Novel Chemically Modified Curcumin (CMC) Analogs Exhibit Anti-Melanogenic Activity in Primary Human Melanocytes.

Hyperpigmentation is a dermatological condition characterized by the overaccumulation and/or oversecretion of melanin pigment. The efficacy of curcumin as an anti-melanogenic therapeutic has been recognized, but the poor stability and solubility that have limited its use have inspired the synthesis of novel curcumin analogs. We have previously reported on comparisons of the anti-melanogenic activity of four novel chemically modified curcumin (CMC) analogs, CMC2.14, CMC2.5, CMC2.23 and CMC2.24, with that of parent curcumin (PC), using a B16F10 mouse melanoma cell model, and we have investigated mechanisms of inhibition. In the current study, we have extended our findings using normal human melanocytes from a darkly pigmented donor (HEMn-DP) and we have begun to study aspects of melanosome export to human keratinocytes. Our results showed that all the CMCs downregulated the protein levels of melanogenic paracrine mediators, endothelin-1 (ET-1) and adrenomedullin (ADM) in HaCaT cells and suppressed the phagocytosis of FluoSphere beads that are considered to be melanosome mimics. All the three CMCs were similarly potent (except CMC2.14, which was highly cytotoxic) in inhibiting melanin production; furthermore, they suppressed dendricity in HEMn-DP cells. CMC2.24 and CMC2.23 robustly suppressed cellular tyrosinase activity but did not alter tyrosinase protein levels, while CMC2.5 did not suppress tyrosinase activity but significantly downregulated tyrosinase protein levels, indicative of a distinctive mode of action for the two structurally related CMCs. Moreover, HEMn-DP cells treated with CMC2.24 or CMC2.23 partially recovered their suppressed tyrosinase activity after cessation of the treatment. All the three CMCs were nontoxic to human dermal fibroblasts while PC was highly cytotoxic. Our results provide a proof-of-principle for the novel use of the CMCs for skin depigmentation, since at low concentrations, ranging from 5 to 25 µM, the CMCs (CMC2.24, CMC2.23 and CMC2.5) were more potent anti-melanogenic agents than PC and tetrahydrocurcumin (THC), both of which were ineffective at melanogenesis at similar doses, as tested in HEMn-DP cells (with PC being highly toxic in dermal fibroblasts and keratinocytes). Further studies to evaluate the efficacy of CMCs in human skin tissue and in vivo studies are warranted.

Chi-miR-30b-5p inhibits dermal papilla cells proliferation by targeting CaMKIIδ gene in cashmere goat.

BACKGROUND: During goat embryonic morphogenesis and postnatal initiation of hair follicle (HF) regeneration, dermal papilla (DP) cells play a vital role in hair formation. Growing evidence shows that microRNAs (miRNAs) participate in HF development and DP cell proliferation. However, the molecular mechanisms have not been thoroughly investigated. RESULT: In this study, we utilized miRNA sequencing (miRNA-Seq) to identify differentially expressed miRNAs at different HF cycling stages (anagen and telogen). MiRNA-Seq has identified 411 annotated miRNAs and 130 novel miRNAs in which 29 miRNAs were up-regulated and 32 miRNAs were down-regulated in the anagen phase compared to the telogen phase. Target gene prediction and functional enrichment analysis indicated some major biological pathways related to hair cycling, such as Wnt signaling pathways, ECM-receptor interaction, VEGF signaling pathway, biosynthesis of amino acids, metabolic pathways, ribosome and oxidative phosphorylation. Also, we explored the function of chi-miR-30b-5p in regulating hair growth cycle. Similar to the HF cycling, DP cells were isolated from skin and used to investigate miRNA functions. The MTT and EdU assays showed that the viability and proliferation of DP cells were inhibited or promoted after the transfection of chi-miR-30b-5p mimic or inhibitor, respectively. Bioinformatics analysis revealed CaMKIIδ as a candidate target gene of chi-miR-30b-5p, and the dual-luciferase and western blot assay demonstrated that chi-miR-30b-5p bound to the 3'UTR of CaMKIIδ and further inhibited its translation. CONCLUSION: Chi-miR-30b-5p was found to be highly expressed in the telogen than that in the anagen phase and could inhibit the proliferation of DP cells by targeting CaMKIIδ. Our study provides new information on the regulatory functions of miRNAs during HF development.

