Biomedical subjects

A M Jetten

Publications and source records attributed to A M Jetten.

At least 37 records · Page 2Linked to original sources

PubMed1998

Regulation of peroxisome proliferator-activated receptor alpha-induced transactivation by the nuclear orphan receptor TAK1/TR4.

Recently, we reported the cloning of the nuclear orphan receptor TAK1. In this study, we characterized the sequence requirements for optimal TAK1 binding and analyzed the repression of the peroxisome proliferator-activated receptor alpha (PPARalpha) signaling pathway by TAK1. Site selection analysis showed that TAK1 has the greatest affinity for direct repeat-1 response elements (RE) containing AGGTCAAAGGTCA (TAK1-RE) to which it binds as a homodimer. TAK1 is a very weak inducer of TAK1-RE-dependent transcriptional activation. We observed that TAK1, as PPARalpha, is expressed within rat hepatocytes and is able to bind the peroxisome proliferator response elements (PPREs) present in the promoter of the PPARalpha target genes rat enoyl-CoA hydratase (HD) and peroxisomal fatty acyl-CoA oxidase (ACOX). TAK1 is unable to induce PPRE-dependent transcriptional activation and represses PPARalpha-mediated transactivation through these elements in a dose-dependent manner. Two-hybrid analysis showed that TAK1 does not form heterodimers with either PPARalpha or retinoid X receptor (RXRalpha), indicating that this repression does not involve a mechanism by which TAK1 titrates out PPARalpha or RXRalpha from PPAR.RXR complexes. Further studies demonstrated that the PPARalpha ligand 8(S)-hydroxyeicosatetraenoic acid strongly promotes the interaction of PPARalpha with the co-activator RIP-140 but decreases the interaction of PPARalpha with the co-repressor SMRT. In contrast, TAK1 interacts with RIP-140 but not with SMRT and competes with PPARalpha for RIP-140 binding. These observations indicated that the antagonistic effects of TAK1 on PPARalpha.RXRalpha transactivation act at least at two levels in the PPARalpha signaling pathway: competition of TAK1 with PPARalpha.RXR for binding to PPREs as well as to common co-activators, such as RIP-140. Our results suggest an important role for TAK1 in modulating PPARalpha-controlled gene expression in hepatocytes.

Animals↗

PubMed1998

Suppression of relaxin gene expression by retinoids in squamous differentiated rabbit tracheal epithelial cells.

Northern blot analysis of total RNA from a variety of rabbit tissues indicated that placenta is the primary site of expression of the protein hormone relaxin (previously called SQ10) in rabbits. Relaxin was not detected by this method in other rabbit tissues, including normal trachea and several squamous tissues. However, relaxin is highly induced during squamous cell differentiation in cultured rabbit tracheal epithelial (RbTE) cells. Retinoic acid and retinoids that selectively bind to the nuclear retinoid receptors, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs), and induce RARE- or RXRE-dependent transactivation as well as repression of AP-1-dependent transactivation, were all effective in suppressing relaxin expression. In addition, the retinoid SR11302, which exhibits only anti-AP-1 activity but does not induce RARE- or RXRE-dependent transactivation, was also able to inhibit relaxin expression. These results suggest that the suppression of relaxin expression is related to the anti-AP-1 activity of retinoids. To determine whether the relaxin gene is regulated by retinoids at the level of transcription, a 4.3 kb fragment of the 5' flanking region of the rabbit relaxin gene was cloned and analyzed. This regulatory region included a classic TATA-box as well as consensus sequences for several transcription factors, including CREB, NF-kappaB and AP-1. The ability of the 4.3 kb regulatory region to control the transcription of a luciferase reporter gene was analyzed in transiently transfected, squamous-differentiated RbTE cells. The results demonstrated that this regulatory region caused strong transactivation of the reporter gene. This transactivation was inhibited by retinoic acid, suggesting retinoid control at the transcriptional level. Deletion analysis indicated that multiple regulatory elements are involved in the regulation of relaxin gene expression during squamous differentiation as well as in the suppression by retinoids.

Animals↗

PubMed1998

Expression of germ cell nuclear factor (GCNF/RTR) during spermatogenesis.

