Keratinocyte Extracellular Matrix-Mediated Regulation of Normal Human Melanocyte Functions

Active roles of cell-cell interaction between melanocytes and neighboring keratinocytes for the regulation of melanocyte functions in the skin have been suggested. We examined substantial regulatory mechanisms of keratinocyte extracellular matrix (kECMs) for normal human melanocyte functions without direct cell-cell contact. We specially devised kECMs from proliferating or differentiating keratinocytes and further treated them with environmental stimulus ultraviolet B (UVB) for skin pigmentary system. Normal human melanocytes (NHM) were cultured on the various keratinocyte ECMs and initially the effects of the kECMs upon melanocyte morphology (dendrite formation and extension), growth, melanin production and expressions of pigmentation-associated protein (MEL-5) and proliferation-associated protein (proliferating cell nuclear antigen; PCNA/cyclin) were studied. Then we compared the effects of these cell-matrix interactions with those of direct melanocyte-keratinocyte, cell-cell contact in co-culture on melanocyte functions. Melanocytes cultured on any types of the kECMs that were tested significantly extended dendrites more than that on plastic cell culture dish without kECM (control). Melanocytes cultured on the kECM prepared from UVB irradiated differentiating keratinocytes resulted in 219% increase in the number of dendrites. The growth of melanocytes on kECMs was also stimulated up to 280% of control. The kECM produced by proliferating keratinocytes had a more significant effect on the growth than kECM from differentiating keratinocytes. This melanocyte growth stimulating effect was decreased with kECM from UVB treated differentiating keratinocytes. The melanin content per melanocyte was constant on any of the kECMs. Expression of pigmentation-associated protein detected by monoclonal antibody, MEL-5, was not changed on the kECM, while it was increased in melanocytes in co-culture with keratinocytes. Expression of PCNA/cyclin in melanocytes cultured on kECMs was generally downregulated on kECM and in co-culture compared to that in a control culture. We demonstrated that the kECMs play important roles in the melanocyte morphology and proliferation. These observations suggest that environmental (UVB) and physiological (Ca⁺⁺) stimuli can regulate melanocyte functions through the keratinocyte extracellular matrix in vivo.     

Another Origin of Coloniality in Volvocaleans: The Phylogenetic Position of Pyrobotrys Arnoldi (Spondylomoraceae,Volvocales)

ABSTRACT. Colonial volvocaleans (Chlorophyceae) are used as a standard model of multicellular evolution. However, the phylogenetic position of the colonial volvocalean family Spondylomoraceae has yet to be resolved. To examine this, the molecular phylogenies of Pyrobotrys stellata and Pyrobotrys squarrosa were analyzed using combined 18S rRNA, RUBISCO large subunit, and P700 chl a‐apoprotein A2 gene sequences. In the phylogenetic trees, Pyrobotrys belonged to the clade Caudivolvoxa and was not closely related to other colonial volvocalean flagellates. The results indicate that colony formation of Spondylomoraceae independently evolved from unicellular volvocaleans. The phylogenetic position of problematic “Pascherina tetras” SAG 159‐1 was also analyzed.     

Late Jurassic decapod crustaceans from northeast Japan

Abstract: Astacidean and thalassinidean macrurans (Glyphea sp., ?Eryma sp. and Protaxius sp.) and a new longodromitid crab, Planoprosopon kashimaensis, are recorded from the Upper Jurassic (upper Kimmeridgian to lower Tithonian) of Fukushima Prefecture, northeast Japan. Material was collected from the Tatenosawa Sandstone Member of the Nakanosawa Formation, Somanakamura Group, from which abundant Tethyan‐type marine invertebrates are known. Planoprosopon kashimaensis sp. nov. closely resembles P. heydeni (von Meyer), a common form in the Upper Jurassic of the Tethyan realm in Europe, and represents the oldest record of a brachyuran from the circum‐Pacific region. Similarities to contemporaneous decapod assemblages in southern Germany indicate that closely comparable, parallel decapod faunas in the Tethyan realm, inclusive of brachyurans, had already been established in the western circum‐Pacific region by the Late Jurassic.     

