Expression of retinoid receptors during the retinoic acid-induced neuronal differentiation of human embryonal carcinoma cells

Retinoic acid (RA), a derivative of vitamin A, is essential for normal patterning and neurogenesis during development. Until recently, studies have been focused on the physiological roles of RA receptors (RARs), one of the two types of nuclear receptors, whereas the functions of the other nuclear receptors, retinoid X receptors (RXRs), have not been explored. Accumulating evidence now suggests that RXRalpha is a critical receptor component mediating the effects of RA during embryonic development. In this study, we have examined the expression profiles of RXRalpha and RARs during the RA-induced neuronal differentiation in a human embryonal carcinoma cell line, NT2. Distinct expression profiles of RXRalpha, RARalpha, RARbeta, and RARgamma were observed following treatment with RA. In particular, we found that RA treatment resulted in a biphasic up-regulation of RXRalpha expression in NT2 cells. The induced RXRalpha was found to bind specifically to the retinoid X response element based on gel mobility retardation assays. Furthermore, immunocytochemical analysis revealed that RXRalpha expression could be localized to the somatoaxonal regions of the NT2 neurons, including the tyrosine hydroxylase- and vasoactive intestinal peptide-positive neurons. Taken together, our findings provide the first demonstration of the cellular localization and regulation of RXRalpha expression in NT2 cells and suggest that RXRalpha might play a crucial role in the cellular functions of human CNS neurons.     

Induction of cAMP-dependent protein kinase I during human monocyte differentiation

Differentiation of human peripheral blood monocytes into macrophages was accompanied by induction of the regulatory subunit of cAMP-dependent protein kinase I as determined by photoaffinity labeling of cytosol proteins with 8-N3-[32P]cAMP and DEAE-Sephacel chromatography. The appearance of cAMP-dependent protein kinase I in macrophages was not due to translocation from the particulate fraction of monocytes. The regulatory subunit of cAMP-dependent protein kinase II was present in both monocytes and in vitro-differentiated macrophages. Protein kinase I in macrophages demonstrated higher affinity for 8-N3-cAMP (KD = 0.7 nM) than did protein kinase II from either monocytes (KD = 14.5 nM) or macrophages (KD = 4.9 nM). These studies demonstrate induction of the regulatory subunit of cAMP-dependent protein kinase I during the differentiation of a normal human cell and support the hypothesis that cAMP may regulate some stages of differentiation.     

Expression of transferrin receptors and intracellular ferritin during terminal differentiation of human monocytes

Human blood monocytes when cultured on hydrophobic Teflon membranes differentiate into mature macrophages. The expression of transferrin receptors was monitored by monoclonal antibody (OKT9) binding as detected by immunoperoxidase staining. Whereas monocytes were negative, an increasing percentage of macrophages, starting from day 2 in culture, labelled with the antitransferrin receptor antibody as these cells undergo differentiation. After completion of maturation more than 90% of macrophages expressed transferrin receptors. While 90-95% of macrophages from broncho-alveolar lavage fluids labelled with the OKT9 antibody, only a minor portion of macrophages obtained from peritoneal and pleural cavities did so. In parallel, intracellular ferritin in cells of the monocyte-macrophage lineage increased from 10 ng/10(6) cells to 350-1,500 ng/10(6) cells during maturation in vitro. Alveolar macrophages proved to have the highest ferritin content which ranged from 355-8,400 ng/10(6). The results may indicate that iron uptake and storage is a function of cells at late stages of macrophage maturation and that the occurrence of surface receptors for transferrin can be regarded as differentiation dependent marker.     

Regulation of oxidative metabolism by interferon-gamma during human monocyte to macrophage differentiation

The capacity of human peripheral blood monocytes to generate highly reactive oxygen-derived molecules was studied during differentiation of the cells to macrophages in vitro. The effect of semipurified native interferon gamma (IFN gamma) on the differentiation-associated production of active oxygen intermediates was assessed by continuous exposure of the cells to IFN gamma or by adding it to the cultures at different stages of in vitro differentiation. Chemiluminescence (CL) response, triggered by opsonised zymosan, was highest in fresh isolated monocytes and fell constantly during a two-week culture. IFN gamma had little effect on CL. Generation of intracellular O2- was determined by the reduction of nitroblue tetrazolium (NBT). Zymosan-induced NBT reduction increased slightly during monocyte to macrophage differentiation and was further enhanced by continuous presence of IFN gamma. Hydrogen peroxide (H2O2) release, triggered by phorbol myristate acetate (PMA), was low in monocytes, increased slightly, reaching a maximum on day 3, and declined thereafter. H2O2 secretion was greatly enhanced by the presence of IFN gamma and remained raised for at least 14 d. When added at intervals to spontaneously matured monocytes, IFN gamma had only modest and transient effects on the generation of intracellular O2- and H2O2. It is concluded that IFN gamma seems so to modulate human mononuclear phagocyte differentiation that they maintain or increase their oxidative metabolic capacity.     

