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D D Bikle

Publications and source records attributed to D D Bikle.

At least 91 records · Page 5Linked to original sources
PubMed1992

Progestin antagonism of estrogen stimulated 1,25-dihydroxyvitamin D levels.

Progestins are frequently used in combination with estrogen to prevent or treat postmenopausal osteoporosis. Progestins protect against the undesirable hyperplastic effects of estrogen on the endometrium. The possibility that progestins might antagonize the beneficial effects of estrogen on calcium homeostasis has received little attention. In this study we determined whether the addition of progestin to estrogen would alter the ability of estrogen to raise serum 1,25-dihydroxyvitamin D [1,25-(OH)2D] levels. Women within 5 yr of menopause were treated with three cycles of oral unopposed estrogen [1 or 2 mg/day (3.67 or 7.34 mumol/day) 17 beta-estradiol (E2) for 25/30 days of each cycle] followed by three cycles of E2 plus progestin [10 mg/day (29 mumol/day) medroxyprogesterone on days 12-25]. E2 increased both total and free 1,25-(OH)2D concentrations in a dose-dependent fashion. These levels increased progressively over the three cycles of unopposed estrogen treatment. In contrast the vitamin D binding protein concentration reached maximal levels after one cycle of E2. With the addition of progestin, the levels of total and free 1,25-(OH)2D returned toward baseline although vitamin D binding protein levels remained elevated. PTH levels rose with both doses of E2 as serum calcium levels fell. Progestin did not significantly alter the effects of E2 on PTH or calcium. Our results raise the possibility that progestin may antagonize part of the salubrious effects of estrogen on calcium homeostasis.

Dihydroxycholecalciferols↗
PubMed1991

Role of intracellular-free calcium in the cornified envelope formation of keratinocytes: differences in the mode of action of extracellular calcium and 1,25 dihydroxyvitamin D3.

Extracellular calcium (Cao) and the steroid hormone 1,25(OH)2D, induce the differentiation of human epidermal cells in culture. Recent studies suggest that increases in intracellular free calcium (Cai) levels may be an initial signal that triggers keratinocyte differentiation. In the present study, we evaluated cornified envelope formation, the terminal event during keratinocyte differentiation, and correlated it with changes in the Cai levels during differentiation of keratinocytes in culture induced by Cao or 1,25(OH)2D. Keratinocytes were grown in different Cao concentrations (0.1 or 1.2 mM) or in the presence of 1,25(OH)2D (10(-11) to 10(-7) M), and the Cai levels were measured using the fluorescent probe Indo-1. Our results suggest that the induction of cornified envelope formation is associated with an increase in Cai level during calcium-induced differentiation. Cao and the calcium ionophore ionomycin acutely increased Cai and cornified envelope formation. In contrast, the effect of 1,25(OH)2D on increasing Cai levels and stimulating cornified envelope formation was long-term, requiring days of treatment with 1,25(OH)2D. Our data are consistent with other recent studies and support the hypothesis that Cao regulates keratinocyte differentiation primarily by acutely increasing their Cai levels. The role of calcium in the mechanism of action of 1,25(OH)2D on keratinocyte differentiation is less clear. The increase in Cai of keratinocytes during 1,25(OH)2D induced differentiation may be essential for or subsequent to its prodifferentiation effects.

Calcitriol↗
PubMed1991

Uncoupling of the calcium-sensing mechanism and differentiation in squamous carcinoma cell lines.

Increases in the intracellular calcium (Cai) levels, induced either by extracellular calcium or by calcium ionophores, stimulate the terminal differentiation of normal human keratinocytes in culture (NHK). Despite extensive differences in phenotypic expression, squamous carcinoma cell lines (SCC lines) display only partial terminal differentiation even in the presence of normal extracellular calcium. Therefore, in this study, we evaluated whether the inability of SCC lines to differentiate normally is due to a defect in achieving adequate levels of Cai. Membrane-bound transglutaminase activity and involucrin levels of the various SCC lines were lower than those of NHK and correlated with their low extent of cornified envelope formation. Ionomycin, a calcium ionophore, acutely increased cornified envelope formation of NHK 60- to 70-fold, but only initiated a 1- to 5-fold increase in SCC lines. Yet resting Cai levels in and the Cai response to various agents of SCC lines were similar or higher than those of NHK. Extracellular calcium evoked a rapid, transient and a slower, sustained increase of Cai. Extracellular ATP increased Cai by a rapid release from intracellular sources. Ionomycin, on the other hand, increased Cai from both intracellular compartments and extracellular sources. Thus, these studies indicate that the abnormalities in differentiation among SCC lines do not appear to involve their calcium-sensing mechanism. An uncoupling of the Cai changes to the synthesis of the precursor molecules required for differentiation may be responsible for the defect in differentiation displayed by these SCC lines.

