Differential effects of selective HDAC inhibitors on macrophage inflammatory responses to the Toll-like receptor 4 agonist LPS.

Broad-spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti- and proinflammatory effects on macrophages in vitro. We report here that several broad-spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS-induced mRNA expression of the proinflammatory mediators Edn-1, Ccl-7/MCP-3, and Il-12p40 but amplified the expression of the proatherogenic factors Cox-2 and Pai-1/serpine1 in primary mouse BMM. Similar effects were also apparent in LPS-stimulated TEPM and HMDM. The pro- and anti-inflammatory effects of TSA were separable over a concentration range, implying that individual HDACs have differential effects on macrophage inflammatory responses. The HDAC1-selective inhibitor, MS-275, retained proinflammatory effects (amplification of LPS-induced expression of Cox-2 and Pai-1 in BMM) but suppressed only some inflammatory responses. In contrast, 17a (a reportedly HDAC6-selective inhibitor) retained anti-inflammatory but not proinflammatory properties. Despite this, HDAC6(-/-) macrophages showed normal LPS-induced expression of HDAC-dependent inflammatory genes, arguing that the anti-inflammatory effects of 17a are not a result of inhibition of HDAC6 alone. Thus, 17a provides a tool to identify individual HDACs with proinflammatory properties.

An autoregulatory loop controlling CYP1A1 gene expression: role of H(2)O(2) and NFI.

Cytochrome P450 1A1 (CYP1A1), like many monooxygenases, can produce reactive oxygen species during its catalytic cycle. Apart from the well-characterized xenobiotic-elicited induction, the regulatory mechanisms involved in the control of the steady-state activity of CYP1A1 have not been elucidated. We show here that reactive oxygen species generated from the activity of CYP1A1 limit the levels of induced CYP1A1 mRNAs. The mechanism involves the repression of the CYP1A1 gene promoter activity in a negative-feedback autoregulatory loop. Indeed, increasing the CYP1A1 activity by transfecting CYP1A1 expression vectors into hepatoma cells elicited an oxidative stress and led to the repression of a reporter gene driven by the CYP1A1 gene promoter. This negative autoregulation is abolished by ellipticine (an inhibitor of CYP1A1) and by catalase (which catalyzes H(2)O(2) catabolism), thus implying that H(2)O(2) is an intermediate. Down-regulation is also abolished by the mutation of the proximal nuclear factor I (NFI) site in the promoter. The transactivating domain of NFI/CTF was found to act in synergy with the arylhydrocarbon receptor pathway during the induction of CYP1A1 by 2,3,7,8-tetrachloro-p-dibenzodioxin. Using an NFI/CTF-Gal4 fusion, we show that NFI/CTF transactivating function is decreased by a high activity of CYP1A1. This regulation is also abolished by catalase or ellipticine. Consistently, the transactivating function of NFI/CTF is repressed in cells treated with H(2)O(2), a novel finding indicating that the transactivating domain of a transcription factor can be targeted by oxidative stress. In conclusion, an autoregulatory loop leads to the fine tuning of the CYP1A1 gene expression through the down-regulation of NFI activity by CYP1A1-based H(2)O(2) production. This mechanism allows a limitation of the potentially toxic CYP1A1 activity within the cell.

Glucose-dependent insulinotropic polypeptide (GIP) and its receptor (GIPR): cellular localization, lesion-affected expression, and impaired regenerative axonal growth.

Glucose-dependent insulinotropic polypeptide (GIP) was initially described to be rapidly regulated by endocrine cells in response to nutrient ingestion, with stimulatory effects on insulin synthesis and release. Previously, we demonstrated a significant up-regulation of GIP mRNA in the rat subiculum after fornix injury. To gain more insight into the lesion-induced expression of GIP and its receptor (GIPR), expression profiles of the mRNAs were studied after rat sciatic nerve crush injury in 1) affected lumbar dorsal root ganglia (DRG), 2) spinal cord segments, and 3) proximal and distal nerve fragments by means of quantitative RT-PCR. Our results clearly identified lesion-induced as well as tissue type-specific mRNA regulation of GIP and its receptor. Furthermore, comprehensive immunohistochemical stainings not only confirmed and exceeded the previous observation of neuronal GIP expression but also revealed corresponding GIPR expression, implying putative modulatory functions of GIP/GIPR signaling in adult neurons. In complement, we also observed expression of GIP and its receptor in myelinating Schwann cells and oligodendrocytes. Polarized localization of GIPR in the abaxonal Schwann cell membranes, plasma membrane-associated GIPR expression of satellite cells, and ependymal GIPR expression strongly suggests complex cell type-specific functions of GIP and GIPR in the adult nervous system that are presumably mediated by autocrine and paracrine interactions, respectively. Notably, in vivo analyses with GIPR-deficient mice suggest a critical role of GIP/GIPR signal transduction in promoting spontaneous recovery after nerve crush, insofar as traumatic injury of GIPR-deficient mouse sciatic nerve revealed impaired axonal regeneration compared with wild-type mice.

Light-induced retinal degeneration correlates with changes in iron metabolism gene expression, ferritin level, and aging.

