Robin Kurfurst

The silver locus product (Silv⁄gp100⁄Pmel17) as a new tool for the analysis of melanosome transfer in human melanocyte-keratinocyte co-culture

Abstract:  Melanosomes are melanocyte‐specific lysosome‐related organelles that are transferred to keratinocytes of the epidermis and anagen hair bulb. Transferred melanin forms supra‐nuclear caps that protect epidermal keratinocytes against UV irradiation. The mechanism(s) responsible for melanosome transfer into keratinocytes and their subsequent intra‐keratinocyte distribution has long remained one of the most enigmatic of heterotypic cell interactions. Although there have been many attempts to study this process, significant progress has been hindered by the absence of an adequate in vitro model. During our ongoing study of melanocyte–keratinocyte interactions in skin and hair follicle, we have developed a novel in vitro assay that exploits the specificity of Silv/Pmel17/gp100 expression for melanosome/melanin granules. Using matched cultures of keratinocytes and melanocytes isolated from normal healthy epidermis together with double immunofluorescence, we have determined that gp100 is a surprisingly useful tracker of transferred melanin. Moreover, transferred gp100 stained melanin granules emit a bright fluorescence signal, facilitating ready quantification of melanin transfer levels between melanocytes and keratinocytes. This quantitative approach was validated using known inducers and inhibitors of the melanocyte phenotype. This assay further confirmed that cytophagocytosis of melanocyte components (e.g. dendrite tips) by keratinocytes is one route for melanin incorporation into keratinocytes. Lastly, a role for the recently proposed filopodium as a direct conduit for melanin transfer was substantiated using this novel approach. In conclusion, this assay promises to significantly aid our investigations of the molecular basis of melanosome transfer and offers a new tool for the clinical evaluation of melanocyte modulators.

Solid-phase preparation of 5′,3′-heterobifunctional oligodeoxyribonucleotides using modified solid supports

Abstract The solid-phase preparation of oligodeoxyribonucleotides attached to intercalator or reactive groups through their 5′- and (or) 3′-ends is reported. These syntheses implicate the introduction of suitable masked functional groups at the 5′-end of the oligonucleotide by the intermediate of their phosphoramidite derivatives or at the 3′-end of the oligonucleotide using modified solid supports. After full deblocking, the functional groups (phosphate, thiophosphate, primary amine or thiol) can be reacted with the suitable reactive group involved in the chosen ligand. These methods allow the preparation of heterobifunctional derivatized oligodeoxyribonucleotides.

Decreased expression of keratinocyte β1 integrins in chronically sun-exposed skin in vivo

Summary Background Chronic exposure to ultraviolet (UV) radiation induces changes in the skin structure which are mostly found in the superficial dermis and at the dermal–epidermal junction. Keratinocytes and fibroblasts contribute both to the synthesis and to the degradation of the molecules important for the integrity of this skin site. While several studies have reported on alterations of dermal components and of the functions of fibroblasts in vivo and in vitro after UV exposure, recent data suggested that keratinocytes could be the main skin cell type involved in the photoageing process.

Oligo-α-deoxyribonucleotides with a modified nucleic base and covalently linked to reactive agents

Abstract Solid-phase preparation of oligo-α-deoxyribonucleotides attached to intercalator, chemically or photochemically reactive groups through either their 5′- or 3′-ends, including use of the 5-methyl-α-deoxycytidine.

Study of fibroblast gene expression in response to oxidative stress induced by hydrogen peroxide or UVA with skin aging.

The skin aging process, implying oxidative stress, is associated with specific gene expression. Ultraviolet A (UVA) and hydrogen peroxide (H(2)O(2)) both generate reactive oxygen species (ROS) making them relevant in the study of skin cell responses to oxidative stresses. To investigate transcript expression associated with chronological skin aging and its modulation by two oxidative stresses, cDNA micro-arrays, composed of a set of 81 expressed sequence tag (EST) clones, were used to probe the patterns of transcript expression in human fibroblasts of five young (< 21 years-old) and five older (> 50 years-old) healthy females at basal levels and 24 h after exposure to UVA (7 J/cm2) and H(2)O(2) (20 mM). At the basal state, 22% of total genes were up-regulated in the older group. Although both stresses led to the same cell mortality, H(2)O(2) induced a stronger modulation of gene expression than UVA, with 19.5% of transcripts up-regulated versus 4%. The aging process affected the response to H(2)O(2) and even though cells from old donors presented higher basal levels of transcripts they were not able to regulate them in response to the stress. Interestingly, UVA had a specific strong inhibitory effect on the expression of chemokine (C-C) motif ligand 2 (CCL2) transcript, suggesting a possible mechanism for its anti-inflammatory and immunoregulatory roles.