Proteinase expression during differentiation of human osteoclasts in vitro

Osteoclasts are macrophage-derived polykaryons that degrade bone in an acidic extracellular space. This differentiation includes expression of proteinases and acid transport proteins, cell fusion, and bone attachment, but the sequence of events is unclear. We studied two proteins expressed at high levels only in the osteoclast, cathepsin K, a thiol proteinase, and tartrate-resistant acid phosphatase (TRAP), and compared this expression with acid transport and bone degradation. Osteoclastic differentiation was studied using human apheresis macrophages cocultured with MG63 osteosarcoma cells, which produce cytokines including RANKL and CSF-1 that mediate efficient osteoclast formation. Immunoreactive cathepsin K appeared at 3-5 days. Cathepsin K activity was seen on bone substrate but not within cells, and cathepsin K increased severalfold during further differentiation and multinucleation from 7 to 14 days. TRAP also appeared at 3-5 d, independently of cell fusion or bone attachment, and TRAP activity reached much higher levels in osteoclasts attached to bone fragments. Two proteinases that occur in the precursor macrophages, cathepsin B, a thiol proteinase related to cathepsin K, and an unrelated lysosomal aspartate proteinase, cathepsin D, were also studied to determine the specificity of the differentiation events. Cathepsin B occurred at all times, but increased two- to threefold in parallel with cathepsin K. Cathepsin D activity did not change with differentiation, and secreted activity was not significant. In situ acid transport measurements showed increased acid accumulation after 7 days either in cells on osteosarcoma matrix or attached to bone, but bone pit activity and maximal acid uptake required 10-14 days. We conclude that TRAP and thiol proteinase expression begin at essentially the same time, and precede cell fusion and bone attachment. However, major increases in acid secretion and proteinases expression continue during cell fusion and bone attachment from 7 to 14 days.     

Expression of angiotensin II receptors type 1 and type 2 in human preadipose cells during differentiation.

Human adipose tissue expresses all the components necessary for the production of angiotensin peptides. Although local effects of angiotensin II on cells from adipose tissue are beginning to be recognised, the expression of angiotensin receptors on human preadipocytes and adipocytes is still controversial. This study addresses the issue by monitoring the mRNA levels as well as the protein production of angiotensin II receptors of type 1 and 2 (AT 1 and AT 2 ) during differentiation of primary human preadipocytes in culture and in mature adipocytes. mRNA levels of the two receptor types are inversely correlated during adipose conversion. AT 1 receptor mRNA is greatly diminished within 12 days after induction of differentiation, while AT 2 receptor mRNA is elevated. mRNA levels of mature adipocytes confirm this trend. The regulation is not seen as strongly on the protein level. The amount of AT 2 receptor protein is increased, correlating well with the rise in specific glycerol-3-phosphate dehydrogenase activity of the cells, but the AT 1 receptor protein does not vary during the whole differentiation period. As the functional role of AT 2 receptors in adipose tissue is not known to date, further studies have to show if the AT 1 -mediated inhibitory actions on adipose conversion are downregulated in differentiating cells through decreased AT 1 /AT 2 receptor ratio.     

Coordinate expression of OPN and associated receptors during monocyte/macrophage differentiation of HL-60 cells

Promyelocytic leukemia HL-60 cells promoted by PMA to differentiate along the monocyte pathway adhere to tissue culture plates. To explore the regulation of adhesion molecules in cells promoted to differentiate, the expression and secretion of osteopontin (OPN) and expression of associated cell surface receptors, CD44 and integrin subunits α_v, β₃, β₁, were examined. Results were as follows: (1) PMA induced OPN mRNA and OPN secretion into media; (2) untreated cells expressed β₁ and CD44 mRNA, and PMA induced α_v and β₃ mRNA and increased β₁ and CD44 mRNA expression; (3) PMA increased levels of α_v, β₃, β₁ and CD44 protein on the cell surface; and (4) retinoic acid, which promotes granulocytic differentiation of HL-60 cells, did not affect OPN, α_v, β₃, β₁, or CD44 mRNA or protein expression. These data suggest that induction of OPN and associated receptors may play a role during monocytic differentiation of HL-60 cells. J. Cell. Physiol. 175:229–237, 1998. © 1998 Wiley-Liss, Inc.     

