Gregor Kirfel

The biological role of the Alzheimer amyloid precursor protein in epithelial cells

Abstract. Proteolytic processing of the Alzheimer amyloid precursor protein (APP) results in the generation of at least two distinct classes of biologically relevant peptides: (1) the amyloid beta peptides which are believed to be involved in the pathogenesis of Alzheimers disease and (2) the soluble N-terminal ectodomain (sAPP) which exhibits a protective but as yet ill-defined effect on neurons and epithelial cells. In this report we present an overview on the functions of sAPP as an epithelial growth factor. This function involves specific binding of sAPP to membrane rafts and results in signal transduction and various physiological effects in epithelial cells as different as keratinocytes and thyrocytes. At nanomolar concentrations sAPP induces a two to fourfold increase in the rate of cell proliferation and cell migration. Specific inhibition of APP expression by antisense techniques results in decreased sAPP release and in reduced proliferative and motogenic activities. Proliferation and migration are known to be part of complex processes such as wound healing which, therefore, might be facilitated by the growth factor function of sAPP.

Connexin Expression and Functional Analysis of Gap Junctional Communication in Mouse Embryonic Stem Cells

Gap junctional intercellular communication (GJIC) has been suggested to be necessary for cellular proliferation and differentiation. We wanted to investigate the function of GJIC in mouse embryonic stem (ES) cells using pharmacological inhibitors or a genetic approach to inhibit the expression of connexins, that is, the subunit proteins of gap junction channels. For this purpose, we have analyzed all known connexin genes in mouse ES cells but found only three of them, Cx31, Cx43, and Cx45, to be expressed as proteins. We have demonstrated by coimmunoprecipitation that Cx31 and Cx43, as well as Cx43 and Cx45, probably form heteromeric gap junction channels, whereas Cx31 and Cx45 do not. The pharmacological inhibitors reduced GJIC between ES cells to approximately 3% and initiated apoptosis, suggesting an antiapoptotic effect of GJIC. In contrast to these results, reduction of GJIC to approximately 5% by decreased expression of Cx31 or Cx45 via RNA interference in homozygous Cx43‐deficient ES cells did not lead to apoptosis. Additional studies suggested that apoptotic death of ES cells and adult stem cells reported in the literature is likely due to a cytotoxic side effect of the inhibitors and not due to a decrease of GJIC. Using the connexin expression pattern in mouse ES cells, as determined in this study, multiple connexin‐deficient ES cells can now be genetically engineered in which the level of GJIC is further decreased, to clarify whether the differentiation of ES cells is qualitatively or quantitatively compromised.

Biological roles of APP in the epidermis.

The amyloid precursor protein (APP) was initially detected in cells of the central nervous system where it is considered to be involved in the pathogenesis of Alzheimers disease. However, APP is also found in peripheral organs with exceptionally strong expression in the mammalian epidermis where it fulfils a variety of distinct biological roles. Full length APP appears to facilitate keratinocyte adhesion due to its ability to interact with the extracellular matrix. The C-terminus of APP also serves as adapter protein for binding the motor protein kinesin thereby mediating the centripetal transport of melanosomes in epidermal melanocytes. By the action of alpha-secretase sAPPalpha, the soluble N-terminal portion of APP, is released. sAPPalpha has been shown to be a potent epidermal growth factor thus stimulating proliferation and migration of keratinocytes as well as the exocytic release of melanin by melanocytes. The release of sAPPalpha can be almost completely blocked by inhibiting alpha-secretase with hydroxamic acid-based zinc metalloproteinase inhibitors. In hyperproliferative keratinocytes from psoriatic skin this inhibition results in normalized growth.

sAPP as a regulator of dendrite motility and melanin release in epidermal melanocytes and melanoma cells

Numerous factors including ultraviolet (UV) radiation and growth factors regulate the specific function of epidermal melanocytes. A recently discovered epidermal growth factor is sAPP, the soluble N‐terminal ectodomain of the β‐amyloid precursor protein (APP). Using whole mount preparations of isolated human epidermis, we detected a small population of basal cells, which expressed exceptionally high levels of APP. These cells were identified as melanocytes, which, similar to keratinocytes and neuronal cells, expressed the three APP isoforms 695, 751, and 770. They differed in their expression pattern from that of neuronal cells by expressing only low levels of APP 695. Melanocytes and melanoma cells in vitro released, in addition to keratinocytes, large quantities of sAPP. Because of its growth factor function, we studied possible effects of sAPP on melanocytes. Recombinant sAPP strongly increased lamellipodia activity at dendritic tips, an effect that coincided with increased release of melanin particles. Our observations point to the possible use of APP as an immunocytochemical marker for melanocytes. They suggest that sAPP derived from keratinocytes and/or melanocytes belongs to a family of factors operating in the paracrine and/or autocrine regulation of melanocyte function.

