The POU transcription factor Oct-1 represses virus-induced interferon A gene expression

Alpha interferon (IFN-alpha) and IFN-beta are able to interfere with viral infection. They exert a vast array of biologic functions, including growth arrest, cell differentiation, and immune system regulation. This regulation extends from innate immunity to cellular and humoral adaptive immune responses. A strict control of expression is needed to prevent detrimental effects of unregulated IFN. Multiple IFN-A subtypes are coordinately induced in human and mouse cells infected by virus and exhibit differences in expression of their individual mRNAs. We demonstrated that the weakly expressed IFN-A11 gene is negatively regulated after viral infection, due to a distal negative regulatory element, binding homeoprotein pituitary homeobox 1 (Pitx1). Here we show that the POU protein Oct-1 binds in vitro and in vivo to the IFN-A11 promoter and represses IFN-A expression upon interferon regulatory factor overexpression. Furthermore, we show that Oct-1-deficient MEFs exhibit increased in vivo IFN-A gene expression and increased antiviral activity. Finally, the IFN-A expression pattern is modified in Oct-1-deficient MEFs. The broad representation of effective and potent octamer-like sequences within IFN-A promoters suggests an important role for Oct-1 in IFN-A regulation.     

Human PIEZO1 Ion Channel Functions as a Split Protein

PIEZO1 is a mechanosensitive eukaryotic cation-selective channel that rapidly inactivates in a voltage-dependent manner. We previously showed that a fluorescent protein could be encoded within the hPIEZO1 sequence without loss of function. In this work, we split the channel into two at this site and asked if coexpression would produce a functional channel or whether gating and permeation might be contained in either segment. The split protein was expressed in two segments by a bicistronic plasmid where the first segment spanned residues 1 to 1591, and the second segment spanned 1592 to 2521. When the “split protein” is coexpressed, the parts associate to form a normal channel. We measured the whole-cell, cell-attached and outside-out patch currents in transfected HEK293 cells. Indentation produced whole-cell currents monotonic with the stimulus. Single channel recordings showed voltage-dependent inactivation. The Boltzmann activation curve for outside-out patches had a slope of 8.6/mmHg vs 8.1 for wild type, and a small leftward shift in the midpoint (32 mmHg vs 41 mmHg). The association of the two channel domains was confirmed by FRET measurements of mCherry on the N-terminus and EGFP on the C-terminus. Neither of the individual protein segments produced current when expressed alone.     

Cyclic AMP Accumulation Induces a Rapid Desensitization of the Cyclic AMP-Dependent Protein Kinase in Mouse Striatal Neurons

Striatal neurons from the mouse brain embryo grown in primary culture express high levels of cyclic AMP (cAMP)-dependent protein kinase (PKA) activity. To study the modulation of PKA in intact neurons, a rapid method based on Zn(2+)-protein precipitation was developed. This strategy allowed analysis of the stimulation of PKA under conditions of intracellular cAMP concentration increases. Whereas increases up to 1 microM lead to an activation, large and sustained accumulations of cAMP result in a loss of the enzyme activity. With 8-bromo-cAMP (8-Br-cAMP) at 100 microM, the PKA refractoriness occurs within 2 min. It is rapidly reversible because incubation of treated neurons in fresh medium leads to a complete recovery of PKA activity within 30 min. The decrease in assayable PKA does not involve an activation of phosphatases because the histone dephosphorylation rate is not affected by 8-Br-cAMP treatment. Finally, not only 8-Br-cAMP- but also forskolin- and vasoactive intestinal peptide-induced increases in intracellular cAMP concentration can lead to the PKA desensitization. Altogether, these data unravel a new mechanism of PKA regulation.     

Cyclic AMP modulates interleukin-1 action in a cytotoxic T-cell hybridoma

The induction of cytolytic activity in PC60, a murine T-cell hybridoma, is paralleled by a rise in the level of BLT-esterase (N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl esterase), a serine esterase specific for activated T-cells. Both interleukin-1 (IL-1) and dibutyryl cAMP were albe to increase the esterase activity in a dose-dependent and saturable manner. When added in combination the two activators showed a strong synergism: BLT-esterase levels were up to three times higher than the sum of the levels due to dibutyryl cAMP and IL-1 added separately. Stimulators of the adenylate cyclase, such as forskolin and cholera toxin, induced a similar enhancement of the BLT-esterase response to IL-1. PC60 cells did not produce any cAMP in response to IL-1. When the two stimuli were added sequentially a second effect for cAMP emerged: preincubation with dibutyryl cAMP or activators of the adenylate cyclase for 4 h or longer completely blocked the action of subsequently added IL-1. Taken together, the data demonstrate a dual modulatory role for cAMP in T-lymphocytes activated by IL-1.