L-tyrosine and L-dihydroxyphenylalanine as hormone-like regulators of melanocyte functions

There is evidence that L-tyrosine and L-dihydroxyphenylalanine (L-DOPA), besides serving as substrates and intermediates of melanogenesis, are also bioregulatory agents acting not only as inducers and positive regulators of melanogenesis but also as regulators of other cellular functions. These can be mediated through action on specific receptors or through non-receptor-mediated mechanisms. The substrate induced (L-tyrosine and/or L-DOPA) melanogenic pathway would autoregulate itself as well as regulate the melanocyte functions through the activity of its structural or regulatory proteins and through intermediates of melanogenesis and melanin itself. Dissection of regulatory and autoregulatory elements of this process may elucidate how substrate-induced autoregulatory pathways have evolved from prokaryotic or simple eukaryotic organisms to complex systems in vertebrates. This could substantiate an older theory proposing that receptors for amino acid-derived hormones arose from the receptors for those amino acids, and that nuclear receptors evolved from primitive intracellular receptors binding nutritional factors or metabolic intermediates.     

The biology of melanocyte and melanocyte stem cell

The melanocyte stem cells of the hair follicle provide an attractive system for the study of the stem cells. Successful regeneration of a functional organ relies on the organized and timely orchestration of molecular events among dis- tinct stem/progenitor cell populations. The stem cells are regulated by communication with their specialized microenvironment known as the niche. Despite remarkable progress in understanding stem cell-intrinsic behavior, the molecular nature of the extrinsic factors provided to the stem cells by the niche microenvironment remains poorly understood. In this regard, the bulge niche of the mammalian hair follicle offers an excellent model for study. It holds two resident populations of SCs： epidermal stem cells and melanocyte stem cells. While their behavior is tightly coordinated, very little of the crosstaik involved is known. This review summarized the recent development in trying to understand the regulation of melanocyte and melanocyte stem cells. A better understanding of the normal regulation and behaviors of the melanocytes and the melanocyte stem cells will help to improve the clinical applications in regenerative medicine, cancer therapy, and aging.     

In vitro behaviour of human osteoblasts on dentin and bone

This investigation studied how the behaviour of isolated osteoblasts on standard tissue culture polystyrene compared with cells cultured on cut surfaces of dentin, a natural calcified material. Cellular attachment, viability and growth were monitored in parallel cultures of human osteosarcoma cell lines (MG63, HOS TE85, SaOS-2) and primary human osteoblast-like cells (HOBs). Culture plastic was either left untreated or roughened with abrasive paper of various grit sizes (4000-1200 grit) in order to obtain a level of roughness comparable to that of the dentin slices. Cell counting and intracellular BCECF staining showed that after an initial incubation of 2 h, the primary cells attached and spread out more quickly on the different substrates than the three cell lines. The primary cells also showed a stronger mitochondrial staining and viability on dentin. During subsequent culture morphological differences appeared with the cells on dentin displaying more cellular extensions. All three cell lines proliferated more slowly on dentin than on plastic. In contrast, the primary HOBs were not significantly affected in their growth by the different substrates. Total and specific alkaline phosphatase (AP) activity of the cell lines was not significantly affected by the different substrates after short-term adhesion, but it was increased for the primary cells on the dentin. However, after 2-3 days of culture, AP was decreased on the dentin slices for both the cell lines and primary HOBs. Plasma treatment of the roughened plastic did not alter cellular viability or AP activity, suggesting that grinding of the surface did not affect the property of the culture plastic to support cell attachment and growth. In conclusion, the results show that not only do osteoblastic cells behave differently on a natural calcified substrate surface than on standard culture plastic, but also that differences were evident between the various cell types, in particular the primary HOB versus the continuous cell lines.     

Functionally distinct melanocyte populations revealed by reconstitution of hair follicles in mice

Hair follicle reconstitution analysis was used to test the contribution of melanocytes or their precursors to regenerated hair follicles. In this study, we first confirmed the process of chimeric hair follicle regeneration by both hair keratinocytes and follicular melanocytes. Then, as first suggested from the differential growth requirements of epidermal skin melanocytes and non‐cutaneous or dermal melanocytes, we confirmed the inability of the latter to be involved as follicular melanocytes to regenerate hair follicles during the hair reconstitution assay. This clear functional discrimination between non‐cutaneous or dermal melanocytes and epidermal melanocytes suggests the presence of two different melanocyte cell lineages, a finding that might be important in the pathogenesis of melanocyte‐related diseases and melanomas.     