Complete loss of murine Xin results in a mild cardiac phenotype with altered distribution of intercalated discs

AIMS Xin is a striated muscle-specific F-actin binding protein that has been implicated in cardiomyopathies. In cardiomyocytes, Xin is localized at intercalated discs (IDs). Mice lacking only two of the three Xin isoforms (XinAB(-/-) mice) develop severe cardiac hypertrophy. To further investigate the function of Xin variants in the mammalian heart, we generated XinABC(-/-) mice deficient in all Xin isoforms. METHODS AND RESULTS XinABC(-/-) mice showed a very mild phenotype: heart weight, heart weight to tibia length ratios, and cardiac dimensions were not altered. Increased perivascular fibrosis was only observed in hearts of young XinABC(-/-) mice. Striking differences were revealed in isolated cardiomyocytes: XinABC(-/-) cells demonstrated a significantly increased number of non-terminally localized ID-like structures. Furthermore, resting sarcomere length was increased, sarcomere shortening, peak shortening at 0.5-1 Hz, and the duration of shortening were decreased, and shortening and relengthening velocities were accelerated at frequencies above 4 Hz in XinABC(-/-) cardiomyocytes. ECG showed a significantly shorter HV interval and a trend towards shorter QRS interval in XinABC(-/-) mice, suggesting a faster conduction velocity of the ventricular-specific conduction system. In human cardiac tissue, expression of XinC protein was detected solely in samples from patients with cardiac hypertrophy. CONCLUSION Total Xin deficiency leads to topographical ID alterations, premature fibrosis and subtle changes in contractile behaviour; this is a milder cardiac phenotype than that observed in XinAB(-/-) mice, which still can express XinC. Together with the finding that XinC is detected solely in cardiomyopathic human tissues, this suggests that its expression is responsible for the stronger dominant phenotype in XinAB(-/-) mice. Furthermore, it indicates that XinC may be involved in the development of human cardiac hypertrophy.

Lysophosphatidylcholine Pretreatment Reduces VLA-4 and P-Selectin–Mediated B16.F10 Melanoma Cell Adhesion In vitro and Inhibits Metastasis-Like Lung Invasion In vivo

Lysophosphatidylcholine (LysoPC) is an important intermediate in degradation and biosynthesis of phosphatidylcholine (PC). Reduced plasma LysoPC levels observed in patients with advanced cancer indicate a deregulation of LysoPC metabolism in metastasis. Recent data showed strong antimetastatic effects of liposomes consisting of saturated PC in a murine pancreatic metastasis model. LysoPC, generated from saturated PC after accumulation of the liposomes in tumor tissue, might be contributing to these effects. Examining effects of high local concentrations of saturated LysoPC and investigating potential molecular mechanisms, fast removal of saturated LysoPC from medium by murine B16.F10 melanoma cells and radical shifts in tumor cell membrane fatty acid (FA) composition toward saturated FAs were observed in vitro. Scanning electron microscopy revealed remarkable morphologic surface changes of LysoPC-treated tumor cells, probably causing their impaired migratory ability on fibronectin. A LysoPC concentration exceeding a threshold of about 400 μmol/L, slightly above physiologic levels, strongly reduced VLA-4–mediated binding of B16.F10 cells to VCAM-1 as well as P-selectin–dependent interaction with activated platelets, although expression levels were not altered. These findings were reflected in a syngenic intravenous lung invasion model using repeatedly ex vivo LysoPC-treated (450 μmol/L) B16.F10 cells, resulting in significantly reduced lung metastasis-like lesions (−48.3%, P = 0.006). Prior application of 50 IU unfractionated heparin further reduced lung invasion (−81.6%, P = 0.043). Our work shows for the first time that saturated LysoPC in high concentrations reduces melanoma cell adhesion in vitro and hematogeneous dissemination in vivo by direct ex vivo tumor cell targeting. Mol Cancer Ther; 10(1); 186–97. ©2011 AACR.

