Signal transducer and activator of transcription (Stat) 5b-mediated inhibition of insulin-like growth factor binding protein-1 gene transcription: a mechanism for repression of gene expression by growth hormone.

GH plays a central role in controlling somatic growth, tissue regeneration, and intermediary metabolism in most vertebrate species through mechanisms dependent on the regulation of gene expression. Recent studies using transcript profiling have identified large cohorts of genes whose expression is induced by GH. Other results have demonstrated that signal transducer and activator of transcription (Stat) 5b, a latent transcription factor activated by the GH receptor-associated protein kinase, Jak2, is a key agent in the GH-stimulated gene activation that leads to somatic growth. By contrast, little is known about the steps through which GH-initiated signaling pathways reduce gene expression. Here we show that Stat5b plays a critical role in the GH-regulated inhibition of IGF binding protein-1 gene transcription by impairing the actions of the FoxO1 transcription factor on the IGF binding protein-1 promoter. Additional observations using transcript profiling in the liver indicate that Stat5b may be a general mediator of GH-initiated gene repression. Our results provide a model for understanding how GH may simultaneously stimulate and inhibit the expression of different cohorts of genes via the same transcription factor, potentially explaining how GH action leads to integrated biological responses in the whole organism.

In vivo transcript profiling and phylogenetic analysis identifies suppressor of cytokine signaling 2 as a direct signal transducer and activator of transcription 5b target in liver.

The GH-activated signal transducer and activator of transcription 5b (STAT5b) is an essential regulator of somatic growth. The transcriptional response to STAT5b in liver is poorly understood. We have combined microarray-based expression profiling and phylogenetic analysis of gene regulatory regions to study the interplay between STAT5b and GH in the regulation of hepatic gene expression. The acute transcriptional response to GH in vivo after a single pulse of GH was studied in the liver of hypophysectomized rats in the presence of either constitutively active or a dominant-negative STAT5b delivered by adenoviral gene transfer. Genes showing differential expression in these two situations were analyzed for the presence of STAT5b binding sites in promoter and intronic regions that are phylogenetically conserved between rats and humans. Using this approach, we showed that most rapid transcriptional effects of GH in the liver are not results of direct actions of STAT5b. In addition, we identified novel STAT5b cis regulatory elements in genes such as Frizzled-4, epithelial membrane protein-1, and the suppressor of cytokine signaling 2 (SOCS2). Detailed analysis of SOCS2 promoter demonstrated its direct transcriptional regulation by STAT5b upon GH stimulation. A novel response element was identified within the first intron of the human SOCS2 gene composed of an E-box followed by tandem STAT5b binding sites, both of which are required for full GH responsiveness. In summary, we demonstrate the power of combining transcript profiling with phylogenetic sequence analysis to define novel regulatory paradigms.

Characterization of distinct Stat5b binding sites that mediate growth hormone-stimulated IGF-I gene transcription.

A key agent in the anabolic actions of growth hormone (GH) is insulin-like growth factor-I (IGF-I), a 70-amino acid secreted protein with direct effects on somatic growth and tissue maintenance and repair. GH rapidly and potently stimulates IGF-I gene transcription by mechanisms independent of new protein synthesis, and recent studies have linked the transcription factor Stat5b to a regulatory network connecting the activated GH receptor on the cell membrane to the IGF-I gene in the nucleus. Here we analyze two distinct conserved GH response elements in the rat IGF-I locus that contain paired Stat5b sites. Each response element binds Stat5b in vivo in a GH-dependent way, as assessed by chromatin immunoprecipitation assays, and consists of one high affinity and one lower affinity Stat5b site, as determined by both qualitative and quantitative protein-DNA binding studies. In biochemical reconstitution experiments, both response elements are able to mediate GH-stimulated and Stat5b-dependent transcription when fused to a reporter gene containing either the major IGF-I promoter or a minimal neutral promoter, although the paired Stat5b sites located in the second IGF-I intron were more than twice as effective as the response element that mapped approximately 73 kb 5' to the IGF-I exon 1. Taken together, our results define the initial molecular architecture of a complicated GH-regulated transcriptional pathway, and suggest that apparently redundant hormone response elements provide a mechanism for amplifying GH action at a physiologically important target gene.

