A sensitive method to quantify the terminal differentiation of cultured epidermal cells

Terminal differentiation of normal and malignant keratinocytes is routinely determined by the ability of these cells to form cornified envelopes after incubation with a calcium ionophore. We have used the human squamous cell carcinoma, SqCC/Y1, to quantify cellular differentiation by the formation of detergent-insoluble protein. The methodology developed employs the metabolic labeling of detergent-insoluble cellular protein with [35S]methionine in the presence of a calcium ionophore. The ratio of filter-retainable radioactivity to that of total cellular protein was shown to be closely correlated to the results obtained by measuring the number of envelope-competent cells when cells were induced to enter a pathway of terminal differentiation in culture by serum deprivation or by treatment with hydrocortisone, and during the inhibition of maturation by either retinoic acid (RA) or epidermal growth factor (EGF). This way of measuring the degree of terminal differentiation of epidermal cells is a relatively simple one that readily allows the simultaneous measurement of multiple samples.     

Annexin I and involucrin are cross-linked by particulate transglutaminase into the cornified cell envelope of squamous cell carcinoma Y1.

The squamous cell carcinoma line, SqCC/Y1, like natural squamous epithelia, forms a cornified cell envelope during differentiation which can be directly correlated with an increase in particulate transglutaminase activity. When transglutaminase is activated in these cells by calcium ionophore X-537A, annexin I and involucrin become incorporated into the cornified cell envelope and cannot be extracted with solutions containing sodium dodecyl sulfate (SDS) and beta-mercaptoethanol. This effect is specific for annexin I; thus, the amounts of annexins II and IV that were extractable from cells by SDS and beta-mercaptoethanol did not change following treatment with ionophore X-537A. Annexin I could be cross-linked in vitro to itself and to other endogenous proteins by transglutaminase extracted from the particulate fraction of SqCC/Y1 cells. Immunofluorescence studies showed that cross-linked annexin I and involucrin form an envelope-like structure in SqCC/Y1 cells during differentiation that cannot be extracted by EGTA and Triton X-100. The amount of staining of this envelope structure corresponded directly to the particulate transglutaminase activity of these cells. Annexin I monoclonal and polyclonal antibodies were shown to bind to purified cornified cell envelopes from SqCC/Y1. These studies suggest that particulate transglutaminase regulates a function of annexin I during the differentiation of SqCC/Y1 cells by covalently cross-linking this protein into the cornified cell envelope.     

Role of lipocortin I in the glucocorticoid induction of the terminal differentiation of a human squamous carcinoma

The human squamous cell carcinoma SqCC/Y1 undergoes spontaneous terminal differentiation in the confluent state. The degree of maturation was markedly increased by glucocorticoids and by both human recombinant and placental lipocortin I. Western analyses demonstrated cellular secretion of lipocortin into the medium. Glucocorticoid-induced maturation was antagonized by a lipocortin I-specific monoclonal antibody, by phospholipase A2 (PLA2), and by arachidonic acid. Induction of the differentiation of SqCC/Y1 cells by lipocortin I was prevented by arachidonic acid. The PLA2 inhibitor, dibromoacetophenone, caused an increase in envelope-competent cells indicating that inhibition of PLA2 results in induction of differentiation. Epidermal growth factor prevented the induction of differentiation by both lipocortin I and by glucocorticoids. The nonsteroidal lipoxygenase/cyclo-oxygenase inhibitor, phenidone, also increased SqCC/Y1 differentiation, suggesting that leukotrienes, thromboxanes, and/or prostaglandins may be involved in lipocortin-mediated regulation of SqCC/Y1 maturation. The findings support a role for lipocortin I in mediating the effects of glucocorticoids on epidermal cell differentiation.     

Retinoic acid-induced transglutaminase in mouse epidermal cells is distinct from epidermal transglutaminase

Elevated transglutaminase activity and formation of cornified envelopes are markers of terminal differentiation in mouse epidermal cells. Epidermal transglutaminase catalyzes cornified envelope formation and in cultured cells is inducible by calcium ion or phorbol ester tumor promoters. Retinoic acid also induces transglutaminase activity but inhibits cross-linked envelope formation. This apparent paradox might be resolved by the observation that the retinoic acid-induced transglutaminase appears to be either a different enzyme or a markedly altered form of the epidermal enzyme. The retinoic acid-induced transglutaminase is soluble in aqueous buffers, is thermolabile at pH 9.0, 37 degrees C, and elutes from an anion exchange column at 0.4 M NaCl. In contrast, the epidermal enzyme is particulate and requires detergent for solubilization, is relatively thermostable, and elutes from the anion exchanger at 0.25 M NaCl. The retinoic acid-induced enzyme is probably identical with the "tissue" transglutaminase present in liver and in other cells. It is proposed that the transglutaminase induced by retinoic acid may play a role in the inhibition by retinoids of calcium and tumor promoter-induced differentiation.     

