No abstract

OBJECTIVE-Endoplasmic reticulum stress increases macrophage apoptosis, contributing to the complications of atherosclerosis. Insulin-resistant macrophages are more susceptible to endoplasmic reticulum stress-associated apoptosis probably contributing to macrophage death and necrotic core formation in atherosclerotic plaques in type 2 diabetes. However, the molecular mechanisms of increased apoptosis in insulin-resistant macrophages remain unclear. RESEARCH DESIGN AND METHODS-The studies were performed in insulin-resistant macrophages isolated from insulin receptor knockout or ob/ob mice. Gain-or loss-of-function approaches were used to evaluate the roles of forkhead transcription factors (FoxOs) in endoplasmic reticulum stress-associated macrophage apoptosis. RESULTS-Insulin-resistant macrophages showed attenuatedAkt activation and increased nuclear localization of FoxO1 during endoplasmic reticulum stress induced by free cholesterol loading. Overexpression of active FoxO1 or FoxO3 failed to induce apoptosis in unchallenged macrophages but exacerbated apoptosis in macrophages with an active endoplasmic reticulum stress response. Conversely, macrophages with genetic knockouts of FoxO1, -3, and -4 were resistant to apoptosis in response to endoplasmic reticulum stress. FoxO1 was shown by chromatin immunoprecipitation and promoter expression analysis to induce inhibitor of Bε gene expression and thereby to attenuate the increase of nuclear p65 and nuclear factor-B activity during endoplasmic reticulum stress, with proapoptotic and anti-inflammatory consequences. CONCLUSIONS-DecreasedAkt and increased FoxO transcription factor activity during the endoplasmic reticulum stress response leads to increased apoptosis of insulin-resistant macrophages. FoxOs may have a dual cellular function, resulting in either proapoptotic or anti-inflammatory effects in an endoplasmic reticulum stress-modulated manner. In the complex plaque milieu, the ultimate effect is likely to be an increase in macrophage apoptosis, plaque inflammation, and destabilization.

To address the role of transient torsional stress in transcription, we have utilized the regulated expression of HO endonuclease in yeast to create double-strand breaks in DNA templates in vivo at preselected sites. Linearization of circular minichromosomes, either 2 kb upstream or immediately downstream of a lacZ reporter gene controlled by the yeast metallothionein gene (CUP1) promoter, did not alter the copper induction profile of lacZ RNA transcripts compared to that of nonlinearized controls. Constructs site-specifically integrated into yeast chromosome II gave similar results. In vivo cross-linking with psoralen as a probe for negative DNA supercoiling demonstrated that template linearization efficiently dissipated DNA supercoiling induced by transcription. Therefore, the efficient transcription of linearized, relaxed templates found here demonstrates that transient torsional tension is not required for transcription of chromatin templates in yeast.Several lines of evidence support the proposal that transient torsional stress may be necessary for optimal transcription in eukaryotic cells (for a review, see reference 7). First, since transcription generates twin domains of DNA supercoiling (9), which leads to the recruitment of DNA topoisomerases (6), relaxation may kinetically lag behind stress generation. Studies with psoralen cross-linking support the idea that torsional tension exists in vivo within several different genes (19,27). Second, the transcriptional activity of circular DNA templates has been reported to be greater than that of their linear counterparts when introduced into animal cells or frog oocytes (11,37,38,41,51). Third, nicking genomes in vivo with X rays leads to inhibition of transcription (28). Finally, adenovirus DNA molecules containing mutant terminal protein, which can no longer bind tightly to the nuclear matrix, are less active in transcription in vivo (43).A requirement for transient torsional stress in optimal transcription could be exhibited at the level of initiation or elongation (or both). The results of several in vitro experiments support a role for negative DNA supercoiling in transcription initiation (for example, see references 10, 29, 30, 34-36, and 45). With respect to transcriptional elongation, it has been conceptually formulated that positive supercoils generated downstream of traversing RNA polymerase may unwind the negative supercoils about the histone octamer and pave the way for transcription (23). Consistent with this view is the observation that positive supercoiling generates a DNase Isensitive state in native chromatin (22). In vitro studies have shown that RNA polymerase traversal through arrays of nucleosomes does not efficiently occur (18), even in the presence of known elongation factors (15, 16). Nucleosomes are potent roadblocks for RNA polymerase passage (reviewed in reference 7), particularly when associated with histone H1 (32). In vivo, however, nucleosome arrays provide no apparent barrier to RNA polymerase passage (1), consistent with a requ...

No abstract