A DNA Nanodevice Simultaneously Activating the EGFR and Integrin for Enhancing Cytoskeletal Activity and Cancer Cell Treatment.

Cell-surface receptors (e.g., EGFR and integrin) and their interactions play determining roles in signal transduction and cytoskeletal activation, which affect cell attachment/detachment, invasion, motility, metastasis (intracellular), and cell-cell signaling. For instance, the interactions between the EGFR and integrin (α6ß4) may cause increased mechanical force and shear stress via enhanced cytoskeleton activation. Here, we design a DNA nanodevice (DNA-ND) that can simultaneously target the EGFR and integrin receptors on the caveolae. The piconewton (pN) forces in response to the EGFR-integrin coactivation can be sensed upon the unfolding of the DNA hairpin structure on the side arm of the device via changes of the fluorescence and plasmonic signals. We find that simultaneous activation of EGFR-integrin receptors causes enhanced signal transduction, contractions of the cells, and initiation of the biochemical pathways, thus resulting in a change of the cell division and endocytosis/exocytosis processes that affect the cell proliferation/apoptosis. The DNA-ND further enables us to visualize the cointernalization and degradation of the receptors by lysosomes, providing a novel approach toward bioimaging and mechano-pharmacology.

Effects of the nanotopographic surface structure of commercially pure titanium following anodization-hydrothermal treatment on gene expression and adhesion in gingival epithelial cells.

The long-term stability and maintenance of endosseous implants with anodized-hydrothermally treated commercially pure titanium surfaces and a nanotopographic structure (SA-treated c.p.Ti) depend on the barrier function provided by the interface between the transmucosal portion of the implant surface and the peri-implant epithelium. This study investigated the effects of extracellular and intracellular gene expression in adherent gingival epithelial cells cultured for 1-7 days on SA-treated c.p.Ti implant surfaces compared to anodic oxide (AO) c.p.Ti and c.p.Ti disks. Scanning electron microscopy (SEM) showed filopodium-like extensions bound closely to the nanotopographic structure of SA-treated c.p.Ti at day 7 of culture. Gene expressions of focal adhesion kinase, integrin-α6ß4, and laminin-5 (α3, ß3, γ2) were significantly higher on SA-treated c.p.Ti than on c.p.Ti or AO c.p.Ti after 7 days (P<0.05). Our results confirmed that gingival epithelial cells adhere to SA-treated c.p.Ti as the transmucosal portion of an implant, and that this interaction markedly improves expression of focal adhesion molecules and enhances the epithelial cell phenotype. The cellular gene expression responses driving extracellular and intracellular molecular interactions thus play an important role in maintenance at the interface between SA-treated c.p.Ti implant surfaces and the gingival epithelial cells.

Novel method for proliferation of oral keratinocyte stem cells.

BACKGROUND AND OBJECTIVE: Stem cell-based tissue engineering offers clear advantages over conventional normal cell approaches. Owing to their specific characteristics, oral keratinocyte stem cells represent an attractive solution for therapeutic applications. However, when cultured in vitro, these cells lose their unique properties, acquiring a limited capacity for self-renewal, and differentiate rapidly into normal functional keratinocytes. The main aim of the present study was to develop an in-vitro method for the expansion of oral keratinocyte stem cells using a biomaterial approach. MATERIAL AND METHODS: Oral keratinocyte stem cells were isolated based on the identification of two surface markers - integrin α6ß4 and CD71 - using a magnetic method. The cells were cultured on specific substrates formed from blends of polymers: poly(lactide-co-glycolide) (PLGA); poly(lactide-co-glycolide) + polyurethane (PLGA + PU); and poly(lactide-co-glycolide) + extracellular matrix (PLGA + ECM). The polymers were deposited using a laser-based technique - matrix-assisted pulsed laser evaporation. The cells were analyzed for cell size, cell proliferation, colony-forming efficiency, cell adhesion markers (such as E-cadherin and beta 1 integrin), keratinocyte stem cells and differentiation markers. The methods included ELISAs, immunofluorescence and atomic force microscopy imaging. RESULTS: After 14 d in culture, cells seeded on PLGA + PU stained positive for p63, cd44H, cytokeratin 19 and integrin α6ß4 and negative for involucrin, cytokeratin 14 and cytokeratin 10. The levels of adhesion molecules were significantly increased in cells grown on PLGA + PU: at 14 d the E-cadherin levels were 5.4 ± 0.2 ng/mL (for cells grown on PLGA + PU) vs. 4.1 ± 0.4 ng/mL (for cells grown on control medium) (n = 5, p < 0.05 Bonferroni). Oral keratinocyte stem cells grown on PLGA + PU had the highest colony-forming efficiency and proliferation rate, together with the smallest cell size, compared with cells grown on control medium or other polymeric substrates. CONCLUSION: The present study demonstrates that by culturing oral keratinocyte stem cells on PLGA blended with PU it is possible to preserve their phenotype in vitro and to guide their short-term expansion and proliferation. Certain stem-cell characteristics are preserved and their short-term expansion may be enhanced.

