Biomedical subjects

X K Zhang

Publications and source records attributed to X K Zhang.

At least 37 records · Page 2Linked to original sources

PubMed1997

Inhibition of trans-retinoic acid-resistant human breast cancer cell growth by retinoid X receptor-selective retinoids.

All-trans-retinoic acid (trans-RA) and other retinoids exert anticancer effects through two types of retinoid receptors, the RA receptors (RARs) and retinoid X receptors (RXRs). Previous studies demonstrated that the growth-inhibitory effects of trans-RA and related retinoids are impaired in certain estrogen-independent breast cancer cell lines due to their lower levels of RAR alpha and RARbeta. In this study, we evaluated several synthetic retinoids for their ability to induce growth inhibition and apoptosis in both trans-RA-sensitive and trans-RA-resistant breast cancer cell lines. Our results demonstrate that RXR-selective retinoids, particularly in combination with RAR-selective retinoids, could significantly induce RARbeta and inhibit the growth and induce the apoptosis of trans-RA-resistant, RAR alpha-deficient MDA-MB-231 cells but had low activity against trans-RA-sensitive ZR-75-1 cells that express high levels of RAR alpha. Using gel retardation and transient transfection assays, we found that the effects of RXR-selective retinoids on MDA-MB-231 cells were most likely mediated by RXR-nur77 heterodimers that bound to the RA response element in the RARbeta promoter and activated the RARbeta promoter in response to RXR-selective retinoids. In contrast, growth inhibition by RAR-selective retinoids in trans-RA-sensitive, RAR alpha-expressing cells most probably occurred through RXR-RAR alpha heterodimers that also bound to and activated the RARbeta promoter. In MDA-MB-231 clones stably expressing RAR alpha, both RARbeta induction and growth inhibition by RXR-selective retinoids were suppressed, while the effects of RAR-selective retinoids were enhanced. Together, our results demonstrate that activation of RXR can inhibit the growth of trans-RA-resistant MDA-MB-231 breast cancer cells and suggest that low cellular RAR alpha may regulate the signaling switch from RAR-mediated to RXR-mediated growth inhibition in breast cancer cells.

Antineoplastic Agents↗

PubMed1996

Rubber stoppers as sources of contaminants in electrospray analysis of peptides and proteins.

Using MS/MS, we have identified ions at m/z 399, 421, 609.4, and 819.9 as those of tris (2-butoxyethyl) phosphate, originating in the gray rubber stoppers commonly used as closures in commercial sources of peptides and proteins. The compound is readily extracted by contact with organic solvents such as alcohol or acetic acid and even by water alone. Zinc salts of proteins are also observed, presumably from zinc oxide fillers present in the stoppers.

Drug Packaging↗

PubMed1996

Retinoid receptors in human lung cancer and breast cancer.

Retinoids, the natural and synthetic vitamin A derivatives, are known to inhibit the proliferation of lung cancer and breast cancer cells and the growth of carcinogen-induced bronchogenic squamous cell carcinoma and mammary tumors, and have been used as chemoprevention agents against both types of cancer. However, clinical trials of retinoids in patients with advanced lung cancer and breast cancer have not been successful. In studying how retinoid sensitivity is lost in cancer cells, we have found that lack of the retinoic acid receptor beta (RAR beta) gene expression and its abnormal regulation by retinoic acid (RA) are common features in human lung cancer and breast cancer cells. The absence and abnormal RA regulation of RAR beta correlates with the loss of anti-proliferation effect of RA in hormone-independent breast cancer cells, and is due to different abnormalities found in cancer cells. Furthermore, expression of RAR beta gene in hormone-independent breast cancer cells restores their RA sensitivity. These data demonstrate that RAR beta can mediate the growth inhibitory effect of RA and suggest that the lack of RAR beta may contribute to retinoid resistance in certain cancer cells.

Breast Neoplasms↗

PubMed1996

The down-regulated in adenoma (DRA) gene encodes an intestine-specific membrane glycoprotein.

