Spermatogenesis and testis development are normal in mice lacking testicular orphan nuclear receptor 2

Early in vitro cell culture studies suggested that testicular orphan nuclear receptor 2 (TR2), a member of the nuclear receptor superfamily, may play important roles in the control of several pathways including retinoic acids, vitamin D, thyroid hormones, and ciliary neurotrophic factor. Here we report the surprising results showing that mice lacking TR2 are viable and have no serious developmental defects. Male mice lacking TR2 have functional testes, including normal sperm number and motility, and both male and female mice lacking TR2 are fertile. In heterozygous TR2(+/-) male mice we found that beta-galactosidase, the indicator of TR2 protein expression, was first detected at the age of 3 weeks and its expression pattern was restricted mainly in the spermatocytes and round spermatids. These protein expression patterns were further confirmed with Northern blot analysis of TR2 mRNA expression. Together, results from TR2-knockout mice suggest that TR2 may not play essential roles in spermatogenesis and normal testis development, function, and maintenance. Alternatively, the roles of TR2 may be redundant and could be played by other close members of the nuclear receptor superfamily such as testicular orphan receptor 4 (TR4) or unidentified orphan receptors that share many similar functions with TR2. Further studies with double knockouts of both orphan nuclear receptors, TR2 and TR4, may reveal their real physiological roles.     

Induction of expression of the alpha v beta 1 and alpha v beta 3 integrin heterodimers during retinoic acid-induced neuronal differentiation of murine embryonal carcinoma cells

All-trans-retinoic acid, an endogenous morphogen, induced neuronal differentiation of P19 murine embryonal carcinoma cells. Peak differentiation, as judged by the elaboration of neuronal processes, occurred 8 days after exposure of the cells to 0.5 mM retinoic acid, a concentration known to induce neuronal differentiation. An examination of the expression of the extracellular matrix receptors, integrins, during this retinoic acid-induced differentiation period, demonstrated a specific and strong induction of expression of two polypeptides (130 and 115 kDa) immunoprecipitated with an anti-human vitronectin receptor antiserum. The expression of a 90-kDa polypeptide, also immunoprecipitating with this antiserum was induced as well, but to a much smaller extent. The expression of a 96-kDa polypeptide immunoprecipitated by this antiserum and present in the untreated cells was not induced by retinoic acid. The increase in the expression of these polypeptides paralleled the neuronal differentiation of the P19 embryonal carcinoma cells. The expression of these integrins was not induced in a variant of the P19 cells, P19RAC65, which are resistant to differentiation induction by retinoic acid. Utilizing integrin subunit-specific anti-cytoplasmic peptide antibodies together with immunoprecipitation and Western blot analysis, the 130- and 115-kDa polypeptides were identified as the integrin alpha v and beta 1 subunits, respectively. The 90-kDa polypeptide, also induced by retinoic acid, was identified as beta 3, whereas the identity of the uninduced 96-kDa polypeptide remains unclear as yet. Peptide map analysis of deglycosylated polypeptides demonstrated that the 90- and 96-kDa polypeptides are distinct proteins and that the 115-kDa polypeptides immunoprecipitated with either anti-alpha v or anti-beta 1 antibodies are identical, further establishing that the 115-kDa polypeptide associating with alpha v is beta 1. The retinoic acid-induced expression of beta 1 occurred at the level of mRNA expression which also paralleled neuronal differentiation, but peaked slightly ahead of the cell surface expression of beta 1. The expression of other beta 1-associated alpha subunits was not induced by retinoic acid in these cells. These data demonstrate that retinoic acid strongly induces the expression of the integrin heterodimer alpha v beta 1 and also, to a smaller extent, the expression of alpha v beta 3. The retinoic acid-induced, high level surface expression of the alpha v beta 1 heterodimer is tightly correlated with the induction of neuronal differentiation by retinoic acid. This finding suggests an important role for the alpha v beta 1 heterodimer in the neuronal differentiation process.     

The nuclear orphan receptor TR4 promotes proliferation of myeloid progenitor cells.

