A potential link between desmoglein 3 and epidermal growth factor receptor in oral squamous cell carcinoma and its effect on cetuximab treatment efficacy.

Desmoglein (DSG) 3 is overexpressed in oral squamous cell carcinoma (OSCC). Epidermal growth factor receptor (EGFR) inhibitor cetuximab is widely used for OSCC treatment. Several evidences suggest a correlation between DSG3 and EGFR in epidermal keratinocytes. EGFR inhibition has been shown to enhance cell-cell adhesion and induce terminal differentiation in epidermal cells. Thus, here we investigated the DSG3-EGFR interaction in OSCC and its effect on cetuximab treatment. Cell lines established from the primary tumor and metastatic lymph nodes of four OSCC patients and three commercial OSCC cell lines were used for the experiments. Cells from metastatic lymph nodes of each patient expressed increased DSG3 and EGFR than cells from the primary tumor in the same patient. Cetuximab treatment increased DSG3 expression by up to 3.5-fold in seven of the 11 cell lines. A high calcium concentration increased the expression of DSG3 and EGFR in a dose-dependent manner. Strikingly, a high calcium-associated DSG3 induction enhanced cetuximab efficacy by up to 23% increase in cetuximab-low-sensitive cell lines. Our findings also suggest a correlation between DSG3 and EGFR in OSCC, and this affects cetuximab treatment efficacy.

Protective Function of Ahnak1 in Vascular Healing after Wire Injury.

AIM: Vascular remodeling following injury substantially accounts for restenosis and adverse clinical outcomes. In this study, we investigated the role of the giant scaffold protein Ahnak1 in vascular healing after endothelial denudation of the murine femoral artery. METHODS: The spatiotemporal expression pattern of Ahnak1 and Ahnak2 was examined using specific antibodies and real-time quantitative PCR. Following wire-mediated endothelial injury of Ahnak1-deficient mice and wild-type (WT) littermates, the processes of vascular healing were analyzed. RESULTS: Ahnak1 and Ahnak2 showed a mutually exclusive vascular expression pattern, with Ahnak1 being expressed in the endothelium and Ahnak2 in the medial cells in naïve WT arteries. After injury, a marked increase of Ahnak1- and Ahnak2-positive cells at the lesion site became evident. Both proteins showed a strong upregulation in neointimal cells 14 days after injury. Ahnak1-deficient mice showed delayed vascular healing and dramatically impaired re-endothelialization that resulted in prolonged adverse vascular remodeling, when compared to the WT littermates. CONCLUSION: The large scaffold and adaptor proteins Ahnak1 and Ahnak2 exhibit differential expression patterns and functions in naïve and injured arteries. Ahnak1 plays a nonredundant protective role in vascular healing.

Targeted delivery of tumor necrosis factor-related apoptosis-inducing ligand to keratinocytes with a pemphigus mAb.

We determined the feasibility of using an anti-desmoglein (Dsg) mAb, Px44, to deliver a biologically active protein to keratinocytes. Recombinantly produced Px44-green fluorescent protein (GFP) injected into mice and skin organ culture delivered GFP to the cell surface of keratinocytes. We replaced GFP with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to produce Px44-TRAIL. We chose TRAIL as a biological model because it inhibits activated lymphocytes and causes apoptosis of hyperproliferative keratinocytes, features of various skin diseases. Px44-TRAIL formed a trimer, the biologically active form of TRAIL. Standard assays of TRAIL activity showed that Px44-TRAIL caused apoptosis of Jurkat cells and inhibited IFN-γ production by activated CD4+ T cells. Enzyme-linked immunoassay with Px44-TRAIL showed delivery of TRAIL to Dsg. Immunofluorescence with Px44-TRAIL incubated on skin sections and cultured keratinocytes or injected into mouse skin, human organ culture, or human xenografts detected TRAIL on keratinocytes. Px44-TRAIL caused apoptosis of the hyperproliferative, but not differentiating, cultured keratinocytes through binding to Dsg3. Foldon, a small trimerization domain, cloned into Px44-TRAIL maintained its stability and biological activity at 37° C for at least 48 hours. These data suggest that such targeted therapy is feasible and may be useful for hyperproliferative and inflamed skin diseases.

