Effect of retinoic acid on the proliferation and phagocytic capability of murine macrophage-like cell lines

Retinoic acid (RA) exerted a variable degree of growth inhibitory activity on the macrophage-like cell lines P388D1, J774.2, WEHI-265, WEHI-3, and PU-5. Comparison of cell proliferation and clonal growth suggests that at concentrations of 10(-9)-10(-6) M the inhibitory activity stems from processes leading to elongation of cell cycle time and not from terminal differentiation processes. RA was shown to be a potent inducer of the development of high-phagocytic phenotypes (assessed by phagocytosis of heat-killed yeast cells) in the P388D1, J774.2, and WEHI-265 cell lines which differ substantially in their proliferative and adherence characteristics. The PU-5 and WEHI-3 cell lines were not induced by RA to express an enhanced phagocytic activity toward heat-killed yeast cells. The augmented phagocytic capability was dose dependent over a wide range of RA concentrations. In P388D1 cells, 2 X 10(-12) M RA already exerted significant phagocytosis augmentation effects, which progressively increased up to 2 X 10(-5) M RA, the highest concentration tested. Retinal, retinyl acetate, and retinol had similar effects to those of RA on both cell adherence and phagocytosis in P388D1 cells, albeit at concentrations four to six orders of magnitude higher. Optimal development of the high-phagocytic phenotype in P388D1, J774.2, and WEHI-265 cells required at least 96 hr of culture in the presence of RA; at 48 hr and 23 hr the effects were already substantial, whereas at 4 hr of exposure to RA no significant enhancement of phagocytosis could be detected. Thus both extended periods of culture in the presence of RA (more than two to three cell cycles) and high concentrations were needed for induction, in more than 90% of the cells, of the expression of a high-phagocytic phenotype. The reversion to a low-phagocytic phenotype upon removal of RA was also rather slow and required several cell cycles. In P388D1 cells RA also enhanced the phagocytosis of latex beads but had no effect on the phagocytosis of starch particles, or the extent of binding of immunoglobulin G-coated sheep red blood cells (SRBC). The expression of receptors for concanavalin A and for nonopsonized SRBC remarkably increased in RA-treated cells, as was the ability to perform Fc-receptor mediated erythrophagocytosis. Both P388D1 cells and WEHI-265 cells were induced by RA to express nitroblue tetrazolium reducing activity. The data suggest that RA induces profound changes in the functional capabilities of macrophage-like cell lines which are apparently not dependent on cessation of growth and terminal differentiation processes.     

Retinoic acid enhances colony-stimulating factor-induced clonal growth of normal human myeloid progenitor cells in vitro

We studied the effect of vitamin A and its analogues (retinoids) on the clonal growth in vitro of normal human myeloid progenitor cells. Normal human bone marrow cells were cultured in soft gel in the presence of a source of colony-stimulating factor (CSF), and various retinoids, and the number of granulocyte-macrophage colonies (CFU-GM) were scored. The addition of 3 × 10^?8 to 3 × 10^?6 M retinoic acid to culture plates containing CSF markedly increased the number of myeloid colonies as compared with culture plates containing CSF alone. Maximal stimulation occurred at a concentration of 3 × 10^?7 M retinoic acid which increased the mean number of colonies by 213 ± 8 % (±S.E.) over plates containing CSF alone. Retinal or retinyl acetate was less potent than retinoic acid, and retinol (vitamin A) had no effect on colony growth. Retinoic acid had no direct CSF activity nor did it stimulate CSF production by the cultured bone marrow cells. Our studies show for the first time that retinoids can stimulate granulopoiesis in vitro and we suggest that this stimulation may be mediated by increased responsiveness of the granulocyte-macrophage progenitors to the action of CSF.     

Regional differences in retinoid release from embryonic neural tissue detected by an in vitro reporter assay.

