The acyl-CoA thioesterase I is regulated by PPARalpha and HNF4alpha via a distal response element in the promoter

The cytosolic acyl-coenzyme A thioesterase I (Acot1) is an enzyme that hydrolyzes long-chain acyl-CoAs of C(12)-C(20)-CoA in chain length to the free fatty acid and CoA. Acot1 was shown previously to be strongly upregulated at the mRNA and protein level in rodents by fibrates. In this study, we show that Acot1 mRNA levels were increased by 90-fold in liver by treatment with Wy-14,643 and that Acot1 mRNA was also increased by 15-fold in the liver of hepatocyte nuclear factor 4alpha (HNF4alpha) knockout animals. Our study identified a direct repeat 1 (DR1) located in the Acot1 gene promoter in mouse, which binds the peroxisome proliferator-activated receptor alpha (PPARalpha) and HNF4alpha. Chromatin immunoprecipitation (ChIP) assay showed that the identified DR1 bound PPARalpha/retinoid X receptor alpha (RXRalpha) and HNF4alpha, whereas the binding in ChIP was abrogated in the PPARalpha and HNF4alpha knockout mouse models. Reporter gene assays showed activation of the Acot1 promoter in cells by the PPARalpha agonist Wy-14,643 after cotransfection with PPARalpha/RXRalpha. However, transfection with a plasmid containing HNF4alpha also resulted in an increase in promoter activity. Together, these data show that Acot1 is under regulation by an interplay between HNF4alpha and PPARalpha.     

Generation of mice with conditional null allele for GdX/Ubl4A

GdX (also named Ubl4A) is a house-keeping gene located on the X chromosome and encodes a protein harboring an ubiquitin-like domain in human and mouse. Although identified in 1988, the function of GdX remains unknown. To elucidate the role of GdX in vivo, we generated a conditional GdX knockout mouse in which Exon 2 was flanked by two loxP sites. We obtained viable and fertile mice with homozygous GdX(flox/flox) or GdX(flox/Y) allele. Germ-line transmission was confirmed by crossing the mouse bearing conditionally targeted allele with an EIIα-Cre transgenic mouse. GdX was successfully depleted in tissues of EIIα-Cre-GdX-null mice. GdX(-/-) and GdX(-/Y) mice are viable and exhibit normal development compared with wild-type littermates within 6 months during our observation. We also observed that GdX knockout male mice were functionally normal in the reproductive system where Ubl4B was specifically expressed. GdX(flox/flox) and GdX(flox/Y) conditional mice provide a tool for further tissue-specific function analysis of the GdX protein under different conditions.     

Genetic dissection of retinoid dehydrogenases

Biochemical studies indicate that alcohol dehydrogenase (ADH) metabolizes retinol to retinal, and that aldehyde dehydrogenase (ALDH) metabolizes retinal to retinoic acid, a molecule essential for growth and development. Summarized herein are several genetic studies supporting in vivo functions for ADH and ALDH in retinoic acid synthesis. Gene targeting was used to create knockout mice for either Adh1 or Adh4. Both knockout mice were viable and fertile without obvious defects. However, when wild-type and Adh4 knockout mice were subjected to vitamin A deficiency during gestation, the survival rate at birth was 3.3-fold lower for Adh4 knockout mice. When adult mice were examined for production of retinoic acid following retinol administration, Adh1 knockout mice exhibited 10-fold lower retinoic acid levels in liver compared with wild-type, whereas Adh4 knockout mice differed from wild-type by less than 2-fold. Thus, Adh1 plays a major role in the metabolism of a large dose of retinol to retinoic acid in adults, whereas Adh4 plays a role in maintaining sufficient retinol metabolism for development during retinol deficiency. ALDHs were examined by overexpression studies in frog embryos. Injection of mRNAs for either mouse Raldh1 or Raldh2 stimulated retinoic acid synthesis in frog embryos at the blastula stage when retinoic acid is normally undetectable. Overexpression of human ALDH2, human ALDH3, and mouse Aldh-pb did not stimulate retinoic acid production. In addition, Raldh2 knockout mice exhibit embryonic lethality with defects in retinoid-dependent tissues. Overall, these studies provide genetic evidence that Adh1, Adh4, Raldh1, and Raldh2 encode retinoid dehydrogenases involved in retinoic acid synthesis in vivo.     