Germ cell nuclear factor (GCNF/RTR), a novel orphan receptor in the nuclear receptor superfamily of ligand-activated transcription factors, is expressed predominantly in developing germ cells. In several mammalian species two GCNF/RTR mRNAs are present in the testis, with the smaller 2.3-kb transcript generally expressed at higher levels than the larger 7.4- or 8.0-kb transcript. In both the mouse and rat, the 2.3- and 7.4-kb GCNF/RTR transcripts were detected in isolated spermatogenic cells, but not in Sertoli cells. Expression of these transcripts is differentially regulated, with the larger 7.4-kb mRNA appearing earlier during testicular development. The major 2.3-kb transcript is expressed predominantly in round spermatids in the mouse and rat. In situ hybridization studies in the rat demonstrated that GCNF/RTR transcripts reach maximal steady-state levels in round spermatids at stages VII and VIII of the spermatogenic cycle, and then decline abruptly as spermatids begin to elongate. RNase protection assays were used to predict the 3' termination site of the 2.3-kb transcript. An alternative polyadenylation signal (AGUAAA) was identified just upstream of this termination site. These studies suggest that GCNF/RTR may regulate transcription during spermatogenesis, particularly in round spermatids just prior to the initiation of nuclear elongation and condensation.

Animals↗

PubMed1998

Induction of apoptosis by the novel retinoid AHPN in human T-cell lymphoma cells involves caspase-dependent and independent pathways.

Retinoids play an important role in the control of lymphocyte function and homeostasis in the thymus. In this study, we show that the induction of growth arrest and apoptosis in a variety of T-cell lymphoma cell lines, including Jurkat and Molt-4 cells, is highly specific for the synthetic retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) since all-trans retinoic acid (RA), the RAR-selective retinoid TTAB, the RXR-selective retinoid SR11217 and the retinoid SR11302 exhibiting selective anti-AP1 activity, do not induce apoptosis or cause growth arrest. These findings support the concept that the effects of AHPN on proliferation and induction of apoptosis are mediated by a novel signaling pathway. AHPN-induced apoptosis is associated with an induction of internucleosomal DNA-fragmentation, increased annexin V binding and a 30-fold stimulation of caspase-3-like activity. Overexpression of Bcl-2 in Molt-4 cells greatly inhibits the induction of apoptosis by AHPN as indicated by the inhibition of DNA-fragmentation, annexin V binding and caspase-3-like activity. However, Bcl-2 overexpression does not interfere with the ability of AHPN to cause growth arrest or accumulation of cells in the early S-phase of the cell cycle, indicating that the effects of AHPN on growth arrest can be uncoupled from the effects on apoptosis. The caspase inhibitor Z-VAD-FMK, at concentrations that totally block caspase activity, delays but does not prevent cell death and does not affect the accumulation of cells in the S-phase of the cell cycle. Our results show that induction of caspase-3-like activity plays an important role in the execution of AHPN-induced apoptosis but cells can undergo cell death in the absence of this activity suggesting that AHPN-induced cell death involves caspase-dependent and -independent mechanisms.

Amino Acid Chloromethyl Ketones↗

PubMed1998

Inhibition of cell proliferation and induction of apoptosis by the retinoid AHPN in human lung carcinoma cells.

In this study, we investigated the effect of the novel retinoid 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN/CD437) on the growth of human lung carcinoma cell lines. AHPN inhibits the proliferation of all cell lines tested, irrespective of the lung tumor type, in a concentration- and time-dependent manner. A dramatic reduction in cell number was observed in adenocarcinoma H460 cells, and was shown to be related to an induction of apoptosis. Bromodeoxyuridine (BrdU) incorporation and flow-cytometric analyses indicated that treatment of H460 cells with AHPN induces cell-cycle arrest at the G1 phase. We therefore investigated the effect of AHPN on several regulatory proteins of the G1 phase of the cell-cycle. The cell-cycle arrest induced by AHPN was accompanied by an inhibition of the hyperphosphorylation of the retinoblastoma (Rb) protein, an indication of G1 arrest. Furthermore, two cyclin-dependent kinases, cdk2 and cdk4, which are normally involved in the phosphorylation of Rb, were shown to have decreased activity. In some cell lines, the decrease in cdk activity may be partly related to an increase in p21(WAF1/Cip1) (p21), an inhibitor of cyclin-dependent kinases. No changes were observed in the cyclin-dependent kinase inhibitor p27(Kip1). The observed increase in p53 in response to AHPN could at least to some extent be responsible for the increased levels of p21. The increase in p53 expression was found to be regulated at a post-transcriptional level. Our results suggest that the growth inhibition of certain lung carcinoma cell lines by AHPN is at least partly related to an increase in p21. However, in other cell lines, different mechanisms appear to be involved. The specificity with which AHPN and other retinoids induce growth arrest and p21 expression indicates that the action of AHPN is not mediated by RAR or RXR receptors, but involves a novel signaling pathway.

