Regulation of gluconeogenesis by Krüppel-like factor 15.

In the postabsorptive state, certain tissues, including the brain, require glucose as the sole source of energy. After an overnight fast, hepatic glycogen stores are depleted, and gluconeogenesis becomes essential for preventing life-threatening hypoglycemia. Mice with a targeted deletion of KLF15, a member of the Krüppel-like family of transcription factors, display severe hypoglycemia after an overnight (18 hr) fast. We provide evidence that defective amino acid catabolism promotes the development of fasting hypoglycemia in KLF15-/- mice by limiting gluconeogenic substrate availability. KLF15-/- liver and skeletal muscle show markedly reduced mRNA expression of amino acid-degrading enzymes. Furthermore, the enzymatic activity of alanine aminotransferase (ALT), which converts the critical gluconeogenic amino acid alanine into pyruvate, is decreased (approximately 50%) in KLF15-/- hepatocytes. Consistent with this observation, intraperitoneal injection of pyruvate, but not alanine, rescues fasting hypoglycemia in KLF15-/- mice. We conclude that KLF15 plays an important role in the regulation of gluconeogenesis.

Antifibrotic properties of c-Ski and its regulation of cardiac myofibroblast phenotype and contractility.

Cardiac myofibroblasts are key players in chronic remodeling of the cardiac extracellular matrix, which is mediated in part by elevated transforming growth factor-ß1 (TGF-ß1). The c-Ski proto-oncoprotein has been shown to modify TGF-ß1 post-receptor signaling through receptor-activated Smads (R-Smads); however, little is known about how c-Ski regulates fibroblast phenotype and function. We sought to elucidate the function of c-Ski in primary cardiac myofibroblasts using a c-Ski overexpression system. Cardiac myofibroblasts expressed three forms of c-Ski with the predominant band at 105 kDa, and adenoviral c-Ski treatment resulted in overexpression of 95-kDa c-Ski in cellular nuclei. Exogenous c-Ski led to significant inhibition of type I collagen secretion and myofibroblast contractility using two-dimensional semifloating gel contraction assay in both basal and with TGF-ß1 (10 ng/ml for 24 h) stimulation. Overexpressed c-Ski did not inhibit nuclear translocation of phosphorylated R-Smad2, despite their binding, as demonstrated by immunoprecipitation. Acute treatment of primary myofibroblasts with TGF-ß1 in vitro revealed a marked nuclear shuttling of c-Ski at 24 and 48 h following stimulation. Remarkably, overexpression of c-Ski led to a stepwise reduction of the myofibroblast marker α-smooth muscle actin with increasing multiplicity of infection, and these results indicate that 95-kDa c-Ski overexpression may effect a loss of the myofibroblastic phenotype. Furthermore, adenovirus (Ad) for hemagglutinin-tagged c-Ski infection led to a reduction in the number of myofibroblasts versus Ad-LacZ-infected and uninfected controls, due to induction of apoptosis. Finally, we observed a significant increase in 105-kDa c-Ski in the cytosolic fraction of cells of the infarct scar and adjacent remnant myocardium vs. noninfarcted controls.

Kruppel-like factor 15 regulates smooth muscle response to vascular injury--brief report.

To determine the role of Kruppel-like factor (KLF) 15, a zinc finger transcriptional factor that is expressed in vascular smooth muscle cells (VSMCs) in vascular biology. VSMCs respond to mechanical injury via a tightly orchestrated series of gene regulatory events. KLF15 is broadly expressed in both arterial and venous vascular beds in a VSMC restricted fashion. KLF15 expression is markedly reduced by both pharmacological and mechanical stimuli. To examine the specific role of KLF15 in the vascular response to injury, we performed femoral artery wire injury in KLF15(-/-) and wild-type mice. KLF15(-/-) mice develop exaggerated neointimal growth, with evidence of increased SMC proliferation and migration within the neointima. In concordance, gain and loss of function studies in isolated VSMCs demonstrate that KLF15 can directly inhibit SMC proliferation and migration. To our knowledge, these data are the first to identify KLF15 as a novel inhibitor of VSMC proliferation and migration and to implicate this factor as a critical regulator of the vascular response to injury.

The genetic differentiation of a cricket (Velarifictorus micado) with two modes of life cycle in East Asia after the middle Pleistocene and the invasion origin of the United States of America.