Nuclear receptors represent key regulators in cell proliferation, differentiation, and development. Here we demonstrate that the nuclear orphan receptor TR4 is highly expressed in hematopoietic cells and tissues and have analyzed the impact of TR4 in this cell compartment. We show that TR4, when ectopically expressed in bone marrow cells via retrovirus vector, promotes proliferation of myeloid progenitor cells. Cells represent promyelocytes as judged by morphological features, expression of cell surface molecules, and specific markers like Mim-1 and CAAT/enhancer binding protein beta. We also demonstrate that the growth promoting activity of TR4 is not exclusively dependent on its association with DNA, because expression of a mutated TR4 version devoid of its DNA binding domain exhibits a similar proliferative potential as wild-type TR4. In conclusion, these data position the orphan receptor TR4 as an important regulator of myeloid progenitor cell proliferation and development.     

Increased expression of cyclin A1 protein is associated with all-trans retinoic acid-induced apoptosis

Deregulated cell growth and inhibition of apoptosis are hallmarks of cancer. All-trans retinoic acid induces clinical remission in patients with acute promyelocytic leukemia by inhibiting cell growth and inducing differentiation and apoptosis of the leukemic blasts. An important role of the cell cycle regulatory protein, cyclin A1, in the development of acute myeloid leukemia has previously been demonstrated in a transgenic mouse model. We have recently shown that there was a direct interaction between cyclin A1 and a major all-trans retinoic acid receptor, RAR alpha, following all-trans retinoic acid treatment of leukemic cells. In the present study, we investigated whether cyclin A1 might be involved in all-trans retinoic acid-induced apoptosis in U-937 leukemic cells. We found that all-trans retinoic acid-induced apoptosis was associated with concomitant increase in cyclin A1 expression. However, there was no induction of cyclin A1 mRNA expression following the all-trans retinoic acid-induced apoptosis. Treatment of cells with a caspase inhibitor was not able to prevent all-trans retinoic acid-induced up-regulation of cyclin A1 expression. Interestingly, induced cyclin A1 expression in U-937 cells led to a significant increase in the proportion of apoptotic cells. Further, U-937 cells overexpressing cyclin A1 appeared to be more sensitive to all-trans retinoic acid-induced apoptosis indicating the ability of cyclin A1 to mediate all-trans retinoic acid-induced apoptosis. Induced cyclin E expression was not able to initiate cell death in U-937 cells. Our results indicate that cyclin A1 might have a role in apoptosis by mediating all-trans retinoic acid-induced apoptosis.     

Loss of testicular orphan receptor 4 impairs normal myelination in mouse forebrain

Testicular orphan nuclear receptor 4 (TR4) has been suggested to play important roles in the development and functioning of the central nervous system (CNS). We find reduced myelination in TR4 knockout (TR4(-/-)) mice, which is particularly obvious in forebrains and in early developmental stages. Further analysis reveals that CC-1-positive (CC-1+) oligodendrocytes are decreased in TR4(-/-) forebrains. The O4+ signals are also reduced in TR4(-/-) forebrains when examined at postnatal d 7. However, the number and proliferation rate of platelet-derived growth factor receptor alpha-positive (PDGFalphaR+) oligodendrocyte precursor cells (OPCs) remain unaffected in these regions, suggesting that loss of TR4 interrupts oligodendrocyte differentiation. This is further supported by the observation that CC-1+ oligodendrocytes derived from 5-bromo-2'-deoxyuridine incorporating OPCs are significantly reduced in TR4(-/-) forebrains. We also find higher Jagged1 expression levels in axon fiber-enriched regions in TR4(-/-) forebrains, suggesting a more activated Notch signaling in these regions that correlates with previous reports showing that Notch activation inhibits oligodendrocyte differentiation. Together, our results suggest that TR4 is required for proper myelination in the CNS and is particularly important for oligodendrocyte differentiation and maturation in the forebrain regions. The altered Jagged1-Notch signaling in TR4(-/-) forebrain underlies a potential mechanism that contributes to the reduced myelination in the forebrain.     

Characterization of an inverted repeat with a zero spacer (IR0)-type retinoic acid response element from the mouse nuclear orphan receptor TR2-11 gene.

