1,25-Dihydroxyvitamin D3-induced differentiation in a human promyelocytic leukemia cell line (HL-60): receptor-mediated maturation to macrophage-like cells.

The human-derived promyelocytic leukemia cell line, HL-60, is known to differentiate into mature myeloid cells in the presence of 1,25-dihydroxyvitamin D3 (1,25[OH]2D3). We investigated differentiation by monitoring 1,25(OH)2D3-exposed HL-60 cells for phagocytic activity, ability to reduce nitroblue tetrazolium, binding of the chemotaxin N-formyl-methionyl-leucyl-[3H]phenylalanine, development of nonspecific acid esterase activity, and morphological maturation of Wright-Giemsa-stained cells. 1,25(OH)2D3 concentrations as low as 10(-10) M caused significant development of phagocytosis, nitroblue tetrazolium reduction, and the emergence of differentiated myeloid cells that had morphological characteristics of both metamyelocytes and monocytes. These cells were conclusively identified as monocytes/macrophages based upon their adherence to the plastic flasks and their content of the macrophage-characteristic nonspecific acid esterase enzyme. The estimated ED50 for 1,25(OH)2D3-induced differentiation based upon nitroblue tetrazolium reduction and N-formyl-methionyl-leucyl-[3H]phenylalanine binding was 5.7 X 10(-9) M. HL-60 cells exhibited a complex growth response with various levels of 1,25(OH)2D3: less than or equal to 10(-10) M had no detectable effect, 10(-9) M stimulated growth, and greater than or equal to 10(-8) M sharply inhibited proliferation. We also detected and quantitated the specific receptor for 1,25(OH)2D3 in HL-60 and HL-60 Blast, a sub-clone resistant to the growth and differentiation effects of 1,25(OH)2D3. The receptor in both lines was characterized as a DNA-binding protein that migrated at 3.3S on high-salt sucrose gradients. Unequivocal identification was provided by selective dissociation of the 1,25(OH)2D3-receptor complex with the mercurial reagent, p-chloromercuribenzenesulfonic acid, and by a shift in its sedimentation position upon complexing with anti-receptor monoclonal antibody. On the basis of labeling of whole cells with 1,25(OH)2[3H]D3 in culture, we found that HL-60 contains approximately 4,000 1,25(OH)2D3 receptor molecules per cell, while the nonresponsive HL-60 Blast is endowed with approximately 8% of that number. The concentration of 1,25(OH)2D3 (5 X 10(-9) M) in complete culture medium, which facilitates the saturation of receptors in HL-60 cells, is virtually identical to the ED50 for the sterol's induction of differentiation. This correspondence, plus the resistance of the relatively receptor-poor HL-60 Blast, indicates that 1,25(OH)2D3-induced differentiation of HL-60 cells to monocytes/macrophages is occurring via receptor-mediated events

Impaired 17,20-lyase activity in male mice lacking cytochrome b5 in Leydig cells

Androgen and estrogen biosynthesis in mammals requires the 17,20-lyase activity of cytochrome P450 17A1 (steroid 17-hydroxylase/17,20-lyase). Maximal 17,20-lyase activity in vitro requires the presence of cytochrome b5 (b5), and rare cases of b5 deficiency in human beings causes isolated 17,20-lyase deficiency. To study the consequences of conditional b5 removal from testicular Leydig cells in an animal model, we generated Cyb5(flox/flox):Sf1-Cre (LeyKO) mice. The LeyKO male mice had normal body weights, testis and sex organ weights, and fertility compared with littermates. Basal serum and urine steroid profiles of LeyKO males were not significantly different than littermates. In contrast, marked 17-hydroxyprogesterone accumulation (100-fold basal) and reduced testosterone synthesis (27% of littermates) were observed after human chorionic gonadotropin stimulation in LeyKO animals. Testis homogenates from LeyKO mice showed reduced 17,20-lyase activity and a 3-fold increased 17-hydroxylase to 17,20-lyase activity ratio, which were restored to normal upon addition of recombinant b5. We conclude that Leydig cell b5 is required for maximal androgen synthesis and to prevent 17-hydroxyprogesterone accumulation in the mouse testis; however, the b5-independent 17,20-lyase activity of mouse steroid 17-hydroxylase/17,20-lyase is sufficient for normal male genital development and fertility. LeyKO male mice are a good model for the biochemistry but not the physiology of isolated 17,20-lyase deficiency in human beings

Expression and functional analysis of Nr2e3, a photoreceptor-specific nuclear receptor, suggest common mechanisms in retinal development between avians and mammals

The photoreceptor-specific nuclear receptor (PNR; Nr2e3) is a transcription factor important for retinal development. We report here the identification and expression analysis of the avian Nr2e3. Nr2e3 mRNA is expressed in the photoreceptor layer of the neural retina during early stages of chick embryogenesis. Its temporal expression is distinct from that of a related nuclear receptor, Tlx. Chick Nr2e3 recognizes and binds to the same target DNA sequence as its vertebrate orthologs. Functional assays revealed that chick Nr2e3 acts as a transcriptional repressor. Our results suggest that Nr2e3 plays a common role in retinal development in vertebrates