Regulated expression of BAFF-binding receptors during human B cell differentiation

BAFF plays a central role in B-lineage cell biology; however, the regulation of BAFF-binding receptor (BBR) expression during B cell activation and differentiation is not completely understood. In this study, we provide a comprehensive ex vivo analysis of BBRs in human B-lineage cells at various stages of maturation, as well as describe the events that drive and regulate receptor expression. Our data reveal that B-lineage cells ranging from naive to plasma cells (PCs), excluding bone marrow PCs, express BAFF-R uniformly. In contrast, only tonsillar memory B cells (MB) and PCs, from both tonsil and bone marrow tissues, express BCMA. Furthermore, we show that TACI is expressed by MB cells and PCs, as well as a subpopulation of activated CD27(neg) B cells. In this regard, we demonstrate that TACI is inducible early upon B cell activation and this is independent of B cell turnover. In addition, we found that TACI expression requires activation of the ERK1/2 pathway, since its expression was blocked by ERK1/2-specific inhibitors. Expression of BAFF-R and B cell maturation Ag (BCMA) is also highly regulated and we demonstrate that BCMA expression is only acquired in MB cells and in a manner accompanied by loss of BAFF-R expression. This inverse expression coincides with MB cell differentiation into Ig-secreting cells (ISC), since blocking differentiation inhibited both induction of BCMA expression and loss of BAFF-R. Collectively, our data suggest that the BBR profile may serve as a footprint of the activation history and stage of differentiation of normal human B cells.     

Terminal differentiation in keratinocytes involves positive as well as negative regulation by retinoic acid receptors and retinoid X receptors at retinoid response elements.

Terminal differentiation of epidermal keratinocytes is inhibited by 1 microM retinoic acid, a concentration which induces differentiation in a number of cell types, including F9 teratocarcinoma cells. The molecular basis for these opposing retinoid responses is unknown, although retinoic acid receptors (RARs) and retinoid X receptors (RXRs) have been detected in both cell types. When F9 cells are stably transfected with a truncated RAR alpha lacking the E/F domain necessary for ligand binding and RAR/RXR dimerization, action at retinoid response elements is suppressed and cells produce a retinoic acid-resistant phenotype; i.e., they are blocked in differentiation (A. S. Espeseth, S. P. Murphy, and E. Linney, Genes Dev. 3:1647-1656, 1989). If retinoid receptors influence epidermal differentiation only in a negative fashion, then suppression of transactivation at retinoid response elements would be expected to enhance, rather than block, keratinocyte differentiation. In this study, we show that surprisingly, even though constitutive expression of an analogous truncated RAR gamma in keratinocytes specifically suppressed transactivation at retinoid response elements, keratinocytes were blocked, rather than enhanced, in their ability to undergo morphological and biochemical features of differentiation. These findings demonstrate a direct and hitherto unrecognized role for RARs and RXRs in positively as well as negatively regulating epidermal differentiation. Additionally, our studies extend those of Espeseth et al. (Genes Dev. 3:1647-1656, 1989), indicating a novel RAR function independent of the E/F domain.     

Expression of tumor necrosis factor alpha and its receptors during cellular differentiation

Tumor necrosis factor alpha (TNFalpha) is a potent proinflammatory cytokine also involved in cellular differentiation processes. TNFalpha and both of its receptors (TNFR1 and TNFR2) can be co-expressed on the same cell, allowing for local signaling. This study has examined the expression of all components necessary for autocrine cytokine regulation during human hematopoietic, epithelial, and mesenchymal models of cellular differentiation. Macrophage and dendritic differentiation of human peripheral blood monocytes decreased their TNFalpha and TNFR2 expression while increasing the TNFR1 mRNA. In colon epithelial cell lines (HT-29 and Caco-2) TNFalpha-, TNFR1-, and TNFR2-expression was decreased upon differentiation. No changes, however, were seen during human skin keratinocyte differentiation. TNFR1 expression was unchanged in all three mesenchymal lineages (adipogenesis, chondrogenesis, osteogenesis) tested. Differentiation decreases the TNFalpha message in adipocytes and the TNFR2 mRNA in adipocytes and osteocytes. Our results demonstrate that there is no general principle for TNFalpha signaling during conversion of cells from progenitor to a more differentiated phenotype. Paracrine signaling by TNFalpha to orchestrate different cell types during tissue development and remodeling, therefore, probably overrides the autocrine regulation of differentiation by TNFalpha. Non-signaling TNF-receptors may protect chondrocytes and osteocytes from the anti-differentiation effects of local TNFalpha production.     

