Modulation of growth and differentiation in normal human keratinocytes by transforming growth factor-beta

The effect of transforming growth factor-type beta 1(TGF-beta) on the growth and differentiation of normal human skin keratinocytes cultured in serum-free medium was investigated. TGF-beta markedly inhibited the growth of keratinocytes at the concentrations greater than 2 ng/ml under low Ca2+ conditions (0.1 mM). Growth inhibition was accompanied by changes in cell functions related to proliferation. Remarkable inhibition of DNA synthesis was demonstrated by the decrease of [3H]thymidine incorporation. The decrease of [3H]thymidine incorporation was observed as early as 3 hr after addition of TGF-beta. TGF-beta also decreased c-myc messenger RNA (mRNA) expression 30 min after addition of TGF-beta. This rapid reduction of c-myc mRNA expression by TGF-beta treatment is possibly one of the main factors in the process of TGF-beta-induced growth inhibition of human keratinocytes. Since growth inhibition and induction of differentiation are closely related in human keratinocytes, the growth-inhibitory effect of TGF-beta under high Ca2+ conditions (1.8 mM Ca2+, differentiation-promoting culture environment) was examined. TGF-beta inhibited the growth of keratinocytes under high Ca2+ conditions in the same manner as under low Ca2+ conditions, suggesting that it is a strong growth inhibitor in both low and high Ca2+ environments. The induction of keratinocyte differentiation was evaluated by measuring involucrin expression and cornified envelope formation: TGF-beta at 20 ng/ml increased involucrin expression from 9.3% to 18.8% under high Ca2+ conditions, while it decreased involucrin expression from 7.0% to 3.3% under low Ca2+ conditions. Cornified envelope formation was modulated in a similar way by addition of TGF-beta: TGF-beta at 20 ng/ml decreased cornified envelope formation by 53% under low Ca2+ conditions, while it enhanced cornified envelope formation by 30.7% under high Ca2+ conditions. Thus, the effect of TGF-beta on keratinocyte differentiation is Ca2+ dependent. It enhances differentiation of human keratinocytes under high Ca2+ conditions, but inhibits differentiation under low Ca2+ conditions. Taken together, there is a clear discrepancy between TGF-beta effects on growth inhibition and induction of differentiation in human keratinocytes. These data indicate that growth inhibition of human keratinocytes by TGF-beta is direct and not induced by differentiation.     

Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor

Effect of hepatocyte growth factor (HGF) on normal human epidermal keratinocytes cultured under conditions of low Ca2+ (0.1 mM, growth-promoting condition) and physiological Ca2+ (1.8 mM, differentiation-promoting condition) was investigated. In low Ca2+, HGF markedly enhanced the migration of keratinocytes while it suppressed cell growth and DNA synthesis in a dose-dependent manner. In contrast, HGF enhanced the migration, cell growth, and DNA synthesis of keratinocytes cultured under conditions of physiological Ca2+. The maximal stimulation of DNA synthesis (2.4-fold stimulation) in physiological Ca2+ was seen at 2.5-5 ng/ml HGF and the stimulatory effect of HGF was suppressed by transforming growth factor-beta 1. Analysis of the HGF receptor using 125I-HGF as a ligand showed that human keratinocytes expressed a single class of specific, saturable receptor for HGF in both low and physiological Ca2+ conditions, exhibiting a Kd = 17.3 pM and approximately 690 binding sites/cell under physiological Ca2+. Thus, HGF is a potent factor which enhances growth and migration of normal human keratinocytes under conditions of physiological Ca2+. HGF may play an important role in epidermal tissue repair as it enhances both the migration and growth of keratinocytes.     

