Jiro Kishimoto

A novel mechanism of matrix metalloproteinase-9 gene expression implies a role for keratinization

To investigate the pathophysiological role of matrix metalloproteinase (MMP)‐9 in the skin, we analyzed MMP‐9 expression from human keratinocytes in culture. MMP‐9 and the terminal differentiation marker involucrin were co‐localized in the same keratinocytes with a high concentration of Ca2+, a potent stimulator of differentiation. We identified the novel KRE‐M9 element, further downstream to the previously reported TPA responsive element in the MMP‐9 promoter, and both of these two elements were shown to be important for MMP‐9 transcription and Ca2+ induction. The concomitant upregulation of MMP‐9 and involucrin transcripts was probably due to the very similar gene regulatory elements, KRE‐M9 and KRE‐4, in their respective promoters. These results indicate a novel mechanism of transcriptional regulation for MMP‐9 in the process of keratinization, implying the probable association of apoptosis and differentiation of keratinocytes in epidermal skin tissue.

Hair‐inducing ability of human dermal papilla cells cultured under Wnt/β‐catenin signalling activation

Abstract:  It is well known that dermal papilla cells (DPCs) play crucial roles in hair follicle induction. In this study, we examined whether Wnt/β‐catenin activation results in maintenance of the hair‐inducing ability of human DPCs. Expression of DPC marker genes was maintained under Wnt/β‐catenin signalling stimulation by GSK‐3β inhibition. Furthermore, human DPCs showed constant hair induction when transplanted with murine epidermal cell fraction. Alu‐positive human DPCs were essentially detected adjacent to the reconstructing epidermal structure positive for P‐cadherin immunoreactivity. The transplanted human DPCs were abundant in the surrounding dermal sheath portion of the fully regenerated hair follicles. These results support the importance of Wnt/β‐catenin signalling in hair follicle induction. This study may provide valuable information to establish a culture method of human DPCs for cell‐based therapy.

Identification of novel hair-growth inducers by means of connectivity mapping

The aim of this study was to identify novel inducers of hair growth using gene expression profiling at various stages of hair‐growth induction. First, we analyzed gene expression at the onset of hair growth in mice induced by cyclosporin A (CsA), a well‐known hair‐growth inducer, using DNA microarray analysis. The results unveiled genes involved in the step‐by‐step progression of hair growth, including increases in melanin biosynthesis and decreases in immune response at d 2 and the subsequent stimulation of cell proliferation at d 4, followed by the up‐regulation of hair specific keratins at d 7 after CsA treatment. With the use of the connectivity map (Cmap), agents that had a similar “gene signature” to that of the profiles of CsA‐treated mice were identified. Several agents, including CsA, were identified by the Cmap and were evaluated for hair induction activity in vivo. One of the proposed agents, fluphenazine (from the d 2 signature) actually induced hair growth in vivo (ED50: 2 mM for single application), and the subsequent application of 5 mM iloprost (from the d 4 signature) significantly enhanced the hair‐growth effect of fluphenazine. From these results, Cmap analysis was proven to be a useful method that connects gene expression profiles of complicated biological processes, such as hair‐growth induction, to effective agents.—Ishimatsu‐Tsuji, Y., Soma, T., Kishimoto, J. Identification of novel hair‐growth inducers by means of connectivity mapping. FASEB J. 24, 1489–1496 (2010). www.fasebj.org

Adenosine increases anagen hair growth and thick hairs in Japanese women with female pattern hair loss: A pilot, double-blind, randomized, placebo-controlled trial

Adenosine upregulates the expression of vascular endothelial growth factor and fibroblast growth factor‐7 in cultured dermal papilla cells. It has been shown that, in Japanese men, adenosine improves androgenetic alopecia due to the thickening of thin hair due to hair follicle miniaturization. To investigate the efficacy and safety of adenosine treatment to improve hair loss in women, 30 Japanese women with female pattern hair loss were recruited for this double‐blind, randomized, placebo‐controlled study. Volunteers used either 0.75% adenosine lotion or a placebo lotion topically twice daily for 12 months. Efficacy was evaluated by dermatologists and by investigators and in phototrichograms. As a result, adenosine was significantly superior to the placebo according to assessments by dermatologists and investigators and by self‐assessments. Adenosine significantly increased the anagen hair growth rate and the thick hair rate. No side‐effects were encountered during the trial. Adenosine improved hair loss in Japanese women by stimulating hair growth and by thickening hair shafts. Adenosine is useful for treating female pattern hair loss in women as well as androgenetic alopecia in men.

Localization of Serine Racemase and Its Role in the Skin

D-Serine is an endogenous coagonist of the N-methyl-D-aspartate (NMDA)–type glutamate receptor in the central nervous system and its synthesis is catalyzed by serine racemase (SR). Recently, the NMDA receptor has been found to be expressed in keratinocytes (KCs) of the skin and involved in the regulation of KC growth and differentiation. However, the localization and role of SR in the skin remain unknown. Here, using SR-knockout (SR-KO) mice as the control, we demonstrated the localization of the SR protein in the granular and cornified layer of the epidermis of wild-type (WT) mice and its appearance in confluent WT KCs. We also demonstrated the existence of a mechanism for conversion of L-serine to D-serine in epidermal KCs. Furthermore, we found increased expression levels of genes involved in the differentiation of epidermal KCs in adult SR-KO mice, and alterations in the barrier function and ultrastructure of the epidermis in postnatal day 5 SR-KO mice. Our findings suggest that SR in the skin epidermis is involved in the differentiation of epidermal KCs and the formation of the skin barrier.

