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Keywords:

  • collagenous repeat containing sequence of 26-kDa protein;
  • adipocyte;
  • peroxisome proliferator-activated receptor γ;
  • adiponectin;
  • resistin

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Objective: Collagenous repeat containing sequence of 26-kDa protein (CORS-26) was identified as a new gene transcript expressed in cartilage with unknown function. It was the aim of this study to investigate expression, regulation, and function of CORS-26 in adipocytes.

Research Methods and Procedures: CORS-26 mRNA and protein expression was studied by reverse transcriptase-polymerase chain reaction, Western blot analysis, and quantitative real-time polymerase chain reaction. Transcriptional regulation was studied by electrophoretic mobility shift assay and luciferase reporter gene assay. The adipocytic secretion of adiponectin and resistin was measured by enzyme-linked immunosorbent assay.

Results: CORS-26 mRNA is absent in 3T3-L1 preadipocytes and adipocytes after 48 hours of differentiation. CORS-26 mRNA was induced from Day 4 to Day 9 of adipocyte differentiation. CORS-26 protein was induced in mature adipocytes. Peroxisome proliferator-activated receptor (PPAR) γ (but not PPARα) in nuclear extracts prepared from adipocytes was shown to bind specifically to a putative peroxisome proliferator response element-one-half-site located at −641/−596 bp. Increasing doses of the ligands troglitazone (1, 10, 20 μM) and fenofibrate (50, 100, 200 μM) but not 15-deoxy-prostaglandin (J2) (0.5, 1.0, 2.5 μM) resulted in a significant reduction of both promoter activity and the amount of mRNA expression. Recombinant CORS-26 significantly stimulated the adipocytic secretion of adiponectin and resistin in a dose-dependent manner.

Discussion: The mRNA and protein expression profile puts CORS-26 in the adipocytokine family. Cartonectin is negatively regulated by exogenous, but not endogenous, PPARγ ligands. Because CORS-26 up-regulates adipokine secretion, it might be involved in metabolic and immunologic pathways.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Recently, a novel murine gene structurally closely related to adiponectin (1, 2) was cloned and termed collagenous repeat-containing sequence of 26-kDa protein (CORS-26)1 (3, 4, 5). Its expression was initially shown to be restricted to the rib growth plate, cartilage, and kidney (3). Compared with adiponectin, the gene encoding CORS-26 has similar structural features, such as an N-terminal collagenous region and a carboxyterminal complement factor C1q globular domain (3). For that reason, both adiponectin and CORS-26 seem to represent members of the C1q-tumor necrosis factor molecular superfamily (1). Although chromosomal localization, promoter sequence, and genomic organization of the human (5) and murine (4) gene were determined, the physiological function and regulation of this novel protein is completely unknown. Because the murine gene on chromosome 15A2 (4) maps to a linkage locus (6, 7) for two animal models of arthritis (proteoglycan-induced arthritis and murine lpr-mutation/lpr arthritis) and because the human gene locus on chromosome 5p13 (5) is affected by chromosomal imbalances in the presence of osteosarcoma (8), a putative function of CORS-26 in the development of mesenchymal tissue can be supposed. In this context, it is important to note that human CORS-26 mRNA is up-regulated in osteosarcoma cells (5).

However, there are additional significant linkage loci that might play a role in the metabolic syndrome and draw attention to adipocyte biology. The location of the murine gene is very close to the microsatellite marker D15Mit225. Shike et al. (9) described an association between fasting glucose levels with the centromeric region on chromosome 15 near D15Mit225, with a maximum logarithmic odds ratio score in the region containing the CORS-26 gene. Hager et al. (10) studied the association of serum leptin levels with genomic loci in French sibpairs suffering from obesity and described a linkage of leptin with the region between microsatellite markers D5S477 and D5S426 containing the CORS-26 gene. Lindsay et al. (11) studied the association of diabetes and BMI with chromosomal loci in Pima Indians and identified a linkage for diabetes with the region between microsatellite markers D5S1470 and D5S426 containing the CORS-26 gene.

Based on these data, it seems reasonable to study the expression, regulation, and function of the CORS-26 gene in the context of adipocyte differentiation and adiopkine secretion such as adiponectin and resistin.