Retinoic acid and related retinoids have been suggested to contribute to the pattern of cell differentiation during vertebrate embryonic development. To identify cell groups that release morphogenetically active retinoids, we have developed a reporter assay that makes use of a retinoic acid inducible response element (RARE) to drive lacZ or luciferase reporter genes in stably transfected cell lines. This reporter gene assay allows detection of retinoids released from embryonic tissues over a range equivalent to that induced by femtomole amounts of retinoic acid. We have used this assay first to determine whether the floor plate, a cell group that has polarizing properties in neural tube and limb bud differentiation, is a local source of retinoids within the spinal cord. We have also examined whether the effects of exogenously administered retinoic acid on anteroposterior patterning of cells in the developing central nervous system correlate with differences in retinoid release from anterior and posterior neural tissue. We find that the release of morphogenetically active retinoids from the floor plate is only about 1.5-fold that of the dorsal spinal cord, which does not have neural tube or limb polarizing activity. These results suggest that the spatial distribution of retinoid release from spinal cord tissues differs from that of the neural and limb polarizing activity. This assay has also shown that retinoids are released from the embryonic spinal cord at much greater levels than from the forebrain. This result, together with previous observations that the development of forebrain structures is suppressed by low concentrations of retinoic acid, suggest that the normal development of forebrain structures is dependent on the maintenance of low concentrations of retinoids in anterior regions of the embryonic axis. This assay has also provided initial evidence that other embryonic tissues with polarizing properties in vivo release retinoids in vitro.     

Comparative role of retinol, retinoic acid and beta-carotene on progesterone secretion by pig corpus luteum in vitro

Quadruplicate wells of pig luteal cells were incubated for 24 h in the presence of different concentrations of retinol, beta-carotene (0, 1 x 10(-5), 1 x 10(-6) and 1 x 10(-7) M) or retinoic acid (0, 1 x 10(-6), 1 x 10(-7) and 1 x 10(-8) M). In addition, the responsiveness of luteal cells to LH challenge was also evaluated. Progesterone was assayed in the media. Cell viability was estimated using trypan blue exclusion and showed over 95% viability. In the presence of LH, progesterone content in the medium was increased by 7-fold. As compared to their respective controls, all concentrations of retinoic acid and beta-carotene increased progesterone content in the media. The highest level of stimulation was observed with 1 x 10(-6) M-retinoic acid (5-fold increase) and 1 x 10(-7) M-beta-carotene (10-fold increase). Only 1 x 10(-5) M-retinol stimulated progesterone secretion (over 3-fold). Therefore, retinol, retinoic acid and beta-carotene stimulate progesterone secretion by pig luteal cells in vitro.     

Characterization of the action of retinoids on mouse fibroblast cell lines

Retinoic acid affects 3T6 and 3T3 cells by inhibiting growth, causing a morphological change and increasing cell-to-substratum adhesiveness. Retinoic acid does not exert such effects on virus-transformed 3T3SV cells. Retinoic acid treatment of 3T6 cells causes a concentration-dependent increase in generation time and a reduction in saturation density. Analysis of cell surface proteins shows that a high molecular weight band of 230 000 D, corresponding to the position of the LETS glycoprotein, is more intensely labeled by iodination of cells treated with retinoic acid compared to control cells. Retinoic acid substantially stimulates the incorporation of ³⁵SO₄ into cell-associated glycosaminoglycans and causes a less dramatic increase in glycosaminoglycans excreted into the medium. The relationship between the increase in these cell surface components and the enhanced adhesiveness is discussed. A retinoic acid binding protein is detectable in the cytosol of 3T6 and 3T3 cells but not in 3T3SV cells, suggesting that the action of retinoids on these cells is mediated via this protein.     

Differential response to retinoic acid of Syrian hamster embryo fibroblasts expressing v-src or v-Ha-ras oncogenes.

It has been shown that treatment of many but not all tumor cell lines with retinoids affects cell proliferation and expression of the transformed phenotype. To determine whether the response of the tumor cell to retinoids is influenced by specific oncogenes activated in the cell, we studied the action of these agents in the immortal, nontumorigenic Syrian hamster embryo cell lines DES-4 and 10W transfected with either v-Ha-ras or v-src oncogenes. In this paper we show that in transformed DES-4 cells expressing v-src, retinoic acid inhibited anchorage-independent growth, reduced saturation density, and inhibited the induction of ornithine decarboxylase by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. In contrast, retinoic acid enhances the expression of the transformed phenotype in DES-4-derived cells that express v-Ha-ras. In these cells retinoic acid increases the number and the average size of colonies formed in soft agar. Moreover, retinoic acid enhances ornithine decarboxylase activity and acts in a synergistic fashion with 12-O-tetradecanoylphorbol-13-acetate. These results indicate that oncogenes activated in cells can indeed influence the response of cells to retinoids. Retinoic acid does not appear to alter the levels of pp60src or p21ras proteins in these cells, suggesting that retinoic acid does not affect the synthesis of these oncogene products. Furthermore, retinoic acid does not affect the protein kinase activity of pp60src. Transformed cell lines derived from 10W cells responded differently, indicating that the presence of a specific oncogene is not the only factor determining the response to retinoids. Possible mechanisms by which retinoic acid may interfere with the expression of the oncogene products are discussed.     

