Embryonic stem (ES) cell lines can be expanded indefinitely in culture while maintaining their potential to differentiate into any cell type. During embryonic development, the skin forms as a result of reciprocal interactions between mesoderm and ectoderm. Here, we report the in vitro differentiation and enrichment of keratinocytes from murine ES cells seeded on extracellular matrix (ECM) in the presence of Bone Morphogenic Protein-4 (BMP-4) or ascorbate. The enriched preparation of keratinocytes was able to form an epidermal equivalent composed of a stratified epithelium when cultured at the air-liquid interface on a collagen-coated acellular substratum. Interestingly, an underlying cellular compartment that belongs to the fibroblast lineage was systematically formed between the reconstituted epidermis and the inert membrane. The resulting tissue displayed morphological patterns similar to normal embryonic skin, as evidenced by light and transmission electron microscopy. Immunohistochemical studies revealed expression patterns of cytokeratins, basement membrane (BM) proteins and late differentiation markers of epidermis, as well as fibroblast markers, similar to native skin. The results demonstrate the capacity of ES cells to reconstitute in vitro a fully differentiated skin. This ES-derived bioengineered skin provides a powerful tool for studying the molecular mechanisms controlling epidermal and dermal commitments.
Melanomas are malignant tumors of melanocytes that, if not detected early, are highly aggressive and poorly treatable. Activation of extracellular signal-regulated (ERK)/mitogen-activated protein (MAP) kinase signaling is commonly found in melanomas mainly through oncogenic mutations of B-Raf. We previously reported that activation of ERK/MAP kinase stimulates synthesis of fibronectin by upregulating the transcription factor early growth response-1 (Egr-1). To further analyze the link between ERK/MAP kinase pathway and fibronectin in melanoma, we have studied the regulation and role of fibronectin produced by melanoma cells bearing oncogenic B-Raf mutation. We show that fibronectin is expressed in situ during tumor progression and that high fibronectin and Egr-1 levels are found in cells expressing this mutation. Expression of active mutants of B-Raf induces fibronectin, whereas endogenous fibronectin is inhibited by small interfering RNA (siRNA)-mediated depletion of B-Raf or Egr-1. In contrast, stimulation of ERK pathway is insufficient to promote fibronectin upregulation in normal melanocytes. Finally, we show that suppression of fibronectin by siRNA leads to decreased melanoma cell invasiveness in vitro. These results reveal a tumor-specific regulation of fibronectin by constitutive ERK/MAP kinase signaling and indicate that self-production of fibronectin may play a role in melanoma tumorigenesis, by promoting tumor cell invasion.
Junctional epidermolysis bullosa (JEB) is a genodermatosis suitable for gene therapy because conventional treatments are ineffective. Here, we elucidate the genetic basis of mild JEB in a breed of dogs that display all the clinical traits observed in JEB patients. The condition is associated with reduced expression of laminin 5 caused by a homozygous insertion (4818+207ins6.5 kb) of repetitive satellite DNA within intron 35 of the gene (lama3) for the laminin alpha3 chain. The intronic mutation interferes with maturation of the alpha3 pre-messenger RNA resulting in the coexpression of a transcript with a 227 nucleotide insertion and a wild-type mRNA that encodes scant amounts of the alpha3 polypeptide. Our results show that the amino acid sequence and structure of the canine and human alpha3 chain are highly conserved and that the reduced expression of laminin 5 affects the adhesion and clonogenic potential of the JEB keratinocytes. These JEB dogs provide the opportunity to perform gene delivery in a naturally occurring genodermatosis and to evaluate host tolerance to recombinant laminin 5.
In their abstract, Davenport et al (1997) state that ''Results consistently demonstrated that all the test sunscreens protected (against immunosuppression) beyond their designated protections factors.'' Protection factors are ratios by definition, and if the above statement by Davenport et al (1997) means anything at all, it is that the sunscreens' immune protection factors were greater than their corresponding sun protection factors (SPF). Chu et al (1998) restate their claim in their recent letter to the Journal of Investigative Dermatology in which they also state that they did not calculate immune protection factors in their original paper but would have to concede, from their own data, that had they done so, as suggested in letters by Gasparro (1998) and Wolf and Kripke (1998), these would have been lower than the corresponding SPF. If the immune protection factor is lower than the SPF, it is just not possible for sunscreens ''to protect beyond their designated protection factors.'' The authors tested products with SPF ranging from 3.6 to 5.7, and defend their original conclusions by arguing that although ex vivo skin exposed to 5 minimal erythema doses (MED) results in 50% immunosuppression, a sunscreen of SPF ϭ 5 completely protects against immunosuppression when skin is exposed to 5 MED. This is hardly surprising as 5 MED with an SPF of 5 is equivalent to 1 MED without a sunscreen, and 1 MED without sunscreen almost certainly does not cause immunosuppression in their model. All the authors can conclude from this analysis is that 1 MED delivered to the skin, with or without sunscreen, is not immunosuppressive. Furthermore, the UV dose-threshold for immunosuppression in Manuscript
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