The protein product of the DRA gene, a gene whose expression is down-regulated in colon adenomas and adenocarcinomas, is a membrane glycoprotein and a member of a family of sulfate transporters. It is expressed in the intestinal tract (duodenum, ileum, cecum, distal colon), but not in the esophagus or stomach. DRA mRNA expression is restricted to the mucosal epithelium, and DRA protein expression is further limited to the columnar epithelial cells, particularly to the brush border. Consistent with its expression in the differentiated columnar epithelium of the adult human colon, DRA is first expressed in the midgut of developing mouse embryos at day 16.5, corresponding with the time of differentiation of the epithelium of the small intestine. A model for the structure of the DRA protein is proposed and its possible role in colon tumorigenesis is discussed.

Animals↗

PubMed1996

Interaction of ETS-1 and ERGB/FLI-1 proteins with DNA is modulated by spacing between multiple binding sites as well as phosphorylation.

ETS is a family of transcription factors that contain a highly conserved ETS DNA binding domain. Various members of the ETS family are expressed in cells of hematopoietic lineage. ETS-1, ETS-2 and ERGB/FLI-1 are expressed at high levels in T-lymphocytes. HIV-1 infects T-cells and it has been shown that its LTR contains binding sites for various transcription factors. In this study we show that the HIV-1 core enhancer is directly regulated by ERGB/FLI-1 protein positively, as well as, negatively, depending upon the presence or absence of accessory factors in different cell types. In addition, we show that the ETS-1 transactivation activity is enhanced upon dephosphorylation of the Calmodulin-dependent Protein Kinase II phosphorylation site located in exon VII. Finally, we demonstrate that the spacing between the two EBS cores in palindromic or direct repeat sites play a crucial role in binding of ETS proteins to DNA.

Animals↗

PubMed1996

Changes in liver membrane potentials after partial hepatectomy in rats.

Changes in potential differences (PD) across hepatocyte membranes after partial hepatectomy may play an important role in hepatic homeostatic mechanisms and regenerative activity. To date, few studies have attempted to describe the extent and duration of such changes. In the present study, we documented PD values immediately before and for a 24-hour period after a partial hepatectomy (PHx) of 70% in healthy adult rats. We also documented PD changes after 30% and 90% PHx and PD values in different lobes of the same liver before and after PHx. Our findings showed that the liver depolarizes promptly (within 5 minutes of PHx) and that changes in PD are sustained for approximately 18 hours before returning to baseline values. We also observed that the magnitude of hepatic depolarization correlates with the extent of PHx. Finally, we did not observe regional differences in PD recordings from various lobes before and after PHx. These findings enhance our understanding of the physiological changes that occur in regenerating livers after PHx in rats.

Animals↗

PubMed1996

Increased expression of the ETS-related transcription factor FLI-1/ERGB correlates with and can induce the megakaryocytic phenotype.

The human leukemia cell line K562 can be induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) to differentiate along the megakaryocytic pathway, generating morphological changes and increased expression of lineage-specific surface markers. We report that TPA-treated K562 cells also express higher levels of FLI-1/ERGB, a member of the ETS family of transcription factors. Furthermore, introduction of a retroviral construct expressing human FLI-1/ERGB into K562 cells induces changes similar to those seen following TPA treatment, including increased adherence to the surface of the culture vessel and altered size and morphology. Infected cells exhibit higher levels of the megakaryocyte marker CD41a and, to a lesser extent, CD49b. These markers, as well as virally encoded FLI-1/ERGB-specific RNA and protein, are expressed at the highest levels in the attached cell population, while the growth rate of adherent cells is reduced, and the fraction of cells in G0-G1 is increased. FLI-1/ERGB virus-infected cells also exhibit increased expression of hemoglobin, a marker of erythroid differentiation. Our results suggest FLI-1/ERGB plays a role in controlling differentiation and gene expression along the megakaryocyte/platelet pathway, and further implicate ETS-related genes in the control of multiple developmentally regulated hematopoietic genes.

Animals↗

PubMed1996

Propranolol and bepridil attenuating levothyroxine-induced rat cardiac hypertrophy and mitochondrial Ca2+ Mg(2+)-ATPase activity elevation.