An inverted repeat with zero nucleotides in the spacer (IR0, 5'-GGGTCA CGAACT-3') element was localized in the proximal promoter region of the mouse TR2-11 gene, and characterized as a functional retinoic acid response element (RARE). In gel mobility shift assays, heterodimers of retinoic acid receptor alpha (RARalpha) and retinoid X receptor beta (RXRbeta) bound specifically to this element. Neither receptor alone was able to bind to this element efficiently. The dissociation constant (Kd) with respect to RAR-RXR binding was estimated to be 8 nM. The biological activity of this IR0 element was assessed in a heterologous reporter system. The IR0-containing reporter was induced by RA in COS-1 cells in the presence of exogenously provided RARalpha and RXRbeta. In addition, the IR0 oligomers could be bound by nuclear extracts isolated from COS-1 cells harboring the expression vectors for RARalpha and RXRbeta, but not by extracts isolated from control COS-1 cells. RA responsiveness of this IR0 was further confirmed in P19 cells that expressed endogenous RARs and RXRs. Collectively, these data demonstrated, for the first time, the presence of a natural RARE of the IR0 type, and suggested a potential cross-talk between nuclear orphan receptor TR2-11 and RAR-RXR families.     

Disruption of TR4 orphan nuclear receptor reduces the expression of liver apolipoprotein E/C-I/C-II gene cluster

Apolipoprotein E (apoE) is synthesized in many tissues, and the liver is the primary site from which apoE redistributes cholesterol and other lipids to peripheral tissues. Here we demonstrate that the TR4 orphan nuclear receptor (TR4) can induce apoE expression in HepG2 cells. This TR4-mediated regulation of apoE gene expression was further confirmed in vivo using TR4 knockout mice. Both serum apoE protein and liver apoE mRNA levels were significantly reduced in TR4 knockout mice. Gel shift and luciferase reporter gene assays further demonstrated that TR4 can induce apoE gene expression via a TR4 response element located in the hepatic control region that is 15 kb downstream of the apoE gene. Furthermore our in vivo data from TR4 knockout mice prove that TR4 can also regulate apolipoprotein C-I and C-II gene expression via the TR4 response element within the hepatic control region. Together our data show that loss of TR4 down-regulates expression of the apoE/C-I/C-II gene cluster in liver cells, demonstrating important roles of TR4 in the modulation of lipoprotein metabolism.     

Constitutive activation of retinoic acid receptor beta2 promoter by orphan nuclear receptor TR2

The orphan nuclear receptor TR2 functions as a constitutive activator for the endogenous retinoic acid receptor beta2 (RAR(beta2)) gene expression in P19 embryonal carcinoma cells and for reporters driven by the RAR(beta2) promoter in COS-1 cells. The activation of RAR(beta2) by TR2 is mediated by the direct repeat-5 (DR5) element located in the RAR(beta2) promoter. Furthermore, cAMP exerts an enhancing effect on the activation of RAR(beta2) by TR2, which is mediated by the cAMP response element located in the 5'-flanking region of the DR5. The constitutive activation function-1 (AF-1) of TR2 is mapped to amino acid residues 10-30 in its N-terminal A segment. A direct molecular interaction occurs between CREMtau and TR2, detected by co-immunoprecipitation, which is mediated by the N-terminal AB segment of TR2. In gel mobility shift assays, TR2 competes with P19 nuclear factor binding to the RAR(beta2) promoter, and TR2 and CREMtau bind simultaneously to this DNA fragment. The role of TR2 in the early events of RA signaling process is discussed.     

Distinct role and functional mode of TR3 and RARalpha in mediating ATRA-induced signalling pathway in breast and gastric cancer cells

All-trans retinoic acid (ATRA) affects cell proliferation, differentiation and apoptosis through its receptors, RARs and RXRs. Besides these, other receptors such as orphan receptor TR3, are also involved in the regulatory process of ATRA. However, how different receptors function in response to ATRA is still largely unknown. In the present study, we found that formation of TR3/RXRalpha heterodimers in the nucleus and their subsequent translocation into the cytoplasm, in association with regulation of apoptosis-related proteins Bcl-2, Bcl-xl and Bax, was critical for apoptosis induction by ATRA in breast cancer cells MCF-7. When such translocation was blocked by Leptomycin B (LMB), ATRA-induced apoptosis was consequently abolished. However, in ATRA-induced gastric cancer cells MGC80-3, RXRalpha heterodimerised with RARalpha but not with TR3, and remained in the nucleus exerting its effect on cell cycle regulation. When transfected with antisense-RARalpha, MGC80-3 cells changed from ATRA-sensitive to ATRA-resistant and most cells were arrested in the S phase, implying the importance of RARalpha in cell cycle regulation. Furthermore, we demonstrated that the effects of ATRA depend on the relative levels of TR3, RARalpha and RXRalpha expression in cancer cells. In ATRA-induced MCF-7 cells, highly expressed TR3 favours the formation of TR3/RXRalpha and promotes the TR3/RXRalpha signalling pathway causing apoptosis; while in ATRA-induced MGC80-3 cells, high expression of RARalpha favours the formation of RARalpha/RXRalpha and promotes the RXRalpha/RARalpha signalling pathway in mediating cell cycle regulation. In conclusion, these results reveal the novel mechanism that cellular expression and location of protein is associated with diverse signalling transduction pathways and the resultant physiological process.     

