Contribution of retinoid X receptor signaling to the specification of skeletal muscle lineage

Pluripotent stem cells possess a tremendous potential for the treatment of many diseases because of their capacity to differentiate into a variety of cell lineages. However, they provide little promise for muscle-related diseases, mainly because of the lack of small molecule inducers to efficiently direct myogenic conversion. Retinoic acid, acting through the retinoic acid receptor (RAR) and retinoid X receptor (RXR), affects stem cell fate determination in a concentration-dependent manner, but it only has a modest efficacy on the commitment of ES cells into skeletal muscle lineage. The RXR is very important for embryonic development but is generally considered to act as a silent partner of RAR in a non-permissive mode. In this study, we have examined whether activation of the RXR by rexinoid or RXR-specific signaling play a role in the specification of stem cells into muscle lineage. Our findings demonstrate that mouse ES cells generate skeletal myocytes effectively upon treatment with rexinoid at the early stage of differentiation and that on a molecular level, rexinoid-enhanced myogenesis simulates the sequential events observed in vivo. Moreover, RXR-mediated myogenic conversion requires the function of β-catenin but not RAR. Our studies establish the feasibility of applying the RXR agonist in cell-based therapies to treat muscle-related diseases. The aptitude of mouse ES cells to generate skeletal myocytes following rexinoid induction also provides a model system to study the convergence of different signaling pathways in myogenesis.     

Activation of RXR and RAR signaling promotes myogenic differentiation of myoblastic C2C12 cells

Differentiation of embryonic and adult myogenic progenitors undergoes a complex series of cell rearrangements and specification events which are controlled by distinct gene regulatory networks. Delineation of the molecular mechanisms that regulate skeletal muscle specification and formation should be important for understanding congenital myopathies and muscular degenerative diseases. Retinoic acid (RA) signaling plays an important role in development. However, the role of RA signaling in adult myogenic progenitors is poorly understood. Here, we investigate the role of RA signaling in regulating myogenic differentiation of myoblastic progenitor cells. Using the mouse myoblast progenitor C2C12 line as a model, we have found that the endogenous expression of most RAR and RXR isotypes is readily detected. While the nuclear receptor co-repressors are highly expressed, two of the three nuclear receptor co-activators and the enzymes involved in RA synthesis are expressed at low level or undetectable, suggesting that the RA signaling pathway may be repressed in myogenic progenitors. Using the α-myosin heavy chain promoter-driven reporter (MyHC-GLuc), we have demonstrated that either ATRA or 9CRA is able to effectively induce myogenic differentiation, which can be synergistically enhanced when both ATRA and 9CRA are used. Upon ATRA and 9CRA treatment of C2C12 cells the expression of late myogenic markers significantly increases. We have further shown that adenovirus-mediated exogenous expression of RARα and/or RXRα is able to effectively induce myogenic differentiation in a ligand-independent fashion. Morphologically, ATRA- and 9CRA-treated C2C12 cells exhibit elongated cell body and become multi-nucleated myoblasts, and even form myoblast fusion. Ultrastructural analysis under transmission electron microscope reveals that RA-treated myogenic progenitor cells exhibit an abundant presence of muscle fibers. Therefore, our results strongly suggest that RA signaling may play an important role in regulating myogenic differentiation.     

High retinoid X receptor expression in JEG-3 choriocarcinoma cells: Involvement in cell function modulation by retinoids

Retinoic acid (RA) is an important mediator of cell differentiation. It stimulates hCG secretion by JEG-3 choriocarcinoma cells in vitro after a time lag. The first aim of this study was to characterize which types of retinoid receptors (RARs and RXRs) are present in JEG-3 cells. Using Western blot analysis and immunocytochemistry with specific antibodies as well as Northern blot analysis, we found that JEG-3 cells expressed RARα and RXRα, the latter being the predominant receptor. We then analyzed the action on cell proliferation and hCG secretion of the physiological retinoids all-trans RA (RA) and 9 cis RA as well as synthetic retinoids with specific affinity for RARα and RXRα. All these retinoids were potent inhibitors of cell growth, maximal inhibition (72 ± 2%) being observed after 4 days of treatment with Ro 25, a RXRα specific ligand. Within 24 h, 9 cis RA and Ro 25 stimulated hCG secretion, and maximal stimulation (1,472 ± 10%) occurred at 48 h with the RXRα-specific ligand. The RARα-specific ligand also stimulated hCG secretion but to a lower extend and after a delay of 48 h. These results suggest a predominant role of RXRα in mediating the biological effects of retinoids on JEG-3 cells and the possible induction by RA itself of the metabolic pathway leading to 9 cis RA. J. Cell. Physiol. 176:595–601, 1998. © 1998 Wiley-Liss, Inc.     

