Adipogenic and myogenic gene expression in rotator cuff muscle of the sheep after tendon tear

Chronic rotator cuff tendon tears lead to fatty infiltration and muscle atrophy with impaired physiological functions of the affected muscles. However, the cellular and molecular mechanisms of corresponding pathophysiological processes remain unknown. The purpose of this study was to characterize the expression pattern of adipogenic (PPARgamma, C/EBPbeta) and myogenic (myostatin, myogenin, Myf-5) transcription factors in infraspinatus muscle of sheep after tenotomy, implantation of a tension device, refixation of the tendon, and rehabilitation, reflecting a model of chronic rotator cuff tears. In contrast to human patients, the presented sheep model allows a temporal evaluation of the expression of a given marker in the same individual over time. Semiquantitative RT/PCR analysis of PPARgammaã, myostatin, myogenin, Myf-5, and C/EBPbeta transcript levels was carried out with sheep muscle biopsy-derived total RNA. We found a significantly increased expression of Myf-5 and PPARgamma after tenotomy and a significant change for Myf-5 and C/EBPbeta after continuous traction and refixation. This experimental sheep model allows the molecular analysis of pathomechanisms of muscular changes after rotator cuff tear. The results point to a crucial role of the transcription factors PPARgamma, C/EBPbeta, and Myf-5 in impairment and regeneration of rotator cuff muscles after tendon tears in sheep.

The tumor suppressor gene hypermethylated in cancer 1 is transcriptionally regulated by E2F1

The Hypermethylated in Cancer 1 (HIC1) gene encodes a zinc finger transcriptional repressor that cooperates with p53 to suppress cancer development. We and others recently showed that HIC1 is a transcriptional target of p53. To identify additional transcriptional regulators of HIC1, we screened a set of transcription factors for regulation of a human HIC1 promoter reporter. We found that E2F1 strongly activates the full-length HIC1 promoter reporter. Promoter deletions and mutations identified two E2F responsive elements in the HIC1 core promoter region. Moreover, in vivo binding of E2F1 to the HIC1 promoter was shown by chromatin immunoprecipitation assays in human TIG3 fibroblasts expressing tamoxifen-activated E2F1. In agreement, activation of E2F1 in TIG3-E2F1 cells markedly increased HIC1 expression. Interestingly, expression of E2F1 in the p53(-/-) hepatocellular carcinoma cell line Hep3B led to an increase of endogenous HIC1 mRNA, although bisulfite genomic sequencing of the HIC1 promoter revealed that the region bearing the two E2F1 binding sites is hypermethylated. In addition, endogenous E2F1 induced by etoposide treatment bound to the HIC1 promoter. Moreover, inhibition of E2F1 strongly reduced the expression of etoposide-induced HIC1. In conclusion, we identified HIC1 as novel E2F1 transcriptional target in DNA damage responses.

Role of DNA methylation in the tissue-specific expression of the CYP17A1 gene for steroidogenesis in rodents

The CYP17A1 gene is the qualitative regulator of steroidogenesis. Depending on the presence or absence of CYP17 activities mineralocorticoids, glucocorticoids or adrenal androgens are produced. The expression of the CYP17A1 gene is tissue as well as species-specific. In contrast to humans, adrenals of rodents do not express the CYP17A1 gene and have therefore no P450c17 enzyme for cortisol production, but produce corticosterone. DNA methylation is involved in the tissue-specific silencing of the CYP17A1 gene in human placental JEG-3 cells. We investigated the role of DNA methylation for the tissue-specific expression of the CYP17A1 gene in rodents. Rats treated with the methyltransferase inhibitor 5-aza-deoxycytidine excreted the cortisol metabolite tetrahydrocortisol in their urine suggesting that treatment induced CYP17 expression and 17alpha-hydroxylase activity through demethylation. Accordingly, bisulfite modification experiments identified a methylated CpG island in the CYP17 promoter in DNA extracted from rat adrenals but not from testes. Both methyltransferase and histone deacetylase inhibitors induced the expression of the CYP17A1 gene in mouse adrenocortical Y1 cells which normally do not express CYP17, indicating that the expression of the mouse CYP17A1 gene is epigenetically controlled. The role of DNA methylation for CYP17 expression was further underlined by the finding that a reporter construct driven by the mouse -1041 bp CYP17 promoter was active in Y1 cells, thus excluding the lack of essential transcription factors for CYP17 expression in these adrenal cells.