Calcium-mediated regulation of the low density lipoprotein receptor and intracellular cholesterol synthesis in human epidermal keratinocytes

Unlike cells cultured under physiological Ca2+ concentrations (1-2 mM), keratinocytes cultured in media containing Ca2+ in low concentrations (less than 0.1 mM) do not stratify. The latter cells also differ with respect to several features of the regulation of cholesterol synthesis. In keratinocytes cultured in medium containing high Ca2+ concentrations (1.6 mM) and fetal calf serum, the rate of cholesterol synthesis was 20-30 times higher than in keratinocytes exposed to a low Ca2+ concentration. The rate of cholesterol synthesis did not change when high-calcium cells were deprived of extracellular sources of cholesterol but increased (8-10 fold) in deprived low-calcium cells. Furthermore, the addition of low density lipoprotein (LDL) reduced cholesterol synthesis markedly in low-calcium cells but had no effect on high-calcium cells. Finally, in keratinocytes cultured at low calcium concentrations the association and degradation of 125I-LDL was 20-30 times higher than in keratinocytes cultured under high-calcium conditions. Switching of the cells from the low-calcium to the high-calcium medium resulted in the induction of terminal differentiation within 15 hours and was accompanied by increased cholesterol and protein synthesis, increased competence of cells to form cornified envelopes, and reduced association of 125I-LDL. A gradual increase of the extracellular Ca2+ concentration was accompanied by a corresponding increase of cholesterol and protein synthesis and a decrease of the response of intracellular cholesterol synthesis to changes in the extracellular concentrations of lipoprotein. Various morphological techniques showed virtually no binding and internalization of LDL by keratinocytes cultured at the high-calcium level, whereas both were observed at the low-calcium level. Once internalized, the LDL was delivered to dense bodies representing lysosomes. It is concluded that in human epidermal keratinocytes, the expression of the LDL receptor and the endogenous synthesis of cholesterol are regulated by the conditions determined by the differentiation stage of the cells.     

Subcellular distribution of protein kinase C/phorbol ester receptors in differentiating mouse keratinocytes

The activation of protein kinase C (PKC) by diacylglycerol or tumor promoters plays a pivotal role in signal transduction and subsequent activation of cellular processes. Since the activity of this enzyme is dependent on its immediate lipid domain, its relative distribution within the cell may be an important regulatory mechanism. We report here a relative decrease in PKC/phorbol ester receptor associated with the particulate fraction of mouse keratinocytes induced to differentiate by two separate systems. First, proliferating keratinocytes maintained in low Ca2+ (0.09 mM) serum-free medium were induced to differentiate rapidly by the addition of Ca2+ (1.8 mM). A 1.4-fold decrease in the percent of total phorbol receptor binding activity present in the particulate fraction and concomitant increase in binding in the cytosol fraction was evident 20 min after the Ca2+ addition. Second, in keratinocytes that differentiate over a 6 day cultivation period in serum-containing medium with Ca2+ concentration of 1.8 mM, a significant decrease in the percent of the phorbol receptor binding activity present in the particulate fraction was observed as the culture begins to differentiate on days 3 and 4. Maximal phorbol ester binding in the particulate fraction corresponded to the proliferative phase of the culture (day 2), while lower levels of PKC/phorbol ester binding to particulate fractions were noted during the early differentiative phase (days 3 and 4). Addition of the synthetic diacylglycerols 1-oleoyl-2-acetylglycerol or L-alpha-1,2 dioctanyl glycerol at 30 micrograms/ml to proliferating keratinocyte cultures induced a modest increase in two markers of terminal differentiation: cornified envelope formation and transglutaminase levels. These findings, taken together, support the hypothesis that PKC activation plays a role in the initial signalling events for keratinocyte differentiation.     

Epidermal growth factor receptor expression related to differentiation capacity in normal and transformed keratinocytes

Epidermal growth factor (EGF) and Ca2+ have been indicated to play a major role in skin development. We have used normal keratinocytes, SV40-transformed keratinocytes (SVK14) and various squamous carcinoma cell (SCC) lines as in vitro model system to study the effect of the extracellular Ca2+ concentration of EGF-receptor expression in relation to the capability of cells to differentiate. The cell lines used exhibit a decreasing capacity to differentiate in the order of keratinocytes approximately SVK14 greater than SCC-12F2 greater than SCC-15 greater than SCC-12B2 greater than SCC-4, as judged from Ca2+-ionophore-induced cornified envelope formation. Under normal Ca2+ conditions, all cell lines (except for SCC-15) exhibited two classes of EGF-binding sites. The number of low-affinity binding sites increased considerably as cells were less able to differentiate, while the apparent dissociation constant (kd) was similar in all cell lines. In contrast, the properties of high-affinity EGF binding varied in the various cell lines without a clear relationship to the degree of differentiation capacity. Lowering the extracellular Ca2+ concentration to 0.06 mM resulted in a decrease of Ca2+ ionophore-induced cornified envelope formation, demonstrating the decreased ability to differentiate under these conditions. The decreased ability to differentiate was accompanied by a marked increase in the number of EGF-binding sites, but without a change of the kd. Furthermore, no high-affinity EGF-binding sites were detectable under these conditions. Finally, addition of Ca2+ to low Ca2+-cultured cells caused a rapid decrease of EGF binding in all cell lines, most prominently in normal keratinocytes and SCC-12F2 cells. The data presented demonstrate: The combination of normal keratinocytes, SVK14 and the various SCC lines provides an attractive model system to study differentiation in vitro; EGF-receptor expression is related to the state of differentiation, both phenomena being sensitive to the external Ca2+ concentration; and EGF-receptor expression is related to the capability of cells to differentiate.     

