Transcriptome correlation analysis identifies two unique craniosynostosis subtypes associated with IRS1 activation.

The discovery of causal mechanisms associated with nonsyndromic craniosynostosis has proven to be a difficult task due to the complex nature of the disease. In this study, differential transcriptome correlation analysis was used to identify two molecularly distinct subtypes of nonsyndromic craniosynostosis, termed subtype A and subtype B. In addition to unique correlation structure, subtype A was also associated with high IGF pathway expression, whereas subtype B was associated with high integrin expression. To identify a pathologic link between altered gene correlation/expression and the disease state, phosphorylation assays were performed on primary osteoblast cell lines derived from cases within subtype A or subtype B, as well as on primary osteoblast cell lines with novel IGF1R variants previously reported by our lab (Cunningham ML, Horst JA, Rieder MJ, Hing AV, Stanaway IB, Park SS, Samudrala R, Speltz ML. Am J Med Genet A 155A: 91-97, 2011). Elevated IRS1 (pan-tyr) and GSK3ß (ser-9) phosphorylation were observed in two novel IGF1R variants with receptor L domain mutations. In subtype A, a hypomineralization phenotype coupled with decreased phosphorylation of IRS1 (ser-312), p38 (thr-180/tyr-182), and p70S6K (thr-412) was observed. In subtype B, decreased phosphorylation of IRS1 (ser-312) as well as increased phosphorylation of Akt (ser-473), GSK3ß (ser-9), IGF1R (tyr-1135/tyr-1136), JNK (thr-183/tyr-187), p70S6K (thr-412), and pRPS6 (ser-235/ser-236) was observed, thus implicating the activation of IRS1-mediated Akt signaling in potentiating craniosynostosis in this subtype. Taken together, these results suggest that despite the stimulation of different pathways, activating phosphorylation patterns for IRS1 were consistent in cell lines from both subtypes and the IGF1R variants, thus implicating a key role for IRS1 in the pathogenesis of nonsyndromic craniosynostosis.

Effects of dietary saturated fat on LDL subclasses and apolipoprotein CIII in men.

BACKGROUND/OBJECTIVES: Small dense low-density lipoprotein (LDL) particles and apolipoprotein (apo) CIII are risk factors for cardiovascular disease (CVD) that can be modulated by diet, but there is little information regarding the effects of dietary saturated fat on their plasma levels. We tested the effects of high vs low saturated fat intake in the context of a high beef protein diet on levels and composition of LDL subclasses and on apoCIII levels in plasma and LDL. SUBJECTS/METHODS: Following consumption of a baseline diet (50% carbohydrate (CHO), 13% protein, 38% total fat, 15% saturated fat) for 3 weeks, 14 healthy men were randomly assigned to two reduced CHO high beef protein diets (31% CHO, 31% protein, 38% fat) that differed in saturated fat content (15% vs 8%) for 3 weeks each in a crossover design. RESULTS: The high saturated fat (HSF) diet resulted in higher mass concentrations of buoyant LDL I, medium density LDL II and dense LDL III, but not the very dense LDL IV; and significant increases in plasma and LDL apoCIII concentration of 9.4% and 33.5%, respectively. The saturated fat-induced changes in LDL apoCIII were specifically correlated with changes in apoCIII content of LDL IV. CONCLUSIONS: Taken together with previous observations, these findings suggest that, at least in the context of a lower CHO high beef protein diet, HSF intake may increase CVD risk by metabolic processes that involve apoCIII.

Characterizing the expression of CYP3A4 and efflux transporters (P-gp, MRP1, and MRP2) in CYP3A4-transfected Caco-2 cells after induction with sodium butyrate and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate.