Apoptosis↗

PubMed1998

Up-regulation of p27Kip1, p21WAF1/Cip1 and p16Ink4a is associated with, but not sufficient for, induction of squamous differentiation.

Irreversible growth arrest is an early and integral part of squamous cell differentiation in normal human epidermal keratinocytes (NHEKs) and is assumed to be linked to the control of expression of differentiation-specific genes. In this study, we examine the link between the molecular events associated with growth arrest and the expression of differentiation genes. NHEKs that have been induced to undergo growth arrest and differentiation by suspension culture contain populations in both G1 and G2/M of the cell cycle. The irreversible growth arrest state in NHEKs is characterized by an accumulation of the hypophosphorylated forms of Rb and p130, with subsequent down-regulation of levels of Rb, up-regulation of p130 and associated down-regulation of E2F-regulated genes such as cyclin A. These events correlate with an inhibition of G1 cdk activity, mediated in part by an increase in the cdk inhibitors p21(WAF1/Cip1), p27(Kip1) and p16(Ink4a). Flow cytometric and immunoblot analysis demonstrated that the timing of the up-regulation of p27, p16 and p130 corresponds closely with the induction of the squamous-specific genes cornifin alpha (SPRR-1) and transglutaminase type I, suggesting a close link between control of growth arrest and differentiation. However, growth arrest induced by over-expression of p27, p21 or p16 by recombinant adenovirus is not sufficient to induce expression of the differentiation genes, or to invoke the pattern of cell cycle regulatory protein expression characteristic of the differentiation-specific irreversible growth arrest. We conclude that growth arrest mediated by activation of the Rb pathway is not sufficient to trigger terminal squamous differentiation and additional signals which can be generated during suspension culture are required to promote the complete differentiation program.

Cell Cycle↗

PubMed1998

Induction of the nuclear orphan receptor RORgamma during adipocyte differentiation of D1 and 3T3-L1 cells.

Here, we analyzed the expression of the three members of the retinoid-like orphan receptor (ROR) nuclear receptor subfamily during adipocyte differentiation. RORalpha and RORgamma mRNA were upregulated during adipocyte differentiation in preadipocyte D1 and 3T3-L1 cells, whereas RORbeta mRNA could not be detected. The induction of RORalpha and RORgamma mRNA succeeded the induction of peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer binding protein alpha and occurred at a similar time interval as did the increase in aP2 and lipoprotein lipase mRNA. Like the expression of PPARgamma and aP2, the induction of RORgamma mRNA was repressed by tumor necrosis factor alpha and transforming growth factor beta. The induction of adipogenesis by prostaglandin D2 and two thiazolidinediones in the multipotent stem cells C3H10T1/2 was also accompanied by an induction in RORgamma mRNA. In contrast to parental cells, clofibrate induces adipogenesis and RORalpha and RORgamma mRNA in BALB/c3T3 cells that ectopically express PPARgamma. RORgamma mediates its effect on transcription through specific response elements. Cotransfection of RORalpha or RORgamma and (RORgamma response element)4-chloramphenicol acetyltransferase into preadipocyte D1 cells induced transactivation of chloramphenicol acetyltransferase about 100-fold, suggesting that ROR plays a role in the regulation of gene expression in adipocytes. The nuclear orphan receptor Rev-ErbAalpha, which did not exhibit transactivation function, was able to inhibit transactivation by RORgamma at two different levels. Our results show that RORgamma is induced during adipocyte differentiation in D1 and 3T3-L1 cells and functions as an active transcription factor, suggesting a role for RORgamma in the regulation of gene expression during this differentiation process.

3T3 Cells↗

PubMed1998

Differentiation-dependent regulation of retinal dehydrogenase gene expression in the trachea.