Lethal respiratory failure and mild primary hypothyroidism in a term girl with a de novo heterozygous mutation in the TITF1/NKX2.1 gene

CONTEXT: Thyroid transcription factor 1 (TITF1/NKX2.1) is expressed in the thyroid, lung, ventral forebrain, and pituitary. In the lung, TITF1/NKX2.1 activates the expression of genes critical for lung development and function. Titf/Nkx2.1(-/-) mice have pituitary and thyroid aplasia but also impairment of pulmonary branching. Humans with heterozygous TITF1/NKX2.1 mutations present with various combinations of primary hypothyroidism, respiratory distress, and neurological disorders. OBJECTIVE: The objective of the study was to report clinical and molecular studies of the first patient with lethal neonatal respiratory distress from a novel heterozygous TITF1/NKX2.1 mutation. Participant: This girl, the first child of healthy nonconsanguineous French-Canadian parents, was born at 41 wk. Birth weight was 3,460 g and Apgar scores were normal. Soon after birth, she developed acute respiratory failure with pulmonary hypertension. At neonatal screening on the second day of life, TSH was 31 mU/liter (N <15) and total T(4) 245 nmol/liter (N = 120-350). Despite mechanical ventilation, thyroxine, surfactant, and pulmonary vasodilators, the patient died on the 40th day. RESULTS: Histopathology revealed pulmonary tissue with low alveolar counts. The thyroid was normal. Sequencing of the patient's lymphocyte DNA revealed a novel heterozygous TITF1/NKX2.1 mutation (I207F). This mutation was not found in either parent. In vitro, the mutant TITF-1 had reduced DNA binding and transactivation capacity. CONCLUSION: This is the first reported case of a heterozygous TITF1/NKX2.1 mutation leading to neonatal death from respiratory failure. The association of severe unexplained respiratory distress in a term neonate with mild primary hypothyroidism is the clue that led to the diagnosis.

Gene array of primary human osteoblasts exposed to enamel matrix derivative in combination with a natural bone mineral

OBJECTIVES The application of an enamel matrix derivative (EMD) for regenerative periodontal surgery has been shown to promote formation of new cementum, periodontal ligament, and alveolar bone. In intrabony defects with a complicated anatomy, the combination of EMD with various bone grafting materials has resulted in additional clinical improvements, but the initial cellular response of osteoblasts coming in contact with these particles have not yet been fully elucidated. The objective of the present study was to evaluate the in vitro effects of EMD combined with a natural bone mineral (NBM) on a wide variety of genes, cytokines, and transcription factors and extracellular matrix proteins on primary human osteoblasts. MATERIAL AND METHODS Primary human osteoblasts were seeded on NBM particles pre-coated with versus without EMD and analyzed for gene differences using a human osteogenesis gene super-array (Applied Biosystems). Osteoblast-related genes include those transcribed during bone mineralization, ossification, bone metabolism, cell growth and differentiation, as well as gene products representing extracellular matrix molecules, transcription factors, and cell adhesion molecules. RESULTS EMD promoted gene expression of various osteoblast differentiation markers including a number of collagen types and isoforms, SMAD intracellular proteins, osteopontin, cadherin, alkaline phosphatase, and bone sialoprotein. EMD also upregulated a variety of growth factors including bone morphogenetic proteins, vascular endothelial growth factors, insulin-like growth factor, transforming growth factor, and their associated receptor proteins. CONCLUSION The results from the present study demonstrate that EMD is capable of activating a wide variety of genes, growth factors, and cytokines when pre-coated onto NBM particles. CLINICAL RELEVANCE The described in vitro effects of EMD on human primary osteoblasts provide further biologic support for the clinical application of a combination of EMD with NBM particles in periodontal and oral regenerative surgery.

Hepatic gene expression involved in glucose and lipid metabolism in transition cows: effects of fat mobilization during early lactation in relation to milk performance and metabolic changes.

