No description available.

Studies on mouse keratinocyte stem cells are hampered by the absence of markers for epidermal stem cells, and there is little information about the <I>in vivo</I> or <I>in vitro</I> micro environment required for maintenance of the murine keratinocyte stem cell. In this study, a unique immortal mouse follicular dermal papilla cell line was generated in order to investigate the interactions <I>in vitro</I> and <I>in vivo</I> between dermal papilla cells and keratinocytes. The cell line was characterised and used for experimental analysis of keratinocyte self-renewal. Characterisation of the DPC1" line included growth properties, morphology and expression at mRNA level of growth factors known to be important in keratinocyte or dermal papilla cell growth. Primary mouse epidermal keratinocytes were cultured in monolayer on the dermal papilla cell line (DPC1") and compared to cultures on other fibroblast lines (A31 3T3 and J2 3T3). The DPC1" supported a more proliferative phenotype of keratinocyte, both in primary culture and after subculture. The keratinocytes were also grown in organotypic culture and the DPC1" cells supported the formation of a multilayered proliferative epidermis-like structure. <I>In vivo</I> it was shown that the keratinocytes were able to reform a self-renewing epidermis in the graft bed when they were grafted in conjunction with DPC1". Finally, keratinocytes were cultured on the protein matrix synthesised by the DPC1" and it was demonstrated that there was greater attachment and proliferation on this matrix when compared to matrix derived from other cell lines. It is proposed that the DPC1" cell line can reproduce at least part of the micro environments required in terms of the protein matrix and growth factors for maintenance of the mouse keratinocyte stem cell.

No description available.

To model sweat gland fluid secretion <I>in vitro</I>, human apocrine glands were isolated from axillary skin and their secretory coil cells were cultured and mounted in an Ussing chamber. Cultured apocrine coil cells exhibited an amiloride-sensitive inward resting short circuit current, suggesting an absorptive phenotype. However, evidence of a chloride-secretory phenotype was also observed: transient inward currents were recorded in response to autonomic secretagogues and inflammatory mediators, and these were not abolished by amiloride, but they were reduced by half in chloride-free buffer. This may indicate a role for chloride secretion in mediating the stimulated currents. Further, sustained inward currents were recorded in 58% of tissues which were attenuated by frusemide, implying the involvement of the Na<SUP>+</SUP>/K<SUP>+</SUP>/Cl<SUP>-</SUP> transporter. Assuming that secondary active chloride transport is responsible for aqueous fluid secretion in the apocrine sweat gland, these data are compatible with some maintenance of an <I>in vivo</I> phenotype in culture. To study the cellular determinants of eccrine sweat gland differentiation, eccrine sweat gland-associated fibroblasts were cultured and compared with derminal fibroblasts from the same subject, demonstrated a different pattern of outgrowth and proliferation. Therefore these cells may represent a novel fibroblast subtype. To improve the differentiation of eccrine sweat gland coil cells <I>in vitro</I>, they were co-cultured with eccrine fibroblasts. Eccrine sweat gland cells grown on a Transwell as a control demonstrated apparent dedifferentiation to a reabsorptive phenotype, indicated by the amiloride-sensitivity of their resting and agonist-stimulated inward short circuit currents. Eccrine cells co-cultured with eccrine fibroblasts, however, demonstrated agonist-stimulated outward currents in the presence of amiloride.

There is little data regarding the action of HLH proteins in the skin. I have used RT-PCR Northern blotting analysis, immunocytochemistry (ICC) and in situ hybridisation (ISH) to determine the expression profiles of Id1, Id2, Id3 and Id4 and the Class A factors E2A, E2-2 and HEB mRNAs and proteins during human keratinocyte differentiation both <I>in vivo</I> and in monolayer and organotype <I>in vitro</I> differentiation culture models, and in the human anagen hair follicle, the rat hair follicle growth cycle, and the human sebaceous gland and eccrine sweat gland. Preliminary RT-PCR analysis determined that the Class A factors, E2A, E2-2 and HEB and Id1, Id2 and Id3, and to a very limited extent Id4 mRNAs, were expressed by the epidermis and skin appendages. Northern blotting analysis demonstrated that Ca<SUP>2+</SUP> induced cell cycle withdrawal and differentiation of primary human keratinocytes, determined by an up regulation of p21<SUP>cip1</SUP> and involucrin mRNA expression, was inversely correlated with Id1, Id2 and Id3 mRNA expression. ICC and ISH analysis of human and rat epidermis confirmed Id protein and mRNA expression in the most proliferative layers of the epidermis. The sub-cellular localisation of Id1 protein was predominantly cytoplasmic, whereas that of Id2 and Id3 proteins was predominantly nuclear. Interestingly, a sub-population of supra-basal, post-mitotic keratinocytes also expressed Id1, Id2 and Id3 proteins in which the sub-cellular localisation of each was predominantly nuclear. Supra-basal Id expressing keratinocytes were often seen arranged in stacks extending from the basal layer to the confirmed layers and these cells may be post-mitotic keratinocytes with maintained capacity to re-enter the cell cycle, and may delineate epidermal proliferative units.