MicroRNAs in melanocyte and melanoma biology

The importance of microRNAs as key molecular components of cellular processes is now being recognized. Recent reports have shown that microRNAs regulate processes as diverse as protein expression and nuclear functions inside cells and are able to signal extracellularly, delivered via exosomes, to influence cell fate at a distance. The versatility of microRNAs as molecular tools inspires the design of novel strategies to control gene expression, protein stability, DNA repair and chromatin accessibility that may prove very useful for therapeutic approaches due to the extensive manageability of these small molecules. However, we still lack a comprehensive understanding of the microRNA network and its interactions with the other layers of regulatory elements in cellular and extracellular functions. This knowledge may be necessary before we exploit microRNA versatility in therapeutic settings. To identify rules of interactions between microRNAs and other regulatory systems, we begin by reviewing microRNA activities in a single cell type: the melanocyte, from development to disease.     

Substrate specificity of human kallikreins 1 and 6 determined by phage display

The human tissue kallikrein (KLK) family contains 15 secreted serine proteases that are expressed in a wide range of tissues and have been implicated in different physiological functions and disease states. Of these, KLK1 has been shown to be involved in the regulation of multiple physiological processes such as blood pressure, smooth muscle contraction, and vascular cell growth. KLK6 is overexpressed in breast and ovarian cancer tissues and has been shown to cleave peptide derived from human myelin protein and Abeta amyloid peptide in vitro. Here we analyzed the substrate specificity of KLK1 and KLK6, by substrate phage display using a random octapeptide library. Consistent with earlier biochemical data, KLK1 was shown to exhibit both trypsin- and chymotrypsin-like selectivities with Tyr/Arg preferred at site P1, Ser/Arg strongly preferred at P1', and Phe/Leu at P2. KLK6 displayed trypsin-like activity, with the P1 position occupied only by Arg and a strong preference for Ser in P1'. Docking simulations of consensus peptide provide information on the identity of the enzyme residues that are responsible for substrate binding. Bioinformatic analysis suggested several putative KLK6 protein substrates, such as ionotropic glutamate receptor (GluR) and synphilin.     

Isolation and characterization of Kit-independent melanocyte precursors induced in the skin of Steel factor transgenic mice

Steel factor (SLF, also called KIT-ligand, mast cell growth factor, or stem cell factor) acting through the tyrosine kinase receptor KIT is thought to be indispensable for the early phase of melanocyte development both in vivo and in vitro . In the present study, Kit-independent precursor cells were generated in mice expressing exogenous SLF in their skin keratinocytes and were detected as pigmented spots after administration of Kit function-blocking antibody. We successfully purified these precursor or stem cells as Kit⁺CD45⁻ cells by flow cytometry. The purified cells showed normal but delayed differentiation into mature melanocytes, indicating the immature nature of Kit-independent precursors. The Kit-independent interfollicular population generated in SLF transgenic mice was suggested to be the counterpart of the follicular melanocyte stem cell based on the Kit-independent nature for their survival.     

Aligned human cardiac syncytium for in vitro analysis of electrical,structural, and mechanical readouts

Human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) have emerged as an exciting new tool for cardiac research and can serve as a preclinical platform for drug development and disease modeling studies. However, these aspirations are limited by current culture methods in which hPSC‐CMs resemble fetal human cardiomyocytes in terms of structure and function. Herein we provide a novel in vitro platform that includes patterned extracellular matrix with physiological substrate stiffness and is amenable to both mechanical and electrical analysis. Micropatterned lanes promote the cellular and myofibril alignment of hPSC‐CMs while the addition of micropatterned bridges enable formation of a functional cardiac syncytium that beats synchronously over a large two‐dimensional area. We investigated the electrophysiological properties of the patterned cardiac constructs and showed they have anisotropic electrical impulse propagation, as occurs in the native myocardium, with speeds 2x faster in the primary direction of the pattern as compared to the transverse direction. Lastly, we interrogated the mechanical function of the pattern constructs and demonstrated the utility of this platform in recording the strength of cardiomyocyte contractions. This biomimetic platform with electrical and mechanical readout capabilities will enable the study of cardiac disease and the influence of pharmaceuticals and toxins on cardiomyocyte function. The platform also holds potential for high throughput evaluation of drug safety and efficacy, thus furthering our understanding of cardiovascular disease and increasing the translational use of hPSC‐CMs.     