Severe alterations in expression and localisation of {alpha}6{beta}4 integrin in salivary gland acini from patients with Sjogren syndrome.

OBJECTIVES: In salivary glands from patients with Sjögren syndrome, overexpression of laminins 1 and 5 and disorganisation of the acinar basal lamina have been reported. Laminin 5 mediates association of the basal lamina with epithelial cells by forming adhesion complexes upon interaction with alpha6beta4 integrin. In the present work, mRNA and protein levels of alpha6beta4 integrin were determined and its localisation in salivary glands evaluated in patients with Sjögren syndrome. METHODS: Salivary glands of 12 patients with Sjögren syndrome and 8 controls were studied. The mRNA and protein levels of alpha6beta4 were determined by semiquantitative reverse transcriptase (RT)-PCR and western blot analysis, respectively. The subcellular localisation of alpha6beta4 and laminin were evaluated by confocal microscopy. RESULTS: In patients, no significant differences in alpha6 and beta4 mRNA levels were detected. However, beta4 integrin protein levels were significantly lower, whereas, changes in alpha6, were highly variable. In controls, alpha6beta4 was detected in the basolateral and basal surface of serous and mucous acini, respectively. In patients, alterations in alpha6beta4 distribution were particularly dramatic for acini with strong basal lamina disorganisation. alpha6beta4 was also detected in the cytoplasm and lateral plasma membrane in serous and mucous acini. CONCLUSION: Mild alterations in the basal lamina correlated with lateral redistribution of alpha6beta4 integrin and the formation of new cell-cell adhesions that help maintain acinar organisation and promote cell survival. Conversely, in cases with severe basal lamina alterations, lateral alpha6beta4 redistribution was no longer sufficient to maintain acinar cell survival. Thus, maintenance of equilibrium between cell-cell and cell-basal lamina attachment is required to sustain gland cell survival.

Involvement of laminin and integrins in adhesion and migration of junctional epithelium cells.

BACKGROUND AND OBJECTIVE: The junctional epithelium attaches to the enamel surface with hemidesmosomes (of which laminin-5 and integrin-alpha(6)beta(4) are the main components) in the internal basal lamina. Laminin-5 is also involved in cell motility with integrin-alpha(3)beta(1), although their functions have not yet been clarified.The purpose of this study was to determine the functions of those adhesive components between the tooth and the junctional epithelium during cell migration.Because an idea has been proposed that directly attached to tooth cells (DAT cells) may not contribute to cell migration, 5-bromo-2-deoxyuridine staining was performed to confirm cell migration. MATERIAL AND METHODS: We investigated laminin-gamma(2) (contained only in laminin-5), integrin-beta(4) (involved in cell-extracellular matrix contact) and integrin-alpha(3) (inducing cell migration) in the junctional epithelium, oral gingival epithelium and gingival sulcus epithelium of 6-wk-old ICR mice using laser microdissection, quantitative real-time reverse transcription-polymerase chain reaction, immunofluorescence and 5-bromo-2-deoxyuridine staining. RESULTS: Laminin and integrins were clearly immuno-localized in the basal lamina of all epithelium. Quantitative analysis of laminin and integrin mRNAs by laser microdissection showed that they were more highly expressed in DAT cells than in basal cells in the oral gingival epithelium. In particular, a 12-fold higher expression of laminin-5 was observed in the junctional epithelium compared with the oral gingival epithelium. 5-Bromo-2-deoxyuridine staining showed rapid coronal migration of DAT cells. CONCLUSION: These results suggest that the abundant expression of laminin-5 and integrin-alpha(6)beta(4) is involved in the attachment of DAT cells to teeth by hemidesmosomes. Abundant expression of laminin-5 and integrin-alpha(3)beta(1) might assist in DAT cell migration, confirmed by 5-bromo-2-deoxyuridine staining during the turnover of junctional epithelium.

Chromogenic in situ hybridization for alpha6beta4 integrin in breast cancer: correlation with protein expression.

The alpha6beta4 integrin is the receptor for the basement membrane protein laminin-5. Recent studies suggest that alpha6beta4 integrin expression in invasive breast carcinomas may be a poor prognostic factor. Because we have not had reliable results with commercially available antibodies for the immunohistochemical detection of alpha6beta4 integrin in archival paraffin-embedded tissues, we designed a probe to detect beta4 integrin subunit mRNA in paraffin sections. In situ hybridization for beta4 mRNA was performed on paraffin-embedded tissue sections of 25 invasive breast carcinomas using a hyperbiotinylated oligonucleotide DNA probe. Immunohistochemical staining was performed on corresponding frozen tumor sections using two commercially available antibodies to the beta4 integrin subunit. All cases positive for beta4 protein by one or both antibodies were also positive for beta4 mRNA by in situ hybridization, but three cases with beta4 mRNA expression were negative by immunohistochemistry with both antibodies. These findings suggest that in situ hybridization appears to be a sensitive method for detecting beta4 integrin mRNA, but it appears to identify some cases that either lack beta4 protein or express variants not recognized with commercial antibodies directed to particular extracellular or cytoplasmic domains.

