Transient activaton of β-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours

When β-catenin signalling is disturbed from mid-gestation onwards lineage commitment is profoundly altered in postnatal mouse epidermis. We have investigated whether adult epidermis has the capacity for β-catenin-induced lineage conversion without prior embryonic priming. We fused N-terminally truncated, stabilised β-catenin to the ligand-binding domain of a mutant oestrogen receptor (ΔNβ -cateninER). ΔNβ-cateninER was expressed in the epidermis of transgenic mice under the control of the keratin 14 promoter and β -catenin activity was induced in adult epidermis by topical application of 4-hydroxytamoxifen (4OHT). Within 7 days of daily 4OHT treatment resting hair follicles were recruited into the hair growth cycle and epithelial outgrowths formed from existing hair follicles and from interfollicular epidermis. The outgrowths expressed Sonic hedgehog, Patched and markers of hair follicle differentiation, indicative of de novo follicle formation. The interfollicular epidermal differentiation program was largely unaffected but after an initial wave of sebaceous gland duplication sebocyte differentiation was inhibited. A single application of 4OHT was as effective as repeated doses in inducing new follicles and growth of existing follicles. Treatment of epidermis with 4OHT for 21 days resulted in conversion of hair follicles to benign tumours resembling trichofolliculomas. The tumours were dependent on continuous activation of β-catenin and by 28 days after removal of the drug they had largely regressed. We conclude that interfollicular epidermis and sebaceous glands retain the ability to be reprogrammed in adult life and that continuous β-catenin signalling is required to maintain hair follicle tumours.Peer reviewedFinal Published versio

Investigation of the effects of injury upon intracellular signalling pathways and expression of inflammatory response genes in articular cartilage

Damage to joints predisposes to osteoarthritis. The mechanism by which injury to cartilage might lead to net matrix loss and cartilage degeneration remains unknown. Following experimental sharp injury to porcine articular cartilage (dissection from, or scoring of the articular surface), our group has previously shown rapid activation of the 3 mitogen activated protein kinase (MAPK) pathways in cartilage. Activation of ERK, and probably also p38 MAPK, is due to release of fibroblast growth factor (FGF) from the matrix after injury. However, despite a long search, the cause of JNK activation following sharp injury remains unknown. I investigated the extent and regulation of inflammatory signalling after cartilage injury and whether it was sufficient to cause expression of inflammatory response genes. In this work, I show that a number of intracellular signalling pathways including PI3 kinase and IκB kinase (IKK) (which leads to activation of NFκB) are activated by sharp injury to cartilage. The signalling following injury was sufficient to induce a wide range of inflammatory response mRNAs, including pro-inflammatory cytokines such as IL-6, COX-2 and proteinases such as MMP-1 and ADAMTS-4 in a pattern which was not entirely IL-1-like. Pharmacological inhibition experiments suggested that the production by injured cartilage of an inflammatory response gene which could be assayed at the protein level, activin A, was regulated by FGF-mediated pathways (ERK) as well as by NFκB and tyrosine kinases (src family kinases). Given these findings, the role of tyrosine kinases in the early response of cartilage to injury was explored. By phosphotyrosine immunoprecipitation and purification from injured cartilage lysates, FAK and its substrate paxillin were identified from silverstained gels by mass spectrometry. The phosphorylation of these src substrates accounted for rapidly inducible bands seen on phosphotyrosine western blotting of injured cartilage lysates. However, no evidence of a role for src kinases in the regulation of MAPK/IKK signalling upon injury was found. In contrast, blockade of another tyrosine kinase, the FGF receptor, led to partial inhibition not only of the ERK pathway following sharp injury, but also the other MAPKs and IKK. Whilst activation of the same pathways was also seen following injury to synovium, FGF receptor inhibition had no effect on this signalling. This suggested that FGF may have a pro-inflammatory action following injury in vivo which is a particular feature of cartilage

Stem cell patterning and fate in human epidermis

AbstractWithin human epidermis there are two types of proliferating keratinocyte: stem cells, which have high proliferative potential, and transit-amplifying cells, which are destined to undergo terminal differentiation after a few rounds of division. We show that, in vivo, stem cells express higher levels of the α2β1, and α3β1 integrins than transit-amplifying cells and that this can be used both to determine the location of stem cells within the epidermis and to isolate them directly from the tissue. The distribution of stem cells and transit-amplifying cells is not random: patches of integrin-bright and integrin-dull cells have a specific location with respect to the epidermal-dermal junction that varies between body sites and that correlates with the distribution of S phase cells. Stem cell patterning can be recreated in culture, in the absence of dermis, and appears to be subject to autoregulation

