Smad3 is acetylated by p300/CBP to regulate its transactivation activity.

Smad proteins are crucial for the intracellular signaling of transforming growth factor-beta (TGF-beta). Upon their receptor-induced activation, Smad proteins are phosphorylated and translocated to the nucleus to activate the transcription of a select set of target genes. Here, we show that the co-activator p300/CBP bound and acetylated Smad3 as well as Smad2 in vivo, and that the acetylation was stimulated by TGF-beta. A major acetylation site of Smad3 by p300/CBP is Lys-378 in the MH2 domain (Smad3C) known to be critical for the regulation of transcriptional activity. Replacement of Lys-378 with Arg decreased the transcriptional activity of GAL4-Smad3C in a luciferase assay. Moreover, p300/CBP potentiated the transcriptional activity of GAL4-Smad3C, but not the acetylation-resistant GAL4-Smad3C(K378R) mutant. These results suggest that acetylation of Smad3 by p300/CBP regulates positively its transcriptional activity.

Effects of high-fat diet, angiotensinogen (agt) gene inactivation, and targeted expression to adipose tissue on lipid metabolism and renal gene expression.

To address the role of angiotensinogen (agt) in lipid metabolism and its potential endocrine effects in vivo, we studied the effects of high-fat diet (HFD) on adult, 28-week-old agt knockout (KO) mice compared to wild type (WT) mice. Recent studies (Massiera et al., 2001) have demonstrated that reexpression of agt in adipose tissue of KO mice normalized adiposity, blood pressure, and kidney abnormalities. We therefore used microarray analysis to investigate changes in gene expression profile in kidneys of KO vs. Tg-KO mice, where agt expression is restricted to adipose tissue. Body weight, adiposity and insulin levels were significantly decreased (p < 0.05) in KO mice on a chow diet (CD) compared to WT mice, while circulating leptin levels were similar. On a high-fat diet, KO mice exhibited significantly lower bodyweight (p < 0.05), adiposity (p < 0.05), leptin, and insulin levels (p < 0.05) compared to WT mice. In agreement with previously reported changes in kidney histology, agt KO mice displayed altered expressions of genes involved in blood pressure regulation and renal function, but these levels were corrected by reexpression of agt in adipose tissue. Collectively, these findings further document important endocrine roles of adipocyte agt, in part via regulation of lipid metabolism and kidney homeostasis.

PODs in the nuclear spot: enigmas in the magician's pot.

The promyelocytic leukemia (PML) nuclear body, also known as the PML oncogenic domain (POD), is implicated in the pathophysiology of PML. These nuclear subcompartments are dynamic structures. The PML protein, which undergoes a fusion event in patients with promyelocytic leukemia, is normally found in PODs. The PML protein may be a major regulator of the constituents of PODs, controlling POD organization and function. Hatta and Fukamizu describe the functions of PML and discuss how the POD structure and organization may be regulated and affect apoptosis, gene expression, and cellular transformation.

Possible involvement of p38 mitogen-activated protein kinase in decidual function in parturition.

We designed the present study to elucidate the molecular mechanism for parturition, focusing on p38 mitogen-activated protein kinase (p38). The kinase activity of p38 in mouse uterus was gestation stage-dependent, and was markedly increased on day 19 of gestation and during labor. Immunohistochemical examination with anti-phospho p38 antibody revealed that activated p38 was predominantly localized in decidual stromal cells stained with anti-prolactin antibody. In human primary cultured decidual cells, a p38 inhibitor, SB202190, significantly inhibited both prostaglandin F(2alpha) production and COX-2 expression induced by stimulation with IL-1beta. These results suggest that the p38 signaling pathway is involved in decidual function at the late stage of gestation and may contribute to parturition.

Angiotensinogen-deficient mice exhibit impairment of diet-induced weight gain with alteration in adipose tissue development and increased locomotor activity.