Accumulation of cholesterol 3-sulfate during in vitro squamous differentiation of rabbit tracheal epithelial cells and its regulation by retinoids

Rabbit tracheal epithelial (RbTE) cells in culture undergo terminal squamous differentiation characterized by enhanced transglutaminase activity, synthesis of specific keratins, and the formation of cross-linked envelopes. The expression of each of these markers of differentiation occurs spontaneously after the cells reach confluency, but this expression can be inhibited by the inclusion of retinoids in the extracellular medium. In the current work, we demonstrate that radioactive sulfate incorporation into the organic phase of a CHCl3/CH3OH (2:1) extract of RbTE cells increases 50- to 100-fold upon differentiation and that this accumulation can be completely blocked by the inclusion of retinoic acid in the culture medium. By the techniques of specific metabolic radiolabeling, thin layer chromatography, gas chromatography-mass spectrometry, and fast atom bombardment-mass spectrometry, the sulfated amphiphile was shown to be cholesterol 3-sulfate. Cholesterol sulfate accumulation begins 1 to 2 days after the RbTE cells reach the stationary phase of growth which is the same time that other differentiated functions begin to be expressed. The inhibition of accumulation by retinoic acid is concentration-dependent and half-maximal at 5 X 10(-11) M. The relative efficacy of a series of synthetic retinoids in inhibiting cholesterol sulfate accumulation correlated with their binding to the cellular retinoic acid-binding protein. These data taken together indicate that cholesterol sulfate is a marker of squamous differentiation in RbTE cells in culture. Possible biochemical mechanisms of the regulation of cholesterol sulfate levels during differentiation are discussed.     

Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis, resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. Retinoic acid, arotinoid ethylsulfone, and hydrocortisone had no effects on LDL binding and only minor effects on LDL degradation. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.     

Incomplete epidermal differentiation of A431 epidermoid carcinoma cells

Summary A431 malignant keratinocytes, although derived from a muco-cutaneous carcinoma of the vulva, fail to achieve terminal epidermal differentiation in culture as shown by their inability to form cornified envelopes. Even after culture in a serum-free medium (MCDB 153) containing no retinoic acid and a high (10⁻³ M) calcium concentration (conditions known to facilitate epidermal differentiation), the cells do not become competent as shown by the fact that subsequent treatment with a calcium ionophore is unable to provoke the formation of cornified envelopes. Nevertheless, A431 cells are able to synthesize the envelope precursor involucrin. The block in formation of cornified envelopes is thus not due to a lack in involucrin. The results described here suggest that the absence of cross-linking of this molecule is due to a lowered epidermal membrane-bound transglutaminase activity in A431 cells, enhances involucrin accumulation in these cells, although in normal human keratinocytes it stimulates growth and reduces involucrin synthesis. These results suggest that involucrin synthesis is triggered by the arrest of growth. EDITOR'S STATEMENT The A431 cell line has been used extensively in the study of EGF receptors and effects, and recently has been employed in studies of surface membrane receptors for other factors, as well as studies of extracellular matrix synthesis and deposition and tumor promoter activities. The expanding use of A431 cells calls for a more thorough understanding of the cell type it represents and the degree to which it represents a general in vitro model of normal or neoplastic epidermal cells. This article addresses some of these questions.     

Retinoic acid induces granulocytic differentiation of myeloid progenitors in congenital agranulocytosis bone marrow cells

Congenital agranulocytosis is a rare fatal infantile disease characterised by recurrent bacterial infections, persistent absence of neutrophils and maturation arrest at the promyelocyte/myelocyte stage. The effectiveness of retinoic acid in inducing differentiation of congenital agranulocytosis marrow myeloid progenitor cells was studied. Non-adherent mononuclear marrow cells were treated in an in vitro culture with retinoic acid at various concentrations from 1nM to 1 microM for seven days. Morphological and functional differentiation into mature granulocytes was induced by retinoic acid in a dose-response stimulation with a maximum response at a concentration of 1 microM. These results suggest a potential therapeutic role for retinoic acid in the treatment of congenital agranulocytosis.     

