A retrovirus-derived vector called self-inactivating (SIN) vector was designed for the transduction of whole genes into mammalian cells.

No abstract

The epidermal-melanin unit is composed of one melanocyte sis theory. PAR-2 controls melanosome ingestion and phagocytosis by keratinocytes and exerts a regulatory role in skin and approximately 36 neighboring keratinocytes, working in pigmentation. Modulation of PAR-2 activity can enhance or synchrony to produce and distribute melanin. Melanin is decrease melanosome transfer and affects pigmentation only synthesized in melanosomes, transferred to the dendrite tips, when there is keratinocyte -melanocyte contact. Moreover, and translocated into keratinocytes, forming caps over the keratinocyte nuclei. The molecular and cellular mechanisms PAR-2 is induced by UV irradiation and inhibition of PAR-2 activation results in the prevention of UVB-induced tanning. involved in melanosome transfer and the keratinocytemelanocyte interactions required for this process are not yetThe role of PAR-2 in mediating UV-induced responses remains to be elucidated. completely understood. Suggested mechanisms of melanosome transfer include melanosome release and endocytosis, direct inoculation ('injection'), keratinocyte-melanocyte membrane Key words: Melanosome transfer, PAR-2, Phagocytosis, Keratinocyte, Melanocyte fusion, and phagocytosis. Studies of the keratinocyte receptor protease-activated receptor-2 (PAR-2) support the phagocytonisms for melanosome transfer. These include the release of melanosomes into intercellular spaces followed by endocytosis, direct inoculation ('injection'), keratinocyte -melanocyte membrane fusion, and phagocytosis. Using time-lapse microscopy, Okazaki et al. (7) observed dendrites enfolded by recipient keratinocytes, which are then pinched off to form a cluster of melanosomes. The internalized dendrites were decomposed, leaving each melanosome aggregate surrounded by a single keratinocyte membrane. Single large melanosomes, or complexes of small melanosomes, were then dispersed from the aggregate into the keratinocyte cytoplasm, in a skin-type-dependent process defined as cytophagocytosis. Electron microscopy studies of photo-damaged Caucasian facial skin (8) suggested a similar mechanism, as well as keratinocyte -melanocyte membrane fusion, and exocytosis of single melanosomes (reviewed in (5)). However, a mechanistic understanding of the cellular interactions involved in pigment transfer has not been described.

The chemical basis of melanogenesis is well documented, but the mechanism of melanosome transfer and the regulation of pigmentation by keratinocyte-melanocyte interactions are not well understood. Therefore we examined the effects of serine protease inhibitors on skin pigmentation and found that the protease-activated receptor 2, expressed on keratinocytes, may regulate pigmentation via keratinocyte-melanocyte interactions. Here we show that modulation of protease-activated receptor 2 activation affects melanosome transfer into keratinocytes, resulting in changes in pigment production and deposition. SLIGRL, the protease-activated receptor 2 activating peptide, enhanced melanosome ingestion by keratinocytes, thus increasing pigment deposition. RWJ-50353, a serine protease inhibitor, led to reduced pigment deposition in melanocytes and depigmentation. Electron microscopy studies illustrated an accumulation of immature melanosomes inside melanocytes and abnormal dendrite dynamics in RWJ-50353-treated epidermal equivalents. RWJ-50353 induced a visible and dose-dependent skin lightening effect in the dark-skinned Yucatan swine. Examinations by electron microscopy indicated that the in vivo transfer of melanosomes from melanocytes to keratinocytes was affected. Our data suggest that modulation of keratinocyte-melanocyte interactions via the protease-activated receptor 2 pathway affects melanosome transfer. The use of RWJ-50353 to modulate protease-activated receptor 2 activation could lead to a new class of depigmenting agents.

Thrombin receptor activation was explored in human epidermal keratinocytes and human dermal fibroblasts, cells that are actively involved in skin tissue repair. The effects of thrombin, trypsin, and the receptor agonist peptides SFLLRN and TFRIFD were assessed in inositolphospholipid hydrolysis and calcium mobilization studies. Thrombin and SFLLRN stimulated fibroblasts in both assays to a similar extent, whereas TFRIFD was less potent. Trypsin demonstrated weak efficacy in these assays in comparison with thrombin. Results in fibroblasts were consistent with human platelet thrombin receptor activation. Keratinocytes, however, exhibited a distinct profile, with trypsin being a far better activator of inositolphospholipid hydrolysis and calcium mobilization than thrombin. Furthermore, SFLLRN was more efficacious than thrombin, whereas no response was observed with TFRIFD. Since our data indicated that keratinocytes possess a trypsin-sensitive receptor, we addressed the possibility that these cells express the human homologue of the newly described murine protease-activated receptor, PAR-2