White adipose tissue is known to contain the components of the renin-angiotensin system, which gives rise to angiotensin II from angiotensinogen (AGT). Recent evidence obtained in vitro and ex vivo is in favor of angiotensin II acting as a trophic factor of adipose tissue development. To determine whether AGT plays a role in vivo in this process, comparative studies were performed in AGT-deficient (agt(-/-)) mice and control wild-type mice. The results showed that agt(-/-) mice gain less weight than wild-type mice in response to a chow or high fat diet. Adipose tissue mass from weaning to adulthood appeared altered rather specifically, as both the size and the weight of other organs were almost unchanged. Food intake was similar for both genotypes, suggesting a decreased metabolic efficiency in agt(-/-) mice. Consistent with this hypothesis, cellularity measurement indicated hypotrophy of adipocytes in agt(-/-) mice with a parallel decrease in the fatty acid synthase activity. Moreover, AGT-deficient mice exhibited a significantly increased locomotor activity, whereas metabolic rate and mRNA levels of uncoupling proteins remained similar in both genotypes. Thus, AGT appears to be involved in the regulation of fat mass through a combination of decreased lipogenesis and increased locomotor activity that may be centrally mediated.

Two domains of Nrf2 cooperatively bind CBP, a CREB binding protein, and synergistically activate transcription.

BACKGROUND: Nrf2 belongs to the Cap-N-Collar (CNC) transcription factor family and is essential for the antioxidant responsive element (ARE)-mediated expression of a group of detoxifying and antioxidant genes. The forced expression of Nrf2 in mammalian cells activates the expression of target genes through the ARE, with Nrf2 showing the highest transactivation activity among the CNC family of transcription factors. To elucidate the molecular mechanisms generating this potent transactivation activity, we examined the functions of the domains within Nrf2. RESULT: We found that Nrf2 contains two transcription activation domains, Neh4 and Neh5, which act synergistically to attain maximum a activation of reporter gene expression. Neh4 and Neh5 both individually and cooperatively bind to CBP (CREB (cAMP Responsive Element Binding protein) Binding Protein). In fact, the specific inhibitor of CBP, adenovirus E1A protein, significantly reduced Nrf2 activity. Importantly, the CBP-binding activity of Nrf2 deletion mutants positively correlated with their transactivation activity. Neh5 contains a motif which is commonly conserved among the CNC factors, whereas Neh4 contains the novel CBP-interacting motif recently identified in p53 and E2F. CONCLUSIONS: Our results indicate that Nrf2 exploits the cooperative binding of two independent transactivation domains to CBP in the acquisition of a potent transactivation activity.

Adipose angiotensinogen is involved in adipose tissue growth and blood pressure regulation.

White adipose tissue and liver are important angiotensinogen (AGT) production sites. Until now, plasma AGT was considered to be a reflection of hepatic production. Because plasma AGT concentration has been reported to correlate with blood pressure, and to be associated with body mass index, we investigated whether adipose AGT is released locally and into the blood stream. For this purpose, we have generated transgenic mice either in which adipose AGT is overexpressed or in which AGT expression is restricted to adipose tissue. This was achieved by the use of the aP2 adipocyte-specific promoter driving the expression of rat agt cDNA in both wild-type and hypotensive AGT-deficient mice. Our results show that in both genotypes, targeted expression of AGT in adipose tissue increases fat mass. Mice whose AGT expression is restricted to adipose tissue have AGT circulating in the blood stream, are normotensive, and exhibit restored renal function compared with AGT-deficient mice. Moreover, mice that overexpress adipose AGT have increased levels of circulating AGT, compared with wild-type mice, and are hypertensive. These animal models demonstrate that AGT produced by adipose tissue plays a role in both local adipose tissue development and in the endocrine system, which supports a role of adipose AGT in hypertensive obese patients.

An essential role of angiotensin II receptor type 1a in recipient kidney, not in transplanted peripheral blood leukocytes, in progressive immune-mediated renal injury.