AIM: To study the effects of propranolol and bepridil on levothyroxine-induced rat cardiac hypertrophy and mitochondrial Ca2+ Mg(2+)-ATPase activity elevation. METHODS: Rat heart hypertrophy was induced by i.p., levothyroxine 1 mg.kg-1.d-1 x 10 d. Then rats were treated by ig propranolol (Pro) or bepridil (Bep) 10 mg.kg-1 daily. Ca2+ Mg(2+)-ATPase activity and enzyme kinetic parameters were assayed. RESULTS: The activity and Vmax of mitochondrial Ca2+ Mg(2+)-ATPase isolated from hypertrophic left ventricle were 25 +/- 4 and 35.1 +/- 0.8 mumol Pi.h-1/mg protein, respectively, those of normal were 6.7 +/- 1.8 and 10 +/- 4 mumol Pi.h-1/mg protein, respectively. Apparent K(m) of the hypertrophic group Ca2+ Mg(2+)-ATPase was 0.4 +/- 0.12 mmol.L-1 ATP, and that of normal was 0.59 +/- 0.22 mmol.L-1 ATP. The total protein quantity of hypertrophic left ventricle was 80 +/- 30 mg, and that of normal was 47 +/- 9 mg. After treated with Pro or Bep (both 10 mg.kg-1 ig), the cardiac hypertrophy was attenuated, the enzyme activity and Vmax as well as total protein quantity of hypertrophic left ventricle were reduced to normal level, but apparent K(m) was not affected. CONCLUSION: Both Pro and Bep prevented the myocardium and its mitochondria from ischemia and overload calcium injury.

Adrenergic beta-Antagonists↗

PubMed1995

Correlation of retinoid binding affinity to retinoic acid receptor alpha with retinoid inhibition of growth of estrogen receptor-positive MCF-7 mammary carcinoma cells.

Both anchorage-dependent growth and anchorage-independent growth of the estrogen receptor-positive mammary carcinoma cell line MCF-7 are inhibited by all-trans-retinoic acid. This cell line has nuclear retinoic acid receptors (RARs) alpha and gamma. The natural retinoids all-trans-retinoic acid and 9-cis-retinoic acid and a series of 12 conformationally restricted retinoids, which showed a range of binding selectivities for these receptors and had either agonist or antagonist activity for gene transcriptional activation by the RARs, were evaluated for their abilities to inhibit anchorage-dependent (adherent) and anchorage-independent (clonal) growth of MCF-7 cells. Correlation analyses were performed to relate growth inhibition by these retinoids with their binding affinity to RAR alpha or RAR gamma. Inhibition of anchorage-dependent growth in culture after 7 days of retinoid treatment correlated with binding to RAR alpha (n = 14; P < or = 0.001) and not to RAR gamma (n = 14; P > 0.1). Both the RAR alpha-selective retinoid agonists and the two RAR antagonists that were evaluated inhibited adherent cell growth. The RAR gamma-selective agonists had very low growth inhibitory activity (< 10%) at concentrations as high as 12.5 microM. These results suggest that RAR alpha is the retinoid receptor involved in the inhibition of adherent cell growth by retinoids and that transcriptional activation by this receptor on a RAR response element does not appear to be required for this process to occur. For this series of retinoids, inhibition of anchorage-independent growth after 21 days of retinoid treatment only correlated (n = 12; P < or = 0.005) with binding affinity to RAR alpha for the retinoid agonists, although the RAR gamma-selective retinoids displayed weak activity. The RAR antagonists were very poor inhibitors of growth. These results suggest that activation of gene transcription by RAR alpha appears to be required for inhibition of anchorage-independent growth by retinoids in this estrogen receptor-positive mammary carcinoma cell line.

Animals↗

PubMed1995

Conformationally defined 6-s-trans-retinoic acid analogs. 2. Selective agonists for nuclear receptor binding and transcriptional activity.