Research Methods and Procedures

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Computer-based Analysis of Putative Transcription Factor Binding Sites

A detailed computer-based analysis using the professional software program TESS (transcription element search software) based on the TRANSFAC database (12) was used to identify putative transcription factor binding sites. The TATA-box containing promoter region of murine CORS-26 was shown to possess a putative peroxisome proliferator response element (PPRE)-one-half-site at −621 to −626 bp relative to the ATG start codon (4, 13).

Adipocyte Cell Culture

3T3-L1-preadipocytes (murine preadipocyte cell line derived from disaggregated mouse embryos) were cultured at a 10% CO2 atmosphere at 37 °C in Dulbecco's modified eagle medium (DMEM; Biowitthaker, Verviers, Belgium) supplemented with 10% newborn calf serum (Sigma Biosciences, Deisenhofen, Germany) and penicillin/streptomycin (GIBCO BRL, Berlin, Germany). At confluence, cells were differentiated into adipocytes by treating them with DMEM/F12/glutamate-medium supplemented with 0.5 mM 3-isobutyl-methyl-xanthine, 10−7 M corticosterone, 10−6 M insulin, 200 mM ascorbate, 2 μg/mL transferrin, 1 mM biotin, 17 mM panthothenate, and 300 mg/L Pedersen-fetuin (14, 15) for 5 days. Thereafter, the cells were exposed to DMEM/F12/glutamate-medium with 10−9 M insulin until they reached the fully differentiated phenotype (16, 17, 18, 19, 20, 21) that was controlled by light microscopy for the existence of a more rounded cell shape and the typical appearance of extensive accumulation of lipid droplets.

Reverse Transcriptase-Polymerase Chain Reaction Analysis

For reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of murine CORS-26 mRNA expression, the upstream primer 5′-CATTTGCCTGAGGCGACCACGGTA-3′ and the downstream primer 5′-CCATAATCTCCATGGCAACACTTGC-3′ spanning a 227-bp cDNA fragment were used (annealing temperature: 63 °C). As a control for mRNA yield, murine β-actin mRNA expression was simultaneously studied using the upstream primer 5′-CCAGGGTGTGATGGTGGGAATG-3′ and the downstream primer 5′-CGCACGATTTCCCTCTCAGCTG-3′ spanning a 353-bp cDNA fragment (annealing temperature: 63 °C). All primer sets were designed by a computer-based software program, and it was checked by a gene bank search whether the sequences were gene-specific and intron-spanning. The amplified DNA product was analyzed by gel electrophoresis and subsequently sequenced.

Isolation of Nuclear Extracts

3T3-L1-adipocytes at Day 7 of differentiation were harvested by centrifugation, washed once with ice-cold phosphate-buffered saline (PBS), and washed twice with wash buffer composed of 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10 mM KCL, 0.5 mM dithiothreitol (DTT), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF). After resuspension in 1 mL ice-cold wash buffer, cells were centrifuged 1 minute at 6000 rpm. Hypotonic buffer containing 0.1% Nonidet P40 was added to lyse the cell pellet. After a 5-minute incubation on ice, nuclei were pelleted by centrifugation for 15 minutes (15,000 rpm) at 4 °C. Nuclei were resuspended in lysis buffer containing 20 mM HEPES (pH 7.9), 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 10% vol/vol glycerol, and incubated at 4 °C for 15 minutes with gentle vortexing. Subsequently, the nuclear debris were pelleted by centrifugation at 4 °C for 15 minutes, and the supernatant was diluted 1:6 with storage buffer composed of 20 mM HEPES (pH 7.9), 0.05 M KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, and 20% vol/vol glycerol. The extracts were aliquoted and stored at −80 °C. Protein concentration was determined using the Bradford method (22).

Western Blot Analysis and Generation of Polyclonal Anti-CORS-26 Antibodies

For detection of the murine CORS-26 protein, chicken antibodies were raised (Pineda-abservice, Berlin, Germany) against a synthetic peptide of the human sequence (NH2-CYSYEMKGKSDTSSNH-CONH2) from the noncollagenous region of the protein (amino acids 193–207) and used for Western blot analysis. These antibodies recognize the corresponding protein of human and mouse origin. Cultured cells were washed twice with PBS, harvested gently with a cell scraper, centrifuged, and resuspended in 100 μL PBS. Equal amounts of total protein were submitted to gel electrophoresis after a 1:1 dilution with Lämmli loading buffer. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed following standard procedures. Proteins were transferred to a Fluotrans Transfer Membrane (Pall Corp., Portsmouth, United Kingdom). Transfer was confirmed by Ponceau red staining of the membrane and Coomassie blue staining of the gel after the electroblot. The CORS-26 primary antibodies were used with a 1:200 dilution, the secondary peroxidase-coupled anti-chicken antibodies of rabbit origin (Sigma-Aldrich, Steinheim, Germany) with a 1:5000 dilution in a 5% nonfat dry milk/PBS suspension. Detection of the immune complexes was carried out with the enhanced chemoluminescence Western blot detection system (Amersham Corp.).

