1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation.

Human foreskin keratinocytes in vitro metabolize 25-hydroxyvitamin D3 to a number of metabolites, including 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). This metabolite remains mostly within the cell and does not accumulate in the medium under the conditions of these experiments. With time, 1,25(OH)2D3 is catabolized, and more polar metabolites appear in both the cells and the medium. The production of 1,25(OH)2D3 has an apparent Michaelis constant (Km) for 25-hydroxyvitamin D3 of 5.4 X 10(-8) M. The levels of 1,25(OH)2D3 within the cell are increased both by increased production and decreased catabolism when parathyroid hormone(1-34) and isobutylmethylxanthine are added. Exogenously added 1,25(OH)2D3 at concentrations as low as 10(-12) M reduces endogenous 1,25(OH)2D3 production, increases 1,25(OH)2D3 catabolism, and increases 24,25-dihydroxyvitamin D3 production by an actinomycin D-sensitive process. These data indicate that the regulation of 1,25(OH)2D3 production by keratinocytes is similar to, but not identical to the regulation of 1,25(OH)2D3 by the kidney.

Neonatal human foreskin keratinocytes produce 1,25-dihydroxyvitamin D3.

Primary cultures of neonatal human foreskin keratinocytes converted 25-hydroxyvitamin D in high yield to a metabolite with the chromatographic behavior of 1,25-dihydroxyvitamin D3. The identity of this metabolite as 1,25-dihydroxyvitamin D3 was confirmed both by its potency in displacing 1,25-dihydroxyvitamin D3 in the chick cytosol receptor assay and by mass spectral analysis. These results suggest that 1,25-dihydroxyvitamin D3 may be formed in the epidermis to regulate vitamin D production by the epidermis and to provide an alternative to 1,25-dihydroxyvitamin D3 production by the kidneys.

In vitro synthesis of vitamin D-3 by cultured human keratinocytes and fibroblasts: action spectrum and effect of AY-9944.

With delineation of the photochemical events occurring in the skin after ultraviolet exposure, there has been increased interest in the skin's role in the vitamin D-3-endocrine system. We provide here in vitro conditions for the generation of both labelled (from [3H]acetate) and unlabelled vitamin D-3 in cultures of human keratinocytes and fibroblasts. Sterol precursors and photoproducts in irradiated and non-irradiated cultures are identified by co-chromatography, ultraviolet absorbance spectra, thermal conversion characteristics of previtamin D-3 and mass spectrometry. Because the conversion of 7-dehydrocholesterol to cholesterol is more efficient in vitro than in vivo, the specific delta 7 inhibitor, AY-9944, was added in non-toxic doses to modulate 7-dehydrocholesterol content. Both cell types were equally capable of generating photoproducts, depending on the amount of 7-dehydrocholesterol present. The 290 +/- 5 and 295 nm filters were much more efficient than the 305 nm filter for generating previtamin D-3 and vitamin D-3 in fibroblasts. In contrast, the 305 nm filter was as efficient as the 290 +/- 5 and 295 nm filters in keratinocytes, where it yielded previtamin D-3, with much less lumisterol and tachysterol than appeared with the shorter-wavelength filters. The amount of lumisterol and tachysterol versus previtamin D-3 formed in both cell types was dependent on the total energy applied, with lower energies (less then 1 J/cm2) favoring previtamin D-3 over the other photoproducts. The use of cultured cells provides a system whereby the regulation of vitamin D-3 synthesis by extracutaneous factors can be studied in a homogeneous setting.

Vitamin D3 production by cultured human keratinocytes and fibroblasts.

We have demonstrated that monolayers of human cultured newborn foreskin keratinocytes and fibroblasts elaborate vitamin D3 following exposure to UV-B. This in vitro system provides a new means to study those factors (hormones, ions, vitamin D3 metabolites, etc.) that regulate the production of vitamin D3 by human skin cells. Vitamin D3 production was enhanced greatly by using cells that were pre-treated with AY-9944, a non-toxic drug that inhibits cholesterologenesis while elevating cellular levels of 7-dehydrocholesterol, the sterol precursor of vitamin D3. The pre-D3 formed within viable, irradiated cells is transformed to D3 within a matter of hours at 37 degrees C, and keratinocytes proved to be more proficient sources of the vitamin and its metabolites than corresponding skin fibroblasts.

