A Mitosis Block Links Active Cell Cycle with Human Epidermal Differentiation and Results in Endoreplication

How human self-renewal tissues co-ordinate proliferation with differentiation is unclear. Human epidermis undergoes continuous cell growth and differentiation and is permanently exposed to mutagenic hazard. Keratinocytes are thought to arrest cell growth and cell cycle prior to terminal differentiation. However, a growing body of evidence does not satisfy this model. For instance, it does not explain how skin maintains tissue structure in hyperproliferative benign lesions. We have developed and applied novel cell cycle techniques to human skin in situ and determined the dynamics of key cell cycle regulators of DNA replication or mitosis, such as cyclins E, A and B, or members of the anaphase promoting complex pathway: cdc14A, Ndc80/Hec1 and Aurora kinase B. The results show that actively cycling keratinocytes initiate terminal differentiation, arrest in mitosis, continue DNA replication in a special G2/M state, and become polyploid by mitotic slippage. They unambiguously demonstrate that cell cycle progression coexists with terminal differentiation, thus explaining how differentiating cells increase in size. Epidermal differentiating cells arrest in mitosis and a genotoxic-induced mitosis block rapidly pushes epidermal basal cells into differentiation and polyploidy. These observations unravel a novel mitosis-differentiation link that provides new insight into skin homeostasis and cancer. It might constitute a self-defence mechanism against oncogenic alterations such as Myc deregulation

Transcriptomic analysis of the temporal host response to skin infestation with the ectoparasitic mite Psoroptes ovis

<p>Abstract</p> <p>Background</p> <p>Infestation of ovine skin with the ectoparasitic mite <it>Psoroptes ovis </it>results in a rapid cutaneous immune response, leading to the crusted skin lesions characteristic of sheep scab. Little is known regarding the mechanisms by which such a profound inflammatory response is instigated and to identify novel vaccine and drug targets a better understanding of the host-parasite relationship is essential. The main objective of this study was to perform a combined network and pathway analysis of the <it>in vivo </it>skin response to infestation with <it>P. ovis </it>to gain a clearer understanding of the mechanisms and signalling pathways involved.</p> <p>Results</p> <p>Infestation with <it>P. </it>ovis resulted in differential expression of 1,552 genes over a 24 hour time course. Clustering by peak gene expression enabled classification of genes into temporally related groupings. Network and pathway analysis of clusters identified key signalling pathways involved in the host response to infestation. The analysis implicated a number of genes with roles in allergy and inflammation, including pro-inflammatory cytokines (<it>IL1A, IL1B, IL6, IL8 </it>and <it>TNF</it>) and factors involved in immune cell activation and recruitment (<it>SELE, SELL, SELP, ICAM1, CSF2, CSF3, CCL2 </it>and <it>CXCL2</it>). The analysis also highlighted the influence of the transcription factors NF-kB and AP-1 in the early pro-inflammatory response, and demonstrated a bias towards a Th2 type immune response.</p> <p>Conclusions</p> <p>This study has provided novel insights into the signalling mechanisms leading to the development of a pro-inflammatory response in sheep scab, whilst providing crucial information regarding the nature of mite factors that may trigger this response. It has enabled the elucidation of the temporal patterns by which the immune system is regulated following exposure to <it>P. ovis</it>, providing novel insights into the mechanisms underlying lesion development. This study has improved our existing knowledge of the host response to <it>P. ovis</it>, including the identification of key parallels between sheep scab and other inflammatory skin disorders and the identification of potential targets for disease control.</p

Effects of UV, 4-NQO and TPA on gene expression in cultured human epidermal keratinocytes.

In an approach to study effects of UV light on gene expression in human epidermal keratinocytes, a cDNA library was constructed from poly(A)RNA isolated after UV irradiation from cultured keratinocytes. The cDNA library was differentially screened with labelled cDNA probes synthesized on poly(A)RNA isolated from UV irradiated or nonirradiated keratinocytes. Forty clones were selected and subjected to further analysis, 31 of them are described in this report. Whereas total mRNA synthesis is reduced after UV irradiation or treatment with 4-NQO Northern blot analysis revealed that there is an at least relative increase in the level of mRNAs corresponding to the majority of the isolated cDNA clones. Among these 15 were identified as corresponding to mRNAs for 50K and 56K keratins and for 50K- and 46K-related keratin. In addition, clones were found corresponding to the proteinase inhibitor cystatin A and to the glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Treatment of keratinocytes with the tumor promoter TPA had no effect on the mRNA level for most of the clones except those corresponding to keratins. Our results indicate that in keratinocytes UV irradiation leads to a relative increase in the level of some mRNAs

Altered distribution of keratinization markers in epidermolytic hyperkeratosis

Epidermolytic hyperkeratosis (EH) is a genetic disorder of keratins associated with epidermal differentiation. Affected individuals carry gene mutations for conserved sequences of keratins K1 or K10. The structural alterations of tonofilaments in EH seem to be a direct consequence of the keratin gene mutations. EH epidermis, however, shows many other unexplained abnormalities including acanthosis, hypergranulosis, and hyperkeratosis. To further elucidate the pathogenetic mechanism of EH, we studied distribution patterns of other keratinization-associated molecules including involucrin, small proline-rich protein (SPRR) 1, loricrin and trichohyalin in the skin of four patients by light and electron microscopic immunohistochemistry in conjunction with conventional transmission electron microscopy. The middle to upper epidermal cells showed moderate to strong immunoreactivities to involucrin, SPRR1 and loricrin antibodies. Both intracellular staining and cell peripheral staining was seen for involucrin and SPRR1 antibodies. Loricrin labelling was prematurely associated with the plasma membrane of granular cells, possibly relating to abnormal keratin filament aggregation and cellular vacuolization. Some loricrin labelling was localized on the keratin aggregates, suggesting intermolecular associations between keratin and loricrin. Trichohyalin, hardly detectable in normal epidermis, was present in some granular and cornified cells in EH in association with keratin filaments, suggesting that it may function as an intermediate filament-associated protein. While cornified cell envelopes were intensely labelled only with loricrin antibodies in normal skin, they were immunoreactive to involucrin, SPRR1 and loricrin antibodies in EH. Sequential change in electron density of the cornified cell envelopes, a constant feature in normal skin, was often absent in EH. These results suggest an altered assembly process of cornified cell envelopes in EH