Retinoic acid (RA), a metabolite of vitamin A, is known to be a key signaling molecule in regulating epithelial cell differentiation. We recently characterized and cloned a retinal dehydrogenase (RALDH) that catalyzes the oxidation of retinal to RA. In this study, we investigated the effects of retinoids on the level of RALDH mRNA and protein as well as RALDH activity in the trachea and cultured tracheal epithelial cells. Vitamin A deficiency induced squamous metaplasia in the tracheal epithelium and down-regulated RALDH expression. Supplementation of retinol and retinoic acid to vitamin A deficient rats restored the normal mucociliary epithelium and up-regulated the RALDH expression. In rat epithelial cells cultured in vitro, RAinhibited squamous differentiation and promoted mucociliary differentiation. Squamous differentiated cultures (RA-) expressed very low levels of RALDH mRNA, whereas mucociliary differentiated cultures (RA+) expressed high levels of RALDH mRNA. Retinal and retinol were poor inducers of mucociliary differentiation as well as RALDH expression. The RALDH expression paralleled the expression of the mucin-1 gene in mucociliary cultures. These results suggest that the expression of RALDH is dependent on the differentiation state of the airway epithelium.

Aldehyde Oxidoreductases↗

PubMed1997

Genomic structure and chromosomal mapping of the nuclear orphan receptor ROR gamma (RORC) gene.

The nuclear orphan receptor subfamily ROR/RZR is part of the steroid and thyroid hormone/retinoid receptor superfamily and consists of three different genes, alpha, beta, and gamma. In this study, we determined the genomic structure of mouse ROR gamma and the chromosomal localization of both mouse ROR gamma and human ROR gamma (HGMW-approved symbol RORC). The genomic structure of the mouse ROR gamma gene was derived from the analysis of P1 vector clones containing large genomic fragments encoding ROR gamma. These results revealed that the mROR gamma gene has a complex structure consisting of 11 exons separated by 10 introns spanning more than 21 kb of genomic DNA. The DNA-binding domain is contained in two exons, 3 and 4, each encoding one zinc-finger. The splice site between exon 3 and exon 4 is identical to that found in RAR and TR3 receptors. ROR gamma is expressed as two mRNAs, 2.3 and 3.0 kb in size, that are derived by the use of alternative polyadenylation signals. We show by fluorescence in situ hybridization that the mouse ROR gamma gene is located on chromosome 3, in a region that corresponds to band 3F2.1-2.2. The human ROR gamma was mapped to chromosome region 1q21. The results demonstrate that the ROR gamma genes are located in chromosomal regions that are syntenic between mouse and human.

Alternative Splicing↗

PubMed1997

The role of p27Kip1 in gamma interferon-mediated growth arrest of mammary epithelial cells and related defects in mammary carcinoma cells.

Interferon gamma (IFNgamma) induces growth arrest in normal human mammary epithelial cells by establishing a block during mid-G1 corresponding to the time when the retinoblastoma protein (Rb) would normally be inactivated by hyperphosphorylation. IFNgamma inhibits the kinase activities of cdk2, cdk4 and cdk6 within 24 h of treatment. Protein levels of the cdks and G1 cyclins do not change within this time period, although cdk4 levels are significantly reduced by 48 h. IFNgamma treatment induces p27Kip1 protein levels, presumably by a post-transcriptional mechanism as no change was observed in the mRNA levels. In addition, IFNgamma-induced inhibition of cdk2 and cyclin E-associated kinase activities is accompanied by a 4.5-fold or greater increase of p27Kip1 in cdk2 complexes. p27 may also have a role in the inhibition of cdk4/6 kinase activities, as p27 protein associated with these complexes was increase by 55-70% after IFNgamma. In mammary carcinoma cell lines which are resistant to growth inhibition by IFNgamma, p27 levels are not induced by IFNgamma nor is cdk2 kinase activity inhibited, despite high baseline levels of p27 in cdk2 complexes. However, exogenous expression of p27 in these cells induces growth arrest. In addition, purified p27 protein added to cdk2 complexes immunoprecipitated from carcinoma cells is able to inhibit the kinase activity in a dose dependent manner. Our results suggest that p27Kip1 has a role in mediating IFNgamma-induced terminal growth arrest. Resistance of mammary carcinomas to growth inhibition by IFNgamma does not appear to involve resistance of cdk2 complexes to the action of p27, but rather an inability to appropriately regulate the balance of cdk2, cyclin E and p27 levels.