Membrane glycoconjugate visualization and biosynthesis in normal and retinoid-treated epidermis.

Cell-membrane glycoconjugates can be visualized for ultrastructural and fluorescence studies with certain surface markers, such as lectins and antisaccharide antibodies. When frozen sections of mammalian epidermis are treated with a battery of rhodamine-conjugated lectins, the cell membranes display a pattern of increased sugar complexity during keratinocyte maturation. Although this vectorial sequence is disrupted following retinoid treatment, these changes occur only at high doses and late in the course of treatment, suggesting that retinoid-induced alterations may be secondary. Seemingly as a result of specific glycosidase activity within the cytosol of both granular and cornified cells, lectin staining suddenly disappears from stratum corneum cell membranes. Although lectins stain membrane glycoconjugates of cultured human keratinocytes, quantitative techniques are required to recognize differences in proliferating versus postmitotic cells and in cultures supplemented with various growth factors. Whereas retinoids consistently depress glycoprotein synthesis in cultured keratinocytes, in organ culture they stimulate epidermal glycoprotein, and particularly glycolipid, biosynthesis. These studies suggest (1) that visualization of membrane glycoconjugates with lectins can reveal important variations in normal and pathologic epidermal differentiation, (2) that lectins may reveal subtle quantitative alterations in differentiation in vitro, (3) that retinoid stimulation of glycoconjugate biosynthesis either displays important species differences or requires a higher level of organization than occurs in cell culture, and (4) that the retinoid effect on glycoconjugate biosynthesis in organ culture may provide another useful bioassay for retinoid potency.

Alterations in membrane sugars during epidermal differentiation: visualization with lectins and role of glycosidases.

Differentiation in keratinizing epithelia involves the orderly transformation of basal germinal cells into an exterior cornified layer. We have employed rhodamine-conjugated lectins to visualize distinctive changes in the localization of keratinocyte membrane glycoconjugates during epidermal differentiation. The dermis, basement membrane, and epidermal cell membranes stained positively for mannose, alpha- and beta-galactose, N-acetyl-glucosamine, and sialic acid. In contrast, only the viable epidermis demonstrated N-acetyl-galactosamine, while alpha-L-fucose staining was limited to the upper stratum spinosum and stratum granulosum. Neuraminidase treatment extended the binding of certain lectins, e.g., peanut agglutinin, to regions of the skin that otherwise did not label. Whereas the granular cell membranes displayed the largest number of carbohydrates, these sugars could no longer be visualized after granular cells differentiated into the stratum corneum. Loss of lectin staining may be attributable to the presence of a family of sugar-specific glycosidases that we obtained from granular and cornified cell cytosol fractions. Finally, as further support for sugar deletion during cornification, we found that glycosphingolipids are hydrolyzed to ceramides coincident with both loss of lectin staining and the emergence of glycosidase activity. These results suggest: 1) that carbohydrates on keratinocyte cell membranes can be used as markers of epidermal differentiation, and 2) that removal of cell surface sugars during cornification may be due to the action of specific glycosidases in the outer epidermis.

Perturbations of membrane glycosylation in retinoid-treated epidermis.

Differentiation of keratinizing epithelial results in the formation of an exterior cornified layer that is both sufficiently cohesive to provide a protective barrier and dyshesive enough to permit orderly, apical desquamation. Membrane glycoconjugates, which are of presumed importance for intercellular adhesion and growth control, have recently been visualized in mouse, rat, and human epidermis. Because retinoids are known to profoundly influence epidermal differentiation, the lectin-staining pattern in normal mouse epidermis was compared to that observed in mice treated with etretinate (Ro 10-9359, 50 mg/kg/day). Within 2 days of treatment with 50 mg/kg/day of etretinate, abnormal transepidermal water loss began, followed by mild acanthosis after 4 to 6 days, and focal loss of stratification by 10 days. Despite these dramatic alterations in structure and function, abnormalities in lectin staining first appeared only after 15 days. Both wheat germ agglutinin and succinyl wheat germ agglutinin stained an etretinate-dependent product localized in the granular layer in samples treated for 21 to 38 days. Both the intensity and the distribution of PNA (galactose-N-acetyl-galactosamine) and Ulex ( alpha -L-fucose) staining were affected by retinoid treatment, but the staining pattern with other lectins was not altered until 40 to 45 days, at which time the sugars in some samples were either restricted to isolated nests of cells or greatly diminished. Although etretinate appeared to provoke selective changes in keratinocyte membrane glycoconjugates, these alterations became apparent only after the retinoid had profoundly perturbed epidermal differentiation. These altered patterns may not only reflect retinoid-induced aberrations in cohesion and barrier function, but also may provide clues about the role of certain membrane sugars in the control of normal epidermal differentiation.

