Izumi Horii

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.

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.

HISTIDINE-RICH PROTEIN AS A POSSIBLE ORIGIN OF FREE AMINO ACIDS OF STRATUM CORNEUM

Pulse‐chase labeling experiments with 3H‐histidine and 3H‐arginine were performed in hairless mice to investigate the origin of free amino acids in the stratum corneum. Time‐course labeling of epidermal proteins was examined by sodium dodecyl sulfate‐polyacryl amide gel electrophoresis (SDS‐PAGE). The radioactivity was also measured in the following three epidermal fractions; 0.1 N HClO4 soluble‐ethanol soluble fraction (Fr. I), 0.1 N HClO4 soluble‐ethanol insoluble fraction (Fr. II, histidine‐rich protein fraction) and 0.1 N HClO4 insoluble‐8M urea soluble fraction (Fr. III).

A Distinct Characteristic of the Quiescent State of Human Dermal Fibroblasts in Contracted Collagen Gel as Revealed by No Response to Epidermal Growth Factor Alone, But a Positive Growth Response to a Combination of the Growth Factor and Saikosaponin b1

It is known that fibroblasts cultured in a reconstituted collagen gel contract the gel, resulting in a high density of collagen fibrils comparable to that in dermis. Our previous study indicated that human fibroblasts in the contracted collagen gel did not proliferate in the presence of 10% serum even though there was no apparent cell-cell contact. We interpreted this cell growth inhibition as being caused by a high level of cell-collagen fibril interactions or cell-matrix contact inhibition. In the present study, the effect of epidermal growth factor (EGF) on fibroblast proliferation in the contracted collagen gel was compared with that on cells in other quiescent states. Non-dividing cells at confluency on a plastic dish or on collagen gel responded to the added EGF and multiplied, while the cells in the contracted collagen gel did not show any growth response to EGF at concentrations up to 100 ng/ml. Binding assay of [125I]-EGF to the cells showed that the number of binding sites and the binding constant obtained from Scatchard analysis were essentially unchanged in the contracted collagen gel, indicating that EGF receptors were not masked by collagen fibrils but that the signal transduction after binding of EGF was blocked. The block in the signal transduction was suppressed by the addition of saikosaponin b1. These results suggested that the quiescent fibroblasts in the contracted collagen gel were in a distinct state from previously known quiescent states of cultured cells, namely quiescent states due to cell-cell contact inhibition at confluency or to deficiency of growth factors. The mechanism of the effect of saikosaponin b1, which has a potent saponin activity, is discussed.

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 column length and elution mechanism on the separation of proteins by reversed-phase high-performance liquid chromatography

Abstract Different theories have been proposed for the elution of proteins in reversed-phase high-performance liquid chromatography. To establish the correct elution mechanism, the effects of column length and the concentration of the organic solvent on column efficiency and the elution of high- and low-molecular-weight compounds were examined. It was concluded that protein elution principally involves the same retention process as for low-molecular-weight compounds, although the influence of partition is small under steep gradient conditions. In accordance with this, wide-pore packings in a short column (35 mm) gave excellent separations of proteins and were usable with a wide range of gradients.

Effect of hyaluronate on physicochemical and biological properties of collagen solution which could be used as collagen filler

The effect of hyaluronate (HA) on the physiochemical and biological properties of collagen solution was examined for two preparations of collagen with different rates of fibril formation. The addition of HA to the collagen preparation with the slower rate of fibril formation caused a prominent acceleration of fibril formation. A differential scanning calorimetric measurement of the collagen preparation demonstrated a stabilizing effect of HA on collagen solution after incubation at 37 degrees C. Histochemical examination of rat dermis after injection of the collagen solution into the tissue revealed the migration of fibroblast-like cells into the region occupied by the injected collagen. The addition of HA to collagen preparation S (slower rate of fibril formation) shortened the time-to-appearance of fibroblast-like cells to a similar value to that observed when collagen preparation F (faster rate of fibril formation) was used. The timing of cell appearance was in accord with the rate of fibril formation in vitro. Fibrils newly formed by injected collagen might provide sites for cell attachment, migration and proliferation.