Desmoglein 3-specific CD4+ T cells induce pemphigus vulgaris and interface dermatitis in mice.

Pemphigus vulgaris (PV) is a severe autoimmune disease involving blistering of the skin and mucous membranes. It is caused by autoantibodies against desmoglein 3 (Dsg3), an adhesion molecule critical for maintaining epithelial integrity in the skin, oral mucosa, and esophagus. Knowing the antigen targeted by the autoantibodies renders PV a valuable model of autoimmunity. Recently, a role for Dsg3-specific CD4+ T helper cells in autoantibody production was demonstrated in a mouse model of PV, but whether these cells exert cytotoxicity in the tissues is unclear. Here, we analyzed 3 Dsg3-specific TCRs using transgenic mice and retrovirus induction. Dsg3-specific transgenic (Dsg3H1) T cells underwent deletion in the presence of Dsg3 in vivo. Dsg3H1 T cells that developed in the absence of Dsg3 elicited a severe pemphigus-like phenotype when cotransferred into immunodeficient mice with B cells from Dsg3-/- mice. Strikingly, in addition to humoral responses, T cell infiltration of Dsg3-expressing tissues led to interface dermatitis, a distinct form of T cell-mediated autoimmunity that causes keratinocyte apoptosis and is seen in various inflammatory/autoimmune skin diseases, including paraneoplastic pemphigus. The use of retrovirally generated Dsg3-specific T cells revealed that interface dermatitis occurred in an IFN-γ- and TCR avidity-dependent manner. This model of autoimmunity demonstrates that T cells specific for a physiological skin-associated autoantigen are capable of inducing interface dermatitis and should provide a valuable tool for further exploring the immunopathophysiology of T cell-mediated skin diseases.

AHNAK1 and AHNAK2 are costameric proteins: AHNAK1 affects transverse skeletal muscle fiber stiffness.

The AHNAK scaffold PDZ-protein family is implicated in various cellular processes including membrane repair; however, AHNAK function and subcellular localization in skeletal muscle are unclear. We used specific AHNAK1 and AHNAK2 antibodies to analyzed the detailed localization of both proteins in mouse skeletal muscle. Co-localization of AHNAK1 and AHNAK2 with vinculin clearly demonstrates that both proteins are components of the costameric network. In contrast, no AHNAK expression was detected in the T-tubule system. A laser wounding assay with AHNAK1-deficient fibers suggests that AHNAK1 is not involved in membrane repair. Using atomic force microscopy (AFM), we observed a significantly higher transverse stiffness of AHNAK1⁻/⁻ fibers. These findings suggest novel functions of AHNAK proteins in skeletal muscle.

Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes.

Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart.

Bloom's syndrome gene-deficient phenotype in mouse primary cells induced by a modified tetracycline-controlled trans-silencer.

We recently reported genome-wide bi-allelic mutagenesis and phenotype-based genetic screening by tetracycline-regulated disruption of the Bloom's syndrome gene (Blm) in mouse embryonic stem (ES) cells. However, the same approach was hampered in mouse tissues owing to leaky expression of the Blm gene, which is the major obstacle in the tetracycline regulatory system. Here we describe a single-chain reverse tetracycline-controlled trans-silencer (sc rtTS) which reduces leaky expression in the tet-off system. The sc rtTS consists of two silencer moieties linked by a 36 amino acid linker. Although the silencer moiety contained a dimerization domain compatible with the tetracycline-controlled transactivator (tTA), heterodimerization with tTA was prevented because intramolecular self-assembly between linked silencer moieties was preferred. The system was applied to mouse splenic lymphocytes and elevation of sister chromatid exchange, the hallmark of Blm dysfunction, was observed in the presence of doxycycline. A cassette containing both sc rtTS and tTA was introduced into the Blm allele in ES cells and reduction of basal activity was observed upon doxycycline treatment. Our data demonstrate effectiveness of sc rtTS in the tet-off system. Application of sc rtTS in mice may allow us to implement bi-allelic mutagenesis in vivo.