Electrophoretic Mobility Shift Assays

Ten milligrams of nuclear extracts was incubated with 2 mg poly-(dI-dC) in a volume of 20 mL binding buffer containing 50 mM HEPES (pH 7.9), 6 mM MgCl2, 50 mM KCl, 5 mM DTT, 100 mg/mL bovine serum albumin, and 0.01% Nonidet P40. [32P] end-labeled synthetic oligonucleotides (30,000 cpm) were added, and the reaction mixture was incubated for 20 minutes at room temperature. For competition experiments, nuclear extracts were preincubated for 10 minutes with a 50-fold molar excess of nonlabeled competitor oligonucleotides. Supershift assays were performed with two different antibodies both recognizing PPARγ12 (purchased from ActiveMotif, Carlsbad, CA, and from Calbiochem, Merck, Darmstadt, Germany). Additionally, antibodies recognizing PPARα (purchased from Affinity BioReagents, Golden, CO) or recognizing both PPARα and PPARγ12 (purchased from Calbiochem, Merck) were used. The DNA-protein complexes were analyzed on a 8% polyacrylamide gel at 80 V using 0.25× tris base, boric acid, EDTA as electrophoresis buffer. Gels were dried and exposed to Kodak X-Omat AR films overnight at −80 °C.

For electrophoretic mobility shift assay (EMSA) analysis, double-strand oligonucleotides were used (oligonucleotides were annealed to their respective complementary olignucleotides to generate double strand oligonucleotides). The promoter fragment −641/−596 (prom −641/−596) carrying the PPRE site at −621 to −626 bp with the sequence 5′-AGTTTACCCTTGAACTCAGGTCAGTCTTTCCCAGTCACCATCTGTAGG-3′ was used for EMSA (binding site in italics).

Luciferase Reporter Gene Assays

To study functional promoter activity, genomic DNA fragments derived from the 5′-proximal region of the CORS-26 gene (fragment luc −844/61 carrying the PPRE site and fragment luc −618/61 devoid of the PPRE site) were amplified and cloned into the luciferase expression vector pGL3-basic (Promega, Madison, WI). The numbering of these constructs determines the 5′-end and the 3′-end of the PCR products relative to the ATG-start codon. All chimeric plasmids were sequenced before transfection. Cells grown to a density of 80% to 90% were transiently transfected with 10 μg plasmid DNA using the Superfect reagent based on activated dendrimer technology (Qiagen, Hilden, Germany) as described by the manufacturer. After 3 hours of incubation, cells were differentiated as described above. Measurement of luciferase activity was performed 48 hours after induction of differentiation. In each experiment, cells were transfected with pGL-3 control plasmids that served as positive controls. The pGL-3 control vector contains the simian virus40 early promoter driving the expression of luciferase mRNA transcripts. To standardize the transfection efficiency, 7.5 μg of pSV b-galactosidase plasmid (Promega) were always cotransfected. Moreover, the amount of measured relative light units (RLUs) was normalized to the total protein content of each cell lysate (mean ± standard error, experiments performed in triplicate). For luciferase assays, transfected cells were harvested 48 hours after differentiation and lysed in 1 mL of reporter lysis buffer (Promega). Twenty microliters of the lysates was mixed with 100 μL luciferase assay reagent containing luciferyl-CoA. The luciferase activity was measured in a Lumat LB9501 (Berthold, Munich, Germany). For stimulation experiments, cells were incubated for 16 hours with 1, 10, and 20 μM troglitazone (Sigma, Taufkirchen, Germany), with 50, 100, and 200 μM fenofibrate (Sigma), and with 0.5, 1.0, and 2.5 μM 15-deoxy-prostaglandin-J2 (15-D-PGJ2; Merck, Schwalbach, Germany).