PURPOSE: Retinal degeneration is associated with iron accumulation in several rodent models in which iron-regulating proteins are impaired. Oxidative stress is catalyzed by unbound iron. METHODS: The role of the heavy chain of ferritin, which sequesters iron, in regulating the thickness of the photoreceptor nuclear layer in the 4- and 16-month-old wild-type H ferritin (HFt(+/+)) and heterozygous H ferritin (HFt(+/-)) mice was investigated, before and 12 days after exposure to 13,000-lux light for 24 hours. The regulation of gene expression of the various proteins involved in iron homeostasis, such as transferrin, transferrin receptor, hephaestin, ferroportin, iron regulatory proteins 1 and 2, hepcidin, ceruloplasmin, and heme-oxygenase 1, was analyzed by quantitative (q)RT-PCR during exposure (2, 12, and 24 hours) and 24 hours after 1 day of exposure in the 4-month-old HFt(+/+) and HFt(+/-) mouse retinas. RESULTS: Retinal degeneration in the 4-month-old HFt(+/-) mice was more extensive than in the HFt(+/+) mice. Yet, it was more extensive in both of the 16-month-old mouse groups, revealing the combined effect of age and excessive light. Injury caused by excessive light modified the temporal gene expression of iron-regulating proteins similarly in the HFt(+/-) and HFt(+/+) mice. CONCLUSIONS: Loss of one allele of H ferritin appears to increase light-induced degeneration. This study highlighted that oxidative stress related to light-induced injury is associated with major changes in gene expression of iron metabolism proteins.

Characterization of the fasting-induced adipose factor FIAF, a novel peroxisome proliferator-activated receptor target gene.

Fasting is associated with significant changes in nutrient metabolism, many of which are governed by transcription factors that regulate the expression of rate-limiting enzymes. One factor that plays an important role in the metabolic response to fasting is the peroxisome proliferator-activated receptor alpha (PPARalpha). To gain more insight into the role of PPARalpha during fasting, and into the regulation of metabolism during fasting in general, a search for unknown PPARalpha target genes was performed. Using subtractive hybridization (SABRE) comparing liver mRNA from wild-type and PPARalpha null mice, we isolated a novel PPARalpha target gene, encoding the secreted protein FIAF (for fasting induced adipose factor), that belongs to the family of fibrinogen/angiopoietin-like proteins. FIAF is predominantly expressed in adipose tissue and is strongly up-regulated by fasting in white adipose tissue and liver. Moreover, FIAF mRNA is decreased in white adipose tissue of PPARgamma +/- mice. FIAF protein can be detected in various tissues and in blood plasma, suggesting that FIAF has an endocrine function. Its plasma abundance is increased by fasting and decreased by chronic high fat feeding. The data suggest that FIAF represents a novel endocrine signal involved in the regulation of metabolism, especially under fasting conditions.

A role for glutamate transporters in the regulation of insulin secretion.

In the brain, glutamate is an extracellular transmitter that mediates cell-to-cell communication. Prior to synaptic release it is pumped into vesicles by vesicular glutamate transporters (VGLUTs). To inactivate glutamate receptor responses after release, glutamate is taken up into glial cells or neurons by excitatory amino acid transporters (EAATs). In the pancreatic islets of Langerhans, glutamate is proposed to act as an intracellular messenger, regulating insulin secretion from β-cells, but the mechanisms involved are unknown. By immunogold cytochemistry we show that insulin containing secretory granules express VGLUT3. Despite the fact that they have a VGLUT, the levels of glutamate in these granules are low, indicating the presence of a protein that can transport glutamate out of the granules. Surprisingly, in β-cells the glutamate transporter EAAT2 is located, not in the plasma membrane as it is in brain cells, but exclusively in insulin-containing secretory granules, together with VGLUT3. In EAAT2 knock out mice, the content of glutamate in secretory granules is higher than in wild type mice. These data imply a glutamate cycle in which glutamate is carried into the granules by VGLUT3 and carried out by EAAT2. Perturbing this cycle by knocking down EAAT2 expression with a small interfering RNA, or by over-expressing EAAT2 or a VGLUT in insulin granules, significantly reduced the rate of granule exocytosis. Simulations of granule energetics suggest that VGLUT3 and EAAT2 may regulate the pH and membrane potential of the granules and thereby regulate insulin secretion. These data suggest that insulin secretion from β-cells is modulated by the flux of glutamate through the secretory granules.

Peroxisome proliferator-activated receptor-alpha-null mice have increased white adipose tissue glucose utilization, GLUT4, and fat mass: Role in liver and brain.