Epidermal stem cells are retained in vivo throughout skin aging

In healthy individuals, skin integrity is maintained by epidermal stem cells which self-renew and generate daughter cells that undergo terminal differentiation. It is currently unknown whether epidermal stem cells influence or are affected by skin aging. We therefore compared young and aged skin stem cell abundance, organization, and proliferation. We discovered that despite age-associated differences in epidermal proliferation, dermal thickness, follicle patterning, and immune cell abundance, epidermal stem cells were maintained at normal levels throughout life. These findings, coupled with observed dermal gene expression changes, suggest that epidermal stem cells themselves are intrinsically aging resistant and that local environmental or systemic factors modulate skin aging

Shape-Induced Terminal Differentiation of Human Epidermal Stem Cells Requires p38 and Is Regulated by Histone Acetylation

Engineered model substrates are powerful tools for examining interactions between stem cells and their microenvironment. Using this approach, we have previously shown that restricted cell adhesion promotes terminal differentiation of human epidermal stem cells via activation of serum response factor (SRF) and transcription of AP-1 genes. Here we investigate the roles of p38 MAPK and histone acetylation. Inhibition of p38 activity impaired SRF transcriptional activity and shape-induced terminal differentiation of human keratinocytes. In addition, inhibiting p38 reduced histone H3 acetylation at the promoters of SRF target genes, FOS and JUNB. Although histone acetylation correlated with SRF transcriptional activity and target gene expression, treatment with the histone de-acetylase inhibitor, trichostatin A (TSA) blocked terminal differentiation on micro-patterned substrates and in suspension. TSA treatment simultaneously maintained expression of LRIG1, TP63, and ITGB1. Therefore, global histone de-acetylation represses stem cell maintenance genes independent of SRF. Our studies establish a novel role for extrinsic physical cues in the regulation of chromatin remodeling, transcription, and differentiation of human epidermal stem cells

Provider and service-user perspectives of volunteer health-worker service provision in Ayeyarwady Region, Myanmar: a qualitative study.

OBJECTIVES: To explore perspectives and reported experiences of service users, community providers and policymakers related to volunteer health-worker services provision in a rural area of Myanmar. METHODS: A qualitative interview study was conducted in rural communities with 54 service users and 17 community providers in Ayeyarwady Region, Myanmar, and with 14 national managers and policymakers in Yangon Myanmar. Topics included reasons for seeking health services, views and experiences, and comparison with experiences of other services. Data were analysed thematically using deductive and inductive coding. RESULTS: Accessibility and affordability were important to all participants. Service users described the particular relevance of trust, familiarity and acceptability in choosing a provider. Perceived quality and effectiveness were necessary for trust to develop. Perceived value of volunteers was a cross-cutting dimension, which was interpreted differently by different participants. CONCLUSIONS: Results suggest that volunteers are appropriate and valued, and support 'availability', 'accessibility' and 'acceptability' as dimensions of health services access in this setting. However, social complexities should be considered to ensure effective service delivery. Further research into trust-building, developing quality perceptions and resulting service-user choices would be useful to inform effective policy and planning

The Interfollicular Epidermis of Adult Mouse Tail Comprises Two Distinct Cell Lineages that Are Differentially Regulated by Wnt, Edaradd, and Lrig1

Current models of how mouse tail interfollicular epidermis (IFE) is maintained overlook the coexistence of two distinct terminal differentiation programs: parakeratotic (scale) and orthokeratotic (interscale). Lineage tracing and clonal analysis revealed that scale and interscale are maintained by unipotent cells in the underlying basal layer, with scale progenitors dividing more rapidly than interscale progenitors. Although scales are pigmented and precisely aligned with hair follicles, melanocytes and follicles were not necessary for scale differentiation. Epidermal Wnt signaling was required for scale enlargement during development and for postnatal maintenance of scale-interscale boundaries. Loss of Edaradd inhibited ventral scale formation, whereas loss of Lrig1 led to scale enlargement and fusion. In wild-type skin, Lrig1 was not expressed in IFE but was selectively upregulated in dermal fibroblasts underlying the interscale. We conclude that the different IFE differentiation compartments are maintained by distinct stem cell populations and are regulated by epidermal and dermal signals