A new method for quantifying keratinocyte differentiation using immunofluorescent staining of involucrin and cytofluorography

Involucrin is a precursor protein of detergent-insoluble cornified envelope and a marker of terminal differentiation of epidermal keratinocytes. To quantify differentiation of cultured human keratinocytes, the population of involucrin-positive cells was estimated by immunofluorescent staining using anti-involucrin antibody and flow cytometry. Normal human keratinocytes were cultured under three conditions for induction of differentiation: low Ca2+ concentration (0.1 mM Ca2+), high Ca2+ concentration (1.8 mM Ca2+), and high Ca2+ concentration with 10% fetal bovine serum (FBS). The relationship between fluorescence intensity and involucrin synthesis was confirmed by visual examination of sorted cells. The population of involucrin-positive cells increased from 7.2 to 18.1% by elevating Ca2+ concentration and to 37.0% by adding FBS. The extent of cornified envelope formation under the same culture conditions was consistent with the estimation of involucrin-positive cells. The cytofluorographic analysis of involucrin synthesis made it possible to determine the number of differentiated cells in a large number of samples precisely and reliably. Thus, it is a useful method for quantifying keratinocyte differentiation.     

Differentiation of human keratinocytes: changes in lipid synthesis, plasma membrane lipid composition, and 125I-EGF binding upon administration of 25-hydroxycholesterol and mevinolin

We have studied the relationship between differentiation capacity, plasma membrane composition, and epidermal growth factor (EGF) receptor expression of normal keratinocytes in vitro. The plasma membrane composition of the cells was modulated experimentally by cholesterol depletion, using specific inhibitors of cholesterol synthesis, such as 25-hydroxycholesterol and mevinolin. Exposure of the cells towards these inhibitors resulted in a drastic decrease of cholesterol biosynthesis, as determined from 14C-acetate incorporation into the various lipid fractions. This effect on cholesterol biosynthesis was reflected by changes in plasma membrane composition, as determined by lipid analysis of isolated plasma membrane fractions, these resulting in a decreased cholesterol-phospholipid ratio. The experimental modulation of plasma membrane composition by 25-hydroxycholesterol or mevinolin were accompanied by a decreased cornified envelope formation and by high expression of EGF binding sites. These phenomena were more pronounced in cells induced to differentiate by exposure of cells grown under low Ca2+ to normal Ca2+ concentrations, as compared to cells grown persistently under low Ca2+ concentrations. These results suggest a close correlation between plasma membrane composition, differentiation capacity, and EGF receptor expression.     

Aluminum fluoride stimulates inositol phosphate metabolism and inhibits expression of differentiation markers in mouse keratinocytes.