Inflammatory Mediator TAK1 Regulates Hair Follicle Morphogenesis and Anagen Induction Shown by Using Keratinocyte-Specific TAK1-Deficient Mice

Transforming growth factor-β-activated kinase 1 (TAK1) is a member of the NF-κB pathway and regulates inflammatory responses. We previously showed that TAK1 also regulates keratinocyte growth, differentiation, and apoptosis. However, it is unknown whether TAK1 has any role in epithelial–mesenchymal interactions. To examine this possibility, we studied the role of TAK1 in mouse hair follicle development and cycling as an instructive model system. By comparing keratinocyte-specific TAK1-deficient mice (Map3k7 fl/flK5-Cre) with control mice, we found that the number of hair germs (hair follicles precursors) in Map3k7 fl/flK5-Cre mice was significantly reduced at E15.5, and that subsequent hair follicle morphogenesis was retarded. Next, we analyzed the role of TAK1 in the cyclic remodeling in follicles by analyzing hair cycle progression in mice with a tamoxifen-inducible keratinocyte-specific TAK1 deficiency (Map3k7 fl/flK14-Cre-ERT2). After active hair growth (anagen) was induced by depilation, TAK1 was deleted by topical tamoxifen application. This resulted in significantly retarded anagen development in TAK1-deficient mice. Deletion of TAK1 in hair follicles that were already in anagen induced premature, apoptosis-driven hair follicle regression, along with hair follicle damage. These studies provide the first evidence that the inflammatory mediator TAK1 regulates hair follicle induction and morphogenesis, and is required for anagen induction and anagen maintenance.

Immunohistochemical survey of the distribution of epidermal melanoblasts and melanocytes during the development of UVB-induced pigmented spots

BACKGROUND Repeated exposures to ultraviolet B radiation (UVB) induce pigmented spots on dorsal skin of (HR-1 x HR/De) F(1) hairless mouse. We showed previously that this mouse is suitable for studies of melanocyte function. OBJECTIVE To clarify the mechanism of development of pigmented spots induced by chronic UVB exposure. METHODS We used light and fluorescence microscopy to quantify changes in the numbers of differentiated melanocytes containing melanin pigments (MM) and melanoblasts/melanocytes immunohistochemically positive for tyrosinase-related protein (TRP)-1, TRP-2 (dopachrome tautomerase), and c-kit in epidermis during the development of pigmented spots in hairless mice chronically exposed to UVB (99 mJ/cm(2), 3 times/week, 8 weeks). RESULTS The change in the number of TRP-1-positive cells during chronic UVB exposure was similar to that of MM: both increased dramatically during the stage of acute pigmentation, then decreased sharply after cessation of UVB, concomitantly with depigmentation; subsequently they increased gradually with the development of pigmented spots. In contrast, after two UVB exposures, no c-kit-positive cells were detected, then the number gradually increased during UVB irradiation, and continued to increase after cessation of irradiation; TRP-2-positive cells showed a rather similar pattern, except that they did not disappear initially. CONCLUSION Our results indicate that chronic UVB irradiation induces differentiation and proliferation of melanoblasts, followed by an increase of differentiated melanocytes, leading to the development of pigmented spots. The sequence of expression of markers appeared to be c-kit, TRP-2, TRP-1, and finally melanin, as it is during normal melanocyte differentiation.

Topical adenosine increases the proportion of thick hair in Caucasian men with androgenetic alopecia

Adenosine is an effective treatment for androgenetic alopecia (AGA) in Japanese men and women. Adenosine exerts its effects by significantly increasing the proportion of thick hair. In this study, we assessed the clinical outcome of adenosine treatment for 6 months in 38 Caucasian men. The change in proportion of thick hair (≥60 μm) compared with baseline in the adenosine group was significantly higher than that in the placebo group (P < 0.0001). The change in vellus hair proportion (<40 μm) was significantly lower in the adenosine group than that in the placebo group (P = 0.0154). The change in hair density compared with baseline of the adenosine group was also significantly higher compared with that of the placebo group (P = 0.0470). No adverse effects due to treatment were noted during this study by dermatological evaluation. Adenosine is effective in increasing the proportion of thick hair in Caucasian men with AGA as well as in Japanese men and women.

Isolation of mesenchymal stem cells from human dermis.

Recent studies revealed that mammalian dermis contains multipotent stem cells such as skin-derived precursors (SKPs). SKPs grow in suspension as spheres. In contrast, mesenchymal stem cells (MSCs) are adherent fibroblastic cells. Here, we describe the procedure to isolate MSCs under low-serum culture conditions. In addition, we explain the method to collect MSCs using magnetic cell sorting.

Marked Changes in Lamellar Granule and Trans-Golgi Network Structure Occur During Epidermal Keratinocyte Differentiation

Epidermal lamellar granules transport various lipids, proteins, and protein inhibitors from the trans-Golgi network to the extracellular space, and play an important role in skin barrier formation. We elucidated the 3-dimensional structure of lamellar granules and the trans-Golgi network in normal human skin by focused ion beam scanning electron microscopy. Reconstructed focused ion beam scanning electron microscopy 3-dimensional images revealed that the overall lamellar granule structure changed from vesicular to reticular within the second layer of the stratum granulosum. Furthermore, the trans-Golgi network was well developed within this layer and spread through the cytoplasm with branched, tubular structures that connected to lamellar granules. Our study reveals the unique overall 3-dimensional structure of lamellar granules and the trans-Golgi network within the cells of the epidermis, and provides the basis for an understanding of the skin barrier formation.