We recently demonstrated in animal models that a new conformationally defined RA isomer (Vaezi et al. J. Med. Chem. 1994, 37, 4499-4507) was as effective as RA in the prevention of skin papillomas but was less toxic. In order to provide more details concerning this improved action, we report here the preparation of a homologous conformationally defined 6-s-trans-retinoid (1) and investigate its ability to interact with proteins and to activate gene expression. Four configurational isomers of 1 were evaluated in binding assays for cellular retinoic acid binding protein, CRABP (isolated from chick skin); CRABP-I and CRABP-II (cloned from mouse); nuclear retinoic acid receptors (RARs); and nuclear retinoid X receptors (RXRs). In each assay the all-E-isomer of this retinoid had an activity that was comparable to that of (all-E)-RA. However, the 9Z-isomer was at least 200-fold less active than (all-E)-RA in binding to different RARs, while it was only 6-20 times less active than (9Z)-RA in binding to different RXRs. In an in vivo transient transfection assay, the all-E-isomer activated a reporter gene containing a retinoic acid response element (RARE) with efficiency similar to (all-E)-RA when expression vectors for either RAR alpha, RAR beta, RAR gamma alone or RAR alpha together with RXR alpha were cotransfected. In contrast, the 9Z-isomer was much less active than (9Z)-RA in the same assay systems. However, (9Z)-1 efficiently enhanced the DNA binding and transactivational activity of RXR alpha homodimers. Taken together, these studies demonstrate that the all-E- and 9Z-isomers of this retinoid are selective and potent agonists of RAR and RXR binding and activation.

Animals↗

PubMed1995

Generation and characterization of monoclonal antibodies against the ERGB/FLI-1 transcription factor.

Five monoclonal antibodies were produced from mice immunized with recombinant full length human ERGB protein. Among these monoclonal antibodies, four clones did not cross react with other ets family proteins and thus are specific for the ERGB protein; however, one clone did react with the ERG protein, which has high amino acid identity with the ERGB protein. The epitope location of these antibodies was studied using bacterially expressed fragments of the human, ERGB protein. These monoclonal antibodies recognized 51 kDa (p51) and 48 kDa (p48), two ERGB gene-encoded proteins, from human, mouse, and rat cell lines. These results suggest that the monoclonal antibodies can be used in human, mouse, or rat cell lines and will be useful for the biochemical and functional analysis of the ERGB protein.

Animals↗

PubMed1995

A retinoic acid response element that overlaps an estrogen response element mediates multihormonal sensitivity in transcriptional activation of the lactoferrin gene.

The lactoferrin gene is highly expressed in many different tissues, and its expression is controlled by different regulators. In this report, we have defined a retinoic acid response element (RARE) in the 5'-flanking region of the lactoferrin gene promoter. The lactoferrin-RARE is composed of two AGGTCA-like motifs arranged as a direct repeat with 1-bp spacing (DR-1). A gel retardation assay demonstrated that it bound strongly with retinoid X receptor (RXR) homodimers and RXR-retinoic acid receptor (RAR) heterodimers as well as chicken ovalbumin upstream promoter transcription factor (COUP-TF) orphan receptor. In CV-1 cells, the lactoferrin-RARE linked with a heterologous thymidine kinase promoter was strongly activated by RXR homodimers in response to 9-cis-retinoic acid (9-cis-RA) but not to all-trans-RA. When the COUP-TF orphan receptor was cotransfected, the 9-cis-RA-induced RXR homodimer activity was strongly repressed. A unique feature of the lactoferrin-RARE is that it has an AGGTCA-like motif in common with an estrogen-responsive element (ERE). The composite RARE/ERE contributes to the functional interaction between retinoid receptors and the estrogen receptor (ER) and their ligands. In CV-1 cells, cotransfection of the retinoid and estrogen receptors led to mutual inhibition of the other's activity, while an RA-dependent inhibition of ER activity was observed in breast cancer cells. Furthermore, the lactoferrin-RARE/ERE showed differential transactivation activity in different cell types. RAs could activate the lactoferrin-RARE/ERE in human leukemia HL-60 cells and U937 cells but not in human breast cancer cells. By gel retardation analyses, we demonstrated that strong binding of the endogenous COUP-TF in breast cancer cells to the composite element contributed to diminished RA response in these cells. Thus, the lactoferrin-RARE/ERE functions as a signaling switch module that mediates multihormonal responsiveness in the regulation of lactoferrin gene expression.