Genetic and Other Contributions to Alcohol Intake in Rhesus Macaques ( Macaca mulatta )

The etiology of alcoholism and alcohol abuse, like many other complex diseases, is heterogeneous and multifactorial. Numerous studies demonstrate a genetic contribution to variation in the expression of alcohol-related disorders in humans. Over the past decade, nonhuman primates have emerged as a valuable model for some aspects of human alcohol abuse because of their phylogenetic proximity to humans. Long-term, longitudinal studies of rhesus macaques ( Macaca mulatta ) have provided much insight into environmental influences, especially early life experiences, on alcohol consumption and behavior patterns that characterize alcohol intake later in life. It is not known, however, whether there is a genetic component as well to the variation seen in alcohol consumption in rhesus macaques. A significant genetic component to variation in alcohol consumption in rhesus macaques would show for the first time that like humans, for nonhuman primates additive genetic influences are important. Moreover, their use as a model for alcohol-related disorders in humans would have even greater relevance and utility for designing experiments incorporating the expanding molecular genetics field, and allow researchers to investigate the interaction among the known environmental influences and various genotypes. Methods : In this study, we investigate factors contributing to variation in alcohol consumption of 156 rhesus macaques collected over 10 years when subjects were adolescent in age, belonging to a single extended pedigree, with each cohort receiving identical early rearing backgrounds and subsequent treatments. To measure alcohol consumption each animal was provided unfettered simultaneous access both to an aspartame-sweetened 8.4% (v/v) alcohol-water solution, the aspartame-sweetened vehicle, and to water for 1 hour each day during the early afternoon between 13:00 and 15:00 in their home cages for a period of 5 to 7 weeks. We use multiple regression to identify factors that significantly affect alcohol consumption among these animals and a maximum likelihood program (ASReml) that, controlling for the significant factors, estimates the genetic contribution to the variance in alcohol consumption. Results : Multiple regression analysis identified test cohort and rearing environment as contributing to 57 and 2%, respectively, of the total variance in alcohol consumption. Of the remaining 41% of the variance about half (19.8%) was attributable to additive genetic effects using a maximum likelihood program. Conclusion : This study demonstrates that, as in humans, there are additive genetic factors that contribute to variation in alcohol consumption in rhesus macaques, with other nongenetic factors accounting for substantial portions of the variance in alcohol consumption, Our findings show the presence of an additive genetic component and suggest the potential utility of the nonhuman primate as a molecular genetics tool for understanding alcohol abuse and alcoholism.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66182/1/j.1530-0277.2006.00044.x.pd

Transcriptional repression of the human collagenase-1 (MMP-1) gene in MDA231 breast cancer cells by all-trans-retinoic acid requires distal regions of the promoter

In the present study, we investigated the mechanisms controlling constitutive transcription of collagenase-1 and its repression by all-trans-retinoic acid (RA) in the highly invasive metastatic and oestrogen-receptor-negative breast cancer cell line MDA231. A combination of in vivo and in vitro experiments that include DNAase I hypersensitivity assays, transient transfection of collagenase-1 promoter constructs, and electrophoretic mobility shift assays implicate several PEA3 sites, binding sites for Ets-related transcription factors, in the constitutive expression of the human collagenase-1 promoter. Transient transfection of promoter constructs linked to the luciferase reporter, along with gel retardation assays, revealed that repression of collagenase-1 transcription by RA is not dependent on the proximal AP-1 site, but, rather, requires sequences located in distal regions of the promoter. Transcriptional analyses and electrophoretic mobility shift assays suggest that the PEA3 site located at –3108 bp facilitates, at least in part, the transcriptional repression of the human collagenase-1 gene in MDA231 cells. We conclude that collagenase-1 repression in MDA231 cells occurs by a novel regulatory pathway that does not depend on the proximal AP-1 site at –73 bp, but does depend on distal regions in the collagenase-1 promoter. © 1999 Cancer Research Campaig

Genome wide analysis of gene expression changes in skin from patients with type 2 diabetes

Non-healing chronic ulcers are a serious complication of diabetes and are a major healthcare problem. While a host of treatments have been explored to heal or prevent these ulcers from forming, these treatments have not been found to be consistently effective in clinical trials. An understanding of the changes in gene expression in the skin of diabetic patients may provide insight into the processes and mechanisms that precede the formation of non-healing ulcers. In this study, we investigated genome wide changes in gene expression in skin between patients with type 2 diabetes and non-diabetic patients using next generation sequencing. We compared the gene expression in skin samples taken from 27 patients (13 with type 2 diabetes and 14 non-diabetic). This information may be useful in identifying the causal factors and potential therapeutic targets for the prevention and treatment of diabetic related diseases

Ablation of the androgen receptor from vascular smooth muscle cells demonstrates a role for testosterone in vascular calcification