Apoptotic cell death in neuronal differentiation of P19 EC cells: Cell death follows reentry into S phase

Apoptotic cell death was observed during aggregate culture of the mouse embryonal carcinoma cell line P19 exposed to all-trans retinoic acid (tRA). This finding was confirmed by genomic DNA agarose gel electrophoresis and transmission electron microscopy. Apoptosis was associated with P19 cell neuronal differentiation; alternative causes of cell death, i.e., cavitation-related, cytotoxicity of tRA, or spontaneous cell death were excluded. Analysis by flow cytometry revealed that the apoptosis was likely to occur in multiplying cells that underwent to reentering into S phase. We therefore examined 5-bromo-2′-deoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression and localization in the aggregates by immunofluorescent staining. Although the P19 cells in the aggregates exposed to tRA incorporated BrdU at an equivalent level to those not exposed to tRA, the cells showed diminished PCNA expression and nuclear accumulation. We propose that P19 apoptosis during neuronal differentiation is a model system in which programmed cell death occurs simultaneously with cell division leading to differentiation. J. Cell. Physiol. 172:25–35, 1997. © 1997 Wiley-Liss, Inc.     

Antisense inhibition of BCL-2 expression induces retinoic acid-mediated cell death during differentiation of human NT2N neurons

Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.     

Phosphorylation of elF-4E and initiation of protein synthesis in P19 embryonal carcinoma cells

Mitogenic stimulation of protein synthesis is accompanied by an increase in elF-4E phosphorylation. The effect on protein synthesis by induction of differentiation is less well known. We treated P19 embryonal carcinoma cells with the differentiating agent retinoic acid and found that protein synthesis increased during the first hour of addition. However, the phosphorylation state, as well as the turnover of phosphate on elF-4E, remained unchanged. Apparently, the change in protein synthesis after RA addition is regulated by another mechanism than elF-4E phosphorylation. By using P19 cells overexpressing the EGF receptor, we show that the signal transduction pathway that leads to phosphorylation of elF-4E is present in P19 cells; the EGF-induced change in phosphorylation of elF-4E in these cells is likely to be regulated by a change in elF-4E phosphatase activity. These results suggest that the onset of retinoic acid-induced differentiation is triggered by a signal transduction pathway which involves changes in protein synthesis, but not elF-4E phosphorylation. © 1995 Wiley-Liss, Inc.     

NR4A orphan nuclear receptors influence retinoic acid and docosahexaenoic acid signaling via up-regulation of fatty acid binding protein 5

The orphan nuclear receptor (NR) Nurr1 is expressed in the developing and adult nervous system and is also induced as an immediate early gene in a variety of cell types. In silico analysis of human promoters identified fatty acid binding protein 5 (FABP5), a protein shown to enhance retinoic acid-mediated PPARβ/δ signaling, as a potential Nurr1 target gene. Nurr1 has previously been implicated in retinoid signaling via its heterodimerization partner RXR. Since NRs are commonly involved in cross-regulatory control we decided to further investigate the regulatory relationship between Nurr1 and FABP5. FABP5 expression was up-regulated by Nurr1 and other NR4A NRs in HEK293 cells, and Nurr1 was shown to activate and bind to the FABP5 promoter, supporting that FABP5 is a direct downstream target of NR4A NRs. We also show that the RXR ligand docosahexaenoic acid (DHA) can induce nuclear translocation of FABP5. Moreover, via up-regulation of FABP5 Nurr1 can enhance retinoic acid-induced signaling of PPARβ/δ and DHA-induced activation of RXR. We also found that other members of the NR4A orphan NRs can up-regulate FABP5. Thus, our findings suggest that NR4A orphan NRs can influence signaling events of other NRs via control of FABP5 expression levels.     

