Transport and Function of Cx26 Mutants Involved in Skin and Deafness Disorders

We examined the subcellular localization and function of several Cx26 mutants that exhibit both sensorineural deafness and various skin disease phenotypes. To facilitate these aims, all Cx26 mutants were tagged at the carboxyl-terminal with green fluorescent protein (GFP), which has previously been shown not to affect Cx26 transport, assembly or function. In this article we focus on two point mutations (R75W and ΔE42) that occur in the first extracellular loop region of Cx26, a region hypothesized to be critical for correct hemichannel docking between contacting cells. In gap junctional intercellular communication (GJIC)-deficient HeLa cells, both R75W-GFP and ΔE42-GFP were transported to the cell surface and assembled into gap junction-like structures. Neither R75W-GFP nor ΔE42-GFP formed gap junctions that were permeable to Lucifer Yellow suggesting they are loss-of-function mutations. We also examined the phenotype of these two mutations in a rat epidermal keratinocyte (REK) cell line that is capable of undergoing differentiation. Using antibodies against several members of the connexin family reportedly expressed by epidermal keratinocytes, we found these cells endogenously expressed Cx43 and Cx26 but not Cx30, Cx32, or Cx37. When expressed in REK cells, similar to in HeLa cells, R75W-GFP and ΔE42-GFP were assembled at the cell surface into structures that resembled gap junctions. Future experiments will examine the effect of the Cx26 mutants on the function and differentiation of these epidermal keratinocytes.     

Junctions and Inflammation in the Skin

Abstract

The skin forms a life-sustaining barrier between the organism and physical environment. The physical barrier of skin is mainly localized in the stratum corneum (SC); however, nucleated epidermis also contributes to the barrier through tight, gap, and adherens junctions (AJs), as well as through desmosomes and cytoskeletal elements. Many inflammatory diseases, such as atopic dermatitis (AD) and psoriasis, are associated with barrier dysfunction. It is becoming increasingly clear that the skin barrier function is not only affected by inflammatory signals but that defects in structural components of the barrier may be the initiating event for inflammatory diseases. This view is supported by findings that mutations in filaggrin, a key structural epidermal barrier protein, cause the inflammatory skin disease AD, and that a loss of AJ components, namely epidermal p120 catenin or α-catenin results in skin inflammation.     

Cx26 Affects the in Vitro Reconstruction of Human Epidermis

To study the function of connexins in human keratinocytes, we have used a three-dimensional culture system, in which a tissue is reconstructed using cells from the outer root sheet of hair follicles. This tissue reproduces in vitro the histological organisation of human epidermis in situ and the normal distribution of several keratinocyte markers. Furthermore, it shows characteristics of a differentiating epidermis, including the expression of connexin26. Connexin26 protein expression is increased under physiological and pathological conditions resulting in increased keratinocyte turnover. Loss of this protein in keratinocytes, obtained from patients carrying a stop mutation, resulted in a reduced stratification of the in vitro reconstructed tissue, probably due to a lower proliferation and migration capacity of the keratinocytes, although dye coupling and persistence of other gap junctions is maintained. No changes were seen in tissues reconstructed with keratinocytes from patients carrying a non stop mutation of connexin30. The data indicate that, at least in vitro, connexin26 affects the function of human keratinocytes, independently of obvious changes in coupling.     

Pannexin1 and Pannexin3 Exhibit Distinct Localization Patterns in Human Skin Appendages and are Regulated during Keratinocyte Differentiation and Carcinogenesis

Having shown that Panx1 and Panx3 are expressed in the epidermis, we investigated their distribution in human skin adnexal structures and skin cancer. Both proteins were found in hair follicles, sebaceous and eccrine glands, as well as blood vessels. Panx1 was detected as punctate or diffuse intracellular labeling, while Panx3 was only observed as diffuse intracellular staining, suggesting different functions. We also identified the Panx3 immunoreactive ～70 kD species modulated during keratinocyte differentiation as Panx3. Since our data indicate that pannexins are regulated during keratinocyte differentiation, we assessed whether their levels are altered under circumstances in which keratinocyte differentiation is compromised. We found that Panx1 and Panx3 levels are highly reduced in human keratinocyte tumors, thus showing for the first time that both pannexins are dysregulated in human cancers. Altogether, these data suggest that Panx1 and Panx3 have distinct and unique functions within the skin in health and disease.     

