We describe software and hardware for a microcomputer-based cyclic strain device which applies programmed cycles of elongation and relaxation to cultured cells. This system has the potential to simulate many of the complex mechanically active environments found in living systems. As a sample application, we use it to simulate the cyclic stresses to which vascular smooth muscle cells in the arterial system are exposed.

Tyrosinase is considered to be the rate-limiting enzyme for the biosynthesis of melanin in epidermal melanocytes, and thus tyrosinase activity is thought to be a major regulatory step in melanogenesis. To determine whether the rate of pigment production was controlled at the level of tyrosinase gene expression, we developed a culture system capable of generating large populations of pure human melanocytes and then measured both melanin content as determined spectrophotometrically by absorption at 475 nm and mRNA levels as detected by hybridization with cloned cDNA Pmel 34, encoding human tyrosinase. We examined the relationship between pigment content and tyrosinase mRNA levels among human melanoma and melanocyte lines with very different levels of basal pigmentation; between two clones of a single human melanoma line, one pigmented and one amelanotic; and sequentially in melanocytes before and after simulation with isobutylmethylxanthine to increase melanin content per cell. Using Northern blot analysis and in-situ hybridization we found no correlation between tyrosinase message levels and melanin content, suggesting that posttranscriptional regulation of tyrosinase and/or other events determine the rate of pigment synthesis in human melanocytes.

Thyroid hormone metabolism was studied in the human Caco-2 colon carcinoma cell line, which at confluence exhibits several functions of differentiated enterocytes. Cells were harvested two to 17 days after reaching confluence. Intact cells and homogenates were tested for deiodination of [125I]-labeled substrates. Small amounts of thyroxine (T4) were converted by homogenates to 3,3′,5′-triiodothyronine (rT3), 3,3′-diiodothyronine (3,3′-T2), and I−, with no detectable production of 3,5,3′-triiodothyronine (T3) by homogenates or cells. rT3 was converted to 3,3′-T2 and I− with an apparent Michaelis constant (Km) for rT3 of 24 nmol/L; 6-n-propyl-2-thiouracil (PTU) had a 50% inhibitory concentration of 30 nmol/L and abolished rT3 5′-deiodination at 1 mmol/L in the presence of 20 mmol/L dithiothreitol (DTT). T3 was deiodinated to 3,3′-T2 and 3′-monoiodothyronine (3′-T1) with an apparent Michaelis constant (Km) for T3 of 5.7 nmol/L; this reaction was not inhibited by 1 mmol/L PTU. Phenolic and tyrosyl ring deiodinating activities were maximal four and six days, respectively, after the cells reached confluence. Homogenates of cells grown in standard medium containing fetal calf serum had fivefold higher rT3 5′-deiodinating activity than cells grown in a serum-free defined culture medium, reflecting a fivefold difference in the apparent Vmax with no difference in the apparent Km for rT3. There was no difference in T3 5-deiodination rates in homogenates of Caco-2 cells grown in the two media until 12 days postconfluence, when cells grown in standard medium had higher activity. These findings indicate that Caco-2 cells have two deiodinating pathways. rT3 5′-deiodination more closely resembles the low Km process described in rat kidney microsomes than classic type I deiodination in either rat or human tissues. T3 5-deiodination occurs via the type III pathway, which is regulated by changes in cell growth and differentiation as in several other cell types.

Epidermal pigmentation involves the synthesis of melanin in melanocytes and its transfer to surrounding keratinocytes, where it functions in photoprotection. To investigate the possible role of the keratinocyte in regulating pigmentation, human keratinocytes were incubated for 24 h in a defined culture medium, which was then transferred to pure human melanocyte cultures. After 1 week, the conditioned medium produced a fourfold increase in melanocyte yield and a seven-fold increase in total melanin. Increased melanocyte dendricity was clearly visible within 24 h as well. Ultrafiltration of the keratinocyte-conditioned medium suggested approximately one-half of the growth promoting activity as well as most of the dendricity and melanization stimulating activities were of low molecular weight (less than 500 Da). High molecular weight fractions stimulated only melanocyte growth. Of the several known keratinocyte-derived factors tested, none could be implicated as a mediator of the observed effects. Basic fibroblast growth factor, known to stimulate melanocyte growth in some culture systems, failed to stimulate growth, dendricity, or melanin content when added to the complete non-conditioned medium. Interleukin-1 alpha, interleukin-1 beta, 12-hydroxyeicosatetraenoic acid, prostaglandin E2, leukotriene B4, and adenosine 3',5'-cyclic monophosphate analogues also had no effect. These studies demonstrate that keratinocytes in vitro release factors that modulate melanocyte behavior and expand our understanding of controls for human epidermal pigmentation.