Retinoic acid induces transglutaminase activity but inhibits cornification of cultured epidermal cells

In cultured mouse epidermal basal cells, retinoic acid is a potent inducer of transglutaminase, the enzyme responsible for isodipeptide bond formation in protein cross-linking in the production of the cornified membrane during terminal differentiation. Paradoxically retinoic acid also inhibits the formation of the cross-linked envelope and greatly reduces the level of dipeptide bond formation in epidermal cells induced to differentiate by calcium. These results suggest a novel mechanism by which retinoids can modify transglutaminase activity and epidermal differentiation.     

Photoisomerization of retinoic acid and its influence on regulation of human keratinocyte growth and differentiation

Retinoic acid constantly undergoes structural inter-conversions among the geometrical isomers (all-trans-retinoic acid, 9-cis-retinoic acid, 11-cis-retinoic acid, 13-cis-retinoic acid and 9-13-di-cis-retinoic acid) by photoisomerization under natural light. Geometric isomers of retinoic acid thus formed showed different effects on human epidermal keratinocyte growth and differentiation. The ability of the isomers to inhibit the synthesis of cornified envelope (terminal event in the keratinocyte differentiation program) changed rapidly when illuminated by white fluorescent light. The 11-cis-retinoic acid had a 3-fold stronger activity to inhibit the growth of keratinocytes than the other geometric isomers. On the other hand, all-trans-retinoic acid, 9-cis-retinoic acid and 9-13-di-cis-retinoic acid exhibited a 3-fold greater ability to inhibit synthesis of involucrin, transglutaminase and the cornified envelopes. The regulation of keratin expression by the geometric isomers of retinoic acids was extremely complex. Level of keratin-1 (K1) mRNA was increased by 11-cis-retinoic acid and 13-cis-retinoic acid, but suppressed by 9,13-di-cis-retinoic acids while all-trans-retinoic acid and 9-cis-retinoic acid had no effect. Level of keratin-10 (K10) mRNA was strongly inhibited by all-trans-retinoic acid, 9-cis-retinoic acid and 11-cis-retinoic acid as compared to 13-cis-retinoic acid and 9,13-di-cis-retinoic acids. The mRNA level of keratin-14 (K14) was suppressed by all-trans-retinoic acid, 9-cis-retinoic acid and 11-cis-retinoic acid but not influenced by 13-cis-retinoic acid and 9,13-di-cis-retinoic acid. Natural light induced structural inter-conversions among the geometric isomers of retinoic acids in tissues-especially the skin, might play a crucial role in the regulation of growth and differentiation of keratinocytes.     

Identification of an interleukin-3-regulated aldoketo reductase gene in myeloid cells which may function in autocrine regulation of myelopoiesis

The EML hematopoietic progenitor cell line is a model system for studying molecular events regulating myeloid commitment and terminal differentiation. We used representational difference analysis to identify genes that are expressed differentially during myeloid differentiation of EML cells. One gene (named mAKRa) encoded a novel member of the aldoketo reductase (AKR) superfamily of cytosolic NAD(P)(H)-dependent oxidoreductases. mAKRa mRNA was detected in murine hematopoietic tissues including bone marrow, spleen, and thymus. In myeloid cell lines, mAKRa was expressed at highest levels in cells representative of promyelocytes. mAKRa mRNA levels increased rapidly in response to interleukin-3 over the first 24 h of EML cell differentiation when the cells undergo lineage commitment and extensive proliferation. mAKRa mRNA levels decreased later in the differentiation process particularly when the EML cells were cultured with granulocyte/macrophage colony-stimulating factor and retinoic acid to induce terminal granulocytic maturation. mAKRa mRNA levels decreased during retinoic acid-induced terminal granulocytic differentiation of the MPRO promyelocyte cell line. AKRs act as molecular switches by catalyzing the interconversion or inactivation of bioactive molecules including steroids and prostaglandins. We propose that mAKRa may catalyze the production or catabolism of autocrine factors that promote the proliferation and/or lineage commitment of early myeloid progenitors.     

Retinoid suppression of transglutaminase activity and envelope competence in cultured human epidermal carcinoma cells

Growth of SCC-13 squamous carcinoma cultures in the presence of retinoids considerably reduced the expression of two differentiation markers, the cellular capability to form cross-linked envelopes, and the enzyme transglutaminase required for cross-linking. A limited survey of retinoids showed that all-trans retinoic acid, 13-cis retinoic acid, and arotinoid Ro 13-6298 were highly effective in the absence of hydrocortisone and were only slightly antagonized by its presence in the medium. In contrast, retinyl acetate, retinol, and retinol bound to its plasma binding protein were quite active in the absence of hydrocortisone but were essentially inactive in its presence. Dexamethasone was also highly effective in antagonizing the suppressive action of retinyl acetate on envelope formation, while the corticosteroid antagonists cortexolone and progesterone were inactive. These results suggest that there are separate pathways, which are differentially regulated by hydrocortisone, for either the metabolism or action of retinol and retinoic acid in SCC-13 cells.     