Ca2 + /calmodulin-dependent protein kinase II mediates apoptosis of P19 cells expressing human tau during neural differentiation with retinoic acid treatment

The involvement of tau phosphorylation in apoptosis resembling Alzheimer's disease (AD) was investigated using a cell model of P19 cells stably expressing human tau441 (tau/P19 cells). Apoptotic cell death was observed specifically in tau/P19 cells during neural differentiation with retinoic acid (RA) treatment. A CaM kinase II inhibitor, KN-93, protected tau/P19 cells from apoptosis, although it stimulated the cell death of wild-type P19 cells (wt/P19 cells). W-7 and calmidazolium, calmodulin antagonists, also specifically inhibited the apoptosis of tau/P19 cells. LiCl, an inhibitor of glycogen synthase 3, a tau kinase, was effective in protecting tau/P19 cells from apoptosis, but the protective effect was less than that of CaM kinase II inhibitor and calmodulin antagonists. Tau in the nuclei of tau/P19 cells was phosphorylated at the sites for CaM kinase II detected by an antibody recognizing a phosphorylated form of tau. These results indicated that CaM kinase II was involved in the apoptosis of tau/P19 cells induced by RA treatment.     

Ca2+/calmodulin-dependent protein kinase II mediates apoptosis of P19 cells expressing human tau during neural differentiation with retinoic acid treatment

The involvement of tau phosphorylation in apoptosis resembling Alzheimer's disease (AD) was investigated using a cell model of P19 cells stably expressing human tau441 (tau/P19 cells). Apoptotic cell death was observed specifically in tau/P19 cells during neural differentiation with retinoic acid (RA) treatment. A CaM kinase II inhibitor, KN-93, protected tau/P19 cells from apoptosis, although it stimulated the cell death of wild-type P19 cells (wt/P19 cells). W-7 and calmidazolium, calmodulin antagonists, also specifically inhibited the apoptosis of tau/P19 cells. LiCl, an inhibitor of glycogen synthase 3, a tau kinase, was effective in protecting tau/P19 cells from apoptosis, but the protective effect was less than that of CaM kinase II inhibitor and calmodulin antagonists. Tau in the nuclei of tau/P19 cells was phosphorylated at the sites for CaM kinase II detected by an antibody recognizing a phosphorylated form of tau. These results indicated that CaM kinase II was involved in the apoptosis of tau/P19 cells induced by RA treatment.     

All-trans-retinoic acid-mediated modulation of p53 during neural differentiation in murine embryonic stem cells

All-trans-retinoic acid (RA) plays an important physiological role in embryonic development and is teratogenic in large doses in almost all species. p53, a tumor suppressor gene encodes phosphoproteins, which regulate cellular proliferation, differentiation, and apoptosis. Temporal modulation of p53 by retinoic acid was investigated in murine embryonic stem cells during differentiation and apoptosis. Undifferentiated embryonic stem cells express a high level of p53 mRNA and protein followed by a decrease in p53 levels as differentiation proceeds. The addition of retinoic acid during 8–10 days of differentiation increased the levels of p53 mRNA and protein, accompanied by accelerated neural differentiation and apoptosis. Marked increase in apoptosis was observed at 10–20 h after retinoic acid treatment when compared with untreated controls. Retinoic acid-induced morphological differentiation resulted in predominantly neural-type cells. Maximum increase in p53 mRNA in retinoic acid-treated cells occurred on day 17, whereas maximum protein synthesis occurred on days 14–17, which coincided with increased neural differentiation and proliferation. Increased p53 levels did not induce p21 transactivation, interestingly a decrease in p21 was observed on day 17 on exposure to retinoic acid. The level of p53 declined by day 21 of differentiation. The results demonstrated that retinoic acid-mediated apoptosis preceded the changes in p53 expression, suggesting that p53 induction does not initiate retinoic acid-induced apoptosis during development. However, retinoic acid accelerated neural differentiation and increased the expression of p53 in proliferating neural cells, corroborated by decreased p21 levels, indicating the importance of cell type and stage specificity of p53 function. This revised version was published online in July 2006 with corrections to the Cover Date.