Mouse keratinocytes are induced to differentiate in vitro by elevating the level of extracellular calcium from 0.05 mM, where keratinocytes express a basal cell phenotype, to greater than 0.10 mM, where they express the differentiated phenotype. This process has been associated with a rapid, sustained increase in inositol phosphate (InsP) turnover, which precedes the expression of differentiation-specific proteins. In 0.05 mM Ca2+ medium, aluminum and fluoride salts (AIF4-), which combine to activate nonspecifically heterotrimeric guanine nucleotide-binding (G) proteins, cause a concentration-dependent increase in InsP metabolism in keratinocytes, and generate elevated intracellular diacylglycerol levels. This is associated with an inhibition of cell growth. Treatment with both AIF4- and Ca2+ greater than 0.10 mM resulted in an additive increase in InsP turnover, implying the presence of at least two responsive InsP pools. AIF4- inhibited the expression of differentiation markers induced by Ca2+ greater than 0.10 mM and altered the morphology of keratinocytes from squamous to dendritic, which was reversible upon withdrawal of AIF4-. Neoplastic keratinocytes, in which basal levels of InsP metabolism are higher than in normal cells, do not differentiate in response to Ca2+. Neoplastic keratinocytes responded to AIF-4 treatment with an even greater rise in InsP metabolism. AIF-4 also inhibited cell growth and reversibly altered morphology in neoplastic keratinocytes. These data suggest that InsP metabolism in keratinocytes is at least partially regulated by a G protein mechanism. Furthermore, an increase in InsP metabolism is not sufficient to stimulate differentiation and may be inhibitory to differentiation if exceeding limited increases. However, these observations cannot exclude the possibility that other AIF-4 stimulated pathways involving G or non-G proteins can also influence keratinocyte biology.     

Coupling of TGF-beta-induced mitogenesis to G-protein activation in AKR-2B cells

We have investigated the signal transduction mechanisms by which TGF-beta stimulates proliferation of AKR-2B murine fibroblasts. Enhanced incorporation of [3H]-thymidine into TGF-beta challenged cells was inhibited in a dose-dependent manner by pertussis toxin. EGF stimulated DNA synthesis was unaffected. Parallel biochemical analysis of pertussis toxin-challenged cells revealed that TGF-beta-induced inhibition of DNA synthesis was associated with ADP-ribosylation of a 41 kDa membrane component and a concomitant decrease in TGF-beta stimulated GTPase activity. These data, along with the observation that Gpp(NH)p decreases the affinity of the TGF-beta receptor for its ligand, strongly suggest that a GTP-binding protein is involved in TGF-beta-induced mitogenesis in AKR-2B cells.     

Growth-inhibitory effects of 1,25-dihydroxyvitamin D3 on normal human keratinocytes cultured in serum-free medium

The effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on the growth of normal human keratinocytes cultured in serum-free medium was investigated. 1,25(OH)2D3 inhibited the cell growth at 10(-7) M by 75.3% and at 10(-6) M almost completely. The growth inhibition was accompanied by changes related to proliferation: (1) remarkable inhibition of DNA synthesis, (2) the decrease in the number of high-affinity receptors for epidermal growth factor, with almost no change in total receptor number, (3) the rapid decrease in c-myc mRNA level. The inhibition of DNA synthesis and the decrease of c-myc mRNA expression occurred at 3 h after the addition of 1,25(OH)2D3. These results suggest that decrease of c-myc mRNA expression is one of the primary effects of 1,25(OH)2D3 in the growth inhibition of human keratinocytes.     

Relationship between glucocorticoid-mediated early decrease of protein synthesis and the steady state decreases of glucocorticoid receptor and TGF-beta activator protein.

The present studies were undertaken to better elucidate the mechanism(s) by which glucocorticoids inhibit the process of tissue repair. The aim was to determine the importance of the effect of glucocorticoids on decreasing the nuclear TGF-beta activator protein. The relationship amongst inhibition of noncollagen protein synthesis and the steady state levels of glucocorticoid receptor and the TGF-beta activator protein was examined. Both collagen and noncollagen synthesis were determined in skin fibroblast cell culture and in dermis. Fetal rat skin fibroblasts were treated for 24 h with dexamethsone. Noncollagen protein synthesis was decreased to approximately one-half that of collagen synthesis. Similar results were obtained in dermis in vivo. At 48 h, dexamethasone treatment resulted in practically no nuclear glucocorticoid receptor being noted and a 40-45% steady state decrease of the TGF-beta activator protein. We have recently reported that the TGF-beta activator protein exists as a protein complex with SP1 and NFKB (p 49). The present data indicate that although the marked decrease of the nuclear glucocorticoid receptor DNA binding following dexamethasone treatment is not comparable to the early 24 h decrease of noncollagen protein synthesis, the decrease of the TGF-beta activator protein complex binding to DNA is. The present studies indicate the importance of the effect of dexamethasone on the steady state level of the TGF-beta activator protein complex in the glucocorticoid-mediated process inhibition of tissue repair and the relationship of this decrease to the earlier inhibition of protein synthesis.     