No description available.

In order to investigate the role of β-catenin in cell fate selection in specific regions of adult epidermis, I used three different promoters to over express N-terminally truncated, stabilised β-catenin fused to the ligand-binding domain of a mutant oestrogen receptor (ΔNβ-cateninER) in the epidermis of transgenic mice. The human keratin 1 (HK1) promoter targets cells in the interfollicular epidermis that are committed to terminal differentiation; the keratin 15 (K15) promoter targets stem cells in the hair bulge; and the truncated keratin 5 (ΔK5) promoter targets the basal layer cells of the hair bulb and sebaceous glands. Using topical applications of 4-hydroxytamoxifen (4OHT) β-catenin was activated at specific stages of the hair cycle and for different lengths of time. Resting hair follicles at K15ΔNβ-cateninER and ΔK5ΔNβ-cateninER mice were recruited into the growth phase of the hair cycle. In addition, ectopic hair follicles formed from the sebaceous glands of ΔK5ΔNβ-cateninER mice. The ectopic hair follicles often formed cyst-like structures, containing highly proliferative cells and cells that expressed hair keratins or sebocyte markers. Whilst a transient activation of β-catenin signalling is sufficient to trigger hair follicle morphogenesis, continuous β-catenin signalling is required to maintain the cyst like structures. No ectopic hair growth was seen in the K15ΔNβ-cateninER mice, although the bulge was seen to proliferate and show induction of β-catenin target genes. The HK1ΔNβ-cateninER mice never presented with a phenotype, which correlated with a failure of the transgene to induce downsteam factors such as Lef1. Wounding or treatment with vitamin A did not influence the response of the epidermis to β-catenin activation. However, vitamin D treatment reduced proliferation in both ΔK5ΔNβ-cateninER and K15ΔNβ-cateninER mice and stimulated differentiation of the ectopic hair follicles in ΔK5ΔNβ-cateninER mice.

Analysis of mouse myoblasts during differentiation showed that ERK5 levels were up-regulated as soon as 12 hours, and ERK5 was specifically activated after 48 hours of differentiation, concomitant with activation of MEF2C. Furthermore, the presence of ERK5 in the nuclei of embryonic mouse muscle cells was shown, suggesting a role in muscle development. When ERK5 activity was increased in myoblasts there was an increase in MEF2C activation and myogenic fusion was enhanced, determined by expression of the muscle-specific genes myosin heavy chain (MHC) and myogenin. Conversely, when a kinase-dead dominant negative ERK5 was expressed, myoblasts showed decreased MEF2C activation and myogenic fusion. Perturbing ERK5 activity levels in this way did not affect proliferation, cell cycle exit or apoptosis of myoblasts, but increased ERK5 activity enhanced myoblast migration. IGF signalling is crucial in coordinating myogenesis. The hypothesis that ERK5 is involved in IGF mediated myogenic signalling was investigated. Addition of IGF-I to myoblasts increased ERK5 activation after 10 minutes, and resulted in ERK5GFP mutated at the activating phosphorylation residues was unable to relocate, demonstrating that phosphorylation of ERK5 is required for relocalisation. My data suggest that ERK5 is important for the formation of muscle tissue in the embryo and of fully differentiated myotubes <i>in vitro</i>, possibly through its interaction with IGF and MEF2C. Further work to delineate the signalling pathways leading to ERK5 activation during myogenesis may have therapeutic applications for muscle disorders such as the muscle-derived tumour rhabdomyosarcoma (RMS), where myoblasts are unable to fuse.

No description available.

No description available.