Monitoring of Quantitative Gene Expression by Real-time RT-PCR

Two microliters of total RNA was reverse transcribed using the Promega Reverse Transcription System (Promega) in a volume of 40 μL. Two microliters of cDNA was subsequently amplified in glass capillaries (LightCycler) using PCR primers specific for CORS-26 and β-actin. The reaction conditions were as follows: 2 μL cDNA, 2 μL 10× LightCycler-Fast Start NA Master SYBR Green I (Roche Diagnostics, Mannheim, Germany), 2.4 μL MgCl2 (25 mM), and 1 μL of each primer (5 pM/μL) in a total volume of 20 μL. The primers for CORS-26 were as follows: CORS-upstream, 5′-AAAGGAGACAAAGGCGACCTAGG-3′; CORS-downstream, 5′-GCACTGCATGGTTGCTGGATGTATCTG-3′. The primers for β-actin were as follows: β-actin-up, 5′-CCAGGGTGTGATGGTGGGAATG-3′; β-actin-down, 5′-CGCACGATTTCCCTCTCAGCT G-3′. Amplification in the LightCycler capillaries was for 45 cycles with initial incubation of 10 minutes at 95 °C for activation of TaqPolymerase. Cycling parameters were 10 seconds at 95 °C, 10 seconds at 63 °C, and 15 seconds at 72 °C. Fluorescence was monitored at 83 °C for CORS-26 and 85 °C for β-actin. The second derivative method was used for quantification with the LightCycler software. For quantification of the results obtained by real-time PCR, the standard curve method was used. For this purpose, a stock of total RNA was serially diluted. A standard curve with 50, 25, 12.5, and 6.25 ng total RNA was generated for CORS-26 and β-actin. The standard curves were used to determine the relative expression of CORS-26 and β-actin mRNA in each sample.

Recombinant CORS-26 Protein Expression

Recombinant CORS-26 protein expression was performed in H5 insect cells (Invitrogen, Karlsruhe, Germany) using the BacPAK Baculovirus Expression System (BD Biosciences, Palo Alto, CA), as published earlier by our group in detail (23). In contrast to Escherichia coli-based expression systems, the recombinant expression in insect cells usually maintains glycosylation and phosphorylation procedures.

Measurement of Adipokines

Adiponectin and resistin were measured in cell culture supernatants using enzyme-linked immunosorbent assay technique (R&D Systems Europe, Abingdon, United Kingdom).

Statistics

For determining mean values, ± standard error, and error bars, a professional software package (SPSS 11.0) was used. Means were compared by the Mann-Whitney U test. A p value < 0.05 (two tailed) was considered to be statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

CORS-26 mRNA Expression During Adipocyte Differentiation

CORS-26 mRNA expression was studied by RT-PCR using RNA purified from 3T3-L1 preadipocytes (Figure 1A, lane 1) and from adipocytes after 48 hours, 4 days, 7 days, and 9 days of hormonally-induced differentiation (Figure 1A, lanes 2 to 5). The murine β-actin gene was used as a control and shown to be expressed similarly during all stages of differentiation (Figure 1A, lanes 1–5). CORS-26 mRNA was not detectable in preadipocytes and adipocytes after 48 hours of differentiation (Figure 1A, lanes 1 and 2). However, CORS-26 mRNA was induced at Day 4 of differentiation (Figure 1A, lane 3) and during late (Days 7 to 9) adipocyte differentiation (Figure 1A, lanes 4 and 5).

image

Figure 1. Expression of murine CORS-26 mRNA and protein during hormonally induced differentiation of 3T3-L1 adipocytes. (A) Expression of murine CORS-26 mRNA by RT-PCR. The murine β-actin gene was used as a positive control (353-bp fragment, indicated by an arrow). RT-PCR for murine CORS-26 mRNA produced a band at 227 bp (indicated by an arrow). λ, molecular weight marker. Lane 1, preadipocytes; lane 2, adipocytes after 48 hours of differentiation; lane 3, adipocytes after 4 days of differentiation; lane 4, adipocytes after 7 days of differentiation; lane 5, adipocytes after 9 days of differentiation. (B) Expression of murine CORS-26 protein by Western blot analysis. Lane 1, preadipocytes; lane 2, adipocytes after 4 days of differentiation; lane 3, adipocytes after 7 days of differentiation; lane 4, adipocytes after 9 days of differentiation; lane 5, human fibroblasts; lane 6, murine fibroblasts.

