Christian Mahé

p63–microRNA feedback in keratinocyte senescence

We investigated the expression of microRNAs (miRNAs) associated with replicative senescence in human primary keratinocytes. A cohort of miRNAs up-regulated in senescence was identified by genome-wide miRNA profiling, and their change in expression was validated in proliferative versus senescent cells. Among these, miRNA (miR)-138, -181a, -181b, and -130b expression increased with serial passages. miR-138, -181a, and -181b, but not miR-130b, overexpression in proliferating cells was sufficient per se to induce senescence, as evaluated by inhibition of BrdU incorporation and quantification of senescence-activated β-galactosidase staining. We identified Sirt1 as a direct target of miR-138, -181a, and -181b, whereas ΔNp63 expression was inhibited by miR-130b. We also found that ΔNp63α inhibits miR-138, -181a, -181b, and -130b expression by binding directly to p63-responsive elements located in close proximity to the genomic loci of these miRNAs in primary keratinocytes. These findings suggest that changes in miRNA expression, by modulating the levels of regulatory proteins such as p63 and Sirt1, strongly contribute to induction of senescence in primary human keratinocytes, thus linking these two proteins. Our data also indicate that suppression of miR-138, -181a, -181b, and -130b expression is part of a growth-promoting strategy of ΔNp63α in epidermal proliferating cells.

Different Gene Expression Patterns in Human Papillary and Reticular Fibroblasts

The dermis contains two distinct layers: the papillary and the reticular layers. In vitro cultures of the fibroblasts from these layers show that they are different. However, no molecular markers to differentiate between the two subtypes of fibroblasts are known. We performed gene expression analysis on cultured fibroblasts isolated from the papillary and reticular dermis. In all, 116 genes were found to be expressed differentially. Of these, 13 were validated by quantitative reverse transcriptase-PCR analysis and two markers could be validated at the protein level in monolayer cultures. Three markers showed differential expression in in vivo skin sections. The identified, characteristic markers of the two fibroblast subpopulations provide useful tools to perform functional studies on reticular and papillary fibroblasts.

MicroRNA-191 triggers keratinocytes senescence by SATB1 and CDK6 downregulation

Highlights ► miR-191 expression is upregulated in senescencent human epidermal keratinocytes. ► miR-191 overexpression is sufficient per se to induce senescence in keratinocytes. ► SATB1 and CDK6 are downregulated in senescence and are direct miR-191 targets. ► SATB1 and CDK6 silencing by siRNA triggers senescence in HEKn cells.

Papillary fibroblasts differentiate into reticular fibroblasts after prolonged in vitro culture.

The dermis can be divided into two morphologically different layers: the papillary and reticular dermis. Fibroblasts isolated from these layers behave differently when cultured in vitro. During skin ageing, the papillary dermis decreases in volume. Based on the functional differences in vitro, it is hypothesized that the loss of papillary fibroblasts contributes to skin ageing. In this study, we aimed to mimic certain aspects of skin ageing by using high‐passage cultures of reticular and papillary fibroblasts and investigated the effect of these cells on skin morphogenesis in reconstructed human skin equivalents. Skin equivalents generated with reticular fibroblasts showed a reduced terminal differentiation and fewer proliferating basal keratinocytes. Aged in vitro papillary fibroblasts had increased expression of biomarkers specific to reticular fibroblasts. The phenotype and morphology of skin equivalents generated with high‐passage papillary fibroblasts resembled that of reticular fibroblasts. This demonstrates that papillary fibroblasts can differentiate into reticular fibroblasts in vitro. Therefore, we hypothesize that papillary fibroblasts represent an undifferentiated phenotype, while reticular fibroblasts represent a more differentiated population. The differentiation process could be a new target for anti‐skin‐ageing strategies.

MicroRNAs in human skin ageing.

The skin protects humans from the surrounding environment. Tissues undergo continuous renewal throughout an individuals lifetime; however, there is a decline in the regenerative potential of tissue with age. The accumulation of senescent cells over time probably reduces tissue regenerative capacity and contributes to the physiological ageing of the tissue itself. The mechanisms that govern ageing remain unclear and are under intense investigation, and insight could be gained by studying the mechanisms involved in cellular senescence. In vitro, keratinocytes and dermal fibroblasts undergo senescence in response to multiple cellular stresses, including the overproduction of reactive oxygen species and the shortening of telomeres, or simply by reaching the end of their replicative potential (i.e., reaching replicative senescence). Recent findings demonstrate that microRNAs play key roles in regulating the balance between a cells proliferative capacity and replicative senescence. Here, we will focus on the molecular mechanisms regulated by senescence-associated microRNAs and their validated targets in both keratinocytes and dermal fibroblasts.