Asoprisnil, a Selective Progesterone Receptor Modulator (SPRM), Inhibits Melanosome Export in B16F10 Cells and HEMn-DP Melanocytes.

Previous studies have reported that estrogen hormone promotes melanogenesis while progesterone inhibits it. A selective estrogen receptor modulator (SERM), tamoxifen, has been shown to promote melanogenesis; however, to date, there have been no reports on the effects of a selective progesterone receptor modulator (SPRM) on melanogenesis. In the present study, we hypothesized that asoprisnil (AP), a SPRM, inhibits melanogenesis. AP was tested for cytotoxicity to B16F10 mouse melanoma cells for screening the nontoxic concentrations using MTS cytotoxicity assay. Extracellular and intracellular melanin levels were estimated at nontoxic concentrations of AP. To evaluate the direct effect of AP on tyrosinase enzyme, tyrosinase activity and copper chelating activities were measured. Next, the effects of AP on melanogenesis were tested in normal human melanocytes, neonatal, darkly pigmented (HEMn-DP). Our results demonstrate that AP was nontoxic at a concentration range of 10-50 µM in B16F10 cells; AP at 50 µM significantly suppressed extracellular melanin levels comparable to kojic acid at 500 µM, with no significant effect on intracellular melanin levels. The mechanism of melanogenesis inhibition was studied to assess if AP downregulated tyrosinase activity in cell lysates or in a cell-free system. However, AP was found to increase intracellular tyrosinase activity without any effect on tyrosinase enzyme activity or copper chelating activity in a cell-free system, indicating that AP inhibits melanogenesis by mechanisms other than direct effects on tyrosinase enzyme activity. The capacity of AP to inhibit melanosome export was further validated in HEMn-DP cells; AP significantly suppressed dendricity at concentrations of 20 and 30 µM in the absence of effects on melanin synthesis or intracellular tyrosinase activity. In addition, AP was nontoxic to human keratinocytes (HaCaT) at these concentrations, validating its safety for topical use. Taken together, our preliminary results demonstrate that AP might be repurposed as a candidate therapeutic for treatment of hyperpigmentation disorders via a unique mechanism, which encompasses a selective inhibition of melanosome export.

CD4- and CD8-expressing cells found in the bovine and porcine anterior chamber of the eye.

The aim of the present study was to investigate whether the anterior chamber constitutes part of the normal migratory pathway of CD4+ and CD8+ lymphocytes in cattle and swine. The cells obtained from aqueous humor of cows and pigs were stained for CD4 and CD8 receptors, and subsequently analyzed with flow cytometry. The mean percentage of CD4+CD8-, CD4-CD8+ and CD4+CD8+ cells within the total lymphocyte population of the bovine anterior chamber was, respectively, 17.88, 12.64 and 27.26%. In turn, the mean values of these parameters in pigs were 1.77, 38.48 and 17.45, respectively. Among bovine and porcine CD4+CD8+ cells prevalent were those displaying CD4lowCD8low and CD4lowCD8high phenotypes, respectively. The results suggest that the anterior chamber in cattle and swine is an element in the normal migratory pathway of CD4+, CD8+ and CD4+CD8+ cells. Furthermore, the contribution of these subsets in the anterior chamber lymphocyte population can differ considerably between animal species.

Comparative study of doxycycline, sancycline, and 4-dedimethylamino sancycline (CMT-3) on epidermal melanogenesis.