Breast↗

PubMed1997

Characterization of the response element and DNA binding properties of the nuclear orphan receptor germ cell nuclear factor/retinoid receptor-related testis-associated receptor.

Recently, we have reported the cloning of the germ cell-specific, nuclear orphan receptor germ cell nuclear factor (GCNF)/RTR. In this study, we characterize the RTR response elements by an electrophoretic mobility shift assay/polymerase chain reaction-based, DNA binding site selection strategy. RTR binds with the greatest affinity to response elements containing TCA(AG(G/T)TCA)2 (consensus RTR response element; conRTRE), to which it binds as a homodimer. RTR is also able to bind as a monomer to a single core motif TCAAG(G/T)TCA, albeit with a lower affinity. Mutation analysis supports the specific requirements of the 5'-flanking sequence and the core motif of the RTRE for optimal binding of RTR. An RTR-specific antiserum (RTR-Ab2) was raised that causes supershift of the RTR-conRTRE complex in EMSA. Based on the sequence of the conRTRE, we located a putative RTRE, referred to as P2-RE, in the 5' promoter-flanking region of the mouse protamine 2 gene, which is induced during the same stage of spermatogenesis as RTR. The ability of RTR-Ab2 to cause a supershift of an RTR-RTRE complex with nuclear extracts from different tissues correlated with the tissue- and development-specific expression of RTR. Transfection of RTR in CV-1 cells was unable to cause RTRE-dependent transactivation of a CAT reporter gene; however, an RTR-VP16 fusion protein could induce transactivation through several RTREs, including P2-RE.

Amino Acid Sequence↗

PubMed1997

cDNA cloning, genomic structure, and chromosome mapping of the human epithelial membrane protein CL-20 gene (EMP1), a member of the PMP22 family.

CL-20 is a novel gene encoding a protein that is structurally related to but distinct from the peripheral myelin protein PMP22. Like PMP22, CL-20 is likely to play important roles in the regulation of cell proliferation, differentiation, and cell death. In this study, we describe the cloning and sequencing of a cDNA encoding the human homologue of CL-20 and characterize the genomic structure of this gene. The hCL-20 gene (HGMW-approved symbol EMP1) encodes a protein of 157 amino acids that exhibits 76% identity to the rabbit CL-20 and to the rat EMP-1, which have been described recently, and 39% identity to human PMP22. CL-20 contains four hydrophobic domains, suggesting that it is an integral membrane protein. In particular the second hydrophobic domain encoded within the fourth exon is highly conserved among CL-20, EMP-1, and PMP22, suggesting a functional role for this region. CL-20 mRNA is abundant in squamous-differentiated bronchial epithelial cells; however, low levels of CL-20 mRNA can be detected in several human tissues by Northern analysis. Retinoic acid, which inhibits squamous differentiation, represses CL-20 expression in normal human bronchial epithelial cells. The genomic structure of the hCL-20 gene was analyzed using a P1 vector containing this gene. The hCL-20 gene contains five exons about 0.2, 0.12, 0.1, 0.14, and 2.2 kb and four introns about 15, 1.9, 0.1, and 0.7 kb. We have mapped the hCL-20 gene to chromosome 12p12 by fluorescence in situ hybridization.

Amino Acid Sequence↗

PubMed1997

Differential expression of human cornifin alpha and beta in squamous differentiating epithelial tissues and several skin lesions.

Cornifins/small proline-rich proteins (SPRRs) belong to a family of proline-rich proteins that function as cornified envelope precursors. We report here an immunohistochemical analysis of human cornifin-alpha and -beta expression in several stratified squamous epithelia. In normal human skin, cornifin-alpha was expressed in the granular layer of the epidermis of palmoplantar skin, in the inner lining cells of the follicular infundibulum, and in the inner root sheath of the hair follicle. It was also expressed in the upper squamous layers of the oral, esophageal, and vaginal epithelia. Cornifin-beta was detected in oral, esophageal, and vaginal epithelia, but not in normal skin. Immunoblot analysis revealed quantitative differences in cornifin-alpha expression in skin from different regions. Studies of specimens from various skin diseases showed that (i) cornifin-alpha was upregulated in inflammatory skin diseases, hyperplastic lesions, and in well-differentiated squamous cell carcinomas (SCCs), (ii) the expression of cornifin-beta was absent in inflammatory skin but was detected in highly differentiated keratinocytes in well-differentiated SCCs of the skin and some other hyperproliferative skin lesions, and in SCCs of the oral mucosa and esophagus. Northern blot analysis revealed that cornifin-alpha mRNA was present in all the squamous epithelial tissues studied, whereas cornifin-beta mRNA was expressed in oral mucosal epithelia and verrucous carcinoma of the skin but neither in normal nor in psoriatic skin. These results indicate that (i) the amount of cornifin alpha/SPRR1 expression in normal human skin depends on the body region, (ii) cornifin-alpha/SPRR1, but not cornifin-beta, contributes to the integrity of the hair follicle, and (iii) the expression of cornifin-beta is induced in some hyperplastic skin diseases only when the keratinocytes undergo extensive squamous differentiation.