Comparison of enzymatic properties between hPADI2 and hPADI4.

In the sera of rheumatoid arthritis (RA) patients, autoantibodies directed to citrullinated proteins are found with high specificity for RA. Peptidylarginine deiminases (PADIs) are enzymes responsible for protein citrullination. Among many isoforms of PADIs, only PADI4 has been identified as an RA-susceptibility gene. To understand the mechanisms of the initiation and progression of RA, we compared the properties of two PADIs, human PADI2 and human PADI4, which are present in the synovial tissues of RA patients. We confirmed their precise distribution in the RA synovium and compared the stability, Ca2+ dependency, optimal pH range, and substrate specificity. Small but significant differences were found in the above-mentioned properties between hPADI2 and hPADI4. Using LC/MS/MS analysis, we identified the sequences in human fibrinogen indicating that hPADI2 and hPADI4 citrullinate in different manners. Our results indicate that hPADI2 and hPADI4 have different roles under physiological and pathological conditions. Further studies are needed for the better understanding of the role of hPADIs in the initiation and progression of RA.

[Angptl3(angiopoietin-like 3)].

Angiopoietin-like 3(Angptl3)-deficiency results in abnormally low lipid levels in mice. Angptl3-deficient mice showed enhanced very low density lipoprotein(VLDL) clearance compared with wild-type mice. Recombinant human ANGPTL3 protein inhibited lipoprotein lipase(LPL) activity in vitro, suggesting that Angptl3 affects VLDL triglyceride clearance by interfering with LPL activity. Liver X receptor(LXR) ligands and LXR-retinoid X receptor complex increased the promoter activity of Angptl3 gene. LXR ligand treatment did not increased plasma triglyceride levels in Angptl3-deficient mice at all, suggesting that hypertriglyceridemia associated with LXR ligand treatment is due to overproduction of Angptl3. Angptl3-deficiency decreases both plasma lipid levels and aorta atherogenic lesions in apoE-deficient mice, suggesting that repression of ANGPTL3 could decrease plasma lipid levels and could be protective against atherosclerosis.

An intronic SNP in a RUNX1 binding site of SLC22A4, encoding an organic cation transporter, is associated with rheumatoid arthritis.

Rheumatoid arthritis is a common inflammatory disease with complex genetic components. We investigated the genetic contribution of the cytokine gene cluster in chromosome 5q31 to susceptibility to rheumatoid arthritis in the Japanese population by case-control linkage disequilibrium (LD) mapping using single nucleotide polymorphisms (SNPs). Here we report that there is significant association between rheumatoid arthritis and the organic cation transporter gene SLC22A4 (P = 0.000034). We show that expression of SLC22A4 is specific to hematological and immunological tissues and that SLC22A4 is also highly expressed in the inflammatory joints of mice with collagen-induced arthritis. A SNP affects the transcriptional efficiency of SLC22A4 in vitro, owing to an allelic difference in affinity to Runt-related transcription factor 1 (RUNX1), a transcriptional regulator in the hematopoietic system. A SNP in RUNX1 is also strongly associated with rheumatoid arthritis (P = 0.00035). Our data indicate that the regulation of SLC22A4 expression by RUNX1 is associated with susceptibility to rheumatoid arthritis, which may represent an example of an epistatic effect of two genes on this disorder.

Protein region important for regulation of lipid metabolism in angiopoietin-like 3 (ANGPTL3): ANGPTL3 is cleaved and activated in vivo.