Adenosine Triphosphate↗
PubMed1991

Limited tissue distribution of the intestinal brush border myosin I protein.

A myosinlike 105-110-kilodalton calmodulin-binding protein, brush border myosin I, found in the intestinal brush border has been linked to two seemingly disparate but possibly interacting functions of the brush border, namely, microvillar motility and vitamin D regulated calcium transport. If brush border myosin I were to function primarily as a myosinlike molecule powering cellular or microvillar motility, one might expect it to be found in a variety of tissues with microvilli such as the renal brush border and bile canaliculus. On the other hand, a more specialized function such as participation in vitamin D regulated calcium transport might dictate a more restricted tissue distribution for brush border myosin I. To determine the tissue distribution of brush border myosin I, we purified this protein to apparent homogeneity, generated antisera to it, and used the antisera to localize the protein within the intestinal epithelial cell by immunocytochemistry. We then screened a variety of other tissues (brain, lung, heart, liver, spleen, pancreas, kidney, and skeletal muscle) both for calmodulin-binding proteins as well as for brush border myosin I using Western blots and immunofluorescence. Our results indicate that the intestinal brush border myosin I is limited in its distribution to the intestinal brush border.

Animals↗
PubMed1991

Squamous carcinoma cell lines produce 1,25 dihydroxyvitamin D, but fail to respond to its prodifferentiating effect.

The active metabolite of vitamin D3, 1,25 dihydroxyvitamin D3 [1,25(OH)2D], is produced by normal human keratinocytes (NKC) and regulates their differentiation. Squamous carcinoma cell (SCC) lines lack the ability to differentiate in vitro, which might involve defective 1,25(OH)2D synthesis or response. To address this possibility we obtained four SCC lines (12F2, 12B2, 25, and A431) and first determined whether they could produce 1,25(OH)2D from its substrate 25 hydroxyvitamin D3 (250HD). All could (12F2 greater than NKC greater than 25 greater than 12B2 greater than A431). Furthermore, exogenously added 1,25(OH)2D inhibited 1,25(OH)2D production and stimulated 24,25 dihydroxyvitamin D3 [24,25(OH)2D] production in all cell lines but with different potency (25 = A431 greater than NKC greater than 12B2 greater than 12F2). Cellular binding studies suggested that the high-affinity binding site for 1,25(OH)2D in NKC is not found in 12F2 and 12B2. When the effect of 1,25(OH)2D on differentiation was determined, only NKC responded with an increase in cornified envelope formation, although some of the cell lines responded to the proliferative [at low 1,25(OH)2D concentration] or antiproliferative [at high 1,25(OH)2D concentration] effect of 1,25(OH)2D. Thus, although SCC lines synthesize 1,25(OH)2D and respond to exogenous 1,25(OH)2D with respect to appropriate regulation of endogenous 250HD metabolism, these cell lines fail to respond to the differentiating influence of this vitamin D metabolite.

Calcitriol↗
PubMed1991

Tumor necrosis factor-alpha regulation of 1,25-dihydroxyvitamin D production by human keratinocytes.

The keratinocyte has receptors for, responds to, and produces both tumor necrosis factor-alpha (TNF) and 1,25-dihydroxyvitamin D [1,25-(OH)2D]. Both TNF and 1,25-(OH)2D facilitate the differentiation of keratinocytes. To explore the possibility that TNF stimulates keratinocyte differentiation at least in part by regulating 1,25-(OH)2D production we examined the effect of TNF on both 1,25-(OH)2D production and differentiation (transglutaminase activity, cornified envelope formation) at different stages of differentiation. 1,25-(OH)2D production, transglutaminase activity, and cornified envelope formation are sequentially increased during differentiation such that 1,25-(OH)2D production occurs first, followed by transglutaminase activity and, finally, cornified envelope formation. In preconfluent cells, TNF stimulated 1,25-(OH)2D production. Interferon-gamma also stimulated 1,25-(OH)2D production at low concentrations, but this was not additive to that by TNF. The stimulatory effect of TNF was maximal after 1-2 days of incubation and decreased with prolonged incubation. TNF had little effect on cornified envelope formation and transglutaminase activity in the preconfluent cells. Once keratinocytes achieved confluence, TNF stimulated transglutaminase activity and cornified envelope formation, but inhibited 1,25-(OH)2D production. Our results suggest that TNF regulates 1,25-(OH)2D production as part of its ability to accelerate keratinocyte differentiation; whether TNF stimulates or inhibits 1,25-(OH)2D production depends on whether TNF is added before or after the cell has reached its maximal capacity to make 1,25-(OH)2D.