Blotting, Northern↗

PubMed1997

Epidermal differentiation and squamous metaplasia: from stem cell to cell death.

Epidermal differentiation is a multi-step process defined by a cascade of interrelated changes in the expression of growth-regulatory and differentiation-specific genes (Fig. 1). Irreversible growth arrest is an early event in epidermal differentiation which occurs when cells transit from the basal to the innermost suprabasal layer of the skin and begin to express squamous-specific genes. In culture, interferon gamma, phorbol esters, confluence and growth in suspension are effective signals to induce irreversible growth arrest and differentiation. The induction of differentiation-specific genes occurs either concomitantly with or following growth arrest and is believed to be linked to the molecular events that control irreversible growth arrest. Such a link has been demonstrated in other cell systems undergoing terminal differentiation, such as myogenesis and adipogenesis. Genes encoding proteins involved in the formation of the cross-linked envelope are one set of squamous-specific genes which are induced in the suprabasal layers and include transglutaminase I and III, involucrin, loricrin and cornifins/small proline-rich proteins. Squamous-specific genes exhibit not only different patterns of tissue-specific expression but are also induced at different stages during differentiation, suggesting that transcription of individual genes is regulated by distinct mechanisms. The latter is supported by the identification of different sets of regulatory elements controlling the transcription of these genes. The importance of understanding both the mechanisms that regulate growth arrest and the differentiation program is emphasized by the association found between specific skin diseases and genetic alterations in growth-regulatory genes as well as differentiation markers. In addition, studies into those mechanisms will provide insight into the control of squamous metaplasia and the development of squamous cell carcinomas.

Amino Acid Sequence↗

PubMed1997

Cloning of the human orphan receptor germ cell nuclear factor/retinoid receptor-related testis-associated receptor and its differential regulation during embryonal carcinoma cell differentiation.

We have cloned a cDNA encoding the full-length coding region of the human homologue of the germ cell nuclear factor (GCNF)/retinoid receptor-related testis-associated receptor (RTR), from a human testis cDNA library. The amino acid sequence of human GCNF/RTR is highly homologous to that of the mouse GCNF/RTR. The largest difference between the two homologues is a 15 amino acid deletion in the human GCNF/RTR at amino acid 47. The GCNF/RTR gene was localized on human chromosome 9. Northern blot analysis using poly(A)+ RNA from different human tissues showed that GCNF/RTR mRNA is most abundantly expressed in the testis. GCNF/RTR was also highly expressed in embryonic stem cells and embryonal carcinoma cells but repressed in its differentiated derivatives. Induction of differentiation of mouse embryonal carcinoma F9 cells and human embryonal carcinoma NTERA-2 clone D1 (NT2/D1) cells by all-trans retinoic acid was accompanied by a down-regulation of GCNF/RTR. Our observations suggest that GCNF/RTR plays a role in the control of gene expression in early embryogenesis and during spermatogenesis.

Amino Acid Sequence↗

PubMed1996

Cloning of a cDNA encoding the murine orphan receptor RZR/ROR gamma and characterization of its response element.