The cricket Velarifictorus micado is widely distributed in East Asia and colonized the United States of America (the USA) in 1959. It has two life cycles: egg and nymph diapause. We aimed to investigate the biogeographic boundary between them and determine when and why V. micado diverged. Mitochondrial fragments including COI and CytB were used for haplotype network, demographic analysis, and divergence time estimation in individuals of East Asia. We selected several samples from the USA to find out the colonization origin. The haplotype network indicated there were three lineages based on COI, NE lineage (the egg diapause and mainly distributed in the northern regions), SE lineage (the egg diapause and mainly distributed in the southern regions), and SN lineage (the nymph diapause and mainly distributed in the southern regions). The molecular chronograms indicated that the first divergence of V. micado into two main lineages, NE and southern lineages (SE and SN), was essentially bounded by the Yangtze River. It occurred around ~0.79 Ma (95% HPD: 1.13-0.46 Ma) in the Middle Pleistocene Transition. This was followed by the divergence of the southern lineage into two sublineages, SE and SN lineage, occurred around ~0.50 Ma (95% HPD: 0.71-0.25 Ma), corresponding to the time of development of glaciers in various parts of the Qinghai-Tibet Plateau (QTP) (0.73-0.46 Ma). SE lineage might originate from southwestern China based on the comparison between the haplotype network based on COI and CytB. Our study suggested that divergences of lineages have twice co-occurred with tendency of cooling climatic in Asia after the Mid-Pleistocene, and the life-history strategy may play an important role in lineage diversification. Additionally, our results indicated that the USA populations were revealed at least twice separate Asian invasions. These both belonged to the egg diapause, which might provide a new perspective for invasion control.

A new species of genus Sichuana from Sichuan, China (Orthoptera: Tettigoniidae: Tettigoniinae).

In this study, we describe one new species, Sichuana feicui He sp. nov., from Mao County, Sichuan Province, China. This new species is different from S. cryptospina in the shape of cercus, and the color pattern of hind femur. The type specimens are deposited in Museum of Biology, East China Normal University (ECNU).

The Kruppel-like factor KLF15 inhibits connective tissue growth factor (CTGF) expression in cardiac fibroblasts.

Cardiac fibrosis is a hallmark feature of pathologic remodeling of the heart in response to hemodynamic or neurohormonal stress. Accumulating evidence implicates connective tissue growth factor (CTGF) as a key mediator of this process. Our group has previously identified Kruppel-Like Factor 15 (KLF15) as an important regulator of cardiac remodeling in response to stress; however, the role of this transcription factor in cardiac fibrosis has not been reported. Here we provide evidence that treatment of neonatal rat ventricular fibroblasts (NRVFs) with the potent pro-fibrotic agent Transforming Growth Factor-beta1 (TGFbeta1) strongly reduces KLF15 expression while inducing the pro-fibrotic factor CTGF. Adenoviral overexpression of KLF15 inhibits basal and TGFbeta1-induced CTGF expression in NRVFs. Furthermore, hearts from KLF15-/- mice subjected to aortic banding exhibited increased CTGF levels and fibrosis. From a mechanistic standpoint, KLF15 inhibits basal and TGFbeta1-mediated induction of the CTGF promoter. Chromatin Immunoprecipitation (ChIP) and electrophoretic mobility shift assays demonstrate that KLF15 inhibits recruitment of the co-activator P/CAF to the CTGF promoter with no significant effect on Smad3-DNA binding. Consistent with this observation, KLF15 mediated repression of the CTGF promoter is rescued by P/CAF overexpression. Our result implicates KLF15 as a novel negative regulator of CTGF expression and cardiac fibrosis.

Transcriptional regulators of angiogenesis.

Angiogenesis, the process by which new blood vessels develop from a pre-existing vascular network, is essential for normal development and in certain physiological states. Inadequate or excessive angiogenesis has been incriminated in a number of pathologic states. For example, vaso-occlusive disease arising from atherosclerosis can lead to ischemia, a situation in which enhanced angiogenesis would be beneficial. Conversely, overzealous angiogenesis can contribute to tumor development and in this case inhibition of angiogenesis is desirable. Thus, strategies to induce or inhibit angiogenesis are of considerable therapeutic interest.