Calcitriol↗
PubMed1990

Calcium regulation of growth and differentiation of normal human keratinocytes: modulation of differentiation competence by stages of growth and extracellular calcium.

In this study we examined the different aspects of the pathway leading to the differentiation of keratinocytes as a function of time in culture and calcium concentration of the culture medium. Human neonatal foreskin keratinocytes were grown in a serum-free, defined medium containing 0.07, 1.2, or 2.4 mM calcium and assayed for the rate of growth and protein synthesis, involucrin content, transglutaminase activity, and cornified envelope formation at preconfluent, confluent, and postconfluent stages of growth. We observed that keratinocytes grown to postconfluence in all calcium concentrations showed an increased protein/DNA ratio and an increased rate of membrane-associated protein synthesis. Extracellular calcium concentrations did not have a clear influence on these parameters. However, preconfluent and confluent keratinocytes grown in 0.07 mM calcium showed markedly retarded differentiation at all steps, i.e., involucrin synthesis, transglutaminase activity, and cornified envelope formation. Within 1 week after achieving confluence, these keratinocytes began synthesizing involucrin and transglutaminase and developed the ability to form cornified envelopes. Cells grown in 1.2 and 2.4 mM calcium synthesized involucrin and transglutaminase prior to confluence and were fully competent to form cornified envelopes by confluence. Thus external calcium-regulated keratinocyte differentiation is not an all or none phenomenon, but rather it is the rate at which keratinocytes differentiate that is controlled by calcium. We conclude that either or both higher extracellular calcium concentration and the achievement of cell-cell contacts lead to a coordinate increase of at least two precursors--involucrin content and transglutaminase activity--required for cornified envelope formation. We speculate that a critical level of cytosolic calcium, achieved by increased extracellular calcium or by achievement of intercellular communication established by cell-cell contact, may trigger mechanisms required for initiation of keratinocyte differentiation.

Calcium↗
PubMed1990

Different responses of trabecular and cortical bone to 1,25(OH)2D3 infusion.

Previous studies regarding the effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on bone have suggested that 1,25(OH)2D3 increases bone mass and calcium. Many of these studies have focused on trabecular or total bone without examining cortical bone per se. To determine whether the response of trabecular bone to 1,25(OH)2D3 differed from the response of cortical bone, we infused 1,25(OH)2D3 into rats and examined bone mass, 45Ca accumulation, and the density distribution of bone particles (as a measure of bone maturation) in both the proximal tibia and shaft. In the proximal tibia 1,25(OH)2D3 decreased 45Ca accumulation, yet increased bone mass and shifted the particle distribution to more mineralized fractions. In the shaft there was a redistribution of bone to less mineralized fractions that was not accompanied by a change in total bone mass or a decrease in 45Ca accumulation. Thus 1,25(OH)2D3 may retard bone maturation and mineralization throughout the tibia, but this effect in the proximal tibia appears to be overshadowed by a reduction in bone resorption resulting in an accumulation of well-mineralized bone in that region. Bone resorption, however, was not measured directly. The net result is an increase in bone mass and density of trabecular bone not seen in cortical bone.

Animals↗
PubMed1989

Isotopic labeling affects 1,25-dihydroxyvitamin D metabolism.

Isotope substitution can change the biochemical properties of vitamin D. To determine the effect of substituting 3H for 1H on the metabolism of 1,25(OH)2D3, we measured the metabolic clearance rate and renal metabolism of unlabeled and 3H-labeled 1,25(OH)2D3. Substitution of 3H for 1H on carbons 26 and 27 [1,25(OH)2[26,27(n)-3H]D3] or on carbons 23 and 24 [1,25(OH)2[23,24(n)-3H]D3] reduced the in vivo metabolic clearance rate of 1,25(OH)2D3 by 36% and 37%, respectively, and reduced the in vitro renal catabolism of 1,25(OH)2D3 by 11% and 54%, respectively. Substitutions of 3H for 1H on carbons 23 and 24 as opposed to carbons 26 and 27 reduced conversion of [3H]1,25(OH)2D3 to [3H]1,24,25(OH)2D3 by 25% and to putative 24-oxo-1,23,25-dihydroxyvitamin D3 by 1600%. These results indicate that substitution of 3H for 1H on carbons 26 and 27 or on carbons 23 and 24 can reduce the metabolic clearance rate and in vitro metabolism of 1,25(OH)2D3 and quantitatively alter the pattern of metabolic products produced.

