Makoto Tsunenaga

Growth and Differentiation Properties of Normal and Transformed Human Keratinocytes in Organotypic Culture

The growth and differentiation of human normal keratinocytes and their transformed counterparts were examined in organotypic cultures in which the keratinocytes were grown at the air‐liquid interface on top of contracted collagen gel containing fibroblasts. We developed a modified culture procedure including the use of a mixed medium for keratinocytes and fibroblasts. Normal keratinocytes formed a three‐dimensional structure of epithelium that closely resembled the epidermis in vivo, consisting of basal, spinous, granular and cornified layers. Cells synthesizing DNA were located in the lowest basal layer facing the collagen gel. Expressions of proteins involved in epidermal differentiation were examined by immunohistochemical staining and compared with those in skin in vivo. In the organotypic culture, transglutaminase, involucrin and filaggrin were expressed, as in the epidermis in vitro, most prominently in the granular layer. Type IV collagen, a component of basement membrane, was expressed at the interface between the keratinocyte sheet and the contracted collagen gel. Keratinocytes transformed by simian virus 40 or human papilloma virus (HPV) exhibited a highly disorganized pattern of squamous differentiation. In particular, HPV‐transformed cells invaded the collagen gel. Organotypic culture is unique in that regulatory mechanisms of growth and differentiation of keratinocytes can be investigated under conditions mimicking those in vivo.

Regulation of creatine phosphokinase B activity by protein kinase C

We previously reported that topical application of 12-o-tetradecanoylphorbol-13-acetate to mouse skin causes phosphorylation of epidermal proteins with molecular weights of 40,000 (p40) and 34,000 (p34). In the accompanying paper, p40 was identified as creatine phosphokinase B. Here we report that both in intact cells and in a cell-free system, phosphorylation of creatine hosphokinase B by protein kinase C resulted in an increase in its ability to catalyze the transfer of the high-energy phosphate of phosphocreatine to ADP, thereby producing ATP. H-7, a specific inhibitor of protein kinase C was found to abolish the increase in enzyme activity. Lineweaver-Burk plot analysis indicated that the increased activity was mostly due to a decreased Km for phosphocreatine. Phosphorylation and activation of creatine phosphokinase B may be a physiological response to maintain ATP balance when a protein kinase C pathway is stimulated.

Purification and identification of creatine phosphokinase B as a substrate of protein kinase C in mouse skin in vivo

We previously described epidermal proteins with molecular weights of 40,000 (p40) and 34,000 (p34) as target proteins of protein kinase C in mouse skin carcinogenesis in vivo. In the present work, p40 was purified from mouse brain by the use of 32P-labeled p40 of BALB/MK-2 cells as a tracer. Following four lines of evidence indicate that p40 is creatine phosphokinase B. 1) The amino acid sequences of all peptide fragments of p40 from mouse brain were located in the primary structure of creatine phosphokinase B. 2) p40 of BALB/MK-2 cells was immunoprecipitated with goat antibody against human creatine phosphokinase B. 3) p40 of BALB/MK-2 cells was absorbed to and eluted from a creatine affinity column. 4) Purified creatine phosphokinase B was phosphorylated in vitro by purified protein kinase C, but not by cAMP-dependent kinase or casein kinase II.

Dissociation of actin microfilament organization from acquisition and maintenance of elongated shape of human dermal fibroblasts in three-dimensional collagen gel

Actin microfilaments of the fibroblasts cultured in a collagen gel were distributed along the inner surface of the entire cell membrane, in either spherical shape at an initial stage of culture or elongated shape at a later stage. The distribution was quite different from that of the fibroblast cultured on a two-dimensional surface, where actin microfilaments were found to be aligned essentially along the inner membrane which is in contact with a flat surface. Timing of morphological change from spherical shape to spread shape or elongated shape was also greatly affected by contact with substrates whether in two-dimension or in three-dimension: distinct morphological change was observed within 6 h on glass or on the collagen gel, and at 30 h or later within the collagen gel. The retardation of cell elongation in the gel was antagonized by a low dose (0.2 microM) of cytochalasin D, although the drug kept the cells in round shape at a concentration of 2 microM. Since a low concentration of cytochalasin was reported to induce actin polymerization in vitro, the organization of actin microfilaments was examined by rhodamine-phalloidin staining. It was found that actin filaments in elongated cells by low cytochalasin D were disrupted. These results suggest that accelerated acquisition of elongated shape by the treatment of a low dose of cytochalasin D might be initiated by destabilization of the actin microfilaments that may scaffold the spherical shape of the cell in the collagen gel. The elongated shape thus formed returned to spherical upon washing of the added free cytochalasin D.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured human dermal papilla cells secrete a chemotactic factor for melanocytes