Biphasic action of retinoids on gonadotropin receptor induction in rat granulosa cells in vitro

Vitamin A (retinol) has been held to be uniquely essential for normal vision and reproduction, all other functions being served by its metabolite retinoic acid. The inability of retinoic acid to maintain adequate serum progesterone is implicated as the cause of fetal resorption. The availability of lipoproteins is a major limiting factor in progesterone production and the ovarian expression of lipoprotein receptors is dependent on the action of luteinizing hormone (LH). Therefore, we investigated the effects of retinol and retinoic acid on LH receptor induction by ovarian cells in an attempt to determine the basis for the reported differences in the gonadal action of these two retinoids. Our results indicate that retinoic acid (10(-10) M) and retinol (10(-8) M) each synergistically enhance the ability of follicle stimulating hormone (FSH) to induce LH-receptors and to stimulate the formation of cyclic adenosine 3',5'-monophosphate (cAMP) and progesterone. However, at higher concentrations, both retinoids inhibited these effects of FSH. For every measured effect, retinoic acid was more potent than retinol. Since retinol is metabolized to retinoic acid in other tissues, these results suggest that retinoic acid may be the mediator of the action of retinol on the ovary and that retinol's unique effect on reproduction needs to be investigated further.     

Stimulation of differentiation of several murine embryonal carcinoma cell lines by retinoic acid

Retinoic acid stimulates several murine embryonal carcinoma (EC) cell lines, even those previously considered to be incapable of differentiating, to give rise to cell types distinguishable from the parental phenotype in morphology, production of plasminogen activator and surface protein properties. Retinoic acid promotes these changes over a range of low concentrations (10⁻⁹–10⁻⁵ M) which are generally non-toxic to the cells. The effects are clearly demonstrated when EC cells are aggregated prior to exposure to retinoic acid. It is concluded that the observed phenotypic alterations induced by retinoic acid reflect differentiation of the EC cells since non-EC cell characteristics are maintained by cloned cells several generations after retinoic acid is removed from the cultures. Our studies suggest that although retinoic acid stimulates the conversion of EC cells to differentiated derivatives, it does not influence the direction of differentiation. Furthermore, the effectiveness of retinoic acid in stimulating differentiation of EC cells from lines such as Nulli-SCC1 raises the question of whether true ‘nullipotent’ EC lines really exist.     

Effects of retinoic acid on the human lymphocyte response to mitogens

Nontoxic concentrations of retinoic acid enhance DNA synthesis of human peripheral blood lymphocytes in response to phytohemagglutinin or rabbit-antihuman thymocyte globulin, whereas the response to concanavalin A or pokeweed mitogen remained unaffected. Retinoic acid-induced stimulation of lymphocyte reactivity to phytohemagglutinin or antithymocyte globulin was most evident in T cell-enriched subpopulations and required the near-concurrent addition of retinoic acid and mitogens. Retinoic acid-mediated enhancement of lymphocyte proliferation in response to phytohemagglutinin or antithymocyte globulin was paralleled by a concomitant suppression of immune interferon production of lymphocytes stimulated with these mitogens. These findings allow further studies on the immunoregulatory action of retinoids in vitro.     

Regulation of the murine alpha(2)(I) collagen promoter by retinoic acid and retinoid X receptors

Retinoic acid decreases collagen production by hepatic stellate cells. This study investigated the effects of retinoic acid receptor beta (RARbeta) and retinoid X receptor alpha (RXRalpha) on the regulation of the alpha(2)(I) collagen promoter. Retinoic acid and the RARbeta and RXRalpha expression vectors suppressed the promoter in transfected stellate cells with maximal suppression obtained when combined. Mutation of the retinoic acid response element (RARE) at -879 to -874 (site 1) enhanced promoter activity and diminished but did not eliminate the suppression by RARbeta and RXRalpha. Mutation of another RARE site (site 2), at -930 to -911, resulted in low activity that was inhibited by retinoic acid. Mutation of the AP-2-binding site enhanced promoter activity that was inhibited by retinoic acid. This study shows that the suppressive effect of retinoic acid on the promoter is maximal with a combination of RARbeta and RXRalpha and occurs at more than one RARE site. The effect of retinoic acid is not mediated by AP-2.     

