Role of retinoic acid receptors alpha1 and gamma in the response of murine limbs to retinol in vitro

BACKGROUND: Derivatives of retinol (vitamin A), commonly referred to as retinoids, signal through retinoic acid and retinoid X receptors (RARs/RXRs) and are essential for normal limb formation. Retinoid imbalances or perturbations in receptor function result in aberrant limb development. To examine the mechanisms underlying retinol-induced limb defects, we determined the responsiveness of limbs from RARalpha1-/-gamma mice to excess retinol in vitro. METHODS: RARalpha1-/-gamma+/- mice were bred and their embryos were recovered at gestational day (GD) 12.5. The forelimbs were excised and cultured in vitro in the presence of all-trans retinol acetate (0, 1.25, 12.5, or 62.5 microM) for 6 days. The expression profiles of genes known to affect chondrogenesis (sox9 and col2a1) and limb outgrowth (meis1, meis2, and pbx1a) were examined by real-time qRT-PCR following retinol exposure for 3 hr. RESULTS: Whereas RARalpha1-/-gamma+/+ and RARalpha1-/-gamma+/- limbs exhibited deleterious effects on limb outgrowth and chondrogenesis in the presence of exogenous retinol, this outcome was significantly attenuated in RARalpha1-/-gamma-/- limbs. The expressions of sox9 and col2a1 were significantly decreased in retinol-exposed RARalpha1-/-gamma+/+ limbs. In contrast, expression was not altered in limbs from their RARalpha1-/-gamma+/- or RARalpha1-/-gamma-/- littermates. Retinol exposure upregulated the expression of meis1 and meis2 in RARalpha1-/-gamma+/+ limbs; however, in RARalpha1-/-gamma-/- limbs the expression of both genes was unresponsive to retinol. Pbx1a remained unresponsive to retinol treatment in all genotypes. CONCLUSION: In the absence of RARalpha1, RARgamma is a functionally important mediator of retinoid-induced limb dysmorphogenesis.     

Regulation of the rat interstitial collagenase promoter by IL-1β, c-Jun,and ras-dependent signaling in growth plate chondrocytes

In an attempt to better define molecular influences on rat interstitial collagenase gene expression in cartilage, the promoter function was characterized using transient transfection assay, electrophoresis mobility shift assay, and genetic analysis in isolated growth plate chondrocytes. Data from 5′-flanking deletion and selected mutations suggest that multiple cis elements in both the proximal and distal regions of the promoter were important in the regulation of promoter activity. A proximal tumor response element (TRE) was shown to be necessary for basal and interleukin (IL)-1β–inducible reporter gene activity. Cells stimulated by IL-1β (1 ng/ml; 18 h) had elevated TRE binding activity, and one of the factors involved was identified as the nuclear protein, c-Jun. Indeed, c-Jun directed antisense oligonucleotides reduced rat interstitial collagenase mRNA. A sense oligonucleotide was ineffective. Regulation of promoter activity was susceptible to Ras-dependent signaling as expression of dominant negative mutant of Ras kinase (pZIP-RasN17) reduced reporter gene activity. In a comparison of proximal promoter reporter plasmid activity between proliferative and hypertrophic cells, inhibition of Ras-dependent signaling was less effective in the later cell type. This study suggests that the activation of nuclear binding proteins that bind TRE may be a common event with IL-1β regulation. Moreover, these data suggest that the regulation of rat interstitial collagenase gene expression is a combinatorial process and multiple cis-acting regulatory sites may interact to exert different effects dependent on the stage of chondrocyte differentiation. J. Cell. Biochem. 67:92–102, 1997. Published 1997 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Synthesis of novel (2R,4R)- and (2S,4S)-iso dideoxynucleosides with exocyclic methylene as potential antiviral agents.