Large numbers of pigmented melanocytes are located in human hair follicles, predominantly around the dermal papillae, and the number of melanocytes and the melanogenic activity of the hair follicles are closely related to the hair cycle. We found that cultured human dermal papilla cells secreted a melanocyte chemoattractant into the medium. Skin fibroblasts also showed weak chemoattraction of melanocytes, while skin keratinocytes and melanocytes did not. Since this chemotactic activity was heat-and protease-sensitive and was present in the relatively high molecular weight fraction (130-200 kDa), it may be due to extracellular matrix (ECM) that proteins secreted from the cultured dermal papilla cells. This chemotactic signal between dermal papillae and melanocytes may control the localization and migration of hair melanocytes in vivo.

Influence of heparan sulfate chains in proteoglycan at the dermal-epidermal junction on epidermal homeostasis.

Abstract:  Basement membrane (BM) plays important roles in skin morphogenesis and homeostasis by controlling dermal–epidermal interactions. However, it remains unclear whether heparan sulfate (HS) chains of proteoglycan in epidermal BM contribute to epidermal homeostasis. To explore the function of HS chains at the dermal–epidermal junction (DEJ), we used a skin equivalent (SE) model. This model lacked HS at the DEJ and showed abnormal expression of the differentiation markers filaggrin and loricrin; similar changes were seen in ultraviolet B‐irradiated human skin. Perlecan (core‐protein of HS proteoglycan) remained localized at the DEJ in both SE and UV‐irradiated human skin. Heparanase, which degrades HS, was increased in epidermis of UV‐irradiated skin, compared with unirradiated skin. We found that deposition of HS at the DEJ in the SE model was markedly augmented by a synthetic heparanase inhibitor, and release of HS into conditioned medium was suppressed. The inhibitor also increased filaggrin and loricrin expression. Moreover, the recovery of HS was associated with an increase of Ki67‐positive basal cells, compared with control SE cultured without inhibitor. Comparative gene expression analysis in epidermis of SE cultured in the presence and absence of heparanase inhibitor, using DNA microarrays, showed that recovery of HS was associated with increased expression of differentiation‐related genes and down‐regulation of degradation‐enzyme‐related genes. These results indicate that degradation of HS at the DEJ by heparanase impairs epidermal homeostasis in SE, leading to abnormal differentiation and proliferation behaviour. Thus, HS chains in epidermal BM appear to play an important role in epidermal homeostasis.

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.

Changes in the expression of epidermal differentiation markers at sites where cultured epithelial autografts were transplanted onto wounds from burn scar excision

This study investigated the recovery process during which grafted cultured epithelium formed normal epidermis. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) after transplantation. These samples were stained for monoclonal antibodies against filaggrin, transglutaminase (TG), cytokeratin 6 and involucrin. Their expressions were examined in the epidermis. The expression patterns were classified using a six‐grade scale. The grades of filaggrin and TG were significantly higher at stage 3 and 4 compared with stage 1. There was a marginally significant increase in the grade of cytokeratin 6 at stage 3 and it was significantly higher at stage 4 compared with stage 1. These results showed that wound healing continued at a molecular level until the end of stage 3. The recovery of involucrin was delayed compared with that of other markers. TG and involucrin are thought to be regulated independently at the grafted sites.

Changes in fibrillin-1 expression, elastin expression and skin surface texture at sites of cultured epithelial autograft transplantation onto wounds from burn scar excision

This study investigated the recovery process during which grafted cultured epithelium generated skin elasticity and skin surface microarchitecture. The subjects were 18 patients whose burn scars were excised at a depth not exposing the fat layer and who subsequently received cultured epithelial autografts. A total of 24 samples were obtained from the grafted sites: 6 samples within 6 weeks (stage 1), 5 samples after 6 weeks and within 6 months (stage 2), 6 samples after 6 months and within 18 months (stage 3) and 7 samples beyond 18 months (stage 4) of transplantation. These samples were evaluated by taking replicas of skin surface, and histological changes of fibrillin‐1 and elastin. The expression patterns were classified using a grading scale. The grade of skin surface texture was significantly higher at stage 3 and marginally significantly higher at stage 4 compared with stage 1. The grade of fibrillin‐1 was marginally significantly higher at stage 3 and significantly higher at stage 4 compared with stage 1. The grade of elastin was marginally significantly higher at stage 4 compared with stage 1. These results showed that it is important for patients to have skin care and avoid external forces for at least 18 months after transplantation.