[Integrin alpha6 beta4 and experimental allergic neuritis].

OBJECTIVE: To explore the relationship between integrins alpha6,beta4 and the process of the injury and repair of the myelin, their expressions in experimental allergic neuritis were analyzed. METHODS: Experimental allergic neuritis (EAN) models were induced in Lewis rats. The expressions of integrins alpha6 and beta 4 and their receptor laminin, both in the acute phase and recovery phase of EANs, were analyzed by in situ hybridization and immunohistochemistry. RESULTS: The expression of beta4 decreased in the acute phase P<0.05 and showed no significant changes in the recovery phase(P=0.6840). There were no significant changes of the expression of alpha6 and laminin both in the acute phase and recovery phase of EANs by immunohistochemistry (alpha6 P=0.0751;laminin P=0.2047). The similar results were obtained by in situ hybridization (beta4 in acute phase P<0.05;beta4 in recovery phase P=0.823;alpha6 P= 0.81). CONCLUSION: The expressions of integrins of Schwann cells were affected by inflammation injury, and their patterns of expression were similar to those of integrins alpha6, beta1 and beta4 during embryogenesis. It is inferred that integrin beta4 and the reformation of myelin have a more imitate relationship. The expression changes of alpha6 and beta4 correlate with the injury and repair of myelin during EAN.

Deletion of a cytoplasmic domain of integrin beta4 causes epidermolysis bullosa simplex.

Integrin alpha6beta4 is a hemidesmosomal transmembrane molecule involved in maintaining basal cell-matrix adhesion through interaction of the large intracytoplasmic tail of the beta4 subunit with the keratin intermediate filament network, at least in part through its binding with plectin and BP180/type XVII collagen. Here we report a patient with predominant features of epidermolysis bullosa simplex due to a mutation in the integrin beta4 gene. The patient, a 49-y-old female, had mild blistering of hands and feet from birth on, dystrophy of the nails with onychogryposis, and enamel hypoplasia. She had no alopecia and no history of pyloric atresia. Electron microscopy and antigen mapping of a skin blister revealed that the level of separation was intraepidermal, low in the basal keratinocytes through the attachment plaque of the hemidesmosome. Immuno-fluorescence microscopy revealed absent binding of monoclonal antibody 450-11 A against the third fibronectin III repeat on the intracellular domain of integrin beta4, whereas binding was reduced with monoclonal antibodies recognizing epitopes on amino-terminal and carboxy-terminal ends of the polypeptide. At the molecular level the phenotype was caused by a novel 2 bp deletion 4733delCT in ITGB4, resulting in in-frame skipping of exon 36 and a deduced 50 amino acid deletion (1450-1499) within the third fibronectin type III repeat in the cytoplasmic domain of the integrin beta4 polypeptide. Immunoblot analysis demonstrated a 5 kDa shorter beta4 polypeptide. The 4733delCT mutation was heterozygously present in the DNA. The patient is also expected to be heterozygous for a null allele, as no full-size protein was detected in vitro and the epitope 450-11 A was absent in vivo. These data show that deletion of the third fibronectin type III repeat in the cytoplasmic domain of integrin beta4, which is thought to interact with BP180/type XVII collagen, is clinically pathogenic and results in a mild phenotype with predominant features of epidermolysis bullosa simplex.

Modulation in vitro of keratinocyte integrins by interferon-alpha and interferon-gamma.

BACKGROUND: Interferon-alpha and -gamma are glycoproteins with antiviral and immunoregulatory properties. In vitro studies have shown a role for these cytokines in the regulation of epidermal keratinocyte growth and differentiation. In the same way, integrins are adhesion molecules which regulate keratinocyte proliferation and differentiation. AIM: To determine whether the regulatory activity of interferons on keratinocyte proliferation and differentiation is related to a modulation of keratinocyte integrins. METHODS: Two different methods were used: monolayers and reconstituted skin, incubated either with 1,200 U/mL interferon-alpha or 500 U/mL interferon-gamma or control medium for 48 h. The integrin expression was assessed by flow cytometry and immunohistochemistry. RESULTS: In monolayers, only the alpha3 subunit was significantly inhibited by interferon-gamma. In reconstituted skin, where keratinocytes are differentiated, both interferons had an inductive effect on beta1 expression and interferon-alpha had an inhibitory effect on alpha6 expression. CONCLUSION: Interferon-alpha and -gamma induce a modulatory effect on alpha3, alpha6 and beta1 which appears to be related to the state of differentiation. Moreover, the decreased expression of alpha6 and alpha3 could be one of the mechanisms involved in the formation of bullous lesions during long-term interferon therapy.