Prospective on cell surface labeling using the hapten-sandwich method: an integrated approach.

Specific molecular units on the surface membranes of cells were tagged with markers visible in fluorescence, transmission and scanning electron microscopy. The Hapten-sandwich method does this by the sequential application of hapten-modified anti-cell-surface antibody, anti-hapten antibody and a hapten-modified marker. Details are given for the preparation and testing of the various reagents used in this method. The specificity and efficiency of Hapten-sandwich coupling of hemocyanin to mammary tumor virus (MTV) budding from breast tumor cells was demonstrated by quantitative transmission electron microscopy. Con A and anti-saccharide antibodies were used to demonstrate the presence of various sugars on the surfaces of the mammary tumor cells using fluorescence, transmission and scannings electron microscopy. Some samples were doubly labeled for the simultaneous presence of two different sugars. Light and electron microscopy were used to show that several hormones (insulin(I), hydrocortisone(F), and prolactin) greatly affect the expression of tumor virus and the kind and density of cell surface sugars on mammary tumor cells. Individual cells produced virus and expressed surface sugars independently. A micro-scanning densitometer was used to quantitate the results from fluorescence microscopy. Future advances in cell labeling including other applications of the Hapten-sandwich method, and problems such as the quantitation of electron microscopy labeling are discussed.

Hapten-sandwich labeling. II. Immunospecific attachment of cell surface markers suitable for scanning electron microscopy.

A hapten-sandwich procedure has been used for immunospecific labeling of cell surface antigens with markers visible by scanning electron microscopy. Antihapten antibody was used to link hapten-modified tobacco mosaic virus, bushy stunt virus, or hemocyanin to hapten-modified human erythrocytes. The antihapten antibody bridge was also used to link the hapten-virus marker to hapten-modified antibodies against mammary tumor virus on mouse mammary tumor cells, or against immunoglobulin receptors on mouse splenic lymphocytes. In all cases, labeling was highly specific. With this technique, it is possible to (a) compare morphological features of cells bearing differing cell surface antigens, and (b) examine the arrangement of specific antigenic sites on a cell surface or their distribution relative to membrane structures such as microvilli.

Cell contacts in the mouse mammary gland. I. Normal gland in postnatal development and the secretory cycle.

The nature and distribution of cell contacts have been examined in thin sections and freeze-fracture replicas of mammary gland samples from female C3H/Crgl mice at stages from birth through pregnancy, lactation, and postweaning involution. Epithelial cells of major mammary ducts at all stages examined are linked at their luminal borders by junctional complexes consisting of tight junctions, variable intermediate junctions, occasional small gap junctions, and one or more series of desmosomes. Scattered desmosomes and gap junctions link ductal epithelial and myoepithelial cells in all combinations; hemidesmosomes attach myoepithelial cells to the basal lamina. Freeze-fracture replicas confirm the erratic distribution of gap junctions and reveal a loose, irregular network of ridges comprising the continuous tight-junctional belts. Alveoli develop early in gestation and initially resemble ducts. Later, as alveoli and small ducts become actively secretory, they lose all desmosomes and most intermediate junctions, whereas tight and gap junctions persist, The tight-junctional network becomes compact and orderly, its undulating ridges oriented predominantly parallel to the luminal surface. It is suggested that these changes in junctional morphology, occurring in secretory cells around parturition, may be related to the greatly enhanced rate of movement of milk precursors and products through the lactating epithelium, or to the profound and recurrent changes in shape of secretory cells that occur in relation to myoepithelial cell contraction, or to both.