Base Sequence↗

PubMed1995

Ciprofloxacin prevents the inhibitory effects of acute ethanol exposure on hepatic regeneration in the rat.

To determine whether the inhibitory effects of ethanol on hepatic regeneration could be prevented by ciprofloxacin, a fluroquinolone antibiotic with gamma-aminobutyric acid (GABAA), receptor antagonist properties, adult, male Sprague-Dawley rats (n = 6-8/group) received intraperitoneal injections of saline, putrescine (a hepatic growth promoter, 50 mg/kg), or ciprofloxacin (100 mg/kg), followed 1 hour later by gastric gavage with saline or ethanol (5 g/kg). One hour post-gavage, all rats underwent a 70% partial hepatectomy (PHx). Hepatic regenerative activity was documented 24 hours post-PHx by 3H-thymidine incorporation into hepatic DNA (DNA synthesis), proliferating cell nuclear antigen staining, and hepatic tissue putrescine levels. Compared with healthy controls, DNA synthesis rates were significantly lower in ethanol-gavaged/saline-treated rats (22.7 +/- 4.4 x 10(3) vs. 12.3 +/- 6.9 x 10(3) DPM/mg DNA, respectively, P < .001) but unaltered in putrescine-(18.8 +/- 3.4 x 10(3) DPM/mg DNA) and ciprofloxacin-treated (18.3 +/- 2.6 x 10(3) DPM/mg DNA) rats. Hepatic proliferating cell nuclear antigen staining supported these findings. Hepatic putrescine levels also correlated with DNA synthesis data, being decreased in ethanol-gavaged/saline-treated rats (86 +/- 14 pmoles/mg tissue) compared with healthy controls (120 +/- 12 pmoles/mg, P < .01), ethanol-gavaged/putrescine-treated (112 +/- 14 pmoles/mg, P < .05) and ethanol-gavaged/ciprofloxacin-treated (125 +/- 17 pmoles/mg, P < .05) rats. To determine whether these effects resulted from GABAA receptor-mediated changes in liver membrane potentials, intracellular membrane potentials were recorded before and 1 hour after PHx in healthy control, ethanol-gavaged/saline-treated and ethanol-gavaged/ciprofloxacin-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

A specific defect in the retinoic acid response associated with human lung cancer cell lines.

The effects of retinoic acid (RA) are mainly mediated by its nuclear receptors, the RA receptors (RARs) and retinoid X receptors (RXRs) that regulate target gene expression by binding to specific RA-response elements (RAREs). RAR beta is the best characterized RA-responsive gene. Due to the presence of a RARE (beta RARE) in its promoter, the expression of the RAR beta 2 is markedly increased in response to RA in most epithelial tissues, including lung. Recently, it was observed that the RAR beta gene is not expressed in a number of human lung cancer cell lines, suggesting a possible correlation between abnormal expression of the RAR beta gene and lung cancer development. In this study, we investigate the RA response in human lung cancer cell lines. Here we report that the expression of the RAR beta gene cannot be regulated by RA in the majority of human lung cancer cell lines examined, while the general response to RA is intact. The nonresponsiveness of the RAR beta gene results from different defects in the response mechanism. Interestingly, we find in some cell lines a differential responsiveness of the beta RARE such that the element is inactive in its natural promoter context but active when linked to the heterologous tk promoter. Importantly, we also observe that the presence of retinoid receptors is not sufficient for the induction of the RAR beta gene. This suggests that specific factors determine the RA responsiveness in the context of its natural promoter. Our observation that the RA nonresponsiveness of the RAR beta promoter is a common feature of human lung cancer cell lines suggests that balanced RAR beta expression is an essential feature for the maintenance of a normal state of lung tissue.