Pigmented Human Skin Equivalent: New Method of Reconstitution by Grafting an Epithelial Sheet Onto a Non-Contractile Dermal Equivalent

We have established a new protocol for reconstituting a pigmented human skin equivalent (PSE) and have evaluated its functional responses to environmental stimulus, UVB. The PSE is reconstituted by grafting an epithelial sheet consisting of keratinocytes and melanocytes onto a porous non-contractile dermal equivalent populated with mitotically and metabolically active fibroblasts. i) The PSE has a multilayered, well-differentiated epidermis with cuboidal basal cells and highly organised dermis with newly synthesised extracellular matrix components. ii) Ki67-positive proliferating keratinocytes (18.1 ± 7.4%) were detected on the basal layer of the epidermis. iii) Melanocytes located exclusively within the basal layer were detected by monoclonal antibody against tyrosinase-related protein (TRP-1). iv) After exposure to UVB (100 mJ/cm² per day) for 7 consecutive days, the intensity of TRP-1 staining was increased in the PSE, showing their functional state, whereas the number of melanocytes was not changed. This non-contractile and functioning new PSE is potentially useful as a model for studying the role of melanocyte-keratinocyte-fibroblast interactions in photoprotection of the skin in more complex cutaneous microenvironment than monolayer culture, and for developing in vitro disease models and therapeutic protocols with genetically altered cells both in epidermis and dermis.     

Prothoracicotropic Hormone Bioassay: Pupal-Adult Bombyx Assay

Blockage of adult development by brain removal and its resumption by application of the prothoracicotropic hormone (PTTH) were studied using pupae of a racial hybrid J-122 × C-115 of Bombyx mori . A log-linear dose-response relationship was obrained after injection of a PTTH solution. The Bombyx -unit of PTTH has been defined from this dose-response curve.     

Species Specificity of the Insect Prothoracicotropic Hormone (PITH): the Presence of Bombyx-and Samia-Specific PTTHs in the Brain of Bombyx mori

Crude extracts of Bombyx mori brains can provoke adult development when injected into brain-removed dormant pupae of Bombyx mori and Samia Cynthia ricini. From this fact the prothoracicotropic hormone (PTTH) of Bombyx has long been thought to be species-nonspecifically active on Samia. Chemical fractionation of Bombyx brain or head extracts by fractional precipitation with acetone, Sephadex G-50 gel-filtration, and DEAE-Sepharose CL-6B chromatography, however, separated the fractions which activated Bombyx brainless pupae from those which activated Samia. Those results reveal the existence of two species-specific PTTHs.     

Arsenic alters uptake and distribution of sulphur in Pteris vittata

Low‐molecular‐weight thiol (LMWT) synthesis has been reported to be directly induced by arsenic (As) in Pteris vittata, an As hyperaccumulator. Sulphur (S) is a critical component of LMWTs. Here, the effect of As treatment on the uptake and distribution of S in P. vittata was investigated. In P. vittata grown under low S conditions, the presence of As in the growth medium enhanced the uptake of SO₄^2?, which was used for LMWT synthesis in fronds. In contrast, As application did not affect SO₄^2? uptake in Nephrolepis exaltata, an As non‐hyperaccumulator. Moreover, the isotope microscope system revealed that S absorbed with As accumulated locally in a vacuole‐like organelle in epidermal cells, whereas S absorbed alone was distributed uniformly. These results suggest that S is involved in As transport and/or accumulation in P. vittata. X‐ray absorption near‐edge structure analysis revealed that the major As species in the fronds and roots of P. vittata were inorganic As(III) and As(V), respectively, and that As–LMWT complexes occurred as a minor species. Consequently, in case of As accumulation in P. vittata, S possibly acts as a temporary ligand for As in the form of LMWTs in intercellular and/or intracellular transport (e.g. vacuolar sequestration).     