Insufficient feed intake during early lactation results in elevated body fat mobilization to meet energy demands for milk production. Hepatic energy metabolism is involved by increasing endogenous glucose production and hepatic glucose output for milk synthesis and by adaptation of postcalving fuel oxidation. Given that cows differ in their degree of fat mobilization around parturition, indicated by variable total liver fat concentration (LFC), the study investigated the influence of peripartum fat mobilization on hepatic gene expression involved in gluconeogenesis, fatty acid oxidation, ketogenesis, and cholesterol synthesis, as well as transcriptional factors referring to energy metabolism. German Holstein cows were grouped according to mean total LFC on d 1, 14, and 28 after parturition as low [<200mg of total fat/g of dry matter (DM); n=10], medium (200-300 mg of total fat/g of DM; n=10), and high (>300 mg of total fat/g of DM; n=7), indicating fat mobilization during early lactation. Cows were fed total mixed rations ad libitum and held under equal conditions. Liver biopsies were taken at d 56 and 15 before and d 1, 14, 28, and 49 after parturition to measure mRNA abundances of pyruvate carboxylase (PC); phosphoenolpyruvate carboxykinase; glucose-6-phosphatase; propionyl-coenzyme A (CoA) carboxylase α; carnitine palmitoyl-transferase 1A (CPT1A); acyl-CoA synthetase, long chain 1 (ASCL1); acyl-CoA dehydrogenase, very long chain; 3-hydroxy-3-methylglutaryl-CoA synthase 1 and 2; sterol regulatory element-binding factor 1; and peroxisome proliferator-activated factor α. Total LFC postpartum differed greatly among cows, and the mRNA abundance of most enzymes and transcription factors changed with time during the experimental period. Abundance of PC mRNA increased at parturition to a greater extent in high- and medium-LFC groups than in the low-LFC group. Significant LFC × time interactions for ACSL1 and CPT1A during the experimental period indicated variable gene expression depending on LFC after parturition. Correlations between hepatic gene expression and performance data and plasma concentrations of metabolites and hormones showed time-specific relations during the transition period. Elevated body fat mobilization during early lactation affected gene expression involved in gluconeogenesis to a greater extent than gene expression involved in lipid metabolism, indicating the dependence of hepatic glucose metabolism on hepatic lipid status and fat mobilization during early lactation.

Promoter- and RNA polymerase II-dependent hsp-16 gene association with nuclear pores in Caenorhabditis elegans.

Some inducible yeast genes relocate to nuclear pores upon activation, but the general relevance of this phenomenon has remained largely unexplored. Here we show that the bidirectional hsp-16.2/41 promoter interacts with the nuclear pore complex upon activation by heat shock in the nematode Caenorhabditis elegans. Direct pore association was confirmed by both super-resolution microscopy and chromatin immunoprecipitation. The hsp-16.2 promoter was sufficient to mediate perinuclear positioning under basal level conditions of expression, both in integrated transgenes carrying from 1 to 74 copies of the promoter and in a single-copy genomic insertion. Perinuclear localization of the uninduced gene depended on promoter elements essential for induction and required the heat-shock transcription factor HSF-1, RNA polymerase II, and ENY-2, a factor that binds both SAGA and the THO/TREX mRNA export complex. After induction, colocalization with nuclear pores increased significantly at the promoter and along the coding sequence, dependent on the same promoter-associated factors, including active RNA polymerase II, and correlated with nascent transcripts.

"Hot spot" in the PROP1 gene responsible for combined pituitary hormone deficiency.

As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). Although pit-1 was 1 of the first factors identified as a cause of CPHD in mice, many other homeodomain and transcription factors have been characterized as being involved in different developmental stages of pituitary gland development, such as prophet of pit-1 (prop-1), P-Lim, ETS-1, and Brn 4. The aims of the present study were first to screen families and patients suffering from different forms of CPHD for PROP1 gene alterations, and second to define possible hot spots and the frequency of the different gene alterations found. Of 73 subjects (36 families) analyzed, we found 35 patients, belonging to 18 unrelated families, with CPHD caused by a PROP1 gene defect. The PROP1 gene alterations included 3 missense mutations, 2 frameshift mutations, and 1 splice site mutation. The 2 reported frameshift mutations could be caused by any 2-bp GA or AG deletion at either the 148-GGA-GGG-153 or 295-CGA-GAG-AGT-303 position. As any combination of a GA or AG deletion yields the same sequencing data, the frameshift mutations were called 149delGA and 296delGA, respectively. All but 1 mutation were located in the PROP1 gene encoding the homeodomain. Importantly, 3 tandem repeats of the dinucleotides GA at location 296-302 in the PROP1 gene represent a hot spot for CPHD. In conclusion, the PROP1 gene seems to be a major candidate gene for CPHD; however, further studies are needed to evaluate other genetic defects involved in pituitary development.

Phenotypic variability in familial combined pituitary hormone deficiency caused by a PROP1 gene mutation resulting in the substitution of Arg-->Cys at codon 120 (R120C).