Despite an intensive effort of elucidating the pathogenic role of angiotensin II (AII) in immune-mediated renal injury, the precise mechanisms are poorly understood. In the present study, we examined the site of AII action, peripheral blood leukocytes or resident renal cells, in immune-mediated renal injury using AII type 1a receptor (AT1a)-deficient homozygous (AT1a -/-) mice and wild-type (AT1a +/+) mice. The AT1a -/- mice showed delayed-type hypersensitivity similar to that of the AT1a +/+ mice, suggesting that the lack of AT1a does not impair a Th1-type cellular immune response of peripheral blood leukocytes involved in immune-mediated renal injury. We then generated the radiation bone marrow chimera mice, WA and AW, which have transplanted peripheral blood leukocytes from the AT1a +/+ and AT1a -/- mice into the AT1a -/- and AT1a +/+ mice, respectively. As controls, WW and AA, the AT1a +/+ and AT1a -/- mice given bone marrow cells from the AT1a +/+ and AT1a -/- mice, respectively, were generated. Seven days after induction of antiglomerular basement membrane nephritis, glomerulosclerosis observed in the WW mice was markedly ameliorated in the WA mice, but not in the AW mice. In addition, the recruitment of monocytes/macrophages and the expressions of monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 in the glomeruli of the AW and WW mice was evident, but such significant phenotypes were not seen in the WA and AA mice, showing a marked amelioration of renal injury dependent on the host AT1a genotype. These results demonstrate an essential role of AT1a in intrinsic renal cells for progressive immune-mediated renal injury and indicate a beneficial effect of blocking the renin-angiotensin system in the treatment of such diseases.

Dimerization of small GTPase Rab5.

Rab proteins are small GTPases, localized to distinct cellular compartments, regulating specific steps of intracellular membrane trafficking. One member of the Rab family, Rab5, consists of three isoforms, Rab5a, Rab5b, and Rab5c, which have been shown to play an important role in early events of endocytosis. Using the yeast two-hybrid system, we have identified several cytosolic proteins that interact with the Rab5b Q79L (decreasing intrinsic and GTPase-activating protein-stimulated GTPase activities). Surprisingly, most positive clones were Rab5b or Rab5c, indicating that Rab5 could dimerize among extra-isoforms in the yeast two-hybrid system. In vitro and in vivo chemical cross-linking assays demonstrated that lipid-unmodified wild-type Rab5b purified from Escherichia coli, wild-type Rab5b, or a dominant active form Rab5b Q79L expressed in human 293T cells dimerized. Furthermore, the same assays using a Rab5b R81A substitution mutant showed that the Arg81 in the Switch II region [the second GTP/GDP binding motif (residues 74-93)] was essential for Rab5b dimerization. These results suggest that Rab5 isoforms can be dimerized depending upon the GTP-bound conformation, but independent upon a lipid modification.

Role of Deltex-1 as a transcriptional regulator downstream of the Notch receptor.

Intercellular signaling through the cell-surface receptor Notch plays important roles in a variety of developmental processes as well as in pathogenesis of some human cancers and genetic disorders. However, the mechanisms by which Notch signals are transduced into cells still remain elusive. Here we investigated the signaling mechanisms for Notch in the cell fate control of neural progenitor cells. We show that Deltex-1 (DTX1), a mammalian homolog of Drosophila Deltex, mediates a Notch signal to block differentiation of neural progenitor cells. We found that a significant fraction of DTX1 proteins were localized in the nucleus and physically interacted with the transcriptional coactivator p300. Through its binding to p300, DTX1 inhibited transcriptional activation by the neural-specific helix-loop-helix-type transcription factor MASH1, and this mechanism is likely responsible for the differentiation inhibition of neural progenitor cells. Our results further suggest that DTX1 regulates transcription independently of the previously characterized Notch signaling pathway involving RBP-J and HES1/HES5. Thus, DTX1 serves as an important signaling component downstream of Notch that regulates transcription in the nucleus.

Role of Notch-1 intracellular domain in activation of rheumatoid synoviocytes.