Retinoic acid and nitric oxide promote cell proliferation and differentially induce neuronal differentiation in vitro in the cnidarian Renilla koellikeri

Retinoic acid (RA) and nitric oxide (NO) are known to promote neuronal development in both vertebrates and invertebrates. Retinoic acid receptors appear to be present in cnidarians and NO plays various physiological roles in several cnidarians, but there is as yet no evidence that these agents have a role in neural development in this basal metazoan phylum. We used primary cultures of cells from the sea pansy Renilla koellikeri to investigate the involvement of these signaling molecules in cnidarian cell differentiation. We found that 9‐cis RA induce cell proliferation in dose‐ and time‐dependent manners in dishes coated with polylysine from the onset of culture. Cells in cultures exposed to RA in dishes devoid of polylysine were observed to differentiate into epithelium‐associated cells, including sensory cells, without net gain in cell density. NO donors also induce cell proliferation in polylysine‐coated dishes, but induce neuronal differentiation and neurite outgrowth in uncoated dishes. No other cell type undergoes differentiation in the presence of NO. These observations suggest that in the sea pansy (1) cell adhesion promotes proliferation without morphogenesis and this proliferation is modulated positively by 9‐cis RA and NO, (2) 9‐cis RA and NO differentially induce neuronal differentiation in nonadherent cells while repressing proliferation, and (3) the involvement of RA and NO in neuronal differentiation appeared early during the evolutionary emergence of nervous systems. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 842–852, 2010     

Regulation of alpha 2(I) collagen expression in stellate cells by retinoic acid and retinoid X receptors through interactions with their cofactors

Retinoic acid (RA) suppresses alpha 2(I) collagen expression in hepatic stellate cells through the binding of retinoic acid receptor beta (RAR beta) and retinoid X receptor alpha (RXR alpha) to RA response elements (RAREs) in the alpha 2(I) collagen promoter. This study determined the influence of coactivators and corepressors to RAR beta and RXR alpha on the regulation of the alpha 2(I) collagen promoter. The coactivators, steroid receptor coactivator-1 (SRC-1) and growth hormone receptor interacting protein-1 (GRIP-1), enhanced, while the nuclear receptor corepressor (N-CoR) abolished the inhibitory effect of RAR beta and RXR alpha on the promoter activity. In the presence of RA, the coactivators SRC-1 and GRIP-1 formed complexes with RAR beta and RXR alpha which are bound to an oligonucleotide specifying a RARE site in the promoter. In conclusion, this study shows that in the presence of retinoic acid, the coactivators SRC-1 and GRIP-1 augment, while the corepressor N-CoR abolishes, the suppressive effects of RAR beta and RXR alpha on alpha 2(I) collagen promoter activity.     

Distribution of serotonin uptake and binding sites in the cnidarian Renilla koellikeri: an autoradiographic study

Using [(3)H]-serotonin ([(3)H]-5-HT) as radiolabel and autoradiography, we have mapped the distribution of 5-HT uptake and binding sites in the sea pansy Renilla koellikeri in order to identify potential cellular pathways of 5-HT inactivation and to identify cellular substrates for the previously characterized 5-HT receptor involved in the modulation of peristaltic behavior. Uptake measured in fresh polyp tissues occurred via two processes: a high affinity (uptake(1)), clomipramine-sensitive process with a K(m) of 0.45 muM, and another of lower affinity (uptake(2)) with a Km of 11.6 muM. Autoradiograms of high affinity uptake sites revealed a diffuse distribution of label with higher density in the ectoderm and endoderm, and lower density in the mesoglea. No subsets of cells, including serotonergic neurons, appeared to retain label preferentially, thus suggesting that removal of 5-HT and its chemical conversion is a general property of sea pansy tissues. Under incubation conditions identical to those used in a previous radiobinding analysis, autoradiograms of binding sites were generated on sections from lightly fixed and cryosectioned polyps. In contrast to uptake sites, binding sites appeared as aggregations of label around neurons of the subectodermal, mesogleal and endodermal nerve nets. In the endoderm where the myoepithelia subtend peristalsis, myoepithelial cells appeared unlabeled, suggesting that 5-HT exerts its modulatory effect on peristalsis principally via neurons. Taken together, these results indicate that 5-HT is released as a neurohormone in the sea pansy and that it may act as a broad-range neuromodulator.     

Selective roles of retinoic acid receptor and retinoid x receptor in the suppression of apoptosis by all-trans-retinoic acid

Retinoic acids exert profound effects on many biological processes including cell proliferation, differentiation, and morphogenesis. We previously reported that all-trans-retinoic acid (t-RA) protected mesangial cells from H(2)O(2)-triggered apoptosis by suppressing the activator protein 1 (AP-1) pathway. It was via inhibition of c-fos and c-jun expression and suppression of c-Jun N-terminal kinase (JNK) activation. In this report, we investigated the involvement of retinoic acid receptor (RAR) and retinoid X receptor (RXR) in the antiapoptotic effect of t-RA in H(2)O(2)-exposed cells. We found that pretreatment with RAR pan-antagonist (AGN193109) or RXR pan-antagonist (HX531) attenuated the antiapoptotic effect of t-RA. Similarly, transient transfection with a dominant-negative mutant of RAR or a dominant-negative RXR diminished the antiapoptotic effect of t-RA. Both RAR and RXR antagonists reversed the suppressive effect of t-RA on AP-1 activity. However, the roles of RAR and RXR in the suppression of AP-1 components by t-RA were found to be different. RAR antagonist reversed the suppressive effect of t-RA on both c-fos and c-jun, whereas RXR antagonist reversed the effect of t-RA on c-fos but not c-jun. Furthermore, suppression of JNK activation by t-RA was observed even in the presence of RAR and RXR antagonists. Consistently, suppression of JNK by t-RA was not affected by overexpression of either the dominant-negative RAR or the dominant-negative RXR. These data elucidated that the antiapoptotic effect of t-RA is mediated by both nuclear receptor-dependent and -independent mechanisms.     

Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.     

Tyrosine hydroxylase and dopamine-β-hydroxylase immunoreactivities in the cnidarian <Emphasis Type="Italic">Renilla koellikeri</Emphasis>

Western Blot and immunohistochemical studies were conducted in the sea pansy Renilla koellikeri, a representative of the earliest multicellular animals with a nervous system, using various antibodies raised against enzymes of the catecholamine biosynthetic pathway. Western blots of sea pansy extracts revealed a protein band that co-migrated with dopamine-beta-hydroxylase (DBH) from mouse adrenal glands. Similar experiments with antisera against tyrosine hydroxylase (TH) revealed several immunoreactive protein bands, all of larger molecular weight than mammalian tyrosine hydroxylase. DBH-like and, to a lesser extent, TH-like and phenylethanolamine N-methyltransferase-like immunoreactivities were detected in ectodermal sensory neurons and associated subectodermal neurites, in neurons of the mesogleal nerve-net and associated amoebocytes, and in some endodermal neurons. While it is still not clear whether the detected TH-immunoreactive proteins represent some form of TH, the presence in sea pansies of a DBH-like protein is in agreement with previously detected norepinephrine-like immunoreactivity in the same species. The widespread distribution of these immunoreactivities in various sea pansy neurons suggests important roles for catecholamines in nerve net activity.     

Expression of Retinoid X Receptors in Human Dermal Fibroblasts

Retinoic acid (RA) is known to exert profound effects on growth and differentiation in human dermal fibroblasts. In the observations presented here, we examined the regulation of expression of members of the RXR multigene family in human dermal fibroblasts. We showed that the messenger RNAs for both RXRα and RXRβ are expressed in human fibroblasts, but that the messenger RNA for RXRγ is not detectable in these cells. Electrophoretic mobility shift studies of binding to the β2RARE in human dermal fibroblasts demonstrated that a single complex binds to β2RARE in the absence of RA. Stimulating cells with all-transRA induced a second complex. An antibody to the RXRβ protein supershifted both complexes, while an antibody to the RXRα S/B protein had no effect on the binding. These data demonstrate that RXRβ plays an important role in retinoid-regulated signal transduction pathways in human dermal fibroblasts and the regulation of expression of the RXR gene family is different from that of the RAR gene family.     

Expression of human cation-independent mannose 6-phosphate receptor cDNA in receptor-negative mouse P388D1 cells following gene transfer

We recently reported the cDNA cloning, sequence, and expression of the human cation-independent mannose 6-phosphate receptor (hCI-MPR) (Oshima, A., Nolan, C. M., Kyle, J. W., Grubb, J. H., and Sly, W. S. (1988) J. Biol. Chem. 263, 2553-2562). The sequence of the hCI-MPR was virtually identical to that of the human insulin-like growth factor II receptor cDNA (Morgan, D. O., Edman, J. C., Standring, D. N., Fried, V. A., Smith, M. C., Roth, R. A., and Rutter, W. J. (1987) Nature 329, 301-307). To test the role of the putative bifunctional receptor in intracellular sorting of acid hydrolases, we studied its effect on lysosomal enzyme transport following gene transfer to receptor-negative cells. Receptor-negative mouse P388D1 cells were transfected with a cDNA construct containing the entire coding sequence of hCI-MPR under the control of the mouse metallothionine I promoter. Stable transformants were isolated and characterized. The expressed hCI-MPR was localized in membranes including the plasma membrane, bound mannose 6-phosphate containing ligands, and mediated endocytosis which could be specifically blocked by mannose 6-phosphate. We next measured the effect of the expressed hCI-MPR on intracellular and secreted acid hydrolases. The intracellular activity of the lysosomal marker enzymes beta-glucuronidase and beta-hexosaminidase increased up to 2-fold following transformation. In addition, expression of the receptor greatly reduced the fraction of acid hydrolases secreted. These phenotypic changes in the transformed cell lines support the proposed role of the cation-independent mannose 6-phosphate receptor in intracellular sorting and targeting of lysosomal enzymes.     

Small-scale mapping of indeterminate arborescent acroporid coral (Acropora cervicornis) patches

Coral Reefs - Western Atlantic populations of the staghorn coral Acropora cervicornis have drastically declined over the past few decades. Hence, interest in its ecology and spatial extent has...