Melanogenesis is regulated by melanocytes, which synthesize the pigment melanin inside melanosomes; these melanosomes are exported through dendritic extensions to adjacent keratinocytes and result in skin coloration. Chemically modified tetracyclines (CMTs) are nonantimicrobial tetracyclines that retain the capacity to inhibit matrix metalloproteinases (MMPs) and have shown several biological benefits; in particular, CMT-3 [(4-dedimethylamino sancycline (SAN)] has emerged as a candidate for therapeutic benefits in our previous studies. However, to date, studies of the effects of CMT-3 or SAN on melanogenesis are lacking. We have previously reported the anti-melanogenic activity of CMT-308 (the 9-amino derivative of CMT-3). Herein, we have compared the three tetracycline analogs, doxycycline (DOX), SAN, and CMT-3, for their effects on melanogenesis using B16F10 mouse melanoma cells and have validated results in primary human melanocytes (HEMn-DP). DOX did not show any significant effects on intracellular melanin or melanosome export in DP cells while SAN was cytotoxic at high doses but without effects on melanogenesis at lower doses. However, CMT-3 showed a robust suppression of dendricity parameters (dendrite number, dendrite length, and proportion of dendritic cells) in DP cells which was associated, at least in part, with a significant reduction of intracellular tyrosinase activity. In spite of its inhibition of tyrosinase activity, CMT-3 had no significant effects on intracellular melanin levels, suggesting that it selectively targets melanosome export. Our results demonstrate a unique structure-activity relationship (SAR) for the effects of these compounds on melanogenesis and support the conclusion that removal of the 4-dimethylamino moiety confers the selective capacity to suppress melanosome export. Collectively, these results indicate that CMT-3 might be a candidate for diminishing hyperpigmentation skin disorders.

Transcriptional Regulation of Early T-Lymphocyte Development in Thymus.

T-lymphocytes play crucial roles for maintaining immune homeostasis by fighting against various pathogenic microorganisms and establishing self-antigen tolerance. They will go through several stages and checkpoints in the thymus from progenitors to mature T cells, from CD4-CD8- double negative (DN) cells to CD4+CD8+ double positive (DP) cells, finally become CD4+ or CD8+ single positive (SP) cells. The mature SP cells then emigrate out of the thymus and further differentiate into distinct subsets under different environment signals to perform specific functions. Each step is regulated by various transcriptional regulators downstream of T cell receptors (TCRs) that have been extensively studied both in vivo and vitro via multiple mouse models and advanced techniques, such as single cell RNA sequencing (scRNA-seq) and Chromatin Immunoprecipitation sequencing (ChIP-seq). This review will summarize the transcriptional regulators participating in the early stage of T cell development reported in the past decade, trying to figure out cascade networks in each process and provide possible research directions in the future.

CMT-308, a Nonantimicrobial Chemically-Modified Tetracycline, Exhibits Anti-Melanogenic Activity by Suppression of Melanosome Export.