Blotting, Northern↗

PubMed1994

Induction of plasmacytomas with silicone gel in genetically susceptible strains of mice.

BACKGROUND: Plasmacytomas can be induced in high frequency in susceptible strains of mice by the intraperitoneal introduction of plastics or paraffin oils, including the chemically defined oil pristane (2,6,10,14-tetramethylpentadecane). These materials persist in the peritoneal cavity, where they induce chronic inflammation during the long periods before plasmacytomas develop. Such plasmacytomas appear to arise from B cells carrying chromosomal translocations that affect c-myc transcription. PURPOSE: Because silicone gels are in widespread medical use and share many of the characteristics of other materials known to be inducers of plasmacytomas, we wished to determine their capacity to induce plasmacytomas in mice. METHODS: In a series of parallel experiments, corn oil, pristane, silicone oil (dimethylpolysiloxane), or silicone gel from commercially obtained mammary implants was injected intraperitoneally into plasmacytoma-susceptible BALB/cAnPt-A and congenic BALB/cAnPt.DBA/2-Idh1-Pep3 mice, as well as into plasmacytoma-resistant C57BL/6N, C3H/HeJ, DBA/2N, and (BALB/c x DBA/2)F1 mice. Mice were examined at least once every 2 weeks for signs of abdominal tumor or weight loss and screened every 4-6 weeks for peritoneal plasmacytoma cells by peritoneal lavage. Tissues were examined by histologic and immunohistochemical techniques. Metaphase chromosome spreads were made from ascitic plasmacytomas without Colcemid treatment, and metaphase plates were G-banded according to standard techniques. The t(12;15) or t(6;15) translocation chromosomes were identified under the microscope in at least five metaphase plates of high banding quality. Mice were autopsied 125-400 days after the injection of test material. Gas chromatography and mass spectrometry were utilized to determine the composition of the silicone oil and silicone gel used in the injections. RESULTS: The silicone gels tested induced plasmacytomas in BALB/cAnPt-A and BALB/cAnPt.DBA/2-Idh1-Pep3 mice. Neither corn oil used as a control nor 1000-centistoke or 12,500-centistoke dimethylpolysiloxane induced plasmacytomas in these mice. The plasmacytomas were transplantable in syngeneic hosts. Cytogenetic studies of 41 silicone-induced plasmacytomas showed that 30 had t(12;15) translocations, eight had t(6;15) translocations, and three had no translocations. CONCLUSIONS: The silicone gels used in mammary implants, which contain a complex mixture of different siloxanes, induced peritoneal plasmacytomas in genetically susceptible mice. Silicone gels provide new chemically defined materials that are effective inducers of plasmacytomas in BALB/cAnPt-A and BALB/cAnPt.DBA/2-Idh1-Pep3 mice. Further studies will be required to determine which of the components of these gels are the active materials.

Animals↗

PubMed1994

A synthetic retinoid antagonist inhibits the human immunodeficiency virus type 1 promoter.

Retinoids regulate a broad range of biological processes and affect cell growth and differentiation of many cell types, including the immune system. Recently, it was reported that human immunodeficiency virus type 1 (HIV-1) expression in macrophages is enhanced by retinoic acid (RA). Retinoid signals are mediated by the RA receptors (RARs) and retinoid X receptors (RXRs) that bind to specific RA responsive elements (RAREs) in the promoter region of susceptible genes. Here, we report on a RARE in the long terminal repeat (LTR) region that allows activation of the HIV-1 LTR. The RARE is composed of two consensus RARE half-sites (A/GGGTCA) arranged as a palindrome separated by 9 nucleotides and is activated by both RAR/RXR heterodimers and RXR homodimers. We show that the COUP (chicken ovalbumin upstream promoter) orphan receptors also bind to the HIV-1 RARE and repress the retinoid response of the HIV-1 RARE or the HIV-1 LTR. Furthermore, a newly discovered synthetic retinoid is shown to be a potent inhibitor of retinoid-induced activation of the HIV-1 RARE. These observations suggest additional approaches for the inhibition of HIV replication.