Ahnak/Desmoyokin is dispensable for proliferation, differentiation, and maintenance of integrity in mouse epidermis.

Desmoyokin was first isolated from bovine muzzle epidermis and thought to be an epidermal desmosome-related protein. We previously demonstrated that the Desmoyokin gene is identical to the Ahnak gene, which is expressed ubiquitously and downregulated in neuroblastomas. It was assumed Ahnak/Desmoyokin was associated with epidermal cell adhesion, tumorigenesis, cell proliferation and differentiation, and embryonic development. To determine the precise biological function of Ahnak/Desmoyokin, we generated a null mutation in ES cells and mice. The resultant Ahnak/Desmoyokin-deficient ES cells normally differentiated into embryoid bodies and neural cells. The mutant mice were viable and fertile and showed no gross developmental defects. Electron microscopic examination of skin sections demonstrated that the ultrastructure of epidermal intercellular junctions, including desmosomes, of the mutant mice was indistinguishable from that of wild-type mice. Two-stage chemical skin carcinogenesis experiments showed no difference in frequency or onset of cutaneous tumor formation between wild-type and mutant mice. Moreover, no tumorigenesis was observed in other tissues and organs of mutant mice up to 2 y of age. These results lead us to conclude that Ahnak/Desmoyokin deficiency has only a minimal effect on epidermal cell adhesion, tumorigenesis, cell proliferation and differentiation, and overall mouse development.

Genome-wide phenotype analysis in ES cells by regulated disruption of Bloom's syndrome gene.

The chief limitation of phenotype-based genetic screening in mammalian systems is the diploid nature of the genome. Cells deficient in the Bloom's syndrome gene (Blm) show an increased rate of loss of heterozygosity. Here we have used a tetracycline-regulated Blm allele (Blm(tet)) to introduce bi-allelic mutations across the genome in mouse embryonic stem (ES) cells. Transient loss of Blm expression induces homologous recombination not only between sister chromatids but also between homologous chromosomes. We considered that the phenotype of ES cells bearing bi-allelic mutations would be maintained after withdrawal of the tetracycline analogue doxycycline. Indeed, a combination of N-ethyl-N-nitrosourea mutagenesis and transient loss of Blm expression enabled us to generate an ES cell library with genome-wide bi-allelic mutations. The library was evaluated by screening for mutants of glycosylphosphatidylinositol-anchor biosynthesis, which involves at least 23 genes distributed throughout the genome. Mutants derived from 12 different genes were obtained and two unknown mutants were simultaneously isolated. Our results indicate that phenotype-based genetic screening with Blm(tet) is very efficient and raises possibilities for identifying gene functions in ES cells.

Identification of a cytokine-induced antiapoptotic molecule anamorsin essential for definitive hematopoiesis.

Many growth factors and cytokines prevent apoptosis. Using an expression cloning method, we identified a novel antiapoptotic molecule named Anamorsin, which does not show any homology to known apoptosis regulatory molecules such as Bcl-2 family, caspase family, or signal transduction molecules. The expression of Anamorsin was completely dependent on stimulation with growth factors such as interleukin 3, stem cell factor, and thrombopoietin in factor-dependent hematopoietic cell lines, and forced expression of Anamorsin conferred resistance to apoptosis caused by growth factor deprivation in vitro. Furthermore, Anamorsin was found to act as an antiapoptotic molecule in vivo because Anamorsin-/- mice die in late gestation due to defective definitive hematopoiesis in the fetal liver (FL). Although the number of hematopoietic stem/progenitor cells in the FL did not decrease in these mice, myeloid, and particularly erythroid colony formation in response to cytokines, was severely disrupted. Also, Anamorsin-/- erythroid cells initiated apoptosis during terminal maturation. As for the mechanism of Anamorsin-mediated cell survival, a microarray analysis revealed that the expression of Bcl-xL and Jak2 was severely impaired in the FL of Anamorsin-/- mice. Thus, Anamorsin is considered to be a necessary molecule for hematopoiesis that mediates antiapoptotic effects of various cytokines.