Reduced expression of the adhesion protein tensin1 in cultured human dermal fibroblasts affects collagen gel contraction

Abstract:  As revealed by immunohistochemistry and RT‐QPCR, the focal adhesion protein tensin1 is expressed in cultured human dermal fibroblasts and reduced by 60% after transfection with tensin1 siRNA. Tensin1 silenced fibroblast exhibited a strongly reduced capacity to contract collagen gels. Aged fibroblasts, generated with the Hayflick replicative senescence model, exhibit as siRNA silences fibroblasts, a reduced tensin1 expression and an impaired gel contraction capacity. Based on these results, we speculate that in human dermal fibroblasts, tensin1 plays an important role in cell–matrix interaction and that a reduced expression might contribute to the dermal alterations observed during skin ageing.

TGF-β1 induces differentiation of papillary fibroblasts to reticular fibroblasts in monolayer culture but not in human skin equivalents

AbstractFibroblasts isolated from the papillary and reticular dermis are different from each other in vitro. If papillary fibroblasts are subjected to prolonged serial passaging they will differentiate into reticular fibroblasts. Reticular fibroblasts have been shown to resemble myofibroblasts in severalways. TGF-β1 is the most important factor involved in myofibroblastdifferentiation.Aimswe investigated if TGF-β1 can induce differentiation of papillary fibroblasts into reticular fibroblasts, in monolayer cultures and in human skin equivalents.MethodMonolayer cultures of and human skin equivalents generated with papillary fibroblasts were stimulated with TGF-β1. The expression of markers specific for reticular and papillary fibroblasts was measured by qPCR and immunohistochemical analysis in monolayer cultures. In human skin equivalents, the morphology and the expression of several markers was analysed and compared to untreated papillary and reticular human skin equivalents.ResultsMonolayer cultures of papillary fibroblasts started to express a reticular marker profile after stimulation with TGF-β1. Human skin equivalents generated with papillary fibroblast and stimulated with TGF-β1 were similar to papillary control equivalents and did not obtain reticular characteristics. Expression of reticular markers was only found in the lower layers of TGF-β1-stimulated papillary skin equivalents.ConclusionsTGF-β1 can induce differentiation to reticular fibroblasts in monolayer cultures of papillary fibroblasts. In skin equivalents no such effects were found. The major difference between these experiments is the presence of extracellular matrix in skin equivalents. Therefore, we hypothesize that the matrix secreted by papillary fibroblasts protects them from TGF-β1 induced differentiation.

miR-330-5p targets tyrosinase and induces depigmentation.

Lin ZX (2009) Study on epidermal permeability barrier function and its associated factors in 160 normal Chinese. Master’s Degree Theme, Fudan University, Shanghai, China. http:// cdmd.cnki.com.cn/Article/CDMD-102462009184487.htm Lin TK, Man MQ, Santiago JL et al. (2013) Topical antihistamines display potent anti-inflammatory activity linked in part to enhanced permeability barrier function. J Invest Dermatol 133:469–78

Differential effect of extracellular matrix derived from papillary and reticular fibroblasts on epidermal development in vitro

BackgroundPapillary and reticular fibroblasts have different effects on keratinocyte proliferation and differentiation.ObjectivesThe aim of this study was to investigate whether these effects are caused by differential secretion of soluble factors or by differential generation of extracellular matrix from papillary and reticular fibroblasts.Materials & MethodsTo study the effect of soluble factors, keratinocyte monolayer cultures were grown in papillary or reticular fibroblast-conditioned medium. To study the effect of extracellular matrix, keratinocytes were grown on papillary or reticular-derived matrix.ResultsConditioned medium from papillary or reticular fibroblasts did not differentially affect keratinocyte viability or epidermal development. However, keratinocyte viability was increased when grown on matrix derived from papillary, compared with reticular, fibroblasts. In addition, the longevity of the epidermis was increased when cultured on papillary fibroblast-derived matrix skin equivalents compared with reticular-derived matrix skin equivalents.ConclusionThe findings indicate that the matrix secreted by papillary and reticular fibroblasts is the main causal factor to account for the differences in keratinocyte growth and viability observed in our study. Differences in response to soluble factors between both populations were less significant. Matrix components specific to the papillary dermis may account for the preferential growth of keratinocytes on papillary dermis.