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CORS-26 Protein Expression During Adipocyte Differentiation

CORS-26 protein expression was studied by Western blot analysis using polyclonal chicken antibodies raised against a synthetic peptide of human origin (Figure 1B). CORS-26 protein is not detectable in preadipocytes (Figure 1B, lane 1). Using cell lysates isolated from adipocytes after 4, 7, and 9 days of differentiation (Figure 1B, lanes 2, 3, and 4), CORS-26 protein is expressed with a molecular weight of 30 kDa. In contrast to the fibroblast-like preadipocytes, human (Figure 1B, lane 5) and murine fibroblasts (Figure 1B, lane 6) are positive for CORS-26 protein.

Functional Assessment of Promoter Activity and Effects of PPAR Ligands

Troglitazone

To study functional promoter activity (Figure 2), 3T3-L1 adipocytes were transfected with promoter constructs with or without the PPRE binding site and stimulated with increasing doses of troglitazone (1, 10, and 20 μM). Transfection with the positive control vector (Figure 2, lane 1) presented with high amounts of RLUs (91 × 106 ± 7.3 × 106 RLU), whereas the negative control vector (Figure 2, lane 2) had only very low activity (0.07 × 106 ± 9203 RLU). Fragment luc844/61 carrying the PPRE site (Figure 3, lane 3) induced a significant induction of promoter activity compared with the control vector (1.9 × 106 ± 0.12 × 106 vs. 0.07 × 106 ± 9203 RLU; p < 0.005). Under basal conditions without PPAR ligands, the fragment luc −618/61 devoid of the PPRE site (Figure 2, lane 4) presented with a similar induction of promoter activity (2.5 × 106 ± 0.41 × 106 RLU) without significant difference compared with the fragment carrying the binding site. However, application of increasing doses of the PPARγ ligand troglitazone (Figure 2, lanes 5 to 7) caused a stepwise reduction of promoter activity using the PPRE site containing fragment (Figure 2, lane 3). Promoter activity was reduced from 1.9 × 106 ± 0.12 × 106 RLU (Figure 2, lane 3) to 1.6 × 106 ± 0.07 × 106 RLU by 1 μM troglitazone (Figure 2, lane 5; p = 0.16), to 0.76 × 106 ± 0.04 × 106 RLU by 10 μM troglitazone (Figure 2, lane 6; p = 0.012) and to 0.36 × 106 ± 0.06 × 106 RLU by 20 μM troglitazone (Figure 2, lane 7; p = 0.007).

image

Figure 2. Effects of troglitazone on promoter activity of murine CORS-26/luciferase gene chimeras. CORS-26 promoter fragments with (luc −844/−61) and without (luc −618/−61) the PPRE binding site, positive control vector (pGL-control), and negative control vector (pGL-basic) were used for transfection. Stimulation experiments were performed with 1, 10, and 20 μM troglitazone for 16 hours. Data are presented as RLUs and were normalized both to pSVb-galactosidase activity and to total protein content. Each experiment was performed in duplicate and is given as mean ± standard error. Lane 1, positive control vector; lane 2, negative control vector; lane 3, PPRE binding site containing vector; lane 4, vector devoid of PPRE binding site; lane 5, PPRE binding site containing vector after stimulation with 1 μM troglitazone; lane 6, PPRE binding site containing vector after stimulation with 10 μM troglitazone; lane 7, PPRE binding site containing vector after stimulation with 20 μM troglitazone.

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image

Figure 3. Effects of fenofibrate on promoter activity of murine CORS-26/luciferase gene chimeras. CORS-26 promoter fragments with the PPRE binding site (luc −844/−61), positive control vector (pGL-control) and negative control vector (pGL-basic) were used for transfection. Stimulation experiments were performed with 50, 100, and 200 μM fenofibrate for 16 hours. Data are presented as RLUs and were normalized both to pSVb-galactosidase activity and to total protein content. Each experiment was performed in duplicate and is given as mean ± standard error. Lane 1, positive control vector; lane 2, negative control vector; lane 3, PPRE binding site containing vector; lane 4, PPRE binding site containing vector after stimulation with 50 μM fenofibrate; lane 5, PPRE binding site containing vector after stimulation with 100 μM fenofibrate; lane 6, PPRE binding site containing vector after stimulation with 200 μM fenofibrate.