PURPOSE: To examine the changes in expression levels of CYP3A4 and efflux transporters in CYP3A4-transfected Caco-2 (colon carcinoma) cells in the presence of the inducers sodium butyrate (NaB) and 12-O-tetradecanoylphorbol-13-acetate (TPA). To characterize the transport of [3H]-digoxin and the metabolism of midazolam in the cells under different inducing conditions. METHODS: CYP3A4-Caco-2 cells were seeded onto cell culture inserts and were grown for 13-14 days. Transport and metabolism studies were performed on cells induced with NaB and/or TPA for 24 h. The expression and localization of P-gp, MRP1, MRP2, and CYP3A4 were examined by Western blot and confocal microscopy. RESULTS: In the presence of both inducers, CYP3A4 protein levels were increased 40-fold over uninduced cells, MRP2 expression was decreased by 90%, and P-gp and MRP1 expression were unchanged. Midazolam 1-OH formation exhibited a rank order correlation with increased CYP3A4 protein, whereas [3H]-digoxin transport (a measure of P-gp activity) was unchanged with induction. P-gp and MRP2 were found on the apical membrane, whereas MRP1 was found perinuclear within the cell. CYP3A4 displayed a punctate pattern of expression consistent with endoplasmic reticulum localization and exhibited preferential polarization towards the apical side of the cell. CONCLUSIONS: The present study characterized CYP3A4-Caco-2 cell monolayers when induced for 24 h in the presence of both NaB and TPA. These conditions provide intact cells with significant CYP3A4 and P-gp expression suitable for the concurrent study of transport and metabolism.

Functional characteristics and steroid hormone-mediated regulation of an organic cation transporter in Madin-Darby canine kidney cells.

Organic cation transporters (OCT1-3) play an important role in renal elimination of many drugs. The goals of this study were to 1) identify a cell culture model which constitutively expressed OCT2 that could be used to study the characteristics and regulation of this transporter, and 2) to study the mechanisms by which xenobiotics and hormones regulate the activity of OCT2. We characterized the endogenous organic cation transporter (OCT) activity in Madin-Darby canine kidney (MDCK) cells. The activity was localized to the basolateral membrane and was pH and membrane potential-dependent. The uptake of the model organic cation, tetraethylammonium, was saturable (Km, 19.5 +/- 4.6 microM; Vmax, 350 +/- 19.4 pmol/mg of protein/10 min) and was inhibited by known OCT inhibitors (e.g., cimetidine and quinidine). A cDNA fragment (711 base pairs) isolated by reverse transcriptase-polymerase chain reaction (RT-PCR) was greater than 83% identical to OCT2 cDNAs from mammalian species; no OCT1 or OCT3 was detected by RT-PCR, suggesting that OCT2 may be the primary basolateral OCT in MDCK. OCT2 mRNA levels were increased significantly following exposure of MDCK to the steroid hormones, dexamethasone (2.0-fold), hydrocortisone (2.4-fold), and testosterone (1.8-fold) with comparable increases in activity. Other compounds tested, including the cytochrome P450 inducers, rifampicin, phenobarbital, and phenytoin, and the OCT substrates, verapamil and metformin, had no inducing effects. Collectively, these data indicate that MDCK can serve as a useful and convenient tool in screening candidate drugs for interaction with OCT2 and for studying the regulation of this transporter. Furthermore, our data demonstrate that steroid hormones induce the transcription of OCT2 in the kidney.

Localization of GFP-tagged concentrative nucleoside transporters in a renal polarized epithelial cell line.

Many nucleosides undergo active reabsorption within the kidney, probably via nucleoside transporters. To date, two concentrative nucleoside transporters have been cloned, the sodium-dependent purine-selective nucleoside transporter (SPNT) and concentrative nucleoside transporter 1 (CNT1). We report the stable expression of green fluorescence protein (GFP)-tagged SPNT and CNT1 in Madin-Darby canine kidney (MDCK) cells, a polarized renal epithelial line. We demonstrate that the GFP tag does not alter the substrate selectivity and only modestly affects the kinetic activity of the transporters. By using confocal microscopy and functional studies, both SPNT and CNT1 are localized primarily to the apical membrane of MDCK and LLC-PK(1) cells. Apical localization of these transporters suggests a role in renal nucleoside reabsorption and regulation of tubular function via the adenosine pathway.