Angiopoietin-like 3 (ANGPTL3) is a secreted protein that is mainly expressed in the liver and regulates lipid metabolism by inhibiting the lipolysis of triglyceriderich lipoproteins. Using deletion mutants of human ANGPTL3, we demonstrated that the N-terminal coiled-coil domain-containing fragment-(17-207) and not the C-terminal fibrinogen-like domain-containing fragment-(207-460) increased the plasma triglyceride levels in mice. We also found that the N-terminal region 17-165 was required to increase plasma triglyceride levels in mice and that a substitution of basic amino acid residues in the region 61-66 of the fragment showed no increase in the plasma triglyceride levels and no inhibition of lipolysis by lipoprotein lipase. In addition, when we analyzed ANGPTL3 in human plasma, we detected cleaved fragments of ANGPTL3. By analyzing recombinant ANGPTL3 in mouse plasma, we found that it was cleaved at two sites, Arg221 downward arrow Ala222 and Arg224 downward arrow Thr225, which are located in the linker region between the coiled-coil domain and the fibrinogen-like domain. Furthermore, a cleavage-resistant mutant of ANGPTL3 was determined to be less active than wild-type ANGPTL3 in increasing mouse plasma triglyceride levels but not in inhibiting lipoprotein lipase activity. These findings suggest that the cleavage of ANGPTL3 is important for the activation of ANGPTL3 in vivo.

Functional haplotypes of PADI4, encoding citrullinating enzyme peptidylarginine deiminase 4, are associated with rheumatoid arthritis.

Individuals with rheumatoid arthritis frequently have autoantibodies to citrullinated peptides, suggesting the involvement of the peptidylarginine deiminases citrullinating enzymes (encoded by PADI genes) in rheumatoid arthritis. Previous linkage studies have shown that a susceptibility locus for rheumatoid arthritis includes four PADI genes but did not establish which PADI gene confers susceptibility to rheumatoid arthritis. We used a case-control linkage disequilibrium study to show that PADI type 4 is a susceptibility locus for rheumatoid arthritis (P = 0.000008). PADI4 was expressed in hematological and rheumatoid arthritis synovial tissues. We also identified a haplotype of PADI4 associated with susceptibility to rheumatoid arthritis that affected stability of transcripts and was associated with levels of antibody to citrullinated peptide in sera from individuals with rheumatoid arthritis. Our results imply that the PADI4 haplotype associated with susceptibility to rheumatoid arthritis increases production of citrullinated peptides acting as autoantigens, resulting in heightened risk of developing the disease.

A decreased expression of angiopoietin-like 3 is protective against atherosclerosis in apoE-deficient mice.

KK/Snk mice (previously KK/San) possessing a recessive mutation (hypl) of the angiopoietin-like 3 (Angptl3) gene homozygously exhibit a marked reduction of VLDL due to the decreased Angptl3 expression. Recently, we proposed that Angptl3 is a new class of lipid metabolism modulator regulating VLDL triglyceride (TG) levels through the inhibition of lipoprotein lipase (LPL) activity. In this study, to elucidate the role of Angptl3 in atherogenesis, we investigated the effects of hypl mutation against hyperlipidemia and atherosclerosis in apolipoprotein E knockout (apoEKO) mice. ApoEKO mice with hypl mutation (apoEKO-hypl) exhibited a significant reduction of VLDL TG, VLDL cholesterol, and plasma apoB levels compared with apoEKO mice. Hepatic VLDL TG secretion was comparable between both apoE-deficient mice. Turnover studies revealed that the clearance of both [3H]TG-labeled and 125I-labeled VLDL was significantly enhanced in apoEKO-hypl mice. Postprandial plasma TG levels also decreased in apoEKO-hypl mice. Both LPL and hepatic lipase activities in the postheparin plasma increased significantly in apoEKO-hypl mice, explaining the enhanced lipid metabolism. Furthermore, apoEKO-hypl mice developed 3-fold smaller atherogenic lesions in the aortic sinus compared with apoEKO mice. Taken together, the reduction of Angptl3 expression is protective against hyperlipidemia and atherosclerosis, even in the absence of apoE, owing to the enhanced catabolism and clearance of TG-rich lipoproteins.

Angiopoietin-like protein 3, a hepatic secretory factor, activates lipolysis in adipocytes.