Transcriptional effect of an Aframomum angustifolium seed extract on human cutaneous cells using low-density DNA chips.

Background  Studying photoexposed and photoprotected skin biopsies from young and aged women, it has been found that a specific zone, composed of the basal layers of the epidermis, the dermal epidermal junction, and the superficial dermis, is major target of aging and reactive oxygen species. We showed that this zone is characterized by significant variations at a transcriptional and/or protein levels.

Oligo-β- and -α-deoxyribonucleotides involving 2-aminopurine and guanine for triple-helix formation

Abstract Purine oligo-β-deoxyribonucleotides and unnatural nuclease-resistant α-anomer derivatives were constructed with 2-aminopurine (amP) guanine (G) in order to recognize purine sequences in double-helical DNA via isomorphous base triples amP.AT and G.GC.

Differential expression of cathepsins K, S and V between young and aged Caucasian women skin epidermis.

Cutaneous aging translates drastic structural and functional alterations in the extracellular matrix (ECM). Multiple mechanisms are involved, including changes in protease levels. We investigated the age-related protein expression and activity of cysteine cathepsins and the expression of two endogenous protein inhibitors in young and aged Caucasian women skin epidermis. Immunofluorescence studies indicate that the expression of cathepsins K, S and V, as well as cystatins A and M/E within keratinocytes is reduced in photoprotected skin of aged women. Furthermore, the overall endopeptidase activity of cysteine cathepsins in epidermis lysates decreased with age. Albeit dermal elastic fiber and laminin expression is reduced in aged skin, staining of nidogen-1, a key protein in BM assembly that is sensitive to proteolysis by cysteine, metallo- and serine proteases, has a similar pattern in both young and aged skin. Since cathepsins contribute to the hydrolysis and turnover of ECM/basement membrane components, the abnormal protein degradation and deposition during aging process may be related in part to a decline of lysosomal/endosomal cathepsin K, S and V activity.

E‐cadherin mediates ultraviolet radiation‐ and calcium‐induced melanin transfer in human skin cells

Skin pigmentation is directed by epidermal melanin units, characterized by long‐lived and dendritic epidermal melanocytes (MC) that interact with viable keratinocytes (KC) to contribute melanin to the epidermis. Previously, we reported that MC:KC contact is required for melanosome transfer that can be enhanced by filopodi, and by UVR/UVA irradiation, which can upregulate melanosome transfer via Myosin X‐mediated control of MC filopodia. Both MC and KC express Ca2+‐dependent E‐cadherins. These homophilic adhesion contacts induce transient increases in intra‐KC Ca2+, while ultraviolet radiation (UVR) raises intra‐MC Ca2+ via calcium‐selective ORAI1 ion channels; both are associated with regulating melanogenesis. However, how Ca2+ triggers melanin transfer remains unclear. Here we evaluated the role of E‐cadherin in UVR‐mediated melanin transfer in human skin cells. MC and KC in human epidermis variably express filopodia‐associated E‐cadherin, Cdc42, VASP and β‐catenin, all of which were upregulated by UVR in human MC in vitro. Knockdown of E‐cadherin revealed that this cadherin is essential for UVR‐induced MC filopodia formation and melanin transfer. Moreover, Ca2+ induced a dose‐dependent increase in filopodia formation and melanin transfer, as well as increased β‐catenin, Cdc42, Myosin X and E‐cadherin expression in these skin cells. Together, these data suggest that filopodial proteins and E‐cadherin, which are upregulated by intracellular (UVR‐stimulated) and extracellular Ca2+ availability, are required for filopodia formation and melanin transfer. This may open new avenues to explore how Ca2+ signalling influences human pigmentation.

Impact of exogenous stress on TGF-β inducible early gene 1 in human skin cells.

Keywords: oxidative stress; skin ageing; skin photoexposure; TGF-β; TGF-β inducible early gene-1