Effects of Calphostin C,Specific PKC Inhibitor on TPA-Induced Normal Human Melanocyte Growth,Morphology and Adhesion

Normal human melanocytes, which rarely undergo mitosis in vivo, require many growth factors and growth-stimulating agents in vitro, such as basic fibroblast growth factor (bFGF) and cyclic adenosine monophosphate-stimulating agents or 12-0-tetrade-canoylphorbol 13-acetate (TPA), to proliferate. TPA, known as a protein kinase C (PKC)-activator, supports normal human melanocyte growth and influences on melanocyte dendrite formation. We have further confirmed the role of the PKC-mediated pathway in the TPA-dependent melanocyte functions—i.e., proliferation, morphology, and adhesion—using Calphostin C (CPC), a highly specific PKC inhibitor. Melanocytes require the continual presence of TPA for growth in culture. Addition of 8 nM TPA to the medium increased melanocyte growth by 198.4 ± 2.3% of that without TPA. The growth induction by TPA was suppressed by the addition of 10 nM CPC at the level comparable to that without TPA without any morphological alterations. Significant levels of PKC were detected in melanocytes chronically exposed to TPA as determined by Western blotting. A long-term exposure to TPA (more than 5 days) resulted in marked reduction of melanocyte adhesion to plastic cell culture dishes, both uncoated and coated with type IV collagen. By the addition of 10 nM CPC in the adhesion assay, the melanocyte adhesion was further inhibited in both conditions. These results indicated the critical involvement of PKC activation in the TPA-dependent melanocyte functions. Continuous activation of PKC by TPA is implicated in melanocyte growth stimulation. TPA also has effects on melanocyte morphology, causing the formation of long extended dendrites with little cytoplasm. However, inhibition of PKC activation by CPC does not affect the melanocyte morphology, and CPC reduces melanocyte adhesion to uncoated or type IV collagen coated plastic cell culture dishes.     

Ca2+ and UVB Radiation Have No Effect on E-Cadherin-Mediated Melanocyte-Keratinocyte Adhesion

Direct cell-cell contact between melanocytes and keratinocytes has been shown to play an important role in the regulation of human melanocyte function and skin pigmentation. An important role for the calcium-dependent epithelium-specific cell adhesion molecule, E-cadherin, in melanocyte-keratinocyte adhesion was suggested previously. To further clarify regulation of E-cadherin-mediated melanocyte-keratinocyte interactions, we investigated the effects of physiological (Ca²⁺) and environmental (ultraviolet B [UVB] radiation) stimuli on the expression and functional activity of E-cadherin in melanocyte-keratinocyte adhesion. Expression of E-cadherin mRNA was detected by Northern blot analysis in cultured normal human melanocytes at levels similar to those in keratinocytes. Flow cytometry analysis with anti-human and anti-mouse-E-cadherin antibodies (anti-uvomorulin and ECCD-2) showed that cultured normal human keratinocytes, melanocytes, and two metastatic melanoma cell lines express E-cadherin strongly on the cell surfaces. Melanocyte adhesion, particularly to differentiating keratinocytes (cultured in 1.2 mM calcium) but not to proliferating keratinocytes or to fibroblasts, was decreased by 41.7 ± 4.5% in the absence of 1 mM Ca²⁺ during the binding assay. Addition of anti-mouse-E-cadherin antibody (ECCD-1) to the binding assay inhibited the adhesion of melanocytes to differentiating keratinocytes by 88.2 ± 1.1%, while addition of anti-P-cadherin antibody (PCD-1) had no effect. The levels of E-cadherin expression in melanocytes were not changed by the presence of calcium (1 mM) in the medium or by UVB irradiation (20 mJ/cm²) for one day before flow cytometry analysis. Moreover, these treatments had no effect on melanocyte-keratinocyte adhesion. These results demonstrate that E-cadherin is strongly involved in melanocyte adhesion to keratinocytes and suggest the implication of E-cadherin in the overall regulation of the skin pigmentary system.