The secretory β-amyloid precursor protein is a motogen for human epidermal keratinocytes

Summary Cell migration is known to be triggered by constituents of the extracellular matrix such as fibronectin and by soluble mediators commonly summarized as motogens. Many growth factors such as the epidermal growth factor (EGF) have been shown to act as motogens. Recently, the secretory N-terminal portion of the β-amyloid precursor protein (sAPP) has been identified as a keratinocyte growth factor. Hence, in this study we analysed whether sAPP stimulates also keratinocyte migration employing the stroboscopic cell motility assay. The migration velocity as well as the frequency of lamellipodia protrusion and ruffle formation were increased about two-fold thus corresponding to the effect of EGF. Using a newly developed β1-integrin migration track assay we observed that sAPP increased the proportion of migrating keratinocytes and their directional persistence. sAPP appeared to operate synergistically with fibronectin with respect to its motogenic effect. Using a modified Boyden chamber assay we showed that sAPP besides its chemokinetic effect functions as a chemoattractant. Like EGF, sAPP exerted its motogenic effect through the activation of Rac kinase but the receptor for sAPP appears to be distinct. The results suggest that sAPP operates as a motogen in the human epidermis, where it may participate in the regulation of reepithelialization during wound healing.

Identification of Xin-repeat proteins as novel ligands of the SH3 domains of nebulin and nebulette and analysis of their interaction during myofibril formation and remodeling

The striated muscle–specific actin-binding proteins Xin and Xirp2 are identified as novel ligands of the SH3 domains of the thin filament ruler nebulin and nebulette. The interaction is spatially restricted to structures associated with myofibril development or remodeling, indicating a role for these proteins in myofibril assembly and repair.

Aciculin interacts with filamin C and Xin and is essential for myofibril assembly, remodeling and maintenance

ABSTRACT Filamin C (FLNc) and Xin actin-binding repeat-containing proteins (XIRPs) are multi-adaptor proteins that are mainly expressed in cardiac and skeletal muscles and which play important roles in the assembly and repair of myofibrils and their attachment to the membrane. We identified the dystrophin-binding protein aciculin (also known as phosphoglucomutase-like protein 5, PGM5) as a new interaction partner of FLNc and Xin. All three proteins colocalized at intercalated discs of cardiac muscle and myotendinous junctions of skeletal muscle, whereas FLNc and aciculin also colocalized in mature Z-discs. Bimolecular fluorescence complementation experiments in developing cultured mammalian skeletal muscle cells demonstrated that Xin and aciculin also interact in FLNc-containing immature myofibrils and areas of myofibrillar remodeling and repair induced by electrical pulse stimulation (EPS). Fluorescence recovery after photobleaching (FRAP) experiments showed that aciculin is a highly dynamic and mobile protein. Aciculin knockdown in myotubes led to failure in myofibril assembly, alignment and membrane attachment, and a massive reduction in myofibril number. A highly similar phenotype was found upon depletion of aciculin in zebrafish embryos. Our results point to a thus far unappreciated, but essential, function of aciculin in myofibril formation, maintenance and remodeling.

Interference with Activator Protein-2 transcription factors leads to induction of apoptosis and an increase in chemo- and radiation-sensitivity in breast cancer cells

BackgroundActivator Protein-2 (AP-2) transcription factors are critically involved in a variety of fundamental cellular processes such as proliferation, differentiation and apoptosis and have also been implicated in carcinogenesis. Expression of the family members AP-2α and AP-2γ is particularly well documented in malignancies of the female breast. Despite increasing evaluation of single AP-2 isoforms in mammary tumors the functional role of concerted expression of multiple AP-2 isoforms in breast cancer remains to be elucidated. AP-2 proteins can form homo- or heterodimers, and there is growing evidence that the net effect whether a cell will proliferate, undergo apoptosis or differentiate is partly dependent on the balance between different AP-2 isoforms.MethodsWe simultaneously interfered with all AP-2 isoforms expressed in ErbB-2-positive murine N202.1A breast cancer cells by conditionally over-expressing a dominant-negative AP-2 mutant.ResultsWe show that interference with AP-2 protein function lead to reduced cell number, induced apoptosis and increased chemo- and radiation-sensitivity. Analysis of global gene expression changes upon interference with AP-2 proteins identified 139 modulated genes (90 up-regulated, 49 down-regulated) compared with control cells. Gene Ontology (GO) investigations for these genes revealed Cell Death and Cell Adhesion and Migration as the main functional categories including 25 and 12 genes, respectively. By using information obtained from Ingenuity Pathway Analysis Systems we were able to present proven or potential connections between AP-2 regulated genes involved in cell death and response to chemo- and radiation therapy, (i.e. Ctgf, Nrp1, Tnfaip3, Gsta3) and AP-2 and other main apoptosis players and to create a unique network.ConclusionsExpression of AP-2 transcription factors in breast cancer cells supports proliferation and contributes to chemo- and radiation-resistance of tumor cells by impairing the ability to induce apoptosis. Therefore, interference with AP-2 function could increase the sensitivity of tumor cells towards therapeutic intervention.