Regulation of the Discoidin I gamma gene in Dictyostelium discoideum: identification of individual promoter elements mediating induction of transcription and repression by cyclic AMP.

We dissected the promoter of the developmentally induced and cyclic AMP-repressed discoidin I gamma gene and identified a sequence element essential for developmental induction. Transfer of the element to an inactive heterologous promoter demonstrated that this sequence is sufficient to confer expression in axenically growing cells and to induce gene activity in development after growth on bacteria. A 16-base-pair sequence within this element was shown to be sufficient for induction in the discoidin promoter context and was used to reactivate different truncated promoter constructs. This led to the localization of an element necessary for down regulation of gene expression by extracellular cyclic AMP.

Disruption of the murine PIASx gene results in reduced testis weight.

PIASx belongs to the PIAS protein family, the members of which modulate activities of several transcription factors and act as E3 ligases in the sumoylation pathway. The PIASx gene is highly expressed in testis, suggesting a role in spermatogenesis. To investigate the function of PIASx in vivo, we have disrupted the PIASx gene in mice. Interestingly, the knockout mice were viable and fertile. Despite the normal fertility, the testis weight of the mutant animals was reduced and their number of apoptotic testicular cells was increased. Also, the sperm count of mutant mice tended to be reduced, but the quality of their sperm cells was normal. No significant changes were observed in the serum levels of LH and FSH or in the intratesticular testosterone concentration between the knockout animals and their wild-type littermates. Compensatory increases in other PIAS protein mRNAs were not observed in the knockout mice. These results imply that PIASx is required quantitatively rather than qualitatively for normal spermatogenesis.

Lovastatin inhibits receptor-stimulated Ca(2+)-influx in retinoic acid differentiated U937 and HL-60 cells.

Lovastatin was used to study the role of isoprenylated proteins on stimulus-induced increase of cytosolic Ca2+ in retinoic acid-differentiated U937 and HL-60 cells. Preincubation of the cells with lovastatin for 11-24 h reduced the Ca(2+)-influx induced by PAF of FMLP. The maximal decrease was 60% in U937 cells and 40% in HL-60 cells. The ID50s of lovastatin in U937 and HL-60 cells were 5 microM and 15 microM, respectively. Lovastatin did not inhibit Ca(2+)-discharge from intracellular stores. Addition of mevalonate to lovastatin-treated cells completely reversed the inhibition of PAF- and FMLP-stimulated Ca(2+)-mobilization. Immunoreactivity of ras-like proteins was decreased in membranes and increased in the cytosol of U937 cells by 1 day treatment with lovastatin. We conclude that isoprenylated proteins are involved in the regulation of receptor-stimulated Ca(2+)-entry of differentiated HL-60 and U937 cells.

Spreading of differentiating human monocytes is associated with a major increase in membrane-bound CDC42.

In a search for a model cell system that might allow studies of the function of the Rho-related GTPase CDC42Hs in human cells, we measured the content and distribution of CDC42Hs in monocytes that were differentiating into macrophages. The total content of this protein increased 5- to 6-fold in phorbol ester-treated human monocytic THP-1 and U-937 cells and increased 13-fold in normal human blood monocytes. Moreover, membrane-associated CDC42Hs in these cells increased 13-fold and 30-fold, respectively, while cytosolic CDC42Hs increased only 3- and 6-fold. Measurements made specifically in U-937 cells showed that the increase in membrane CDC42Hs correlated closely with an increase in cell spreading. The changes in CDC42Hs in U-937 cells probably depended on increased mRNA translation and/or decreased protein degradation, since no change in CDC42Hs mRNA could be detected. Finally, the changes in CDC42Hs were relatively specific, since contents of the CDC42Hs-binding protein Rho-GDI and the Rho-related protein Rac2 were unaffected and no change in CDC42Hs occurred when the cells were stimulated by agonists that induce monocytes to differentiate into nonadherent cells. These findings show that marked changes in content and distribution of CDC42Hs occur when monocytes differentiate into macrophages, suggesting that membrane CDC42Hs may play a role in cell spreading.

Platelet signal transduction mechanisms induced by eicosanoids.

Platelets are in many ways involved in the pathogenesis of artherosclerosis and the development of coronary heart disease, and play a role in acute myocardial infarction (8). They are also an useful model to study cellular activation mechanisms: their isolation from peripheral venous blood is simple and rapid and their physiological responses such as shape change, aggregation and secretion can be easily measured (10). This chapter is focused on our results on the mechanisms of platelet activation and inhibition induced by eicosanoids.

A novel family of repeat sequences in the mouse genome responsive to retinoic acid.

Repetitive DNA sequences form a substantial portion of eukaryotic genomes and exist as members of families that differ in copy number, length, and sequence. Various functions, including chromosomal integrity, gene regulation, and gene rearrangement have been ascribed to repetitive DNA. Although there is evidence that some repetitive sequences may participate in gene regulation, little is known about how their own expression may be regulated. During the course of gene trapping experiments with embryonic stem (ES) cells, we identified a novel class of expressed repetitive sequences in the mouse, using 5' rapid amplification of cDNA ends-polymerase chain reaction (5' RACE-PCR) to clone fusion transcripts from these lines. The expression of these repeats was induced by retinoic acid (RA) in cultured ES cells examined by Northern blot analyses. In vivo, their expression was spatially restricted in embryos and in the adult brain as determined by RNA in situ hybridization. We designated this family of sequences as Dr (developmentally regulated) repeats. The members of the Dr family, identified by cDNA cloning and through database search, are highly similar in sequence and show peculiar structural features. Our results suggest the expression of Dr-containing transcripts may be part of an ES cell differentiation program triggered by RA.