44 and p38 as critical for MMP-13 expression may indicate the mechanistic contribution of mechano-transducing molecules on executioners of ECM homeostasis.</p

Multi-chromatic control of mammalian gene expression and signaling

The emergence and future of mammalian synthetic biology depends on technologies for orchestrating and custom tailoring complementary gene expression and signaling processes in a predictable manner. Here, we demonstrate for the first time multi-chromatic expression control in mammalian cells by differentially inducing up to three genes in a single cell culture in response to light of different wavelengths. To this end, we developed an ultraviolet B (UVB)-inducible expression system by designing a UVB-responsive split transcription factor based on the Arabidopsis thaliana UVB receptor UVR8 and the WD40 domain of COP1. The system allowed high (up to 800-fold) UVB-induced gene expression in human, monkey, hamster and mouse cells. Based on a quantitative model, we determined critical system parameters. By combining this UVB-responsive system with blue and red light-inducible gene control technology, we demonstrate multi-chromatic multi-gene control by differentially expressing three genes in a single cell culture in mammalian cells, and we apply this system for the multi-chromatic control of angiogenic signaling processes. This portfolio of optogenetic tools enables the design and implementation of synthetic biological networks showing unmatched spatiotemporal precision for future research and biomedical application

Concerted Action of Androgens and Mechanical Strain Shifts Bone Metabolism from High Turnover into an Osteoanabolic Mode

Adhesion of bone cells to the extracellular matrix is a crucial requirement for osteoblastic development and function. Adhesion receptors connect the extracellular matrix with the cyto-skeleton and convey matrix deformation into the cell. We tested the hypothesis that sex hormones modulate mechanoperception of human osteoblastic cells (HOB) by affecting expression of adhesion molecules like fibronectin and the fibronectin receptor. Only dihydrotestosterone (DHT), but not 17β-estradiol, stimulated fibronectin (137%) and fibronectin receptor (252%) protein expression. The effects of deformation strain on HOB metabolism were investigated in a FlexerCell® strain unit. Cyclically applied strain (2.5% elongation) increased DNA synthesis (125%) and interleukin-6 (IL-6) production (170%) without significantly affecting alkaline phosphatase (AP) activity, type I collagen (PICP), or osteoprotegerin (OPG) secretion. 10 nM DHT pretreatment abolished the mitogenic response of HOB to strain and increased AP activity (119%), PICP (163%), and OPG production (204%). In conclusion, mechanical strain stimulates bone remodeling by increasing HOB mitosis and IL-6 production. DHT enhances the osteoanabolic impact of deformation strain by increasing bone formation via increased AP activity and PICP production. At the same time, bone resorption is inhibited by decreased IL-6 and increased OPG secretion into the bone microenvironment

A red/far-red light-responsive bi-stable toggle switch to control gene expression in mammalian cells

Growth and differentiation of multicellular systems is orchestrated by spatially restricted gene expression programs in specialized subpopulations. The targeted manipulation of such processes by synthetic tools with high-spatiotemporal resolution could, therefore, enable a deepened understanding of developmental processes and open new opportunities in tissue engineering. Here, we describe the first red/far-red light-triggered gene switch for mammalian cells for achieving gene expression control in time and space. We show that the system can reversibly be toggled between stable on- and off-states using short light pulses at 660 or 740 nm. Red light-induced gene expression was shown to correlate with the applied photon number and was compatible with different mammalian cell lines, including human primary cells. The light-induced expression kinetics were quantitatively analyzed by a mathematical model. We apply the system for the spatially controlled engineering of angiogenesis in chicken embryos. The system's performance combined with cell- and tissue-compatible regulating red light will enable unprecedented spatiotemporally controlled molecular interventions in mammalian cells, tissues and organism

Physical Aspects of Cancer Invasion

Invasiveness, one of the hallmarks of tumor progression, represents the tumor's ability to expand into the host tissue by means of several complex biochemical and biomechanical processes. Since certain aspects of the problem present a striking resemblance with well known physical mechanisms, such as the mechanical insertion of a solid inclusion in an elastic material specimen [1, 2] or a water drop impinging on a surface [3], we propose here an analogy between these physical processes and a cancer system's invasive branching into the surrounding tissue. Accounting for its solid and viscous properties, we present a unifying concept that the tumor behaves as a granular solid. While our model has been explicitly formulated for multicellular tumor spheroids in vitro, it should also contribute to a better understanding of tumor invasion in vivo.Comment: 20 pages, 2 figure

H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent.Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras), were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63), basement-membrane formation (collagen IV, laminin-5), and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin) were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes.The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression