Glycerol-3-phosphate acyltransferases 3 and 4 direct glycerolipid synthesis and affect functionality in activated macrophages.

Macrophage classical M1 activation via TLR4 triggers a variety of responses to achieve the elimination of foreign pathogens. During this process, there is also an increase in lipid droplets which contain large quantities of triacylglycerol (TAG) and phospholipid (PL). The functional consequences of this increment in lipid mass are poorly understood. Here, we studied the contribution of glycerolipid synthesis to lipid accumulation, focusing specifically on the first and rate-limiting enzyme of the pathway: glycerol-3-phosphate acyltransferase (GPAT). Using bone marrow-derived macrophages (BMDMs) treated with Kdo2-lipid A, we showed that glycerolipid synthesis is induced during macrophage activation. GPAT4 protein level and GPAT3/GPAT4 enzymatic activity increase during this process, and these two isoforms were required for the accumulation of cell TAG and PL. The phagocytic capacity of Gpat3-/- and Gpat4-/- BMDM was impaired. Additionally, inhibiting fatty acid ß-oxidation reduced phagocytosis only partially, suggesting that lipid accumulation is not necessary for the energy requirements for phagocytosis. Finally, Gpat4-/- BMDM expressed and released more pro-inflammatory cytokines and chemokines after macrophage activation, suggesting a role for GPAT4 in suppressing inflammatory responses. Together, these results provide evidence that glycerolipid synthesis directed by GPAT4 is important for the attenuation of the inflammatory response in activated macrophages.

Construction and evaluation of an adenoviral vector for the liver-specific expression of the serine/arginine-rich splicing factor, SRSF3.

Serine/arginine-rich splicing factor-3 (SRSF3), alternatively known as SRp20, is a member of the highly-conserved SR protein family of mRNA splicing factors. SRSF3 generally functions as an enhancer of mRNA splicing by binding to transcripts in a sequence-specific manner to both recruit and stabilize the binding of spliceosomal components to the mRNA. In liver, expression of SRSF3 is relatively low and its activity is increased in response to insulin and feeding a high carbohydrate diet. We sought to over-express SRSF3 in primary rat hepatocytes to identify regulatory targets. A standard adenoviral shuttle vector system containing an epitope-tagged SRSF3 under the transcriptional control of the CMV promoter could not be used to produce infectious adenoviral particles. SRSF3 over-expression in the packaging cell line prevented the production of infectious adenovirus particles by interfering with the viral splicing program. To circumvent this issue, SRSF3 expression from the shuttle vector was blocked by placing its expression under the control of the liver-specific albumin promoter. In this system, the FLAG-SRSF3 transgene is only expressed in the target cells (hepatocytes) but not in the packaging cell line. An additional benefit of the albumin promoter is that expression of the transgene does not require the addition of hormones or antibiotics to drive SRSF3 expression in the hepatocytes. Robust expression of FLAG-SRSF3 protein is detected in both HepG2 cells and primary rat hepatocytes infected with adenovirus prepared from this new shuttle vector. Furthermore, abundances of several known and suspected mRNA targets of SRSF3 action are increased in response to over-expression using this virus. This report details the construction of the albumin promoter-driven adenoviral shuttle vector, termed pmAlbAd5-FLAG.SRSF3, that can be used to generate functional adenovirus to express FLAG-SRSF3 specifically in liver. This vector would be suitable for over-expression of other splicing factors that could inhibit virus production. In addition, this vector would allow only liver-specific expression of other cargo genes when used in a whole-animal paradigm.

Serine arginine splicing factor 3 is involved in enhanced splicing of glucose-6-phosphate dehydrogenase RNA in response to nutrients and hormones in liver.

Expression of G6PD is controlled by changes in the degree of splicing of the G6PD mRNA in response to nutrients in the diet. This regulation involves an exonic splicing enhancer (ESE) in exon 12 of the mRNA. Using the G6PD model, we demonstrate that nutrients and hormones control the activity of serine-arginine-rich (SR) proteins, a family of splicing co-activators, and thereby regulate the splicing of G6PD mRNA. In primary rat hepatocyte cultures, insulin increased the amount of phosphorylated SR proteins, and this effect was counteracted by arachidonic acid. The results of RNA affinity analysis with nuclear extracts from intact liver demonstrated that the SR splicing factor proteins SRSF3 and SRSF4 bound to the G6PD ESE. Consequently, siRNA-mediated depletion of SRSF3, but not SRSF4, in liver cells inhibited accumulation of both mRNA expressed from a minigene containing exon 12 and the endogenous G6PD mRNA. Consistent with the functional role of SRSF3 in regulating splicing, SRSF3 was observed to bind to the ESE in both intact cells and in animals using RNA immunoprecipitation analysis. Furthermore, refeeding significantly increased the binding of SRSF3 coincident with increased splicing and expression of G6PD. Together, these data establish that nutritional regulation of SRSF3 activity is involved in the differential splicing of the G6PD transcript in response to nutrients. Nutritional regulation of other SR proteins presents a regulatory mechanism that could cause widespread changes in mRNA splicing. Nutrients are therefore novel regulators of mRNA splicing.

Identification of radiation-induced expression changes in nonimmortalized human T cells.

In the event of a radiation accident or attack, it will be imperative to quickly assess the amount of radiation exposure to accurately triage victims for appropriate care. RNA-based radiation dosimetry assays offer the potential to rapidly screen thousands of individuals in an efficient and cost-effective manner. However, prior to the development of these assays, it will be critical to identify those genes that will be most useful to delineate different radiation doses. Using global expression profiling, we examined expression changes in nonimmortalized T cells across a wide range of doses (0.15-12 Gy). Because many radiation responses are highly dependent on time, expression changes were examined at three different times (3, 8, and 24 h). Analyses identified 61, 512 and 1310 genes with significant linear dose-dependent expression changes at 3, 8 and 24 h, respectively. Using a stepwise regression procedure, a model was developed to estimate in vitro radiation exposures using the expression of three genes (CDKN1A, PSRC1 and TNFSF4) and validated in an independent test set with 86% accuracy. These findings suggest that RNA-based expression assays for a small subset of genes can be employed to develop clinical biodosimetry assays to be used in assessments of radiation exposure and toxicity.