Cultured human keratinocytes converted T4 to T3 by type II iodothyronine deiodination. Homogenates of keratinocytes cultured from neonatal foreskin or adult arm skin had similar mean T4 5'-deiodinating activities. Conversion of T4 to T3 by intact cells was demonstrable in cultures from neonatal and adult donors. Only phenolic ring deiodination occurred in the cultured cells and their homogenates, the apparent Michaelis constant for T4 was 12 nmol/L, and T4 and rT3 each inhibited 5'-deiodination of the other. T4 5'-deiodination was unaffected by addition to the assay mixture of 1 mumol/L T3, but was inhibited less than 10% by 1 mmol/L 6-n-propyl-2-thiouracil, 50% by 270 nmol/L iopanoic acid, 50% by 9.4 mumol/L 3,5-diiodo- 3',5'-dimethyl-L-thyronine, and 33% by 42 mumol/L amiodarone. When keratinocytes were cultured for 3-4 days in medium containing iodothyronine-free fetal calf serum, the T4 5'-deiodination rates in homogenates doubled; this increase was prevented by restoring a physiological free T4 concentration, but not by a supraphysiological T3 concentration. Homogenates of fresh whole skin or fetal cadaveric epidermis did not convert T4 to T3 in measureable amounts, although one epidermal homogenate had low level T3 typrosyl-ring deiodinating activity. These results suggest that human epidermal type II iodothyronine deiodination in man might conceivably contribute to the intracellular T3 content of the skin and even to serum T3 concentrations, especially in hypothyroidism.

Nutrient requirements for proliferation and differentiated function of individual cell types can be determined using cell culture methodologies. Human epidermal keratinocytes are stimulated to grow by choline supplementation in the presence of myo-inositol when grown in a commercial nutrient medium containing six other defined supplements. The optimal range of choline concentrations varied among donor cell lines, but consistently fell between 36 µM and 180 µM. Addition of 72 µM choline increased cell yield to 250 ± 38% of that produced by myo-inositol supplementation alone and 92 ± 8% of that produced by addition of a highly mitogenic hypothalamic extract, which was previously required for good growth in this culture system. Supplementation of the basal medium with both the extract and choline resulted in 165 ± 13% of the cell yield observed with the extract addition alone. Supplementation with other phospholipid precursors did not further increase keratinocyte growth. Neither dermal fibroblasts nor epidermal melanocytes were stimulated by supplementation with choline, suggesting the high keratinocyte requirement is unusual. This completely defined culture medium for keratinocyte growth should prove useful in analyzing the role of phospholipids and other nutrients in human epidermis.

Annals of the New York Academy of SciencesVolume 548, Issue 1 p. 56-65 Keratinocytes Convert Thyroxine to Triiodothyroninea MICHAEL M. KAPLAN, Corresponding Author MICHAEL M. KAPLAN Endcrinology Division Department of Medicine New England Medical Center Hospital and Tufts University School of Medicine Boston, Massachusetts 02111Address for correspondence: Farmbrook Medical Two, Suite 303, 29877 Telegraph Rd., Southfield, MI 48034.Search for more papers by this authorPHILIP R. GORDON, PHILIP R. GORDON United States Department of Agriculture Human Nutrition Research Center on Aging Tufts University Boston, Massachusetts 02111Search for more papers by this authorCHANGYU PAN, CHANGYU PAN Endcrinology Division Department of Medicine New England Medical Center Hospital and Tufts University School of Medicine Boston, Massachusetts 02111Search for more papers by this authorJENN-KUEN LEE, JENN-KUEN LEE Endcrinology Division Department of Medicine New England Medical Center Hospital and Tufts University School of Medicine Boston, Massachusetts 02111Search for more papers by this authorBARBARA A. GILCHREST, BARBARA A. GILCHREST United States Department of Agriculture Human Nutrition Research Center on Aging Tufts University Boston, Massachusetts 02111Search for more papers by this author MICHAEL M. KAPLAN, Corresponding Author MICHAEL M. KAPLAN Endcrinology Division Department of Medicine New England Medical Center Hospital and Tufts University School of Medicine Boston, Massachusetts 02111Address for correspondence: Farmbrook Medical Two, Suite 303, 29877 Telegraph Rd., Southfield, MI 48034.Search for more papers by this authorPHILIP R. GORDON, PHILIP R. GORDON United States Department of Agriculture Human Nutrition Research Center on Aging Tufts University Boston, Massachusetts 02111Search for more papers by this authorCHANGYU PAN, CHANGYU PAN Endcrinology Division Department of Medicine New England Medical Center Hospital and Tufts University School of Medicine Boston, Massachusetts 02111Search for more papers by this authorJENN-KUEN LEE, JENN-KUEN LEE Endcrinology Division Department of Medicine New England Medical Center Hospital and Tufts University School of Medicine Boston, Massachusetts 02111Search for more papers by this authorBARBARA A. GILCHREST, BARBARA A. GILCHREST United States Department of Agriculture Human Nutrition Research Center on Aging Tufts University Boston, Massachusetts 02111Search for more papers by this author First published: December 1988 https://doi.org/10.1111/j.1749-6632.1988.tb18792.xCitations: 7 a Supported in part by a grant from the Tufts University Charlton Fund and by the United States Department of Agriculture Agricultural Research Service. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume548, Issue1Endocrine, Metabolic and Immunological Functions of KeratinocytesDecember 1988Pages 56-65 RelatedInformation