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Fenofibrate

Transfection with the positive control vector (Figure 3, lane 1) revealed high amounts of RLU (1.0 × 108 ± 6.4 × 106 RLU), whereas the negative control vector (Figure 4, lane 2) had only very low activity (0.09 × 106 ± 11,852 RLU). Fragment luc −844/61 carrying the PPRE site (Figure 3, lane 3) induced a significant induction of promoter activity compared with the control vector (1.7 × 106 ± 0.14 × 106 vs. 0.09 × 106 ± 11,852 RLU; p = 0.002). Stimulation with increasing doses of the specific PPARα ligand fenofibrate (Figure 3, lanes 4 to 6) caused a stepwise reduction of promoter activity using the PPRE site containing fragment (Figure 3, lane 3). Promoter activity was reduced from 1.7 × 106 ± 0.14 × 106 RLU (Figure 3, lane 3) to 0.9 × 106 ± 0.1 × 106 RLU by 50 μM fenofibrate (Figure 3, lane 4; p = 0.02) to 0.6 × 106 ± 0.03 × 106 RLU by 100 μM fenofibrate (Figure 3, lane 5; p = 0.005) and to 0.47 × 106 ± 0.05 × 106 RLU by 200 μM fenofibrate (Figure 3, lane 6; p = 0.007).

image

Figure 4. Effects of 15-D-PGJ2 on promoter activity of murine CORS-26/luciferase gene chimeras. CORS-26 promoter fragments with the PPRE binding site (luc −844/−61), positive control vector (pGL-control), and negative control vector (pGL-basic) were used for transfection. Stimulation experiments were performed with 0.5, 1.0, and 2.5 μM 15-D-PGJ2 for 16 hours. Data are presented as RLUs and were normalized both to pSVb-galactosidase activity and to total protein content. Each experiment was performed in duplicate and is given as mean ± standard error. Lane 1, positive control vector; lane 2, negative control vector; lane 3, PPRE binding site containing vector; lane 4, PPRE binding site containing vector after stimulation with 0.5 μM 15-D-PGJ2; lane 5, PPRE binding site containing vector after stimulation with 1.0 μM 15-D-PGJ2; lane 6, PPRE binding site containing vector after stimulation with 2.5 μM 15-D-PGJ2.

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15-D-PGJ2

Transfection with the positive control vector (Figure 4, lane 1) presented with high amounts of RLU (1.5 × 108 ± 11.5 × 106 RLU), whereas the negative control vector (Figure 4, lane 2) had only very low activity (0.13 × 106 ± 4965 RLU). Fragment luc −844/61 carrying the PPRE site (Figure 4, lane 3) induced a significant induction of promoter activity compared with the control vector (2.6 × 106 ± 6806 vs. 0.13 × 106 ± 4965 RLU, p = 0.002). Stimulation with increasing doses of the PPARγ ligand 15-D-PGJ2 (Figure 4, lanes 4 to 6) did not cause significant changes of promoter activity (Figure 4, lane 3). Promoter activity was 2.2 × 106 ± 0.15 × 106 RLU after stimulation with 0.5 μM 15-D-PGJ2 (Figure 4, lane 4), 2.5 × 106 ± 0.14 × 106 RLU after stimulation with 1.0 μM 15-D-PGJ2 (Figure 4, lane 5), and 2.2 × 106 ± 0.04 × 106 RLU after stimulation with 2.5 μM 15-D-PGJ2 (Figure 4, lane 6).

Quantitative CORS-26-mRNA Expression After Stimulation of Adipocytes with Troglitazone and Fenofibrate

Because both troglitazone and fenofibrate but not 15-D-PGJ2 reduced promoter activity, quantitative CORS-26 mRNA expression was studied after stimulation (48 hours) of adipocytes with troglitazone and fenofibrate (24 hours; Figure 5). Stimulation with 5 μM (Figure 5, lane 2) troglitazone led to a nonsignificant, 1-fold reduction of CORS-26 mRNA expression compared with control/vehicle (Figure 5, lane 1). However, stimulation with 10 μM troglitazone (Figure 5, lane 3) caused a −6.6 ± 0.5-fold reduction of CORS-26 mRNA expression (p = 0.036).