CMT-308 is a nonantimicrobial chemically-modified tetracycline (CMT), which we have previously shown exhibits antifungal activity and pleiotropic anti-inflammatory activities, including inhibition of the enzymatic activity of matrix metalloproteinases (MMPs). Based on its chemical structure, we hypothesized that CMT-308 could inhibit melanogenesis and might be a candidate for the treatment of skin hyperpigmentation disorders which occur due to unregulated melanin biosynthesis and/or transport. CMT-308 was first studied for any effects on activity of the enzyme tyrosinase in vitro using a purified preparation of mushroom tyrosinase; the mode of inhibition of the soluble fungal enzyme was evaluated by Lineweaver-Burk and Dixon plots as well as by non-linear least squares fitting. Next, the effects of CMT-308 were tested in mammalian cell cultures using B16F10 mouse melanoma cells and further validated in darkly-pigmented human melanocytes (HEMn-DP). Our results showed that micromolar concentrations of CMT-308 inhibited mushroom tyrosinase enzyme activity, using the first two substrates in the melanogenesis pathway (l-tyrosine and l-3,4-dihydroxyphenylalanine (l-DOPA)); CMT-308 inhibited mushroom tyrosinase primarily via a mixed mode of inhibition, with the major contribution from a competitive mode. In B16F10 cell cultures, CMT-308 (10 µM) significantly diminished total melanin levels with a selective reduction of extracellular melanin levels, under both basal and hormone-stimulated conditions without any cytotoxicity over a duration of 72 h. Studies of potential mechanisms of inhibition of melanogenesis in B16F10 cells showed that, in mammalian cells, CMT-308 did not inhibit intracellular tyrosinase activity or the activity of α-glucosidase, an enzyme that regulates maturation of tyrosinase. However, CMT-308 suppressed MITF protein expression in B16F10 cells and showed copper chelating activity and antioxidant activity in a cell-free system. The significantly lower extracellular melanin levels obtained at 10 µM indicate that CMT-308's anti-melanogenic action may be attributed to a selective inhibition of melanosome export with the perinuclear aggregation of melanosomes, rather than a direct effect on the tyrosinase-catalyzed steps in melanin biosynthesis. These results were validated in HEMn-DP cells where CMT-308 suppressed dendricity in a fully reversible manner without affecting intracellular melanin synthesis. Furthermore, the capacity of CMT-308 to inhibit melanosome export was retained in cocultures of HEMn-DP and HaCaT. In summary, our results offer promise for therapeutic strategies to combat the effects of hyperpigmentation by use of CMT-308 at low micromolar concentrations.

CRISPR/Cas9-mediated VDR knockout plays an essential role in the growth of dermal papilla cells through enhanced relative genes.

BACKGROUND: Hair follicles in cashmere goats are divided into primary and secondary hair follicles (HFs). HF development, which determines the morphological structure, is regulated by a large number of vital genes; however, the key functional genes and their interaction networks are still unclear. Although the vitamin D receptor (VDR) is related to cashmere goat HF formation, its precise effects are largely unknown. In the present study, we verified the functions of key genes identified in previous studies using hair dermal papilla (DP) cells as an experimental model. Furthermore, we used CRISPR/Cas9 technology to modify the VDR in DP cells to dissect the molecular mechanism underlying HF formation in cashmere goats. RESULTS: The VDR expression levels in nine tissues of Shaanbei white cashmere goats differed significantly between embryonic day 60 (E60) and embryonic day 120 (E120). At E120, VDR expression was highest in the skin. At the newborn and E120 stages, the VDR protein was highly expressed in the root sheath and hair ball region of Shaanbei cashmere goats. We cloned the complete CDS of VDR in the Shaanbei white cashmere goat and constructed a VDR-deficient DP cell model by CRISPR/Cas9. Heterozygous and homozygous mutant DP cells were produced. The growth rate of mutant DP cells was significantly lower than that of wild-type DP cells (P < 0.05) and VDR mRNA levels in DP cells decreased significantly after VDR knockdown (P < 0.05). Further, the expression levels of VGF, Noggin, Lef1, and ß-catenin were significantly downregulated (P < 0.05). CONCLUSIONS: Our results indicated that VDR has a vital role in DP cells, and that its effects are mediated by Wnt and BMP4 signaling.

Generation of iPS Cells from Human Hair Follice Dermal Papilla Cells.

Dermal papilla (DP) cells are unique regional stem cells of the skin that induce formation of a hair follicle and its regeneration cycle. DP are multipotent stem cells; therefore we supposed that the efficiency of DPC reprogramming could exceed that of dermal fibroblasts reprogramming. We generated induced pluripotent stem cells from human DP cells using lentiviral transfection with Oct4, Sox2, Klf4, and c-Myc, and cultivation of cells both in a medium supplemented with valproic acid and at a physiological level of oxygen (5%). The efficiency of DP cells reprogramming was ~0.03%, while the efficiency of dermal fibroblast reprogramming under the same conditions was ~0.01%. Therefore, we demonstrated the suitability of DP cells as an alternative source of iPS cells.