Animals↗
PubMed1989

Bone response to normal weight bearing after a period of skeletal unloading.

Skeletal unloading in the growing rat induces a temporary inhibition of bone formation and thereby a deficit in bone calcium compared with age-matched, normally loaded animals. To determine whether this deficit can be restored by skeletal reloading we measured bone formation rate at the tibiofibular junction and total bone calcium in the tibia and lumbar vertebra in rats whose hindlimbs were unloaded for 2 wk and then reloaded by return to normal weight bearing. Continuously loaded or unloaded animals were also studied. Skeletal unloading reduced bone formation by 34% and tibial and vertebral calcium by 12 and 22%, respectively. Reloading significantly increased the rates of bone formation and calcium accretion 30-34% above normally loaded animals, and by 2 wk had decreased the deficit in tibial and vertebral calcium by 36 and 23%, respectively. These data indicate that the deficit in bone calcium induced by skeletal unloading in the growing rat can be restored in part by return to normal weight bearing. However, the time required to restore bone calcium exceeds the time required to produce the original calcium deficit.

Animals↗
PubMed1989

Effects of simulated weightlessness on rat osteocalcin and bone calcium.

Some of the musculoskeletal changes that occur in growing rats during spaceflight are simulated by a model that selectively unloads the hindlimbs while maintaining normal weight bearing on the forelimbs. Using this model we studied the response of mineral and the mineral-binding protein osteocalcin (OC) in the third lumbar vertebra (L3) and the femoral midshaft to periods of unweighting from 2 to 28 days. Serum OC decreased by 25%, consistent with a decreased rate of bone growth, during the first week of suspension and returned toward control values after 15 days. The L3 and femur weighed 20% less than control bones after 10-28 days. OC content of L3 and femur diaphysis were lower after 7 days of suspension and returned to normal levels at 28 days, whereas Ca content rose slightly at 5 days then decreased sharply. OC:Ca ratio was also affected. The data suggest that unweighting affects formation and deposition of OC and Ca differently depending on bone location and duration of unweighting. Both serum and bone OC are highly sensitive indicators of disruption of osteoblast activity by altered skeletal loading.

Animals↗
PubMed1989

Binding and biological effects of tumor necrosis factor alpha on cultured human neonatal foreskin keratinocytes.

Tumor necrosis factor alpha (TNF alpha) localizes to the epidermis when injected in vivo, but its role in the skin has heretofore not been evaluated. As a first approach to assessing the role of TNF alpha in the skin, we evaluated the binding and biological effects of TNF alpha on human neonatal foreskin keratinocytes maintained in culture. We found that TNF alpha at 0.3-1.0 nM inhibited proliferation of keratinocytes in a reversible fashion as demonstrated by a reduction in total DNA content and clonal growth. The antiproliferative effects were most marked when TNF alpha was added in the preconfluent stages of cell growth. Accompanying this antiproliferative effect was a stimulation by TNF alpha of differentiation of keratinocytes as indicated by the stimulation of cornified envelope formation. Keratinocytes specifically bound TNF alpha, reaching maximal binding in 2 h at 34 degrees C or 8 h at 4 degrees C. Much of the apparent binding at 34 degrees C was due to internalization of the TNF alpha. At 4 degrees C the rate of internalization was much less. Confluent keratinocytes showed a single class of high-affinity receptors with 1,250 receptors/cell and a Kd of 0.28 nM. These data suggest a role for TNF alpha in the growth and differentiation of the epidermis.

Cell Differentiation↗
PubMed1989

Transpleural gradient of 1,25-dihydroxyvitamin D in tuberculous pleuritis.