The effect of substrate and epidermal growth factor on human placental trophoblast cells in culture

Summary Attempts were made to select for trophoblast cells in cultures of mixed cell populations derived from preterm (7 to 12 wk) or term human placentas. Epidermal growth factor added to cultures on solid or porous supports caused proliferation of epithelial-type cells to give a confluent monolayer but did not increase the expression of differentiated function. The presence or absence of placental basement membrane collagen as substrate made little apparent difference; however a porous basement membrane collagen support led to increased differentiated function. Initial production of human chorionic gonadotrophin was increased and after 4 wk in culture a substantial proportion of the cells exhibited alkaline phosphatase activity. Epidermal growth factor and a substrate of placental basement membrane collagen on a porous support favorably influence the growth and differentiation of human trophoblast cells in culture. This work was supported by funds from the Medical Research Council of New Zealand which also provided support for Dr. Truman as a Postdoctoral Fellow.     

Restoration of cutaneous pigmentation by transplantation to mice of isogeneic human melanocytes in dermal–epidermal engineered skin substitutes

Autologous engineered skin substitutes (ESS) containing melanocytes (hM) may restore pigmentation and photoprotection after grafting to full‐thickness skin wounds. In this study, normal hM were isolated from discard skin, propagated with or without tyrosinase inhibitors, cryopreserved, recovered into culture, and added to ESS (ESS‐P) before transplantation. ESS‐P were incubated in either UCMC160/161 or UCDM1 medium, scored for hM densities, and grafted to mice. The results showed that sufficient hM can be propagated to expand donor tissue by 100‐fold; incubation of hM in tyrosinase inhibitors reduced pigment levels but did not change hM recovery after cryopreservation; hM densities in ESS‐P were greater after incubation in UCDM1 than UCMC160 medium; hM were localized to the dermal–epidermal junction of ESS‐P; and UCDM1 medium promoted earlier pigment distribution and density. These results indicate that hM can be incorporated into ESS‐P efficiently to restore cutaneous pigmentation and UV photoprotection after full‐thickness skin loss conditions.     

Induced pluripotent stem cell‐derived melanocyte precursor cells undergoing differentiation into melanocytes

Induced pluripotent stem cell (iPSC) technology offers a novel approach for conversion of human primary fibroblasts into melanocytes. During attempts to explore various protocols for differentiation of iPSCs into melanocytes, we found a distinct and self‐renewing cell lineage that could differentiate into melanocytes, named as melanocyte precursor cells (MPCs). The MPCs exhibited a morphology distinctive from that of melanocytes, in lacking either the melanosomal structure or the melanocyte‐specific marker genes MITF, TYR, and SOX10. In addition, gene expression studies in the MPCs showed high‐level expression of WNT5A, ROR2, which are non‐canonical WNT pathway markers, and its related receptor TGFβR2. In contrast, MPC differentiation into melanocytes was achieved by activating the canonical WNT pathway using the GSK3β inhibitor. Our data demonstrated the distinct characteristic of MPCs' ability to differentiate into melanocytes, and the underlying mechanism of interfacing between canonical WNT signaling pathway and non‐canonical WNT signaling pathway.     

Some differentiating effects of selenium on the cultured human hepatoma cells and human pulmonary adenocarcinoma cells in vitro

Two human hepatoma cell lines, QGY-7703 and SMMC-7721, and two human pulmonary adenocarcinoma cell lines, LTEP-a-2 and SPC-A-1, were found to respond to 1 μg/mL Na₂SeO₃, 24 h, in-vitro treatment by decreasing its confluent saturation density. The same treatment was found to cause an increase in the adhesiveness of cells measured as resistance to detachment by trypsin/EDTA. The pathological features of tumors after heterotransplantation of treated and untreated cells were similar, but the size of tumor grown from treated cells was much smaller.     

In vitro cultivation of human renal cell cancer

Summary A technique for initiating and propagating epithelial cell cultures of human renal cell cancer and adjacent nontumor kidney is described. Seventy-five percent of the tumors and 79% of the adjacent kidney specimens cultured with this method have shown initial outgrowth and have been subcultured at least once. Two renal cell cancer cultures initiated by this method have now been in continuous culture more than 6 months, have been subcultured 27 and 19 times, and now appear to be stable lines. The ability to establish long-term in vitro cultures of human renal cell cancers will facilitate studies concerning the immunoreactivity, cholesterol metabolism, the isolation of renal-cell-cancer-specific antigens, and in vitro chemotherapy testing and will further our understanding of the basic biology of human renal cell cancer. American Cancer Society Clinical Fellow 1975–1976. Supported in part by Grants CA 13095-03 and CA 15551-03 from the National Cancer Institute.     