Novel (2R,4R)- and (2S,4S)-iso dideoxynucleosides with exocyclic methylene have been designed and synthesized, based on the lead BMS-200475 (3) which exhibited potent anti-HBV activity. For the synthesis of D types of (2R,4R)-nucleosides, L-xylose was converted to the key intermediate 14. The intermediate 14 was converted to the uracil derivative 4a and the cytosine derivative 4b. Compound 14 was also converted to the purine derivatives such as adenine derivative 4c, hypoxanthine derivative 4d, and guanine derivative 4e. The corresponding L types of (2S,4S)-enantiomers were more efficiently synthesized from the commercially available 1,2-isopropylidene-D-xylose (20) than the synthetic method used in the synthesis of (2R,4R)-nucleosides. The key intermediate 25 was converted to the pyrimidine analogues 5a and 5b and the purine derivatives 5c, 5d, and 5e using the similar method used in the preparation of 4c, 4d, and 4e. The synthesized final (2R,4R)- and (2S,4S)-nucleosides were tested against several viruses such as HIV-1, HSV-1, HSV-2, HCMV and HBV. (2R,4R)-Adenine analogue 4c exhibited potent anti-HBV activity (EC(50)=1.5 microM in 2.2.15 cells) among compounds tested, while (2R,4R)-uracil derivative 4a was the most active against HCMV among compounds tested and (2R,4R)-adenine derivative 4c was found to be moderately active against the same virus. However, the corresponding (2S,4S)-isomers were found to be totally inactive against all tested viruses. Both (2R,4R)-adenine derivative 4c and (2S,4S)-adenine analogue 5c were totally resistant to the adenosine deaminase like iso-ddA (1). From the molecular modeling study the hydroxymethyl side chains of BMS-200475 (3) and 4c were almost overlapped, indicating that 4c may be suitable for phosphorylation by cellular kinases like the lead 3, but some discrepancy between two bases was observed, indicating why 4c is less potent against HBV than 3. It is concluded that discovery of (2R,4R)-adenine analogue 4c as potent anti-HBV agent suggested that the sugar moiety of this series can be regarded as a novel template for the development of new anti-HBV agent and oxygen atom can be acted as a bioisostere of C-OH.     

Trypanocidal activity of (-)-cubebin derivatives against free amastigote forms of Trypanosoma cruzi

Five (-)-cubebin derivative compounds, (-)-O-acetyl cubebin (3), (-)-O-benzyl cubebin (4), (-)-O-(N,N-dimethylaminoethyl)-cubebin (5), (-)-hinokinin (6) and (-)-6,6'-dinitrohinokinin (7), previously synthesised by our research group, were evaluated on in vitro assay against free amastigote forms of Trypanosoma cruzi, the asogic agent of Chagas' disease. It was observed that 6 was the most active compound (IC(50)=0.7 microM), and that 4 and 5 displayed moderate activity against the parasite, giving IC(50) values of 5.7 and 4.7 microM, respectively. In contrast, it was observed that compound 3 was inactive and that 7 displayed low activity with IC(50) values of congruent with 1.5 x 10(4) and 95.3 microM, respectively.     

Synthesis, cytotoxicity, and anti-inflammatory evaluation of 2-(furan-2-yl)-4-(phenoxy)quinoline derivatives. Part 4

A number of 2-(furan-2-yl)-4-phenoxyquinoline derivatives have been synthesized and evaluated for anti-inflammatory evaluation. 4-[(2-Furan-2-yl)quinolin-4-yloxy]benzaldehyde (8), with an IC(50) value of 5.0 microM against beta-glucuronidase release, was more potent than its tricyclic furo[2,3-b]quinoline isomer 3a (>30 microM), its 4'-COMe counterpart 7 (7.5 microM), and its oxime derivative 13a (11.4 microM) and methyloxime derivative 13b (>30 microM). For the inhibition of lysozyme release, however, oxime derivative 12a (8.9 microM) and methyloxime derivative 12b (10.4 microM) are more potent than their ketone precursor 7 and their respective tricyclic furo[2,3-b]quinoline counterparts 4a and 4b. Among them, 4-[4-[(2-furan-2-yl)-quinolin-4-yloxy]phenyl]but-3-en-2-one (10) is the most active against lysozyme release with an IC(50) value of 4.6 microM, while 8 is the most active against beta-glucuronidase release with an IC(50) value of 5.0 microM. (E)-1-[3-[(2-Furan-2-yl)quinolin-4-yloxy]phenyl] ethanone oxime (11a) is capable of inhibiting both lysozyme and beta-glucuronidase release with IC(50) values of 7.1 and 9.5 microM, respectively. For the inhibition of TNF-alpha formation, 1-[3-[(2-furan-2-yl)quinolin-4-yloxy]phenyl]ethanone (6) is the most potent with an IC(50) value of 2.3 microM which is more potent than genistein (9.1 microM). For the inhibitory activity of fMLP-induced superoxide anion generation, 11a (2.7 microM), 11b (2.8 microM), and 13b (2.2 microM) are three of the most active. None of above compounds exhibited significant cytotoxicity.     