We used tuberculous pleuritis as a model to study the compartmentalization and potential immunoregulatory role of 1,25-dihydroxyvitamin D [1,25-(OH)2-D] in human granulomatous disease. In tuberculous pleuritis, mean concentrations of total 1,25-(OH)2-D were elevated in pleural fluid, compared to blood (67 pg/ml vs. 35 pg/ml). Concentrations of albumin, protein and 25-hydroxyvitamin D (25-OH-D) were lower in pleural fluid than blood, suggesting that accumulation of binding proteins does not explain the transpleural gradient of 1,25-(OH)2-D. The mean free 1,25-(OH)2-D concentration in pleural fluid was increased 5.3-fold over that in serum. 1,25-(OH)2-D3 inhibited PPD-induced proliferation of pleural fluid mononuclear cells, antigen-reactive lines and T lymphocyte clones derived from a single cell. Patient-derived PPD-reactive lines expressed a high-affinity intracellular binding moiety for 1,25-(OH)2-D3. Pleural fluid mononuclear cells and PPD-reactive lines did not metabolize 25-OH-D3 to 1,25-(OH)2-D3. The sum of these data suggests that concentration of 1,25-(OH)2-D in pleural fluid of tuberculosis patients is probably due to local hormone production by pleural tissue-based inflammatory cells that are not present in significant numbers in pleural fluid. Elevated concentrations of 1,25-(OH)2-D in pleural fluid may exert receptor-mediated inhibition of antigen-induced proliferation by pleural fluid lymphocytes. Inhibition of lymphocyte proliferation and lymphokine production may prevent tissue destruction from an uncontrolled inflammatory response.

Calcitriol↗
PubMed1989

Free, and not total, 1,25-dihydroxyvitamin D regulates 25-hydroxyvitamin D metabolism by keratinocytes.

Greater than 99% of the total circulating 1,25-dihydroxyvitamin D [1,25-(OH)2D] is bound to proteins such as the vitamin D-binding protein (DBP) and albumin; in the normal human only 0.4% of the circulating 1,25-(OH)2D is free. Although it is often assumed that only the free concentration of 1,25-(OH)2D is available to cells, this has not been demonstrated. In particular, it is not clear whether the DBPs facilitate 1,25-(OH)2D entry into target cells or serve only to transport these metabolites within the circulation. To address this question, we evaluated one of the best characterized target tissue responses to 1,25-(OH)2D, namely its ability to inhibit its own production and induce that of 24,25-(OH)2D, using one of the most sensitive cells, the human foreskin keratinocyte. We incubated keratinocytes in the presence of 1,25-(OH)2D (from 10(-11)-10(-8) M) in medium containing albumin (from 0.1-10%) or serum (from 0.1-10%) for 4 h [to inhibit the 25-hydroxyvitamin D (250HD) 1 alpha-hydroxylase] or 16 h (to induce the 250HD 24-hydroxylase) before evaluating [3H]250HD metabolism by these cells during a 1-h incubation in serum- and albumin-free medium. The free fraction of 1,25-(OH)2D was determined in each medium by centrifugal ultrafiltration and varied from 36% to 0.57% in direct proportion to the albumin or serum in the medium. Increasing the serum or albumin concentration in the medium increased the concentration of total 1,25-(OH)2D needed to inhibit its own production or stimulate that of 24,25-(OH)2D. In contrast, the concentration of free 1,25-(OH)2D needed to half-maximally inhibit its own production or induce 24,25-(OH)2D production remained constant at approximately 10(-11) M. We conclude that the free, not the total, 1,25-(OH)2D concentration regulates 250HD metabolism by keratinocytes, that DBPs do not facilitate 1,25-(OH)2D entry into the cell, and that these cells sense only free 1,25-(OH)2D.

Cells, Cultured↗
PubMed1989

Regulation of 1,25-dihydroxyvitamin D production in human keratinocytes by interferon-gamma.

Interferon-gamma (IFN gamma) and 1,25-dihydroxyvitamin D [1,25-(OH)2D] each have potent antiproliferative and prodifferentiating effects on keratinocytes. Since keratinocytes produce 1,25-(OH)2D, we explored the possibility that IFN gamma acted on keratinocytes in part by regulating 1,25-(OH)2D production. We cultured human neonatal foreskin keratinocytes for various periods of time in the presence of various concentrations of IFN gamma before assessing their ability to produce 1,25-(OH)2D. The production of 1,25-(OH)2D by preconfluent keratinocytes grown in the presence of serum (which retards differentiation) was stimulated by 1.8 nM IFN gamma. Postconfluent keratinocytes did not respond to 1.8 nM IFN gamma. The production of 1,25-(OH)2D by keratinocytes grown in serum-free medium was maximally stimulated by 0.006 nM IFN gamma and inhibited at concentrations greater than 0.06 nM. Keratinocytes grown in 0.1 mM calcium serum-free medium (which prevents differentiation) were more sensitive to both the stimulatory and inhibitory effects of IFN gamma than keratinocytes grown in 1.2 mM calcium serum-free medium (which permits differentiation). The stimulatory effect of IFN gamma on 1,25-(OH)2D production was maximal after 2 days of incubation. Incubations longer than 2 days showed increasingly less stimulation at the low IFN gamma concentrations and increasingly greater inhibition at the higher IFN gamma concentrations. The inhibitory effects of IFN gamma on 1,25-(OH)2D production paralleled the inhibitory effects of IFN gamma on cell growth. Thus, IFN gamma does regulate 1,25-(OH)2D production by keratinocytes. However, this regulation is modulated by the state of keratinocyte proliferation and differentiation and is influenced by calcium and undefined factors in serum. The data are consistent with the possibility that IFN gamma alters keratinocyte differentiation in part by regulating 1,25-(OH)2D production.