Activation of the peroxisome proliferator-activated receptor (PPAR)-alpha increases lipid catabolism and lowers the concentration of circulating lipid, but its role in the control of glucose metabolism is not as clearly established. Here we compared PPARalpha knockout mice with wild type and confirmed that the former developed hypoglycemia during fasting. This was associated with only a slight increase in insulin sensitivity but a dramatic increase in whole-body and adipose tissue glucose use rates in the fasting state. The white sc and visceral fat depots were larger due to an increase in the size and number of adipocytes, and their level of GLUT4 expression was higher and no longer regulated by the fed-to-fast transition. To evaluate whether these adipocyte deregulations were secondary to the absence of PPARalpha from liver, we reexpresssed this transcription factor in the liver of knockout mice using recombinant adenoviruses. Whereas more than 90% of the hepatocytes were infected and PPARalpha expression was restored to normal levels, the whole-body glucose use rate remained elevated. Next, to evaluate whether brain PPARalpha could affect glucose homeostasis, we activated brain PPARalpha in wild-type mice by infusing WY14643 into the lateral ventricle and showed that whole-body glucose use was reduced. Hence, our data show that PPARalpha is involved in the regulation of glucose homeostasis, insulin sensitivity, fat accumulation, and adipose tissue glucose use by a mechanism that does not require PPARalpha expression in the liver. By contrast, activation of PPARalpha in the brain stimulates peripheral glucose use. This suggests that the alteration in adipocyte glucose metabolism in the knockout mice may result from the absence of PPARalpha in the brain.

Nucleotide sequence of the 5' noncoding region and part of the gag gene of mouse mammary tumor virus; identification of the 5' splicing site for subgenomic mRNAs.

We have determined the sequence of the first 1371 nucleotides at the 5' end of the genome of mouse mammary tumor virus using molecularly cloned proviral DNA of the GR virus strain. The most likely initiation codon used for the gag gene of mouse mammary tumor virus is the first one, located 312 nucleotides from the 5' end of the viral RNA. The 5' splicing site for the subgenomic mRNA's is located approximately 288 nucleotides downstream from the 5' end of the viral RNA. From the DNA sequence the amino acid sequence of the N-terminal half of the gag precursor protein, including p10 and p21, was deduced (353 amino acids).

The T cell-specific CXC chemokines IP-10, Mig, and I-TAC are expressed by activated human bronchial epithelial cells.

Recruitment of activated T cells to mucosal surfaces, such as the airway epithelium, is important in host defense and for the development of inflammatory diseases at these sites. We therefore asked whether the CXC chemokines IFN-induced protein of 10 kDa (IP-10), monokine induced by IFN-gamma (Mig), and IFN-inducible T-cell alpha-chemoattractant (I-TAC), which specifically chemoattract activated T cells by signaling through the chemokine receptor CXCR3, were inducible in respiratory epithelial cells. The effects of proinflammatory cytokines, including IFN-gamma (Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and dexamethasone were studied in normal human bronchial epithelial cells (NHBEC) and in two human respiratory epithelial cell lines, A549 and BEAS-2B. We found that IFN-gamma, but not TNF-alpha or IL-1 beta, strongly induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and that TNF-alpha and IL-1 beta synergized with IFN-gamma induction in all three cell types. High levels of IP-10 protein (> 800 ng/ml) were detected in supernatants of IFN-gamma/TNF-alpha-stimulated NHBEC. Neither dexamethasone nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since IFN-gamma is up-regulated in tuberculosis (TB), using in situ hybridization we studied the expression of IP-10 in the airways of TB patients and found that IP-10 mRNA was expressed in the bronchial epithelium. In addition, IP-10-positive cells obtained by bronchoalveolar lavage were significantly increased in TB patients compared with normal controls. These results show that activated bronchial epithelium is an important source of IP-10, Mig, and I-TAC, which may, in pulmonary diseases such as TB (in which IFN-gamma is highly expressed) play an important role in the recruitment of activated T cells.

CYP3A5 and ABCB1 genes and hypertension.

Hypertension is the first single modifiable cause of disease burden worldwide. Genes encoding proteins that are involved in the metabolism (CYP3A5) and transport (ABCB1) of drugs and hormones might contribute to blood pressure control in humans. Indeed, recent data have suggested that CYP3A5 and ABCB1 gene polymorphisms are associated with blood pressure in the rat as well as in humans. Interestingly, the effects of these genes on blood pressure appear to be modified by dietary salt intake. This review summarizes what is known regarding the relationships of the ABCB1 and CYP3A5 genes with blood pressure, and discusses the potential underlying mechanisms of the association. If the role of these genes in blood pressure control is confirmed in other populations and other ethnic groups, these findings would point toward a new pathway for blood pressure control in humans.

APRIL, a new ligand of the tumor necrosis factor family, stimulates tumor cell growth.

Members of the tumor necrosis factor (TNF) family induce pleiotropic biological responses, including cell growth, differentiation, and even death. Here we describe a novel member of the TNF family designated APRIL (for a proliferation-inducing ligand). Although transcripts of APRIL are of low abundance in normal tissues, high levels of mRNA are detected in transformed cell lines, and in human cancers of colon, thyroid, and lymphoid tissues in vivo. The addition of recombinant APRIL to various tumor cells stimulates their proliferation. Moreover, APRIL-transfected NIH-3T3 cells show an increased rate of tumor growth in nude mice compared with the parental cell line. These findings suggest that APRIL may be implicated in the regulation of tumor cell growth.

Main Street Messenger, Summer 2001

Newsletter for Economic Development

Main Street Messenger, Spring, 2002

Newsletter for Economic Development

Main Street Messenger, Autumn 2002

Newsletter for Economic Development