Vascular calcification powerfully predicts mortality and morbidity from cardiovascular disease. Men have a greater risk of cardiovascular disease, compared to women of a similar age. These gender disparities suggest an influence of sex hormones. Testosterone is the primary and most well-recognised androgen in men. Therefore, we addressed the hypothesis that exogenous androgen treatment induces vascular calcification. Immunohistochemical analysis revealed expression of androgen receptor (AR) in the calcified media of human femoral artery tissue and calcified human valves. Furthermore, in vitro studies revealed increased phosphate (Pi)-induced mouse vascular smooth muscle cell (VSMC) calcification following either testosterone or dihydrotestosterone (DHT) treatment for 9 days. Testosterone and DHT treatment increased tissue non-specific alkaline phosphatase (Alpl) mRNA expression. Testosterone-induced calcification was blunted in VSMC-specific AR-ablated (SM-ARKO) VSMCs compared to WT. Consistent with these data, SM-ARKO VSMCs showed a reduction in Osterix mRNA expression. However, intriguingly, a counter-intuitive increase in Alpl was observed. These novel data demonstrate that androgens play a role in inducing vascular calcification through the AR. Androgen signalling may represent a novel potential therapeutic target for clinical intervention

Modulation of COUP-TF Expression in a Cnidarian by Ectopic Wnt Signalling and Allorecognition

COUP transcription factors are required for the regulation of gene expression underlying development, differentiation, and homeostasis. They have an evolutionarily conserved function, being a known marker for neurogenesis from cnidarians to vertebrates. A homologue of this gene was shown previously to be a neuronal and nematocyte differentiation marker in Hydra. However, COUP-TFs had not previously been studied in a colonial cnidarian.We cloned a COUP-TF homologue from the colonial marine cnidarian Hydractinia echinata. Expression of the gene was analysed during normal development, allorecognition events and ectopic Wnt activation, using in situ hybridisation and quantitative PCR. During normal Hydractinia development, the gene was first expressed in post-gastrula stages. It was undetectable in larvae, and its mRNA was present again in putative differentiating neurons and nematocytes in post-metamorphic stages. Global activation of canonical Wnt signalling in adult animals resulted in the upregulation of COUP-TF. We also monitored a strong COUP-TF upregulation in stolons undergoing allogeneic interactions. COUP-TF mRNA was most concentrated in the tissues that contacted allogeneic, non-self tissues, and decreased in a gradient away from the contact area. Interestingly, the gene was transiently upregulated during initial contact of self stolons, but dissipated rapidly following self recognition, while in non-self contacts high expression levels were maintained.We conclude that COUP-TF is likely involved in neuronal/nematocyte differentiation in a variety of contexts. This has now been shown to include allorecognition, where COUP-TF is thought to have been co-opted to mediate allorejection by recruiting stinging cells that are the effectors of cytotoxic rejection of allogeneic tissue. Our findings that Wnt activation upregulates COUP-TF expression suggests that Wnts' role in neuronal differentiation could be mediated through COUP-TF

Molecular targeting of retinoic acid metabolism in neuroblastoma: the role of the CYP26 inhibitor R116010 in vitro and in vivo

Isomerisation to all-trans-retinoic acid (ATRA) is widely accepted as the key mechanism underlying the favourable clinical properties of 13-cis-retinoic acid (13cisRA). As intracellular metabolism of ATRA by CYP26 may result in clinical resistance to 13cisRA, an increase in efficacy may be achieved through modulation of this metabolic pathway. We have evaluated the effect of the CYP26 inhibitor R116010 on retinoid metabolism in neuroblastoma cell lines and a xenograft model. In neuroblastoma cells, which showed a high level of CYP26 induction in response to ATRA, R116010 selectively inhibited ATRA metabolism. In addition, siRNA-mediated knockdown of CYP26 selectively increased ATRA levels and the expression of retinoid-responsive marker genes was potentiated by R116010. Treatment of mice bearing SH-SY5Y xenografts with 13cisRA (100 mg kg−1) revealed substantial levels (16%) of intratumoral ATRA after 6 h, despite plasma ATRA levels representing only 1% total retinoids under these conditions. Co-administration of R116010 with 13cisRA in this mouse model resulted in significant increases in plasma ATRA and 13cisRA concentrations. Furthermore, R116010 induced significant decreases in levels of 4-oxo metabolites in hepatic tissue after co-administration with either ATRA or 13cisRA. These data suggest considerable potential for CYP26 inhibitors in the future treatment of neuroblastoma with 13cisRA

Comparative Developmental Expression Profiling of Two C. elegans Isolates

Gene expression is known to change during development and to vary among genetically diverse strains. Previous studies of temporal patterns of gene expression during C. elegans development were incomplete, and little is known about how these patterns change as a function of genetic background. We used microarrays that comprehensively cover known and predicted worm genes to compare the landscape of genetic variation over developmental time between two isolates of C. elegans. We show that most genes vary in expression during development from egg to young adult, many genes vary in expression between the two isolates, and a subset of these genes exhibit isolate-specific changes during some developmental stages. This subset is strongly enriched for genes with roles in innate immunity. We identify several novel motifs that appear to play a role in regulating gene expression during development, and we propose functional annotations for many previously unannotated genes. These results improve our understanding of gene expression and function during worm development and lay the foundation for linkage studies of the genetic basis of developmental variation in gene expression in this important model organism