Nobuko Akutsu

Response to growth factors of human dermal fibroblasts in a quiescent state owing to cell-matrix contact inhibition.

Mitogenic responses to various growth factors were compared for quiescent human dermal fibroblasts cultured under three different conditions; serum depletion, cell-cell contact inhibition and cell-matrix contact inhibition. The non-dividing fibroblasts cultured under a low serum condition (0.2% fetal bovine serum, FBS) or in a confluent culture with 10% FBS resumed multiplying upon exposure to any one of or any combination of the growth factors examined; epidermal growth factor (EGF), basic fibroblast growth factor (b-FGF), platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta). The only exception was the lack of effect of TGF-beta on the cell under a low serum condition. In contrast, the proliferation of fibroblasts which were growth-arrested in contracted collagen gel by cell-matrix contact inhibition was not stimulated by any of the growth factors examined except for PDGF. It is currently accepted that the mechanism of growth stimulation or signal transduction after binding of each growth factor to the specific receptor depends on the kind of growth factor. The results suggest that the signal transductions delivered by EGF, b-FGF or TGF-beta are inactivated by a high level of interaction of collagen fibrils with the cell membrane (under the condition of cell-matrix contact inhibition); whereas the signal transduction by PDGF is unaffected. The finding supports the existence of a specific growth stimulation pathway for PDGF.

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)

Effect of type XII or XIV collagen NC‐3 domain on the human dermal fibroblast migration into reconstituted collagen gel

Abstract: Type XII and XIV collagens localize near the surface of banded collagen fibrils and most likely work as a molecular bridge between collagen fibrils. We have shown that both collagens can modulate the interactions between collagen fibrils, allowing fibroblasts to act upon the fibrils to vary the deformability. In the present study the effect of the globular domains (collagenase‐resistant domains) of type XII and XIV collagens (XII‐NC‐3 and XIV‐NC‐3) on the migration of fibroblasts into the reconstituted type I collagen gel was investigated. Cell attachment and proliferation on the collagen gel were unaffected. The migration of fibroblasts into the gel was increased proportionally to the concentration of collagen. We found that XII‐NC‐3 and XIV‐NC‐3 domains caused decreases in the numbers of fibroblasts that migrated into the gel. Heat treatment of XII‐NC‐3 and XIV‐NC‐3 or the addition of polyclonal antibodies eliminated the suppressive activity on fibroblast migration, showing that the intact conformation of NC‐3 domain is important for suppression of migration. The results suggest that both NC‐3 domains influence the deformability of type I collagen fibril networks, which may cause the change in fibroblast migration.

Quantitative analysis of the synthesis and secretion of type VII collagen in cultured human dermal fibroblasts with a sensitive sandwich enzyme-linked immunoassay

Abstract:  Type VII collagen is the major component of anchoring fibrils in the epidermal basement membrane. Its expression has been analyzed by immunostaining or Northern blotting, but rarely at the protein level. In this study, we have quantitatively examined the effects of ascorbic acid and various cytokines/growth factors on the protein synthesis and secretion of type VII collagen by human dermal fibroblasts in culture, using a developed, highly sensitive sandwich enzyme‐linked immunoassay with two kinds of specific monoclonal antibodies against the non‐collagenous domain‐1. Ascorbic acid and its derivative induced a twofold increase in type VII collagen synthesis, and markedly increased the secretion of type VII collagen into the medium when compared with the control culture. This effect was not influenced by the presence of transforming growth factor‐β1 (TGF‐β1). The synthesis of type VII collagen was elevated by TGF‐β1, platelet‐derived growth factor, tumor necrosis factor‐α, and interleukin‐1β, but not by TGF‐α. Thus, our data indicate that the synthesis and secretion of type VII collagen in human dermal fibroblasts are regulated by ascorbate and the enhancement of type VII collagen gene expression by cytokines/growth factors is accompanied with elevated production of type VII collagen at the protein level.

Quantitative analysis of laminin 5 gene expression in human keratinocytes.

Abstract:  To examine the expression of laminin 5 genes (LAMA3, LAMB3, and LAMC2) encoding the three polypeptide chains α3, β3, and γ2, respectively, in human keratinocytes, we developed novel quantitative polymerase chain reaction (PCR) methods utilizing Thermus aquaticus DNA polymerase, specific primers, and fluorescein‐labeled probes with the ABI PRISM™ 7700 sequence detector system. Gene expression levels of LAMA3, LAMB3, and LAMC2 and glyceraldehyde‐3‐phosphate dehydrogenase were quantitated reproducibly and sensitively in the range from 1 × 102 to 1 × 108 gene copies. Basal gene expression level of LAMB3 was about one‐tenth of that of LAMA3 or LAMC2 in human keratinocytes, although there was no clear difference among immunoprecipitated protein levels of α3, β3, and γ2 synthesized in radio‐labeled keratinocytes. Human serum augmented gene expressions of LAMA3, LAMB3, and LAMC2 in human keratinocytes to almost the same extent, and this was associated with an increase of the laminin 5 protein content measured by a specific sandwich enzyme‐linked immunosorbent assay. These results demonstrate that the absolute mRNA levels generated from the laminin 5 genes do not determine the translated protein levels of the laminin 5 chains in keratinocytes, and indicate that the expression of the laminin 5 genes may be controlled by common regulation mechanisms.

Repeated folding stress-induced morphological changes in the dermal equivalent

Repeated mechanical stresses applied to the same region of the skin are thought to induce morphological changes known as wrinkle. However, the underlying mechanisms are not fully understood. To study the mechanisms, we examined effects of repeated mechanical stress on the dermal equivalent.