Animals↗

PubMed1994

Mutations that alter ligand-induced switches and dimerization activities in the retinoid X receptor.

The retinoid X receptor (RXR) heterodimerizes with a variety of nuclear receptors. In addition, RXR forms homodimers in the presence of its ligand, 9-cis-retinoic acid. From deletion and point mutation analysis we present evidence that a short region (amino acids 413 to 443) in the carboxy terminus of RXR alpha is critical for both homo- and heterodimeric interactions as well as for diverse functional activities. In addition, we present evidence that homo- and heterodimer functions can be separated. The deletion of 19 amino acids from the C-terminal end of RXR dramatically reduced the transcriptional activation function of RXR. The removal of 10 additional amino acids resulted in a receptor (delta RXR3) that had completely lost its ligand-dependent homodimer function but retained its heterodimer activities. Heterodimer function was abolished by the deletion of an additional 20 amino acids. Single amino acid substitutions in the region generated receptors with altered RXR homodimer DNA binding, while simultaneous mutation of three Leu residues (Leu-418, -419 and -422) completely abolished both RXR homodimer and heterodimer DNA binding activities. Mutation of Leu-430 to Phe (L430-F) resulted in a receptor that bound to DNA strongly as homodimers in a ligand-independent manner, while another single amino acid exchange (L422-Q) led to a mutant that behaved in a manner exactly opposite to that of wild-type RXR in that the homodimerization of the mutant occurred in the absence of ligand and was inhibited by 9-cis-retinoic acid. In transfection assays, both L422-Q and L430-F failed to act as homodimers but retained their heterodimer function. Our studies demonstrate the unique properties of the RXR ligand binding domain and point to specific residues that mediate homo- and heterodimer activities and ligand-induced conformational switches.

Amino Acid Sequence↗

PubMed1994

The EndoA enhancer contains multiple ETS binding site repeats and is regulated by ETS proteins.

EndoA is a type II keratin and with EndoB (type I keratin), constitutes intermediate filaments in various simple epithelial tissues. EndoA is developmentally regulated and has an enhancer that is located at the 3'- end of the gene. This enhancer contains two single and five dual Ets binding sites. Thus far, no other promoter or enhancer has been shown to contain as many potential clustered Ets binding sites. To study the transcriptional regulation of EndoA by the ETS family proteins, we amplified the EndoA enhancer fragment from mouse genomic DNA by PCR, and cloned it into the pBLCAT2 vector upstream from the CAT reporter gene. Several pBLCAT-ENDOA clones were sequenced to verify the presence of all the ETS binding sites. Clones that did not show any point mutations in the ETS binding sites were chosen to study the transcription regulation by ETS1, ETS2 and ERGB/FLI-1 gene products. EMSA results indicated that the ETS1, ETS2 and ERGB/FLI-1 proteins bind to the enhancer sequence, and DNase I protection data demonstrated that the ETS proteins protect all seven EBS core sequences. Cotransfection of the COS cells with the pBLCAT-ENDOA construct, along with increasing amounts of different ETS expression vectors, resulted in a significant induction of CAT reporter gene expression. Previously, we have shown that the overexpression of the ETS1 gene transforms NIH3T3, and these transformed cells (7AQS2.1) produce high levels of ETS1 protein (Seth & Papas, 1990). In this report, we show that the undifferentiated P19 EC cells do not express detectable levels of ETS1; however, an elevated level of ETS1 is expressed in differentiated derivatives of these cells. We therefore used these two cell lines to examine the activity of the EndoA enhancer with the ETS1 product. Transfection of the pBLCAT-ENDOA construct alone in undifferentiated P19 EC cells results in very low CAT gene expression; however, upon differentiation with retinoic acid the level of CAT gene activity increases dramatically. Similarly, an increase in CAT expression from the same construct (pBLCAT-ENDOA) was also observed in 7AQS2.1 cells. Our results therefore indicate that the EndoA enhancer is regulated by ETS proteins via interaction with multiple ETS-binding site sequences.

Animals↗