Absence of the SP/SP receptor circuitry in the substance P-precursor knockout mice or SP receptor, neurokinin (NK)1 knockout mice leads to an inhibited cytokine response in granulomas associated with murine Taenia crassiceps infection

Neurocysticercosis, caused by the cestode Taenia solium, is the most common parasitic infection of the human central nervous system that leads to seizures. Taenia crassiceps cysticercosis in mice is an experimental model for Taenia solium cysticercosis. Similar to the human infection, live parasites cause little or no granulomatous inflammation. Dying parasites initiate a granulomatous reaction. The neuropeptide, substance P (SP), stimulates T-helper (TH) 1 cytokine production. In the current studies, we determined whether absence of SP/SP receptor circuitry in the SP-precursor, preprotachykinin, knockout or SP-receptor, neurokinin (NK) 1, knockout mice affected granuloma cytokine production. We hence compared the levels of Th1 cytokines interleukin (IL)-2 and interferon (IFN)-gamma, and levels of Th2/immunoregulatory cytokines IL-4 and IL-10, by enzyme-linked immunosorbent assay in T. crassiceps-induced granulomas derived from infected C57BL/6 wild type (WT) versus SP-precursor knockout and NK1 knockout mice. We found that mean levels of IL-2, IFN-gamma, IL-4, and IL-10 in infected WT-derived granulomas were significantly higher than those of granulomas derived from infected SP-precursor knockout or the NK1 receptor (NKIR)knockout mice. Levels of Th2/immunoregulatory cytokines, IL-4 and IL-10 were higher in early stage granulomas (histologically-staged on basis of evidence of parasite remnants) versus late stage granulomas (no parasite-remnants) of both knockouts, whereas the reverse was noted in WT-derived granulomas. These study established that the absence of an SP/SP receptor circuitry in the SP precursor knockout mice or NK1 receptor knockout mice led to an inhibited cytokine response.     

A nuclear receptor,hepatocyte nuclear factor 4, differently contributes to the human and mouse angiotensinogen promoter activities

Angiotensinogen (AGT), mainly produced in the liver, is the precursor of angiotensin II, an important regulator of blood pressure and electrolyte homeostasis. We previously showed, in hepatoma-derived HepG2 cells that a hepatocyte nuclear factor 4 (HNF4) potentiated human AGT (hAGT) promoter activity and identified its binding sites (termed regions C and J) in the hAGT promoter region. We also showed in transgenic mouse (TgM) that the hAGT is abundantly expressed in the kidney where the level of endogenous mouse AGT (mAGT) expression is low. To elucidate molecular mechanisms of the AGT gene activation in the kidney, we first investigated the HNF4 and AGT expression in the mouse kidney. Northern blot, in situ hybridization and immunohistochemical analyses revealed that the hAGT and HNF4 were both expressed in the proximal tubular (PT) cells of the kidney. We then transfected the hAGT reporter constructs into immortalized mouse PT (mProx) cells and found that regions C and J contributed additively to the HNF4-potentiated hAGT promoter activity. Curiously, no obvious HNF4 binding motif was found in the corresponding region of the mAGT promoter and co-transfected HNF4 failed to activate this promoter in neither HepG2 nor mProx cells. These results suggest that the high-level hAGT expression in the TgM kidney is, at least in part, due to a presence of high-affinity HNF4 binding sites in its promoter.     

Lung defects in neonatal and adult stromal-derived factor–1 conditional knockout mice

Stromal-derived factor (SDF)-1/CXCL12 is a cytokine that is involved in organogenesis, hematopoiesis, chemoattraction, and wound healing. An SDF-1 knockout mouse (SDF-1^-/-) has provided important insights into the role of SDF-1 in fetal development. Because the SDF-1 knockout is lethal in the perinatal period, we have created a conditional SDF-1 knockout mouse. In the present study, we induced conditionally knocked out SDF-1 in neonatal mice and found that lung development was compromised; neonatal lungs showed increased alveolar airspace and abnormal ultrastructure. Conditional knockout of SDF-1 in adult mice resulted in an emphysemic morphology, with increased alveolar airspace and thickened alveolar septa. Fluorescence angiography showed pulmonary vessel hyperdilation. To determine whether the hyperdilation involved nitric oxide, we inhibited endothelial nitric oxide synthase (eNOS) with N (G)-nitro-L- arginine methyl ester. This resulted in the inhibition of pulmonary vessel hyperdilation. Western blot results showed increased phosphorylation of eNOS in our induced SDF-1 knockout mice, indicating that eNOS is normally repressed in the presence of SDF-1, and that activation of eNOS contributes to pulmonary pathology. Thus, a conditional knockout mouse has been successsfully created for SDF-1; initial characterization indicates that SDF-1 is intimately involved in lung development and physiology.     