Laminin 332 in junctional epidermolysis bullosa

Laminin 332 is an essential component of the dermal-epidermal junction, a highly specialized basement membrane zone that attaches the epidermis to the dermis and thereby provides skin integrity and resistance to external mechanical forces. Mutations in the LAMA3, LAMB3 and LAMC2 genes that encode the three constituent polypeptide chains, α3, β3 and γ2, abrogate or perturb the functions of laminin 332. The phenotypic consequences are diminished dermal-epidermal adhesion and, as clinical symptoms, skin fragility and mechanically induced blistering. The disorder is designated as junctional epidermolysis bullosa (JEB). This article delineates the signs and symptoms of the different forms of JEB, the mutational spectrum, genotype-phenotype correlations as well as perspectives for future molecular therapies.     

Minimal Erythema Dose (MED) Testing

Ultraviolet radiation (UV) therapy is sometimes used as a treatment for various common skin conditions, including psoriasis, acne, and eczema. The dosage of UV light is prescribed according to an individual''s skin sensitivity. Thus, to establish the proper dosage of UV light to administer to a patient, the patient is sometimes screened to determine a minimal erythema dose (MED), which is the amount of UV radiation that will produce minimal erythema (sunburn or redness caused by engorgement of capillaries) of an individual''s skin within a few hours following exposure. This article describes how to conduct minimal erythema dose (MED) testing. There is currently no easy way to determine an appropriate UV dose for clinical or research purposes without conducting formal MED testing, requiring observation hours after testing, or informal trial and error testing with the risks of under- or over-dosing. However, some alternative methods are discussed.     

Treatment of Osteochondral Defects in the Rabbit's Knee Joint by Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface ¹. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential ². In the last decades, several surgical treatment options have emerged and have already been clinically established ^3-6.Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface ^3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation ^9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone ¹¹.The sandwich-technique combines bone grafting with current approaches in Tissue Engineering ^5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing ¹².Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity ¹¹.Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential ^13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results ^1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies ^19-21 and even first human trials ²².The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit''s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit''s knee joint will be described.     

Mechanobiological dysregulation of the epidermis and dermis in skin disorders and in degeneration

During growth and development, the skin expands to cover the growing skeleton and soft tissues by constantly responding to the intrinsic forces of underlying skeletal growth as well as to the extrinsic mechanical forces from body movements and external supports. Mechanical forces can be perceived by two types of skin receptors: (1) cellular mechanoreceptors/mechanosensors, such as the cytoskeleton, cell adhesion molecules and mechanosensitive (MS) ion channels, and (2) sensory nerve fibres that produce the somatic sensation of mechanical force. Skin disorders in which there is an abnormality of collagen [e.g. Ehlers–Danlos syndrome (EDS)] or elastic (e.g. cutis laxa) fibres or a malfunction of cutaneous nerve fibres (e.g. neurofibroma, leprosy and diabetes mellitus) are also characterized to some extent by deficiencies in mechanobiological processes. Recent studies have shown that mechanotransduction is crucial for skin development, especially hemidesmosome maturation, which implies that the pathogenesis of skin disorders such as bullous pemphigoid is related to skin mechanobiology. Similarly, autoimmune diseases, including scleroderma and mixed connective tissue disease, and pathological scarring in the form of keloids and hypertrophic scars would seem to be clearly associated with the mechanobiological dysfunction of the skin. Finally, skin ageing can also be considered as a degenerative process associated with mechanobiological dysfunction. Clinically, a therapeutic strategy involving mechanoreceptors or MS nociceptor inhibition or acceleration together with a reduction or augmentation in the relevant mechanical forces is likely to be successful. The development of novel approaches such as these will allow the treatment of a broad range of cutaneous diseases.     

Molecular conformation changes along the malignancy revealed by optical nanosensors

An interdisciplinary approach employing functionalized nanoparticles and ultrasensitive spectroscopic techniques is reported here to track the molecular changes in early stage of malignancy. Melanoma tissue tracking at molecular level using both labelled and unlabelled silver and gold nanoparticles has been achieved using surface enhanced Raman scattering (SERS) technique. We used skin tissue from ex vivo mice with induced melanoma. Raman and SERS molecular characterization of melanoma tissue is proposed here for the first time. Optical nanosensors based on Ag and Au nanoparticles with chemisorbed cresyl violet molecular species as labels revealed sensitive capability to tissues tagging and local molecular characterization. Sensitive information originating from surrounding native biological molecules is provided by the tissue SERS spectra obtained either with visible or NIR laser line. Labelled nanoparticles introduced systematic differences in tissue response compared with unlabelled ones, suggesting that the label functional groups tag specific tissue components revealed by proteins or nucleic acids bands. Vibrational data collected from tissue are presented in conjunction with the immunohistochemical analysis. The results obtained here open perspectives in applied plasmonic nanoparticles and SERS for the early cancer diagnostic based on the appropriate spectral databank.