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Figure 5. Effect of troglitazone and fenofibrate on quantitative CORS-26 mRNA expression. Quantitative CORS-26 mRNA expression was studied by quantitative real-time PCR analysis. The amount of CORS-26 mRNA was normalized to β-actin. Vehicle/control-induced CORS-26 mRNA expression was set as +1. Changes in mRNA expression are given as x-fold reduction compared with vehicle/control experiments. Each experiment was performed in triplicate and mean values ± standard error were used for calculation of the relative change. Lane 1, vehicle/control without specific stimulation; lane 2, 5 μM troglitazone; lane 3, 10 μM troglitazone; lane 4, 50 μM fenofibrate; lane 5, 100 μM fenofibrate. n.s., not significant; *compared with vehicle/control.

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Both stimulation with 50 (Figure 5, lane 4) and 100 μM (Figure 5, lane 5) fenofibrate caused a significant, 9-fold reduction of CORS-26 mRNA expression (50 μM: −9.5 ± 3.3-fold; p = 0.037 and 100 μM: −9.6 ± 2.2-fold, p = 0.037).

Specific and Competitive Binding of Adipocytic Nuclear Extracts to the PPRE Recognition Element Within the CORS-26 Promoter

The binding of nuclear extracts isolated from mature adipocytes to the putative PPRE binding site after hormonally induced differentiation of preadipocytes was investigated by EMSA (Figure 6). Incubation of the radiolabeled promoter fragment prom −641/−595 (Figure 6, lane 1: free probe) with nuclear extracts from 3T3-L1-adipocytes produced a band shift (Figure 6, lane 2) that can be competed by using 50-fold (Figure 6, lane 3) and 100-fold (Figure 6, lane 4) molar excess of unlabeled probe as specific competitor. Coincubation of the radiolabeled promoter fragment prom −641/−595 with nuclear extracts and specific PPARα antibodies did not generate a supershift band. However, coincubation of the radiolabeled promoter fragment prom −641/−595 with nuclear extracts and two different, but specific PPARγ antibodies (Figure 6, lanes 6 and 8) resulted in the appearance of a specific supershift band. The same band could be observed by coincubation with unspecific PPARα/γ antibodies (Figure 6, lane 7).

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Figure 6. Binding of PPARγ, but not PPARα, to the CORS-26 promoter (EMSA). The binding of nuclear extracts (NEs) from 3T3-L1 adipocytes to the PPRE binding site at −621 to −626 bp was studied. Shifted complexes are marked with brackets. The bands marked with a cross are specific low molecular weight complexes that have not been identified. The supershifted complexes are indicated by an arrow. Competition experiments were performed using unlabeled probe. Lane 1, free probe; lane 2, NE; lane 3, NE preincubated with a 50-fold molar excess of unlabeled probe; lane 4, NE preincubated with a 100-fold molar excess of unlabeled probe; lane 5, NE + specific PPARα antibodies; lane 6, NE + specific PPARγ antibodies; lane 7, NE + unspecific PPARα/PPARγ antibodies; lane 8, NE + PPARγ antibodies.

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Effects of Recombinant CORS-26 Protein on the Adipocytic Secretion of Adiponectin and Resistin

Because adiponectin and resistin represent important adipocyte-derived mediators regulating insulin sensitivity, we decided to study the effects of recombinant CORS-26 stimulation on the adipocytic secretion of these both adipokines.

Adiponectin Secretion (Figure 7A)
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Figure 7. CORS-26-induced secretion of adiponectin and resistin by mature adipocytes. (A) CORS-26-induced secretion of adiponectin. (B) CORS-26-induced secretion of resistin. Data are given as box plots. Median (black line), 25th and 75th quartiles (gray box), and range (upper and lower extreme values) are depicted. 1, control; 2, 1 ng/mL CORS-26; 3, 50 ng/mL CORS-26; 4, 100 ng/mL CORS-26; 5, 250 ng/mL CORS-26.

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The basal adiponectin secretion was 10.2 ± 0.7 ng/mL. Whereas stimulation of adipocytes with 1 ng/mL CORS-26 had no effect on adiponectin secretion (11.1 ± 0.4 ng/mL), higher doses significantly up-regulated adiponectin secretion to 20.6 ± 1.2 ng/mL (10 ng/mL CORS-26; p < 0.001), to 20.0 ± 1.4 ng/mL (100 ng/mL CORS-26; p < 0.001), and to 23.5 ± 0.9 ng/mL (250 ng/mL CORS-26; p < 0001), respectively.