Analysis of comparative genomic hybridization and loss of heterozygosity in 43 primary gastric carcinomas.

OBJECTIVE: To investigate common chromosomal changes and the LOH frequency of microsatellite loci in primary gastric cancer samples in order to locate the deleted regions in which human gastric cancer related genes might exist. METHODS: Comparative genomic hybridization (CGH) was used to define global chromosomal aberrations in 43 primary gastric tumors. Based on the results of CGH, analysis of loss of heterozygosity (LOH) was performed in chromosome 19 in which the loss was first discovered in the gastric cancers. The PCR-based approach was used to investigate 22 loci, which are spaced at 1.1 - 10.9 cM intervals throughout chromosome 19. The amplified PCR fragments were subjected to electrophoresis in PAGE gel and analyzed with Genescan trade mark and Genotyper trade mark. RESULTS: CGH analysis revealed gains in chromosome 3p (8/43), 8q (8/43), 20 [20 (9/43), 20p (7/43), 20q (4/43)], 12q (16/43), 13q (12/43) and losses in 19 [19 (15/43)], 7 [17 (8/43), 17p (10/43)], 16 (10/43) and 1p (11/43). Among the 43 evaluated samples, the most frequent LOH was detected at locus D19S571 (27.81%). CONCLUSIONS: The tumorigenesis of gastric cancer includes several chromosomal changes. The aberration of chromosome 19 was the first common change founded in gastric cancer. The region near the D19S571 might harbor potential genes related to the tumorigenesis of gastric cancer.

[Genescan analysis of non-small cell lung cancer in the long arm of chromosome 6].

OBJECTIVE: To investigate if there are microsatellite loci in the long arm of chromosome 6 that have close relationship with non-small cell lung cancer. METHODS: Multiple PCR approach was used to analyze the 18 loci in the long arm of chromosome 6. The PCR products were analyzed in PAGE, and then the electrophoresis maps were analyzed with Gene Scan(TM) and Genotyper(TM). RESULTS: There were different frequencies of loss of heterozygosity (LOH) in different loci (varying from 3.85% to 38.45%). The total frequency of LOH in 41 gastric cancers was 58.5%(24/41). Eight loci with the LOH frequency higher than 20% were mainly located in 2 regions: 6q24 and 6q27. The accurate location is 6q24-6q25.3 [D6S1699(35%), D6S409(23.33%), D6S441(33.33%)] and 6q26-27 [D6S1550(38.45%), D6S264(20%), D6S1585(25%), D6S446(33.33%), D6S281(30.77%)]. CONCLUSION: There may be tumor suppressor genes located in the region of 6q24 and 6q27, which have close relationship with non-small cell lung cancer.

Regulation of collagen synthesis by inhibitory Smad7 in cardiac myofibroblasts.

Transforming growth factor-beta(1) (TGF-beta(1)) signal and downstream Smads play an important role in tissue fibrosis and matrix remodeling in various etiologies of heart failure. Inhibitory Smad7 (I-Smad7) is an inducible regulatory Smad protein that antagonizes TGF-beta(1) signal mediated via direct abrogation of R-Smad phosphorylation. The effect of ectopic I-Smad7 on net collagen production was investigated using hydroxyproline assay. Adenovirus-mediated I-Smad7 gene (at 100 multiplicity of infection) transfer was associated with significant decrease of collagen synthesis in the presence and absence of TGF-beta(1) in primary rat cardiac myofibroblasts. In I-Smad7-infected cells, we also observed the ablation of TGF-beta(1)-induced R-Smad2 phosphorylation vs. LacZ controls. Overdriven I-Smad7 was associated with significantly increased expression of immunoreactive 65-kDa matrix metalloproteinase-2 (MMP-2) protein in culture medium of myofibroblast compared with LacZ-infected cells. Expression of the 72-kDa MMP-2 variant, e.g., the inactive form, was not altered by exogenous I-Smad7 transfection/overexpression. Furthermore, I-Smad7 overexpression was associated with a significant increase and decrease in expression of p27 and phospho-Rb protein, respectively, as well as reduced [(3)H]thymidine incorporation vs. Ad-LacZ-infected controls. We suggest that negative modulation of R-Smad phosphorylation by ectopic I-Smad7 may contribute to the downregulation of collagen in cardiac myofibroblasts and may suppress the proliferation of these cells. Thus treatments targeting the collagen deposition by overexpression of I-Smad7 may provide a new therapeutic strategy for cardiac fibrosis.