In this study, we describe the cloning of the mouse homologue of the orphan receptor, RZR/ROR gamma, a member of the nuclear receptor superfamily, from a mouse muscle cDNA library. The amino acid sequence of mouse ROR gamma (mROR gamma) is highly homologous to that of human ROR gamma, with an overall identity of 88%. Northern blot analysis using RNA from different tissues showed that mROR gamma was found to be highly expressed in skeletal muscle, liver and kidney. Analysis of the ROR gamma-response element using in vitro synthesized ROR gamma revealed that it binds as a monomer to response elements composed of a single core motif GGTCA preceded by a 6 bp AT-rich sequence. The ROR gamma-binding specificity was further defined by mutational analysis of the consensus RORE. ROR gamma was able to activate RORE-dependent transcription of the CAT reporter gene in mouse fibroblast D1 cells. ROR alpha 1 and ROR gamma inhibit the transactivation induced by GAL4(DBD)-ROR gamma in fibroblast D1 cells suggesting that these receptors compete for binding to the same coactivators.

Amino Acid Sequence↗

PubMed1996

Regulation of cornifin alpha expression in the vaginal and uterine epithelium by estrogen and retinoic acid.

In this study, we analyze the regulation of the squamous-specific gene, cornifin alpha, by estrogen and retinoic acid in vaginal and uterine epithelial cells. In ovariectomized animals, the vaginal epithelium consists of a stratified, nonkeratinizing epithelium which changes into a highly-stratified, keratinizing epithelium upon treatment with estradiol. This transition is accompanied by a dramatic induction of the crosslinked envelope precursor, cornifin alpha. An increase in cornifin mRNA can be detected as early as 3 h after treatment. A similar effect is observed for the synthetic estrogenic agent diethylstilbestrol while other steroid hormones, including testosterone, progesterone or dexamethasone have little effect on cornifin expression. In contrast to the vagina, estradiol induces neither squamous differentiation nor expression of cornifin alpha in the uterine epithelium. Similar to the action of estradiol, vitamin A-deficiency greatly enhances squamous differentiation and keratinization in the vaginal epithelium. But unlike estradiol, it induces squamous metaplasia in the normally columnar, uterine epithelium, which eventually is replaced by a keratinizing epithelium in severe deficiency. This transition is associated with an induction of cornifin alpha expression. Immunohistochemical and in situ hybridization analysis localizes cornifin protein and mRNA in the suprabasal layers of the squamous epithelium. Our results demonstrate that estrogen and retinoids play key roles in the regulation of differentiation and cornifin alpha expression in the uterine and vaginal epithelium.

Animals↗

PubMed1996

Retinoid-induced differentiation of acute promyelocytic leukemia involves PML-RARalpha-mediated increase of type II transglutaminase.

All-trans retinoic acid (t-RA) administration leads to complete remission in acute promyelocytic leukemia (APL) patients by inducing growth arrest and differentiation of the leukemic clone. In the present study, we show that t-RA treatment dramatically induced type II transglutaminase (type II TGase) expression in cells carrying the t(15;17) translocation and expressing the PML-RARalpha product such as the APL-derived NB4 cell line and fresh leukemic cells from APL patients. This induction correlated with t-RA-induced growth arrest, granulocytic differentiation, and upregulation of the leukocyte adherence receptor beta subunit (CD18) gene expression. The increase in type II TGase was not abolished by cycloheximide treatment, suggesting that synthesis of a protein intermediate was not required for the induction. t-RA did not significantly alter the rate of growth arrest and did not stimulate differentiation and type II TGase activity in NB4.306 cells, a t-RA-resistant subclone of the NB4 cell line, or in leukemic cells derived from two patients morphologically defined as APL but lacking the t(15;17). However, in NB4.306 cells, t-RA treatment was able to increase CD18 mRNA expression in a manner similar to NB4 cells. The molecular mechanisms involved in the induction of these genes were investigated. In NB4 cells, using novel receptor-selective ligands such as 9-cis-RA, TTNPB, AM580, and SR11217, we found that RAR- and RARalpha-selective retinoids were able to induce growth arrest, granulocytic differentiation, and type II TGase, whereas the RXR-selective retinoid SR11217 was inactive. Moreover, an RAR alpha-antagonist completely inhibited the expression of type II TGase and CD18 induced by these selective retinoids in NB4 cells. In NB4.306 cells, an RARalpha-dependent signaling pathway was found involved in the modulation of CD18 expression. In addition, expression of the PML-RARalpha gene in myeloid U937 precursor cells resulted in the ability of these cells to induce type II TGase in response to t-RA. On the basis of these results we hypothesize a specific involvement of a signaling pathway involving PML-RAR alpha for the induction of growth arrest, granulocytic differentiation, and type II TGase by retinoids in APL cells.

Apoptosis↗