Induction of the collagenase phorbol ester response element by staurosporine

The indol alkaloid staurosporine is a potent inhibitor of protein kinase C, but has also been shown to have certain effects paradoxically similar to those of protein kinase C–activating phorbol esters. We show here that collagenase mRNA expression is stimulated by 10 nM staurosporine in normal and ras-oncogene–transformed rat fibroblasts. The kinetics of collagenase mRNA induction by staurosporine were slow compared to induction by phorbol ester. Staurosporine induction of the collagenase promoter appeared to be mediated via the TPA response element (TRE). Induction did not involve any increase in jun mRNA expression and did not require expression of c-Jun. Prolonged treatment with phorbol ester to deplete protein kinase C did not inhibit stimulation of the collagenase promoter by staurosporine. Instead, involvement of cAMP-dependent protein kinase (PKA) was indicated by inhibition of staurosporine induction by the PKA inhibitor H-89. In addition, raised levels of cAMP were observed during the first hour of staurosporine treatment. Altogether, our data indicate that staurosporine induces a PKA-dependent pathway leading to c-Jun–independent activation of the collagenase TRE element. © 1994 Wiley-Liss, Inc.     

Vitamin A is a key regulator for cell growth, cytokine production, and differentiation in normal B cells.

In the present paper we demonstrate that retinol-retinol-binding protein and chylomicron remnant retinyl esters in concentrations normally found in human plasma inhibit growth of normal human B lymphocytes. Physiological concentrations of retinoic acid (about 30 nM) were less active than physiological concentrations of retinol (about 3 microM). Pharmacological concentrations of retinol and retinoic acid were more active than the concentrations normally found in plasma. Retinol (3 microM) inhibited anti-IgM-mediated DNA synthesis as measured by [3H]thymidine uptake at 72 h by 78%. Furthermore, we found that the cells were blocked in the mid-G1 phase of the cell cycle. Thus, neither MYC up-regulation measured at 3 h nor the expression of the early activation antigen 4F2 was reduced by retinol, whereas the late activation markers (transferrin receptor expression and actinomycin D staining at 48 h of stimulation) were markedly inhibited. Retinol reduced the interleukin 6 production induced by anti-IgM and interleukin 4 after 48 h, whereas the induction of interleukin 6 and tumor necrosis factor by O-tetradecanoylphorbol-13-acetate and ionomycin was less affected. We also noted that the retinoids reduced the formation of plaque-forming cells (i.e. Ig synthesis). These data imply that vitamin A present in human plasma is a normal modulator of B cell function.     

Synthesis and CYP24 inhibitory activity of 2-substituted-3,4-dihydro-2H-naphthalen-1-one (tetralone) derivatives

The synthesis of novel 2-benzyl- and 2-benzylidene-3,4-dihydro-2H-naphthalen-1-one (tetralone) derivatives and their inhibitory activity versus kidney mitochondrial 25-hydroxyvitamin D(3) 24-hydroxylase (CYP24) is described. The 2-benzylidenetetralone derivatives were found to be very weak inhibitors (IC(50) 20 >100 microM), whereas the 2-benzyltetralone derivatives showed promising inhibitory activity (IC(50) 0.9 microM for the most active derivative) compared with ketoconazole (IC(50) 20 microM).     

Up-regulation of acid sphingomyelinase during retinoic acid-induced myeloid differentiation of NB4, a human acute promyelocytic leukemia cell line.

All-trans-retinoic acid (ATRA) induces myeloid differentiation of a human promyelocytic leukemia cell line, NB4, but does not affect its subclone NB4/RA harboring a point-mutated ligand-binding domain (AF2) in retinoic acid receptor alpha (RARalpha) gene. We found that ATRA induced the 4-fold elevation of acid sphingomyelinase (ASMase) activity 24 h after treatment in NB4 cells, but not in NB4/RA cells. ATRA did not affect neutral sphingomyelinase activity in either NB4 or NB4/RA. Upon treatment with ATRA, ceramide, the product of an ASMase reaction, accumulated in NB4 cells. Northern blot analysis showed a marked elevation of the ASMase mRNA 8 h after ATRA treatment, reaching a plateau at 24 h. Regulation of ASMase gene expression was studied by a promoter analysis using luciferase reporter assay. The 5'-upstream flanking region of human ASMase gene (-519/+300) conjugated with the luciferase gene was introduced into COS-7 cells. Luciferase activity in transformed cells markedly increased in response to ATRA stimulation when the wild type RARalpha or the PML/RARalpha hybrid protein was co-expressed. Deletion experiments revealed that a short sequence at the 5'-end (-519/-485) was indispensable for the ATRA response. Within this short region, two retinoic acid-responsive element-like motifs (TGCCCG and TCTCCT) and one AP2-like motif (CCCTTCCC) were identified. Deletion and base-substitution experiments showed that all three motifs are required for the full expression induced by ATRA. Electrophoresis mobility shift assays with the nuclear extract of ATRA-treated NB4 cells showed that proteins were bound specifically to the probe being mediated by all three motifs in the promoter sequence.