Mononuclear cell-mediated modulation of synovial cell metabolism. I. Collagen synthesis

Human PHA-stimulated mononuclear cells produce a factor which inhibits synovial cell collagen and non-collagen protein synthesis, whereas it enhances hyaluronic acid (HA) production. Indomethacin (10(-4)-10(-6) M), a cyclo-oxygenase inhibitor, suppresses this effect, suggesting that the mechanism is prostaglandin-mediated. The active material, of apparent molecular weight 12 000-20 000, also displays the properties of the mononuclear cell factor (MCF) previously described by others, since its stimulates collagenase and PGE2 release by the cultured synovial cells. Furthermore, it co-purifies with interleukin 1 (IL 1) as shown by lymphocyte-activating factor activity. This strongly suggests that IL 1 could be responsible for some (or all) the effects observed on MCF-exposed synovial cells. From these data, we deduce the possibility that mononuclear cells may participate in limiting synovial collagen deposition in rheumatoid arthritis.     

Retinoic acid modulates dome formation by MDCK cells in defined medium

Retinoic acid dramatically increases the size of domes in confluent MDCK monolayers in a hormonally defined medium (medium K-1). After 4-5 days of retinoic acid treatment, enlarged domes began to appear in confluent MDCK monolayers. After 7 days with 3 x 10(-7) M retinoic acid, the majority of the domes in the monolayers were between 27 and 80 x 10(-3) microns 2 in area, whereas in control medium the majority of the domes were between 0 and 9 x 10(-3) microns 2 in area. The dependence of the retinoic acid effect on prostaglandin E1 (PGE1) was examined. In normal MDCK cells, the effects of retinoic acid on dome size were observed only in medium K-1 supplemented with PGE1. This observation indicated that retinoic acid did not elicit its effects simply by stimulating PGE production. In contrast, in monolayers of PGE1-independent MDCK cells, retinoic acid treatment resulted in an increase in dome frequency even in medium K-1 lacking PGE1. This observation can be explained by the elevated cyclic adenosine monophosphate (cAMP) levels in these PGE1-independent MDCK cells. Dibutyryl cAMP-resistant MDCK cells, which normally do not form domes in medium K-1, were also studied. Remarkably, the dibutyryl cAMP-resistant MDCK cells were observed to form domes at a significant frequency when medium K-1 was supplemented with retinoic acid. However in medium K-1 lacking PGE1, an effect of retinoic acid on dome formation by dibutyryl cAMP-resistant MDCK monolayers was not observed. The inability of dibutyryl cAMP-resistant MDCK cells to form domes in medium K-1 has previously been attributed to their decreased cAMP-dependent protein kinase activity. The stimulatory effects of retinoic acid on dome formation may possibly be due to an increase in the activity of a particular cAMP-dependent protein kinase or activation of a separate pathway.     

Localization of specific retinoid-binding sites and expression of cellular retinoic-acid-binding protein (CRABP) in the early mouse embryo

Retinoids (vitamin A derivatives) are important for normal embryogenesis and retinoic acid, an acidic derivative of vitamin A, was recently proposed to be an endogenous morphogen. Several retinoids are also potent teratogens. Using an autoradiographic technique, we have identified tissues and cells in early mouse embryos that are able to specifically accumulate a radiolabelled synthetic derivative of retinoic acid. Strong accumulation of radioactivity was seen in several neural crest derivatives and in specific areas of the CNS. Gel filtration analyses of cytosols from embryos that received the radiolabelled retinoid in utero suggested that cellular retinoic acid-binding protein (CRABP) was involved in the accumulation mechanism. Immunohistochemical localization confirmed that cells accumulating retinoids also expressed CRABP. Strong CRABP immunoreactivity was found in neural crest-derived mesenchyme of the craniofacial area, in visceral arches, in dorsal root ganglia and in cells along the gut and the major vessels of the trunk region. In CNS, CRABP expression and retinoid binding was largely restricted to the hindbrain, to a single layer of cells in the roof of the midbrain and to cells in the mantle layer of the neural tube. Our data suggest that cells in the embryo expressing CRABP are target cells for exogenous retinoids as well as endogenous retinoic acid. Retinoic acid may thus play an essential role in normal development of the CNS and of tissues derived from the neural crest. We propose that the teratogenic effects of exogenous retinoids are due to an interference with mechanisms by which endogenous retinoic acid regulates differentiation and pattern formation in these tissues.     