Expression and localization of P1 promoter-driven hepatocyte nuclear factor-4α (HNF4α) isoforms in human and rats

BACKGROUND: Hepatocyte nuclear factor-4alpha (HNF4alpha; NR2A1) is an orphan member of the nuclear receptor superfamily involved in various processes that could influence endoderm development, glucose and lipid metabolism. A loss-of-function mutation in human HNF4alpha causes one form of diabetes mellitus called maturity-onset diabetes of the young type 1 (MODY1) which is characterized in part by a diminished insulin secretory response to glucose. The expression of HNF4alpha in a variety of tissues has been examined predominantly at the mRNA level, and there is little information regarding the cellular localization of the endogenous HNF4alpha protein, due, in part, to the limited availability of human HNF4alpha-specific antibodies. RESULTS: Monoclonal antibodies have been produced using baculovirus particles displaying gp64-HNF4alpha fusion proteins as the immunizing agent. The mouse anti-human HNF4alpha monoclonal antibody (K9218) generated against human HNF4alpha1/alpha2/alpha3 amino acids 3-49 was shown to recognize not only the transfected and expressed P1 promoter-driven HNF4alpha proteins, but also endogenous proteins. Western blot analysis with whole cell extracts from Hep G2, Huh7 and Caco-2 showed the expression of HNF4alpha protein, but HEK293 showed no expression of HNF4alpha protein. Nuclear-specific localization of the HNF4alpha protein was observed in the hepatocytes of liver cells, proximal tubular epithelial cells of kidney, and mucosal epithelial cells of small intestine and colon, but no HNF4alpha protein was detected in the stomach, pancreas, glomerulus, and distal and collecting tubular epithelial cells of kidney. The same tissue distribution of HNF4alpha protein was observed in humans and rats. Electron microscopic immunohistochemistry showed a chromatin-like localization of HNF4alpha in the liver and kidney. As in the immunohistochemical investigation using K9218, HNF4alpha mRNA was found to be localized primarily to liver, kidney, small intestine and colon by RT-PCR and GeneChip analysis. CONCLUSION: These results suggest that this method has the potential to produce valuable antibodies without the need for a protein purification step. Immunohistochemical studies indicate the tissue and subcellular specific localization of HNF4alpha and demonstrate the utility of K9218 for the detection of P1 promoter-driven HNF4alpha isoforms in humans and in several other mammalian species.     

Group IVA phospholipase A2 regulates testosterone biosynthesis by murine Leydig cells and is required for timely sexual maturation

In the present paper, we report that PLA2G4A (Group IVA phospholipase A2) is important in the development and function of rodent testes. Interstitial cells of rat testes had high PLA2 (phospholipase A2) activity that was very sensitive to the PLA2G4A-preferential inhibitor AACOCF3 (arachidonyl trifluoromethyl ketone). PLA2G4A protein was expressed primarily in the interstitial cells of wild-type mouse testes throughout maturation. Although Pla2g4a knockout (Pla2g4a-/-) male mice are fertile, their sexual maturation was delayed, as indicated by cauda epididymal sperm count and seminal vesicle development. Delayed function of Pla2g4a-/- mice testes was associated with histological abnormalities including disorganized architecture, swollen appearance and fewer interstitial cells. Basal secretion of testosterone was attenuated significantly and steroidogenic response to hCG (human chorionic gonadotropin) treatment was reduced in Pla2g4a-/- mice compared with their Pla2g4a+/+ littermates during the sexual maturation period. Chemical inhibition of PLA2G4A activity by AACOCF3 or pyrrophenone significantly reduced hCG-stimulated testosterone production in cultured rat interstitial cells. AACOCF3 inhibited forskolin- and cAMP analogue-stimulated testosterone production. These results provide the first evidence that PLA2G4A plays a role in male testes physiology and development. These results may have implications for the potential clinical use of PLA2G4A inhibitors.