An inactive X specific replication origin associated with a matrix attachment region in the human X linked HPRT gene

Early in female mammalian embryogenesis, one of the two X chromosomes is inactivated to compensate the gene dosage between males and females. One of the features of X chromosome inactivation (XCI) is the late replication of the inactivated X chromosome. This study reports the identification, by competitive PCR of nascent DNA, of a replication origin in intron 2 of the human X-linked HPRT gene, that is functional only on the inactive X. Features frequently associated with replication origins, including a peak of enhanced DNA flexibility, a perfect match to the yeast ACS sequence, a 14/15 match to the Drosophila topoisomerase II consensus, and a 20/21 match to an initiation region consensus sequence, were identified close to the replication origin. The origin is located approximately 2 kb upstream of a matrix attachment region (MAR) and also contains two A:T-rich elements, thought to facilitate DNA unwinding.     

Preservation of microplate-attached human hepatoma cells and their use in cytotoxicity tests

We investigated the feasibility of hypothermic- orcryogenically-preserved human hepatoma Hep G2 cell preculturedin 96-well plates in cytotoxicity testings. First, we observedthat microplates precoated with both collagen (CN) and pronectin (PN) showed significantly improved living cell adhesion (71.0 ± 5.5%) after 48 hr of cryopreservation with 10%-DMSO containing culture medium, whereas non-coated surfaces gave very low living cell adhesion (33.5 ± 2.1%). Hypothermic preservation was most suitable for short-term storage, and cryogenic preservation at –20 °C allowed cells to be used within a week of the storage period. Only cryopreservation in a deep freezer (–85 °C) gave satisfactory results in much longer period of storage. Second, we evaluated the cytotoxicity of ten chemicals during 48 hr of exposure using hypothermically – (4 °C for 2 days) or cryogenically – (–85 °C for 7 days) preserved cells cultured inCN/PN-precoated microplates in comparison with results fromfreshly inoculated cells. Although almost the same LD₅₀values were obtained, LD₁₀ values of relatively hydrophilic chemicals obtained with cryopreserved cell were significantly lowered. These results shown that CN/PN-precoating is effective in keeping cells attached even in recultivation of preserved cells and that the toxicities of relatively hydrophilic chemicals tend to be overestimated when we use preserved cells in that manner.     

牛乳腺核基质附着区的克隆及其功能研究

从奶牛的全血中提取基因组DNA,参照GenBank已有序列,通过Primer5.0和Vector 7.0辅助设计一对引物,克隆了牛乳腺核基质附着区(BMRs)。利用生物学软件对其进行初步分析后,TA克隆于pMD－18T vector上。上下游引物5′端分别引入Kpn2Ⅰ和XhoⅠ酶切位点,将所得的BMRs克隆到pEGFP-C1的报告基因下游,构建成表达载体BE,脂质体法转染牛耳成纤维细胞。以转染pEGFP-C1的牛耳成纤维细胞作为对照,发现所克隆的BMRs功能明显,在消除位置效应、增强报告基因表达方面具有一定的作用。     

Expression of α2-macroglobulin receptor-associated protein in normal human epidermal melanocytes and human melanoma cell lines

α₂-Macroglobulin receptor/low-density lipoprotein receptor-related protein is a multifunctional cell surface receptor known to bind and internalize a large number of ligands. α₂-Macroglobulin receptor-associated protein acts as an intracellular “chaperone” for this receptor, and it has been shown to inhibit binding of all its known ligands. In this paper, we characterize the expression of the receptor-associated protein in both normal human epidermal melanocytes and in six different human melanoma cell lines, by the use of flow cytometry and Western blotting analysis. We show that all the melanoma cell lines and the normal melanocytes express the receptor-associated protein at similar levels, with most located intracellularly. No receptor-associated protein was detected at the cell surface in the melanocytes or in three of the cell lines. However, in two of the melanoma cell lines, large amounts of receptor-associated protein were found on the cell surface, these having the largest amounts of it reported to date; in a further melanoma cell line, there was a small amount at the cell surface. We have also shown that the melanocytes and all the melanoma cell lines express the receptor itself at a wide range of levels, the highest levels of both the cell surface receptor and the cell surface receptor-associated protein being found in one particular melanoma cell line. By growing the cell lines under controlled conditions, we have demonstrated that, although the total cellular content of the receptor is markedly increased at high cell culture density, this treatment has no effect on the level of expression of the receptor-associated protein. J. Cell. Biochem. 71:149–157, 1998. © 1998 Wiley-Liss, Inc.