Expression of vimentin and nuclear lamins during the in vitro differentiation of human promyelocytic leukemia cells HL-60

We have reported previously that the human promyelocytic leukemia cell line HL-60, in its undifferentiated state, is devoid of cytoplasmic intermediate filament proteins and nuclear lamins A and C, but does express lamin B. Using immunofluorescence and immunoblotting techniques, we have further investigated the expression of vimentin and lamins A and C during differentiation of these tumor cells along the macrophage or granulocytic pathway in response to the inducing effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or dimethyl sulfoxide. Our results show that, while the expression of lamin B remains largely unchanged, the synthesis of vimentin and lamins A and C is dramatically enhanced during the maturation of HL-60 cells along both hemopoietic pathways. Northern blot analysis of cellular RNAs isolated from untreated and TPA-treated HL-60 cell populations as well as from control HeLa cells was performed using two oligonucleotides, one complementary to the 5' region common to human lamin A/C mRNAs and the other to the 5' region of hamster vimentin mRNA. Very low but still detectable amounts of vimentin and lamin A/C mRNAs were found in untreated HL-60 cell population, in accordance with the detection of small quantities of vimentin and lamins A and C in these populations. This is probably due to the presence of a small number of spontaneously differentiating cells. On the other hand, strong signals comparable to those obtained with RNA from control HeLa cells were detected for the three mRNA species from TPA-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of estrogen receptors during growth of human pancreatic adenocarcinoma cells (Capan-1)-relationship with differentiation

Summary In steroid target tissues, the presence of the corresponding hormone receptors is indicative of hormone dependence. In an attempt to assess the possible role of steroid hormones in the mechanism of growth and/or differentiation of cancerous pancreatic duct cells, the expression of estrogen receptor (ERα) was evaluated in human cancerous pancreatic duct cells (Capan-1) maintained in culture. These cells were selected as they acquire progressively a high degree of differentiation during growth in culture. In the present study, we showed that Capan-1 cells during growth in steroid-free medium associate spontaneously, become polarized, and form duct-like structures, features that are indicative of a high degree of differentiation. Capan-1 cells were also found to express ERα and progesterone receptor (PR). Immunoenzymatic assay showed maximal expression of ERα (236 ± 55 fmol/mg protein) on the first day of the exponential growth phase, followed by a marked fall in expression (76.3%). At the onset of the stationary phase (Day 5), ERα levels were below 10 fmol/mg protein, becoming undetectable by Day 7. A similar time course was observed for PR: 18 ± 0.9 fmol/mg protein at the onset of the exponential growth phase and no expression during the stationary phase. Addition of estradiol to 1-d-old cultures resulted in a twofold increase in PR expression, suggesting an induction of PR expression by estrogen. Immunocytochemical analysis with anti-ERα-1D5 antibodies showed nuclear and cytoplasmic localization of ERα in Capan-1 cells in the first 24 h of culture followed by a progressive disappearance thereafter. We also showed that cellular multiplication was increased by estradiol and progesterone during the exponential growth phase, pointing to the involvement of steroid hormones in the proliferation of nonpolarized Capan-1 cells. These results indicate that the expression of ERα is linked to the state of differentiation of the cells and make Capan-1 cells a model of choice to study ER regulation in nontarget tissues.     

Expression of the estrogen receptor during differentiation of human osteoclasts

Estrogens play a key role in bone structural integrity, which is maintained by the opposing activity of bone forming osteoblasts and bone resorbing osteoclasts. The cellular effects of estrogens are mediated by estrogen receptors, however, the detailed molecular mechanism of ER regulation in osteoclasts has not yet been elucidated. We provide here a detailed analysis of the expression profile and functionality of ER during osteoclast differentiation. We employed a human primary osteoclast cell culture model to evaluate the regulation of estrogen receptor (ER) variant expression. We characterized the expression profile of estrogen receptors and studied the regulation of the predominant estrogen receptor-alpha (ER-alpha) during differentiation into osteoclasts. In addition to the full-length ER-alpha, a shorter ER-alpha mRNA variant is expressed and both ER-alpha variants are regulated during osteoclastogenesis. Furthermore, we show that the pS2 gene is an estrogen-regulated gene in osteoclasts. Analysis of the activity of the pS2 gene throughout differentiation, using chromatin immunoprecipitation (ChIP), revealed the functionality of ER-alpha during differentiation and shows that the occupancy of ER-alpha and activated polymerase II on the pS2 promoter decrease with time and can be blocked by the ER antagonist ICI 182780. These results help to dissect the molecular events relevant to estrogen signaling and provide a better understanding of the role of ER-alpha regulation during bone resorption mediated by osteoclasts.     

Gene targeting of retinoid receptors

Gene targeting in embryonic stem (ES) cells has been employed to investigate the role of the retinoid receptors and binding proteins both in the mouse as well as in embryocarcinoma cells. It is a powerful technique for the modification of the mouse genome. With more recent refinements in gene targeting technology, it is now possible to introduce more subtle mutations in the murine genome, as well as to investigate gene function in a tissue and temporally-restricted manner. It should also be possible to modify genes in diverse diploid cell lines, to generate diverse model systems for analysis of retinoid receptor function. In this article, some of the basic principles for gene targeting are described.