Participation of type II protein kinase A in the retinoic acid-induced growth inhibition of SH-SY5Y human neuroblastoma cells

To examine the role of protein kinase A (EC 2.7.1.37) isozymes in the retinoic acid-induced growth inhibition and neuronal differentiation, we investigated the changes of protein kinase A isozyme patterns in retinoic acid-treated SH-SY5Y human neuroblastoma cells. Retinoic acid induced growth inhibition and neuronal differentiation of SH-SY5Y cells in a dose- and time-dependent manner. Neuronal differentiation was evidenced by extensive neurite outgrowth, decrease of N-Myc oncoprotein, and increase of GAP-43 mRNA. Type II protein kinase A activity increased by 1.5-fold in differentiated SH-SY5Y cells by retinoic acid treatment. The increase of type II protein kinase A was due to the increase of RIIbeta and Calpha subunits. Since type II protein kinase A and RIIbeta have been known to play important role(s) in the growth inhibition and differentiation of cancer cells, we further investigated the role of the increased type II protein kinase A by overexpressing RIIbeta in SH-SY5Y cells. The growth of RIIbeta-overexpressing cells was slower than that of parental cells, being comparable to that of retinoic acid-treated cells. Retinoic acid treatment further increased the RIIbeta level and further inhibited the growth of RIIbeta-overexpressing cells, showing strong correlation between the level of RIIbeta and growth inhibition. However, RIIbeta-overexpressing cells did not show any sign of neuronal differentiation and responded to retinoic acid in the same way as parental cells. These data suggest that protein kinase A participates in the retinoic acid-induced growth inhibition through the up-regulation of RIIbeta/type II protein kinase A.     

Synergistic anti-proliferative effects of vitamin D derivatives and 9-cis retinoic acid in SH-SY5Y human neuroblastoma cells.

This study examines the effect of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], 24,25-dihydroxyvitamin D₃ [24,25(OH)₂D₃], two vitamin D analogues (KH 1060 and EB 1089, which are 20-epi-22-oxa and 22,24-diene-analogues, respectively), 9-cis retinoic acid and all-trans retinoic acid on proliferation of SH-SY5Y human neuroblastoma cells, after treatment for 7 days. Cell number did not change when the cells were incubated with 1, 10 or 100 nM 1,25(OH)₂D₃ or its derivatives, but significantly decreased in the presence of the two retinoids (0.001–10 μM final concentration). A synergistic inhibition was observed, when SH-SY5Y cells were treated combining 0.1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060, and 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089. Acetylcholinesterase activity showed a significant increase, in comparison with controls, after treatment of the cells for 7 days with 0.1 or 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. This increase was synergistic, combining 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or EB 1089. The levels of the c-myc encoded protein remarkably decreased after treatment of SH-SY5Y cells for 1, 3, 7 days with 0.1 and 1 μM 9-cis retinoic acid, alone or combined with 10 nM 1,25(OH)₂D₃ or 10 nM KH 1060 or 10 nM EB 1089. In particular, the association of 1 μM 9-cis retinoic acid and 10 nM 1,25(OH)₂D₃ or 10 nM EB 1089 resulted in a synergistic c-myc inhibition, in comparison with that obtained in the presence of the retinoid alone. These findings may have therapeutic implications in human neuroblastoma.     

Sodium butyrate selectively antagonizes the inhibitory effect of retinoids on cornified envelope formation in cultured human keratinocytes

Sodium butyrate affects cell differentiation in confluent epidermal keratinocyte cultures by considerably increasing the spontaneous formation of cross-linked envelopes in normal human keratinocytes (NHK). It also favors the development of envelope competence in the Simian virus-40 (SV-40)-transformed human foreskin keratinocyte line SV-K14. It completely abolishes the inhibitory effect of serum and retinoic acid on the expression of plasma membrane-associated transglutaminase. However, other markers of epidermal differentiation that are also under the control of retinoids such as keratins or the enzyme cholesterol sulfotransferase are not affected by butyrate. The level of the cellular retinoic acid binding protein (CRABP) is considerably increased in its presence. Butyrate does not interfere with the binding of retinoids to their cellular binding proteins. Our observations suggest that sodium butyrate stimulates cornified envelope formation via the induction of the plasma membrane-associated transglutaminase required for cornified envelope synthesis and, additionally, by abolishing the inhibitory effect of retinoids on the expression of this enzyme.     