OBJECTIVE: Notch family proteins are transmembrane receptors that control cell fate and proliferation. Rheumatoid arthritis (RA) is characterized by activation and abnormal proliferation/differentiation of synoviocytes. We examined the expression of Notch-1 and its role in the activation of RA synoviocytes. METHODS: The expression of Notch-1 protein was detected by a specific antibody raised against the Notch-1 intracellular domain. Notch-1 messenger RNA (mRNA) expression in synoviocytes was analyzed by Northern blotting. Notch-1 protein expression was confirmed by Western blotting with anti-Notch-1 antibody. To analyze the role of Notch-1 in synoviocyte proliferation, we examined the effects of antisense Notch-1 oligonucleotides (ODNs) and MW167, a gamma-secretase inhibitor. RESULTS: Notch-1 protein and mRNA were detected in synovium from all study subjects. The nucleus of RA synoviocytes showed strong staining with anti-Notch-1 antibody, whereas there was predominantly cytoplasmic staining of normal and osteoarthritis (OA) synoviocytes. Western blotting showed a distinct approximately 63-kd protein detected by anti-Notch-1 antibody in nuclear extracts from RA synoviocytes, indicating that nuclear staining of RA synovium and synoviocytes is likely to be the result of nuclear localization of Notch-1 intracellular domain (NICD). Furthermore, tumor necrosis factor alpha (TNFalpha) increased NICD nuclear translocation in a dose-dependent manner. Antisense Notch-1 ODNs partially blocked the proliferation of RA synoviocytes and inhibited TNFalpha-induced proliferation in both OA and RA synoviocytes. In addition, gamma-secretase inhibitor, which blocks the production of NICD, also inhibited TNFalpha-induced proliferation of RA synoviocytes. CONCLUSION: Our results demonstrate the expression of Notch-1 in synoviocytes and the presence of Notch-1 fragment in the nuclei of RA synoviocytes and suggest the involvement of Notch-1 signaling in the TNFalpha-induced proliferation of RA synoviocytes.

Dual roles of RNA helicase A in CREB-dependent transcription.

RNA helicase A (RHA) is a member of an ATPase/DNA and RNA helicase family and is a homologue of Drosophila maleless protein (MLE), which regulates X-linked gene expression. RHA is also a component of holo-RNA polymerase II (Pol II) complexes and recruits Pol II to the CREB binding protein (CBP). The ATPase and/or helicase activity of RHA is required for CREB-dependent transcription. To further understand the role of RHA on gene expression, we have identified a 50-amino-acid transactivation domain that interacts with Pol II and termed it the minimal transactivation domain (MTAD). The protein sequence of this region contains six hydrophobic residues and is unique to RHA homologues and well conserved. A mutant with this region deleted from full-length RHA decreased transcriptional activity in CREB-dependent transcription. In addition, mutational analyses revealed that several tryptophan residues in MTAD are important for the interaction with Pol II and transactivation. These mutants had ATP binding and ATPase activities comparable to those of wild-type RHA. A mutant lacking ATP binding activity was still able to interact with Pol II. In CREB-dependent transcription, the transcriptional activity of each of these mutants was less than that of wild-type RHA. The activity of the double mutant lacking both functions was significantly lower than that of each mutant alone, and the double mutant had a dominant negative effect. These results suggest that RHA could independently regulate CREB-dependent transcription either through recruitment of Pol II or by ATP-dependent mechanisms.

Characterization of RGS5 in regulation of G protein-coupled receptor signaling.