Our previous work identified a genetic mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/Snk mice (previously KK/San), a mutant strain of KK obese mice. KK/Snk had significantly lower plasma triglyceride and free fatty acid (FFA) than KK mice. Human ANGPTL3 treatment increased both plasma triglyceride and FFA. ANGPTL3 inhibited the activity of lipoprotein lipase, which accounted for the increase of plasma triglyceride. The mechanism how ANGPTL3 affects plasma FFA has not been known. The current study reveals that ANGPTL3 targets on adipose cells and induces lipolysis. Both plasma FFA and glycerol decreased in KK/Snk and increased by the treatment of human ANGPTL3. Specific bindings of ANGPTL3 to adipose cells were shown using fluorescence-labeled protein visually and 125I-labeled protein by the binding analysis. Furthermore, ANGPTL3 activated the lipolysis to stimulate the release of FFA and glycerol from adipocytes. We conclude that ANGPTL3 is a liver-derived lipolytic factor targeting on adipocyte.

Angiopoietin-like protein 4 is a potent hyperlipidemia-inducing factor in mice and inhibitor of lipoprotein lipase.

Studies with KK/San, obese and diabetic model mice having a unique hypotriglyceridemia phenotype, revealed that angiopoietin-like protein 3 (ANGPTL3) regulates lipid metabolism in mice. To determine the lipid-modulating role of other ANGPTLs, we focused on ANGPTL4, which overall shows a significant similarity to ANGPTL3. Surprisingly, an intravenous injection of the ANGPTL4 protein in KK/San mice rapidly increased the circulating plasma lipid levels at a higher rate than ANGPTL3 protein. Furthermore, the ANGPTL4 protein inhibited the lipoprotein lipase activity in vitro.

ANGPTL3 decreases very low density lipoprotein triglyceride clearance by inhibition of lipoprotein lipase.

KK/San is a mutant mouse strain established in our laboratory from KK obese mice. KK/San mice show low plasma lipid levels compared with wild-type KK mice despite showing signs of hyperglycemia and hyperinsulinemia. Recently, we identified a mutation in the gene encoding angiopoietin-like protein 3 (Angptl3) in KK/San mice, and injection of adenoviruses encoding Angptl3 or recombinant ANGPTL3 protein to mutant KK/San mice raised plasma lipid levels. To elucidate the regulatory mechanism of ANGPTL3 on lipid metabolism, we focused on the metabolic pathways of triglyceride in the present study. Overexpression of Angptl3 in KK/San mice resulted in a marked increase of triglyceride-enriched very low density lipoprotein (VLDL). In vivo studies using Triton WR1339 revealed that there is no significant difference between mutant and wild-type KK mice in the hepatic VLDL triglyceride secretion rate. However, turnover studies using radiolabeled VLDL revealed that the clearance of (3)H-triglyceride-labeled VLDL was significantly enhanced in KK/San mice, whereas the clearance of (125)I-labeled VLDL was only slightly enhanced. In vitro analysis of recombinant protein revealed that ANGPTL3 directly inhibits LPL activity. These data strongly support the hypothesis that ANGPTL3 is a new class of lipid metabolism modulator, which regulates VLDL triglyceride levels through the inhibition of LPL activity.

Angptl3 regulates lipid metabolism in mice.

The KK obese mouse is moderately obese and has abnormally high levels of plasma insulin (hyperinsulinemia), glucose (hyperglycemia) and lipids (hyperlipidemia). In one strain (KK/San), we observed abnormally low plasma lipid levels (hypolipidemia). This mutant phenotype is inherited recessively as a mendelian trait. Here we report the mapping of the hypolipidemia (hypl) locus to the middle of chromosome 4 and positional cloning of the autosomal recessive mutation responsible for the hypolipidemia. The hypl locus encodes a unique angiopoietin-like lipoprotein modulator, which we named Allm1. It is identical to angiopoietin-like protein 3, encoded by Angptl3, and has a highly conserved counterpart in humans. Overexpression of Angptl3 or intravenous injection of the purified protein in KK/San mice elicited an increase in circulating plasma lipid levels. This increase was also observed in C57BL/6J normal mice. Taken together, these data suggest that Angptl3 regulates lipid metabolism in animals.