The 9-cis retinoic acid signaling pathway and its regulation of prostaglandin-endoperoxide synthase 2 during in vitro maturation of pig cumulus cell-oocyte complexes and effects on parthenogenetic embryo production

The addition of 9-cis retinoic acid to the oocyte maturation culture medium has a beneficial effect on in vitro fertilized embryos. However, the mechanism of this activity is not known. Therefore, this study was done to elucidate the effect of 9-cis retinoic acid on parthenogenetic embryo production and its signaling pathway and molecular function during in vitro maturation of porcine cumulus cell-oocyte complexes (COCs). Concentrations of 0, 5, 50, and 500 nM 9-cis retinoic acid were added to the in vitro maturation medium, and the embryos were assessed after parthenogenetic activation. Cumulus cells and oocytes from the in vitro matured COCs were separated and subjected to RT-PCR and real-time RT-PCR for detecting retinoic acid receptors and measuring expression of prostaglandin-endoperoxide synthase1 and 2. The addition of 5 nM 9-cis retinoic acid to the maturation medium was beneficial for parthenogenetic embryo production. The effect of 9-cis retinoic acid was exerted directly through the oocytes via the retinoic acid receptor alpha and retinoid X receptor gamma signaling pathways and indirectly through the cumulus cells by the retinoic acid receptor beta and gamma and retinoid X receptor alpha and beta signaling pathways. The addition of 5 nM 9-cis retinoic acid-stimulated cumulus cells reaches full expansion by suppressing their excessive expression of prostaglandin-endoperoxide synthase 2. This study shows that 9-cis retinoic acid can exert its beneficial effect on parthenogenetic embryo production in pigs by multidimensional pathways affecting oocyte maturation.     

Human myelogenous leukemia cell line HL-60 cells resistant to differentiation induction by retinoic acid. Decreased content of glycosphingolipids and granulocytic differentiation by neolacto series gangliosides

We have recently reported that neolacto series gangliosides (NeuAc-nLc) are increased during granulocytic differentiation of human myelogenous leukemia cell line HL-60 cells induced by retinoic acid and that HL-60 cells are differentiated into mature granulocytes when the cells are cultivated with NeuAc-nLc (Nojiri, H., Kitagawa, S., Nakamura, M., Kirito, K., Enomoto, Y., and Saito, M. (1988) J. Biol. Chem. 263, 7443-7446). In contrast to these wild-type-HL-60 cells, HL-60 cells resistant to differentiation induction by retinoic acid showed a markedly decreased content of gangliosides, especially NeuAc-nLc, and did not show any increase in the content of gangliosides when cultivated with retinoic acid. Neutral glycosphingolipids, the precursors of gangliosides, were not accumulated in these resistant cells. When retinoic acid-resistant HL-60 cells were cultivated in the presence of NeuAc-nLc, the cells were found to be differentiated into mature granulocytes on morphological and functional criteria. The differentiation of cells was dependent on the concentration of gangliosides and was accompanied by inhibition of cell growth. Wild-type HL-60 cells differentiated by NeuAc-nLc showed the changes in ganglioside composition, which were similar to those in wild-type HL-60 cells differentiated by retinoic acid; among the gangliosides changed, 2----3 sialylparagloboside and 2----3 sialylnorhexaosylceramide were increased. These findings suggest (a) that the synthesis of particular NeuAc-nLe molecules is an important step for retinoic acid-induced granulocytic differentiation and this step could be bypassed or replaced by exogenous NeuAc-nLc, and (b) that the defective synthesis of particular NeuAc-nLc molecules is responsible for the failure of differentiation induction in retinoic acid-resistant HL-60 cells by retinoic acid.     

The response of embryonal carcinoma cells to retinoic acid depends on colony size

Single cells of the feeder-layer-dependent mouse embryonal carcinoma (EC) cell line, NG2, can spontaneously give rise to colonies containing a wide variety of differentiated cell types in vitro. When cultured with retinoic acid at a concentration of 10(-7) M, single NG2 cells irreversibly differentiated to parietal endoderm, as identified by morphological criteria and immunohistochemical staining. Parietal endoderm was also the first product of spontaneous differentiation. However, when retinoic acid was added to monolayer groups of NG2 cells, not all of the cells were induced to differentiate. The parietal-endoderm cells which did form were generally found at the periphery of cell colonies, as is the case during spontaneous differentiation. Differentiation in the centre of these colonies yielded a variety of cell types over a 21-day period. These results are consistent with the hypothesis that retinoic acid induces the differentiation of EC cells by accelerating cellular response to intrinsic stimuli, rather than by overriding these stimuli.