As pituitary function depends on the integrity of the hypothalamic-pituitary axis, any defect in the development and organogenesis of this gland may account for a form of combined pituitary hormone deficiency (CPHD). A mutation in a novel, tissue-specific, paired-like homeodomain transcription factor, termed Prophet of Pit-1 (PROP1), has been identified as causing the Ames dwarf (df) mouse phenotype, and thereafter, different PROP1 gene alterations have been found in humans with CPHD. We report on the follow-up of two consanguineous families (n = 12), with five subjects affected with CPHD (three males and two females) caused by the same nucleotide C to T transition, resulting in the substitution of Arg-->Cys in PROP1 at codon 120. Importantly, there is a variability of phenotype, even among patients with the same mutation. The age at diagnosis was dependent on the severity of symptoms, ranging from 9 months to 8 yr. Although in one patient TSH deficiency was the first symptom of the disorder, all patients became symptomatic by exhibiting severe growth retardation and failure to thrive, which was mainly caused by GH deficiency (n = 4). The secretion of the pituitary-derived hormones (GH, PRL, TSH, LH, and FSH) declined gradually with age, following a different pattern in each individual; therefore, the deficiencies developed over a variable period of time. All of the subjects entered puberty spontaneously, and the two females also experienced menarche and periods before a replacement therapy was necessary.

The orf13 T-DNA gene of Agrobacterium rhizogenes confers meristematic competence to differentiated cells

Plant infections by the soil bacterium Agrobacterium rhizogenes result in neoplastic disease with the formation of hairy roots at the site of infection. Expression of a set of oncogenes residing on the stably integrated T-DNA is responsible for the disease symptoms. Besides the rol (root locus) genes, which are essential for the formation of hairy roots, the open reading frame orf13 mediates cytokinin-like effects, suggesting an interaction with hormone signaling pathways. Here we show that ORF13 induced ectopic expression of KNOX (KNOTTED1-like homeobox) class transcription factors, as well as of several genes involved in cell cycle control in tomato (Lycopersicon esculentum). ORF13 has a retinoblastoma (RB)-binding motif and interacted with maize (Zea mays) RB in vitro, whereas ORF13, bearing a point mutation in the RB-binding motif (ORF13*), did not. Increased cell divisions in the vegetative shoot apical meristem and accelerated formation of leaf primordia were observed in plants expressing orf13, whereas the expression of orf13* had no influence on cell division rates in the shoot apical meristem, suggesting a role of RB in the regulation of the cell cycle in meristematic tissues. On the other hand, ectopic expression of LeT6 was not dependent on a functional RB-binding motif. Hormone homeostasis was only altered in explants of leaves, whereas in the root no effects were observed. We suggest that ORF13 confers meristematic competence to cells infected by A. rhizogenes by inducing the expression of KNOX genes and promotes the transition of infected cells from the G1 to the S phase by binding to RB.

Shoot meristem maintenance is controlled by a GRAS-gene mediated signal from differentiating cells

Plant shoot development depends on the perpetuation of a group of undifferentiated cells in the shoot apical meristem (SAM). In the Petunia mutant hairy meristem (ham), shoot meristems differentiate postembryonically as continuations of the subtending stem. HAM encodes a putative transcription factor of the GRAS family, which acts non-cell-autonomously from L3-derived tissue of lateral organ primordia and stem provasculature. HAM acts in parallel with TERMINATOR (PhWUSCHEL) and is required for continued cellular response to TERMINATOR and SHOOTMERISTEMLESS (PhSTM). This reveals a novel mechanism by which signals from differentiating tissues extrinsically control stem cell fate in the shoot apex.

Looking the cow in the eye: deletion in the NID1 gene is associated with recessive inherited cataract in Romagnola cattle

Cataract is a known condition leading to opacification of the eye lens causing partial or total blindness. Mutations are known to cause autosomal dominant or recessive inherited forms of cataracts in humans, mice, rats, guinea pigs and dogs. The use of large-sized animal models instead of those using mice for the study of this condition has been discussed due to the small size of rodent lenses. Four juvenile-onset cases of bilateral incomplete immature nuclear cataract were recently observed in Romagnola cattle. Pedigree analysis suggested a monogenic autosomal recessive inheritance. In addition to the cataract, one of the cases displayed abnormal head movements. Genome-wide association and homozygosity mapping and subsequent whole genome sequencing of a single case identified two perfectly associated sequence variants in a critical interval of 7.2 Mb on cattle chromosome 28: a missense point mutation located in an uncharacterized locus and an 855 bp deletion across the exon 19/intron 19 border of the bovine nidogen 1 (NID1) gene (c.3579_3604+829del). RT-PCR showed that NID1 is expressed in bovine lenses while the transcript of the second locus was absent. The NID1 deletion leads to the skipping of exon 19 during transcription and is therefore predicted to cause a frameshift and premature stop codon (p.1164fs27X). The truncated protein lacks a C-terminal domain essential for binding with matrix assembly complexes. Nidogen 1 deficient mice show neurological abnormalities and highly irregular crystal lens alterations. This study adds NID1 to the list of candidate genes for inherited cataract in humans and is the first report of a naturally occurring mutation leading to non-syndromic catarct in cattle provides a potential large animal model for human cataract.