Cells, Cultured↗
PubMed1988

1,25-Dihydroxyvitamin D production and receptor binding in human keratinocytes varies with differentiation.

Human foreskin keratinocytes in culture produce 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) and 24,25-dihydroxycholecalciferol (24,25-(OH)2D3) from 25-hydroxycholecalciferol (25-(OH)D3). The production of 1,25-(OH)2D3 by these cells correlated with the early events of differentiation such as expression of transglutaminase activity and the levels of a precursor protein for the cornified envelopes, involucrin. In contrast, the increased production of 24,25-(OH)2D3, as 1,25-(OH)2D3 production declined, correlated with the terminal differentiation marker, cornified envelope formation. Exogenous 1,25-(OH)2D3 (10(-11)-10(-9) M) inhibited the 1-alpha-hydroxylase at all stages of growth of these cells. Keratinocytes in culture expressed receptors for 1,25-(OH)2D3 which had similar sedimentation behavior in sucrose density gradients as chick intestinal cytosol receptors. Cells in early stages of growth (preconfluent and confluent) contained higher numbers of receptors (26-27 fmol/mg protein) than post-confluent cells. The dissociation constant (237-278 pM) of these receptors for 1,25-(OH)2D3 was not consistently altered by differentiation. Since 1,25-(OH)2D3 is a potent stimulator of cell differentiation in a variety of systems including the epidermis, our results suggest the possibility that endogenous 1,25-(OH)2D3 production may participate in the differentiation of keratinocytes in culture and, perhaps, in vivo.

24,25-Dihydroxyvitamin D 3↗
PubMed1988

Biochemical and morphological characterization of growth and differentiation of normal human neonatal keratinocytes in a serum-free medium.

Growth and differentiation of keratinocytes in a serum-free medium (keratinocyte growth medium or KGM) was studied and compared to that under conditions in which serum and feeder cell layers were used. Cells were grown in KGM containing 0.1 mM calcium (KGM/low calcium), KGM containing 1.2 mM calcium (KGM/normal calcium), or Dulbecco's modified Eagles medium containing 5% fetal calf serum and 1.8 mM calcium in presence of mitomycin treated 3T3 M cells (DMEM/5% FCS). Plating efficiency and rate of growth were similar in the three media till confluence. In postconfluent cultures, protein and DNA content of cells attached to the plate in KGM/low-calcium dishes decreased as an increased number of cells were shed into the medium. Cell shedding was much less evident in the presence of normal calcium. Cells grown in KGM/low calcium had a higher rate of cell proliferation (3H-thymidine incorporation into cellular DNA) than cells grown in normal calcium. Transglutaminase activity, involucrin content, and cornified envelope formation were greatest in cells grown in KGM/normal calcium, intermediate in cells grown in DMEM/5% FCS, and least in cells grown in KGM/low calcium. Keratin profiles from cells grown in KGM/low calcium showed a lower percentage of high molecular weight bands compared to the keratin profiles from cells grown in the presence of normal calcium. Keratinocytes in KGM/low calcium grew as a monolayer of cuboidal cells with few features of differentiation, whereas cells grown in KGM/normal calcium stratified into multilayered islands (3-5 layers) surmounted by 2-4 layers of enucleated cells with thickened cornified envelopes. Cells grown in KGM/normal calcium also contained tonofilaments and lamellar bodies unlike cells grown in KGM/low calcium. Cells grown in DMEM/5% FCS also formed stratified layers comparable to cells grown in KGM/normal calcium but lacked cornified cells, keratohyalin granules, tonofilament bundles, and lamellar bodies. These studies indicate the usefulness of serum-free conditions for the culture of human keratinocytes and confirm the importance of extracellular calcium in keratinocyte differentiation.

Cell Differentiation↗