Kruppel-like factor 15 is a regulator of cardiomyocyte hypertrophy.

Cardiac hypertrophy is a common response to injury and hemodynamic stress and an important harbinger of heart failure and death. Herein, we identify the Kruppel-like factor 15 (KLF15) as an inhibitor of cardiac hypertrophy. Myocardial expression of KLF15 is reduced in rodent models of hypertrophy and in biopsy samples from patients with pressure-overload induced by chronic valvular aortic stenosis. Overexpression of KLF15 in neonatal rat ventricular cardiomyocytes inhibits cell size, protein synthesis and hypertrophic gene expression. KLF15-null mice are viable but, in response to pressure overload, develop an eccentric form of cardiac hypertrophy characterized by increased heart weight, exaggerated expression of hypertrophic genes, left ventricular cavity dilatation with increased myocyte size, and reduced left ventricular systolic function. Mechanistically, a combination of promoter analyses and gel-shift studies suggest that KLF15 can inhibit GATA4 and myocyte enhancer factor 2 function. These studies identify KLF15 as part of a heretofore unrecognized pathway regulating the cardiac response to hemodynamic stress.

Decreased Smad 7 expression contributes to cardiac fibrosis in the infarcted rat heart.

We examined the role of the transforming growth factor (TGF)-beta(1) signaling inhibitor Smad 7 in cardiac fibrosis. TGF-beta(1) (10 ng/ml) was found to increase cytosolic Smad 7 expression in primary adult rat fibroblasts and induce rapid nuclear export of exogenous Smad 7 in COS-7 cells. Furthermore, overexpression of Smad 7 in primary adult fibroblasts was associated with suppressed collagen type I and III expression. We detected Smad 7, phosphorylated Smad 2, TGF-beta type I receptor (TbetaRI), and TGF-beta(1) proteins in postmyocardial infarct (MI) rat hearts. In 2 and 4 wk post-MI hearts, Smad 7 and TbetaRI expression were decreased in scar tissue, whereas TGF-beta(1) expression was increased in scar and viable tissue. In the 8 wk post-MI heart, Smad 7 expression was decreased in both scar tissue and myocardium remote to the infarct scar. Finally, we confirmed that these changes are paralleled by decreased expression of cytosolic phosphorylated receptor-regulated Smad 2 in 4-wk viable myocardium and in 2- and 4-wk infarct scar tissues. Taken together, our data imply that decreased inhibitory Smad 7 signal in cardiac fibroblasts may play a role in the pathogenesis of cardiac fibrosis in the post-MI heart.

A new haplogroup pattern displayed in Fujian Han in China.

Human Y-chromosomal binary polymorphisms have been considered to preserve the paternal genetic legacy and provide evidence on human evolution and the genetic relationships among and demographic history of different populations. To reveal the genetic origin and immigration of the Fujian Han, 13 binary markers on the Y chromosome were used to screen Fujian Han by allele-specific polymerase chain reaction. The results indicated that the M9G marker was highly prevalent (96.20%), suggesting a significant genetic drift. In addition, M122C frequency was only 22.78%, and M45A and M103T were default. The distinctive haplogroup frequencies (H1, H5, and H6/7/8) imply that the haplogroup pattern is a relatively ancestral and interim type.

Distribution of the CCR5 gene 32-basepair deletion in 11 Chinese populations.

A mutant allele of the chemokine receptor gene CCR5 bearing a 32-basepair deletion (delta 32CCR5) could increase the resistance to HIV-1 infection or delayed progression to AIDS. The frequency of this mutation is higher in Europeans than in Asians. To investigate the distribution of this polymorphism in China, 715 individuals from 11 Chinese populations were screened by PCR, including the Han and 10 other ethnic groups. The delta 32CCR5 gene was found in 16 individuals from 5 ethnic groups. All of them were heterozygous. The frequency of the mutant alleles of delta 32CCR5 is low in China and reflects (or might reflect) ancestral gene flow from Europe to Chinese ethnic groups and recent intermarriage within the ethnic groups.