Induction of the autophagy-associated gene MAP1S via PU.1 supports APL differentiation

The PU.1 transcription factor is essential for myeloid development. We investigated if the microtubule-associated protein 1S (MAP1S) is a novel PU.1 target with a link to autophagy, a cellular recycling pathway. Comparable to PU.1, MAP1S expression was significantly repressed in primary AML blasts as compared to mature neutrophils. Accordingly, MAP1S expression was induced during neutrophil differentiation of CD34(+) progenitor and APL cells. Moreover, PU.1 bound to the MAP1S promoter and induced MAP1S expression during APL differentiation. Inhibiting MAP1S resulted in aberrant neutrophil differentiation and autophagy. Taken together, our findings implicate the PU.1-regulated MAP1S gene in neutrophil differentiation and autophagy control.

Stress-induced modulation of NF-κB activation, inflammation-associated gene expression, and cytokine levels in blood of healthy men

Acute psychosocial stress stimulates transient increases in circulating pro-inflammatory plasma cytokines, but little is known about stress effects on anti-inflammatory cytokines or underlying mechanisms. We investigated the stress kinetics and interrelations of pro- and anti-inflammatory measures on the transcriptional and protein level. Forty-five healthy men were randomly assigned to either a stress or control group. While the stress group underwent an acute psychosocial stress task, the second group participated in a non-stress control condition. We repeatedly measured before and up to 120min after stress DNA binding activity of the pro-inflammatory transcription factor NF-κB (NF-κB-BA) in peripheral blood mononuclear cells, whole-blood mRNA levels of NF-κB, its inhibitor IκBα, and of the pro-inflammatory cytokines interleukin (IL)-1ß and IL-6, and the anti-inflammatory cytokine IL-10. We also repeatedly measured plasma levels of IL-1ß, IL-6, and IL-10. Compared to non-stress, acute stress induced significant and rapid increases in NF-κB-BA and delayed increases in plasma IL-6 and mRNA of IL-1ß, IL-6, and IκBα (p's<.045). In the stress group, significant increases over time were also observed for NF-κB mRNA and plasma IL-1ß and IL-10 (p's<.055). NF-κB-BA correlated significantly with mRNA of IL-1β (r=.52, p=.002), NF-κB (r=.48, p=.004), and IκBα (r=.42, p=.013), and marginally with IL-6 mRNA (r=.31, p=.11). Plasma cytokines did not relate to NF-κB-BA or mRNA levels of the respective cytokines. Our data suggest that stress induces increases in NF-κB-BA that relate to subsequent mRNA expression of pro-inflammatory, but not anti-inflammatory cytokines, and of regulatory-cytoplasmic-proteins. The stress-induced increases in plasma cytokines do not seem to derive from de novo synthesis in circulating blood cells.

Insulin, CCAAT/enhancer-binding proteins and lactate regulate the human 11β-hydroxysteroid dehydrogenase type 2 gene expression in colon cancer cell lines.

11β-Hydroxysteroid dehydrogenases (11beta-HSD) modulate mineralocorticoid receptor transactivation by glucocorticoids and regulate access to the glucocorticoid receptor. The isozyme 11beta-HSD2 is selectively expressed in mineralocorticoid target tissues and its activity is reduced in various disease states with abnormal sodium retention and hypertension, including the apparent mineralocorticoid excess. As 50% of patients with essential hypertension are insulin resistant and hyperinsulinemic, we hypothesized that insulin downregulates the 11beta-HSD2 activity. In the present study we show that insulin reduced the 11beta-HSD2 activity in cancer colon cell lines (HCT116, SW620 and HT-29) at the transcriptional level, in a time and dose dependent manner. The downregulation was reversible and required new protein synthesis. Pathway analysis using mRNA profiling revealed that insulin treatment modified the expression of the transcription factor family C/EBPs (CCAAT/enhancer-binding proteins) but also of glycolysis related enzymes. Western blot and real time PCR confirmed an upregulation of C/EBP beta isoforms (LAP and LIP) with a more pronounced increase in the inhibitory isoform LIP. EMSA and reporter gene assays demonstrated the role of C/EBP beta isoforms in HSD11B2 gene expression regulation. In addition, secretion of lactate, a byproduct of glycolysis, was shown to mediate insulin-dependent HSD11B2 downregulation. In summary, we demonstrate that insulin downregulates HSD11B2 through increased LIP expression and augmented lactate secretion. Such mechanisms are of interest and potential significance for sodium reabsorption in the colon.