Decrease in thromboxane A2 receptor expression by differentiation with dibutyryl cyclic AMP in 1321N1 human astrocytoma cells.

Thromboxane A2 (TXA2) receptor expression with its signaling was investigated in 1321N1 human astrocytoma cells differentiated with dibutyryl cyclic AMP (dbcAMP). The cells cultured in 0.5% fetal calf serum containing 0.5 mM dbcAMP for 3 days showed the star-shaped morphology, accompanied with the reduction of a TXA2 mimetic U46619-induced phosphoinositide hydrolysis and Ca2+ mobilization. Immunoblotting analysis revealed that human astrocytoma cells expressed phospholipase C (PLC)-beta1 and -beta3, but not PLC-beta2. The contents of PLC-beta1 and beta3 were not changed by the differentiation. The alpha subunit of Gq/ll bound to TXA2-receptor was reduced by the differentiation, determined by immunoblotting after immunoprecipitation with an anti-TXA2-receptor antibody. Scatchard analysis of the binding of [3H]SQ29548, a TXA2 receptor antagonist, to the membranes revealed that the maximum binding site was reduced by the differentiation. The expression of TXA2 receptor mRNA also was reduced by the differentiation, determined by reverse-transcribed-polymerase chain reaction. Although placental type of TXA2 receptor mRNA expression increased after the differentiation, endothelial type of TXA2 receptor mRNA expression slightly decreased. The results suggest that 1321N1 human astrocytoma cells differentiated with dbcAMP show impaired TXA2 receptor-mediated phosphoinositide hydrolysis and Ca2+ mobilization, due to the decrease in TXA2 receptor number.     

Extracellular calcium alters the effects of retinoic acid on DNA synthesis in cultured murine keratinocytes

The rate of proliferation of epidermal keratinocytes was manipulated by growing the cells in medium containing high or low concentrations of calcium. Keratinocytes cultured in high extracellular Ca++ (1.4 mM and 2.8 mM) proliferated twice as fast as those grown in low Ca++ medium (0.09 mM) as measured by incorporation of [3H]thymidine into DNA. Exposure of high calcium keratinocytes to all-trans retinoic acid for 4 days caused a dose-related inhibition of DNA synthesis with an IC50 of about 10 microM. In contrast, incubating low calcium keratinocytes with all-trans retinoic acid caused a dose-related stimulation of DNA synthesis with maximum increase of 278% over control at 10 microM. This increase was accompanied by increases in culture confluency with maximum increase of 109% in cell number over control at 10 microM. These results are of importance since they suggest Ca++ may influence the effect of retinoids on keratinocytes.     

Independent changes in type I and type II receptors for transforming growth factor beta induced by bone morphogenetic protein 2 parallel expression of the osteoblast phenotype.

Transforming growth factor beta (TGF-beta), a potent regulator of bone formation, has bifunctional effects on osteoblast replication and biochemical activity that appear differentiation dependent. We now show that cell surface binding sites for TGF-beta vary markedly among fibroblasts, bone-derived cells, and highly differentiated osteosarcoma cultures from fetal rats. Expression of betaglycan and type II receptors decline relative to type I receptor expression in parallel with an increase in osteoblast-like activity, predicting that the ratio among various TGF-beta binding sites could influence how its signals are perceived. Bone morphogenetic protein 2 (BMP-2), which induces osteoblast function, does not alter TGF-beta binding or biochemical activity in fibroblasts and has only small effects in less differentiated bone cells. In contrast, BMP-2 rapidly reduces TGF-beta binding to betaglycan and type II receptors in osteoblast-enriched primary cell cultures and increases its relative binding to type I receptors in these cells and in ROS 17/2.8 cultures. Pretreatment with BMP-2 diminishes TGF-beta-induced DNA synthesis in osteoblast-enriched cultures but synergistically enhances its stimulatory effects on either collagen synthesis or alkaline phosphatase activity, depending on the present state of bone cell differentiation. Therefore, BMP-2 shifts the TGF-beta binding profile on bone cells in ways that are consistent with progressive expression of osteoblast phenotype, and these changes distinguish the biochemical effects mediated by each receptor. Our observations indicate specific stepwise actions by TGF-beta family members during osteoblast differentiation, developing in part from changes imprinted by BMP-2 on TGF-beta receptor stoichiometry.     