RGS proteins (regulators of G protein signaling) serve as GTPase-activating proteins (GAPs) for G alpha subunits and negatively regulate G protein-coupled receptor signaling. In this study, we characterized biochemical properties of RGS5 and its N terminal (1-33)-deleted mutant (deltaN-RGS5). RGS5 bound to G alpha(i1), G alpha(i2), G alpha(i3), G alpha(o) and G alpha(q) but not to G alpha(s) and G alpha13 in the presence of GDP/AIF4-, and accelerated the catalytic rate of GTP hydrolysis of G alpha(i3) subunit. When expressed in 293T cells stably expressing angiotensin (Ang) AT1a receptors (AT1a-293T cells), RGS5 suppressed Ang II- and endothelin (ET)-1-induced intracellular Ca2+ transients. The effect of RGS5 was concentration-dependent, and the slope of the concentration-response relationship showed that a 10-fold increase in amounts of RGS5 induced about 20-25% reduction of the Ca2+ signaling. Furthermore, a comparison study of three sets of 293T cells with different expression levels of AT1a receptors showed that RGS5 inhibited Ang II-induced responses more effectively in 293T cells with the lower density of AT1a receptors, suggesting that the degree of inhibition by RGS proteins reflects the ratio of amounts of RGS proteins to those of activated G alpha subunits after receptor stimulation by agonists. When expressed in AT1a-293T cells, deltaN-RGS5 was localized almost exclusively in the cytosolic fraction, and exerted the inhibitory effects as potently as RGS5 which was present in both membrane and cytosolic fractions. Studies on relationship between subcellular localization and inhibitory effects of RGS5 and deltaN-RGS5 revealed that the N terminal (1-33) of RGS5 plays a role in targeting this protein to membranes, and that the N terminal region of RGS5 is not essential for exerting activities.

A Role of RNA Helicase A in cis-Acting Transactivation Response Element-mediated Transcriptional Regulation of Human Immunodeficiency Virus Type 1.

RNA helicase A (RHA) has two double-stranded (ds) RNA-binding domains (dsRBD1 and dsRBD2). These domains are conserved with the cis-acting transactivation response element (TAR)-binding protein (TRBP) and dsRNA-activated protein kinase (PKR). TRBP and PKR are involved in the regulation of HIV-1 gene expression through their binding to TAR RNA. This study shows that RHA also plays an important role in TAR-mediated HIV-1 gene expression. Wild-type RHA preferably bound to TAR RNA in vitro and in vivo. Overexpression of wild type RHA strongly enhanced viral mRNA synthesis and virion production as well as HIV-1 long terminal repeat-directed reporter (luciferase) gene expression. Substitution of lysine for glutamate at residue 236 in dsRBD2 (RHA(K236E)) reduced its affinity for TAR RNA and impaired HIV-1 transcriptional activity. These results indicate that TAR RNA is a preferred target of RHA dsRBDs and that RHA enhances HIV-1 transcription in vivo in part through the TAR-binding of RHA.

NFkappaB2 (p52) promoter activation via Notch signaling pathway in rheumatoid synoviocytes.

Rheumatoid synoviocytes produce inflammatory cytokines and exhibit strong proliferation activity, which cause severe cartilage destruction in the joints. Previously, we reported that NFkappaB, a transcriptional factor that activated by mitogenic signals, was activated in rheumatoid synoviocytes. In addition, we revealed that Notch-1, the transcriptional factor that is involved in the developmental stages, was abnormally activated in rheumatoid synoviocytes. In this study, as one of the roles of Notch-1 for the chronic inflammation in RA, we examined its implication to NFkappaB2 activation in rheumatoid synoviocytes. Western blotting analysis showed that NFkappaB2 expression was elevated in rheumatoid synoviocytes compared with patients with osteoarthritis studied as control. We then analyzed NFkappaB2 binding activity to the promoter and revealed that kappaB binding complexes to the NFkappaB2 promoter was elevated in rheumatoid synoviocytes. We analyzed implication of Notch-1 signaling pathway on NFkappaB2 activation, and found that Notch-1 made a complex with recombination binding protein Jkappa (RBPJkappa), a repressor for NFkappaB2 promoter, and blocked binding of RBPJkappa to the promoter. These results indicated that as one of the mechanisms for nuclear translocated Notch-1, complex formation with RBPJkappa induces blocking of the NFkappaB2 promoter suppression, which might cause NFkappaB2 promoter activation.

Effects of interaction between parvovirus minute virus of mice NS1 and coactivator CBP on NS1- and p53-transactivation.

