Unknown,Transcriptomics,Genomics,Proteomics

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Microarray identification of functional modules involving Haptoglobin and/or Hemopexin

ABSTRACT: Haptoglobin and Hemopexin are plasma acute phase proteins that bind with high affinity hemoglobin and heme, respectively. Several studies have demonstrated that they have a key role in the protection against oxidative stress and inflammation. However, little is known about the functional modules in which they are involved. To gain insights into this issue, we integrated bioinformatic and experimental approaches to identify genes coexpressed with Haptoglobin or Hemopexin and modulated in Haptoglobin and/or Hemopexin knock-out mice. These genes have a high probability to be functionally related to Haptoglobin and/or Hemopexin. We performed a gene expression analysis of the livers of Hp- and/or Hx-null mice compared to wild-type controls. The mice used in the following experiments, i.e. wild-type (Hp+/Hx+/), Hp-null (Hp-/-Hx+/), Hx-null (Hp+/Hx-/-), and HpHx-null (Hp-/-Hx-/-), were littermates derived by breeding F1 double heterozygous Hp+/-Hx+/- mice in the mixed genetic background C57/BLJ6 X 129Sv. We have three experimental points: Hx-null mice, Hp-null mice and HpHx-null mice. At least 5 adult mice per genotype were perfused via aorta with PBS, sacrificed and their liver pooled. Thirty microgram of total RNA were subjected to direct labeling reaction by incorporation of cyanin 3 (Cy3) or cyanin 5 (Cy5) fluorescent dyes into the cDNA by priming with oligo(dT). Four replicates were set up for each experimental point. In order to exclude artifacts resulting from different dye usage, we employed the dye-swap approach.

ORGANISM(S): Mus musculus

SUBMITTER: Stefano Volinia

PROVIDER: E-GEOD-3656 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Trypanosoma brucei requires haemoglobin for survival in the mammalian bloodstream. Haptoglobin (Hp), a host protein known to bind haemoglobin, was determined as a key component for haemoglobin acquisition by the parasite. Here, we investigated the impact of haptoglobin deficiency on T. brucei brucei infection and the parasite´s capacity to internalise haemoglobin in the Hp-/- mouse model. The infected Hp-/- mice exhibited normal disease progression, with minimal weight loss and no apparent organ pathology similarly to control mice. While the proteomic profile of mouse sera significantly changed in response to T. b. brucei, no differences in the infection response markers of blood plasma have been observed between Hp-/- and control Black mice. Similarly, very few quantitative differences have been observed between the proteomes of parasites harvested from Hp-/- and Black mice, entailing both endogenous proteins and internalised host proteins. While haptoglobin was indeed absent from parasites isolated from Hp-/-mice, haemoglobin peptides were unexpectedly detected in parasites from both Hp-/- and Black mice. This was verified by western blotting, EM immunolabeling of parasites cryosections, and analytical quantification of haem cofactors in the parasites extracts. Combined, the data support the Hp dispensability for haemoglobin internalisation by T. b. brucei during infection in mice. To further explore this phenomenon, we have generated T. b. brucei haptoglobin-haemoglobin receptor (HpHbR) knock-outs and used them to infect Hp-/- and control mice. The HpHbR-knock-out trypanosomes internalised significantly less haemoglobin from Hp-/- mice compared to those isolated from Black mice strongly suggesting that T. b. brucei employs an HpHbR-independent Hp-mediated mode for haemoglobin internalisation. Our study reveals a so-far hidden flexibility of haemoglobin acquisition by T. b. brucei and offers novel insights into alternative haemoglobin uptake pathways.

Project description:Sparc null mutants have been generated independently via two targeted mutations in exons 4 and 6. Previous studies have identified low turnover osteopenia in the exon 4 knockout (on a mixed 129Sv/C57Bl/6 background). Since both Sparc null mutations have been shown to result in complete absence of Sparc protein, similar phenotypic outcomes are likely. However, genetic background and/or linkage disequilibrium effects can influence phenotype; abnormalities attributed to a given mutation have often been later found to be caused by closely linked genetic anomalies or strain effects. Ideally therefore, different inactivating mutations should be tested in various mouse strains; similar phenotypic outcomes can then confidently be assigned to the mutated gene. We have evaluated the bone phenotype in the Sparctm1cam exon 6 knockout on a purebred 129Sv/Ev background at 4 and 9 months, using physical measurement, mechanical strength tests and DXA scanning. We have also quantified bone marrow adiposity and circulating leptin levels to assess adipose tissue regulation anomalies in these mice. 129Sv/EvSparctm1cam null mice show decreased bone mineral density and bone mineral content. Increased mechanical fragility of bone was also evident, largely in line with previous studies. However, differences were also noted. Increased body weight and levels of bone marrow adiposity, but decreased circulating leptin concentrations were identified at 4 months, but not at 9 months. Circulating leptin concentrations were reportedly increased in Sparc exon 4 null animals, suggesting that leptin concentrations may be influenced more by genetic background than by the absence of Sparc. 129Sv/EvSparctm1cam null mice have shorter femurs; this difference was not noted in prior studies, possibly due to increased variation owing to use of a hybrid strain. Molecular phenotyping was carried out using mouse HGMP NIA microarrays with cortical femur samples at various ages, using semi-quantitative RT PCR validation. Array studies have identified 429 genes expressed in normal bone. Six genes (Sparc, Zfp162, Bysl, E2F4 and two ESTs) are differentially regulated in 129Sv/EvSparctm1cam cortical femur versus 129Sv/Ev controls. We confirm low turnover osteopenia as a feature of the Sparc null phenotype, identifying the usefulness of this mouse as a model for human osteoporosis. Keywords: disease state analysis, time course, genetic modification, osteoporosis, knockout mouse

Project description:Zinc finger and SCAN domain containing 10 (Zscan10) was identified as a novel transcription factor in osteoclast differentiation by our previous study. However, the biological functions of Zscan10 have not been fully understood except its role in maintenance of genome stability and pluripotency in embryonic stem cells. Therefore, the purpose of this study is clarification of Zscan10 function in somatic cells, especially during osteoclast differentiation. First, Zscan10 KO RAW264 (KO) cells were established using CRISPR/Cas9 system and single cell sorting. Then, Control (Ctrl) and KO cells were differentiated into osteoclast by RANKL stimulation. As a result, TRAP activity and expression levels of differentiation marker genes, such as Nfatc1, were significantly increased and the expression of inhibitory factors, such as Irf8, was decreased in KO cells as compared to Ctrl cells. These results suggested that Zscan10 might regulate transcription of the genes which negatively control osteoclastogenesis. To understand the transcriptomes controlled by Zscan10, RNA-seq was performed and the stringent analyses identified significantly down-regulated Haptoglobin (Hp) in KO cells. Additionally, Zscan10 binding sequence was located near the genomic region of Hp gene locus. ChIP against Zscan10 followed by qPCR for the region revealed that Zscan10 binds to the region located near Hp gene locus and transcript start site, suggesting that Zscan10 may regulate transcription of Hp. Next, to examine the effect of Hp under Zscan10 mediated osteoclastogenesis, KO cells were treated with recombinant Hp (rHp). As a result, Hp treatment could suppress the elevated TRAP activity of KO cells without affecting cell viability. Furthermore, Hp KO mice exhibit decreased bone mass and increased osteoclast number, and Hp has been reported to be involved in suppression of osteoclastogenesis. Also, In hemolytic disease, Hp had been decreased and also bone density. These facts suggested that Zscan10 negatively regulates osteoclast differentiation through transcription of Hp.

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Microarray identification of functional modules involving Haptoglobin and/or Hemopexin

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