Retinol metabolism in LLC-PK1 Cells. Characterization of retinoic acid synthesis by an established mammalian cell line

Specific assays, based on gas chromatography-mass spectrometry and high-performance liquid chromatography, were used to quantify the conversion of retinol and retinal into retinoic acid by the pig kidney cell line LLC-PK1. Retinoic acid synthesis was linear for 2-4 h as well as with graded amounts of either substrate to at least 50 microM. Retinoic acid concentrations increased through 6-8 h, but decreased thereafter because of substrate depletion (t1/2 of retinol = 13 h) and product metabolism (1/2 = 2.3 h). Retinoic acid metabolism was accelerated by treating cells with 100 nM retinoic acid for 10 h (t1/2 = 1.7 h) and was inhibited by the antimycotic imidazole ketoconazole. Feedback inhibition was not indicated since retinoic acid up to 100 nM did not inhibit its own synthesis. Retinol dehydrogenation was rate-limiting. The reduction and dehydrogenation of retinal were 4-8-fold and 30-60-fold faster, respectively. Greater than 95% of retinol was converted into metabolites other than retinoic acid, whereas the major metabolite of retinal was retinoic acid. The synthetic retinoid 13-cis-N-ethylretinamide inhibited retinoic acid synthesis, but 4-hydroxylphenylretinamide did not. 4'-(9-Acridinylamino)methanesulfon-m-anisidide, an inhibitor of aldehyde oxidase, and ethanol did not inhibit retinoic acid synthesis. 4-Methylpyrazole was a weak inhibitor: disulfiram was a potent inhibitor. These data indicate that retinol dehydrogenase is a sulfhydryl group-dependent enzyme, distinct from ethanol dehydrogenase. Homogenates of LLC-PK1 cells converted retinol into retinoic acid and retinyl palmitate and hydrolyzed retinyl palmitate. This report suggests that substrate availability, relative to enzyme activity/amount, is a primary determinant of the rate of retinoic acid synthesis, identifies inhibitors of retinoic acid synthesis, and places retinoic acid synthesis into perspective with several other known pathways of retinoid metabolism.     

Lipopolysaccharide-induced production of cytokines by bone marrow-derived macrophages: dissociation between intracellular interleukin 1 production and interleukin 1 release

We investigated the capacity of mouse bone marrow-derived macrophages (BMDM) to produce interleukin 1 (IL 1), interleukin-6 (IL 6), and tumor necrosis factor (TNF) upon lipopolysaccharide (LPS) stimulation. BMDM were allowed to differentiate either in the presence of conditioned medium (from WEHI-3 or L cells), or in the presence of recombinant cytokines (IL 3, macrophage-colony stimulating factor [M-CSF], or granulocyte/macrophage-colony stimulating factor [GM-CSF]). Cells were maintained in culture up to 3 weeks and tested at different times. Significant spontaneous cytokine production was never observed. BMDM rapidly acquired the capacity to elaborate cytokine upon LPS activation. LPS-triggered BMDM were able to produce IL 1, IL 6, and TNF, throughout the culture period, although 2- to 3-week-old cells lost their ability to release IL 1 while accumulation of intracellular IL 1 remained unchanged. The dissociation between synthesis and release of IL 1 was not correlated with a significant modification of the specific binding of LPS onto the cell surface.     

Signaling pathways in retinoid chemoprevention and treatment of cancer

The Vitamin A metabolite, retinoic acid, has been shown to have chemopreventive and therapeutic activity for certain cancers such as head and neck, cervical, neuroblastoma and promyelocytic leukemia. Retinoic acid achieves these activities by inducing differentiation and/or growth arrest. A large number of studies have investigated the mechanism(s) by which retinoic acid alters the behavior of premalignant and tumor cells. Although much important data has been obtained, the exact signaling pathways required for retinoic acid to exert its biological effects remains elusive. In this review, we outline the role and function of retinoid nuclear receptors, followed by a discussion of how major signaling pathways are affected in different tumor types by retinoids. We conclude by examining the effect of retinoic acid on G1 cell cycle regulatory proteins in various tumors.