The non-structural protein NS1, encoded by the parvovirus minute virus of mice (MVM), is a potent regulator of viral gene expression in addition to prominent roles in viral replication and cytopathic effects associated with parvoviral infection. Although NS1 involves the modulation of viral and cellular transcription, the primary activation mechanism of MVM NS1 remains unclear. In the present study, we show here that the coactivator CREB binding protein, CBP, could potentiate NS1-mediated transcription as measured on the P38 promoter, which drives expression of the MVM capsid genes. NS1 bound to the two related cysteine-histidine-rich regions of CBP, referred to as C/H1 and C/H3, the former of which has an antagonistic function to CBP upon the NS1-transactivation. Furthermore, NS1 inhibited the synergistic transactivation by CBP and p53. These findings suggested that CBP as a transcriptional coactivator is required for NS1-mediated viral and cellular transcription in parvovirus-infected cells, resulting in cell proliferation and differentiation to achieve its lytic cycle.

Angiotensinogen, angiotensin II and adipose tissue development.

Adipose tissue is an important source of angiotensinogen (AGT). Recent evidence shows that a local renin-angiotensinogen system (RAS) is present in human adipose tissue and may act as a distinct system from plasma RAS. In obese patients, the involvement of angiotensin II (angII) as a consequence of increased plasma AGT secreted from adipose tissue has been proposed in the development of hypertension. Another role of AGT via angII in the development of adipose tissue is supported by the following: (i) in vitro, angII stimulates the production and release of prostacyclin from adipocytes, which in turn promotes the differentiation of precursor cells into adipocytes; (ii) ex vivo and in vivo, both angII and (carba)prostacyclin promote the formation of new fat cells; and (iii) AGT -/- mice exhibit a slowing down of adipose tissue development, as compared to wild-type mice. Altogether the data are consistent with an autocrine/paracrine mechanism implicating AGT, angII and prostacyclin in adipose tissue development.

Genomic expression systems on hierarchy and network leading to hypertension: long on history, short on facts.

Genomic research that is part of current DNA sequencing projects is having a great impact in many fields of study. With the discovery of sequence information for human and mouse genomes, an explosion of genetical and functional data will be revolutionizing mechanistic studies of hypertension syndrome. Assuming the availability of genome sequences, the challenge includes identifying genes, predicting the proteins they encode, determining when and where genes are expressed and how they interact, and learning how these expression and interaction profiles change in response to environmental signals. As the hypertension research community enters the post-genome era, the study of gene expression patterns and phenotypes in model animals will be a part of analyzing the role of genes involved in the pathogenesis of hypertension. The molecular dissection of hypertension genetics is a complex multidisciplinary challenge and a medical imperative. The understanding of complex disorders such as hypertension has advanced considerably through the use of genetically engineered mice. Studies of hypertension using transgenic and knockout mice have uncovered multiple potential functions of the regulatory systems controlling blood pressure. In this review, I discuss the molecular basis of the hierarchy and network of genomic expression cascades of hypertension by focusing on the functional significance of the renin-angiotensin system.

Identification of a novel isoform of poly(A) polymerase, TPAP, specifically present in the cytoplasm of spermatogenic cells.

We have identified cDNA clones encoding a testis-specific poly(A) polymerase, termed TPAP, a candidate molecule responsible for cytoplasmic polyadenylation of preexisting mRNAs in male haploid germ cells. The TPAP gene was most abundantly expressed coincident with the additional elongation of mRNA poly(A) tails in round spermatids. The amino acid sequence of TPAP contained 642 residues, and shared a high degree of identity (86%) with that of a nuclear poly(A) polymerase, PAP II. Despite the sequence conservation of functional elements, including three catalytic Asp residues, an ATP-binding site, and an RNA-binding domain, TPAP lacked an approximately 100-residue C-terminal sequence carrying one of two bipartite-type nuclear localization signals, and part of a Ser/Thr-rich domain found in PAP II. Recombinant TPAP produced by an in vitro transcription/translation system was capable of incorporating the AMP moiety from ATP into an oligo(A)(12) RNA primer in the presence of MnCl(2). Moreover, an affinity-purified antibody against the 12-residue C-terminal sequence of TPAP recognized a 70-kDa protein in the cytoplasm of spermatogenic cells. These results suggest that TPAP may participate in the additional extension of mRNA poly(A) tails in the cytoplasm of male germ cells, and may play an important role in spermiogenesis, probably through the stabilization of mRNAs.