Cripto-1 alters keratinocyte differentiation via blockade of transforming growth factor-beta1 signaling: role in skin carcinogenesis

Cripto-1 is an epidermal growth factor-Cripto/FRL1/Cryptic family member that plays a role in early embryogenesis as a coreceptor for Nodal and is overexpressed in human tumors. Here we report that in the two-stage mouse skin carcinogenesis model, Cripto-1 is highly up-regulated in tumor promoter-treated normal skin and in benign papillomas. Treatment of primary mouse keratinocytes with Cripto-1 stimulated proliferation and induced expression of keratin 8 but blocked induction of the normal epidermal differentiation marker keratin 1, changes that are hallmarks of tumor progression in squamous cancer. Chemical or genetic blockade of the transforming growth factor (TGF)-beta1 signaling pathway using the ALK5 kinase inhibitor SB431542 and dominant negative TGF-beta type II receptor, respectively, had similar effects on keratinocyte differentiation. Our results show that Cripto-1 could block TGF-beta1 receptor binding, phosphorylation of Smad2 and Smad3, TGF-beta-responsive luciferase reporter activity, and TGF-beta1-mediated senescence of keratinocytes. We suggest that inhibition of TGF-beta1 by Cripto-1 may play an important role in altering the differentiation state of keratinocytes and promoting outgrowth of squamous tumors in the mouse epidermis.     

The extracellular calcium-sensing receptor is required for calcium-induced differentiation in human keratinocytes

In cultured keratinocytes, the acute increase of the extracellular calcium concentration above 0.03 mM leads to a rapid increase in intracellular calcium concentration ([Ca(2+)]i) and inositol trisphosphate production and, subsequently, to the expression of differentiation-related genes. Previous studies demonstrated that human keratinocytes express the full-length extracellular calcium-sensing receptor (CaR) and an alternatively spliced variant lacking exon 5 and suggested their involvement in calcium regulation of keratinocyte differentiation. To understand the role of the CaR, we transfected keratinocytes with an antisense human CaR cDNA construct and examined its impact on calcium signaling and calcium-induced differentiation. The antisense CaR cDNA significantly reduced the protein level of endogenous CaRs. These cells displayed a marked reduction in the rise in [Ca(2+)]i in response to extracellular calcium or to NPS R-467, a CaR activator, whereas the ATP-evoked rise in [Ca(2+)]i was not affected. Calcium-induced inhibition of cell proliferation and calcium-stimulated expression of the differentiation markers involucrin and transglutaminase were also blocked by the antisense CaR cDNA. When cotransfected with luciferase reporter vectors containing either the involucrin or transglutaminase promoter, the antisense CaR cDNA suppressed the calcium-stimulated promoter activities. These results indicate that CaR is required for mediating calcium signaling and calcium-induced differentiation in keratinocytes.     

Lateral mobility of plasma membrane lipids in normal and transformed keratinocytes

In this study we have examined possible differentiation-dependent modulations in plasma membrane lipid properties in normal keratinocytes, SV-40 transformed keratinocytes (SVK14) and a number of squamous carcinoma (SCC) cells. In normal keratinocytes the lateral diffusion coefficient of plasma membrane lipids (D) differs significantly for cells cultured permanently under low and normal Ca2+-conditions (5.16 x 10(-9) and 3.27 x 10(-9) cm2/s, respectively). When differentiation is induced by exposing low Ca2+-cultured cells to normal Ca2+ concentrations D increases to 7.07 x 10(-9) cm2/s during the initial hours of differentiation followed by a gradual sustained decrease to values also observed in cells cultured permanently under normal Ca2+-conditions. In SCC and SVK14 cells a similar initial transient increase in lateral lipid mobility is observed upon initiation of differentiation, but, in contrast to normal keratinocytes, no sustained decrease in D is seen upon prolonged culturing under normal Ca2+ conditions. The results indicate that the deficiency of the transformed cells to respond to Ca2+-induced differentiation might involve transformation-dependent alterations in membrane structure and function.