Resistin Secretion (Figure 7B)

The basal resistin secretion was 3589 ± 233 pg/mL and could not be stimulated using 1 ng/mL CORS-26 (3609 ± 241 pg/mL). However, increasing doses of CORS-26 significantly stimulated resistin secretion up to 4653 ± 335 pg/mL (10 ng/mL CORS-26; p < 0.001), to 4622 ± 224 pg/mL (100 ng/mL CORS-26; p < 0.001) and to 4189 ± 181 pg/mL (250 ng/mL CORS-26; p < 0.001), respectively.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Because antibodies against CORS-26 or enzyme-linked immunosorbent assay-based detection systems are currently not available, data on the regulation and the protein expression profile of CORS-26 during adipocyte differentiation are lacking. These results clearly indicate that CORS-26 mRNA and protein are completely absent in preadipocytes and become induced during late adipocyte differentiation in mature adipocytes. Although preadipocytes are morphologically very similar to fibroblasts and both cell types develop from mesenchymal precursor cells, preadipocytes are negative in contrast to normal skin fibroblasts that are positive for CORS-26 expression. The expression profile during late adipocyte differentiation resembles those of other adipocyte secretory proteins (adipocytokines), for example, adiponectin (1, 2), leptin (24, 25), or resistin (26).

The PPARs are members of the thyroid/steroid/retinoid nuclear receptor superfamily of ligand-activated transcription factors. The three isotypes PPARα, PPARγ, and PPARβ are able to bind the same consensus response element PPRE, although with different affinities. These PPREs consist of a direct repeat of a consensus one-half-site recognition hexamer (AGGTCA or TGACCT) separated by one nucleotide (27, 28, 29). These direct repeat-1 motifs can also be found in the binding sites for other nuclear receptors complexes, such as RXRα dimers (30). Species differences in gene expression in response to PPAR ligands occur at the level of the gene promoter sequence (31). Polarity also plays a role, and thus, each one-half-site can confer unique properties to the bound transcription factor with respect to ligand binding and interaction with potential corepressors and coactivators (32).

AGGTCA one-half-site-containing PPRE/RXRα motifs have been shown to regulate transcriptional activity of human and murine genes such as acyl coenzyme A oxidase (31), ABCD2, ABCD3 (33), CYP4A6 (30), and malic enzyme (34) in response to fibrates and other ligands. AGGTCA one-half-sites are also found in estrogen response element-related palindromic response elements (35). Because the significance of the AGGTCA recognition one-half-site within the murine CORS-26 promoter was unclear, we aimed to test this putative binding site for adipocyte-derived nuclear factor binding and for its role in ligand-induced transcriptional regulation.

While most of the prostaglandins exert their effects through G protein-coupled receptors, the J2 series of prostaglandins are efficacious activators of PPARγ (36). 15-D-PGJ2 represents an endogenous, natural ligand of PPARγ, whereas the glitazones such as troglitazone belong to the synthetic class of exogenous PPARγ ligands (37). In contrast, fibrates such as fenofibrate are typical activators of PPARα mainly controlling hepatic enzymes of fatty acid metabolism (31, 34).

In this study, fenofibrate, 15-D-PGJ2, and troglitazone were used to study effects of PPARα activation (fenofibrate) and PPARγ activation (troglitazone, 15-D-PGJ2) on transcriptional regulation and mRNA expression of the CORS-26 gene. Both fenofibrate and troglitazone caused a stepwise, dose-dependent, and significant reduction of promoter activity and mRNA expression, whereas 15-D-PGJ2 had no effect on promoter activity.

To study whether adipocytic nuclear extracts can bind to the PPRE, EMSAs were performed using specific and unspecific antibodies for PPARα and PPARγ. The effects of the PPARγ ligand troglitazone can be explained by the binding of ligand-activated PPARγ to the PPRE, because specific PPARγ antibodies show that adipocyte-derived PPARγ can bind to the PPRE (specific supershift band in EMSA). In contrast, the effects of the PPARα ligand fenofibrate on promoter activity and mRNA expression must be caused by secondary cellular effects of fenofibrate, because specific PPARα antibodies are not sufficient to produce supershift bands in EMSA and because unspecific PPARα/γ antibodies do not result in the appearance of additional supershift bands.

Taken together, only the exogenous PPARγ ligand troglitazone but not the endogenous ligand 15-D-PGJ2 or the PPARα ligand fenofibrate can exert transcriptional repression of the CORS-26 gene through the PPRE.

Most adipocyte-specific genes such as adiponectin, aP2, phosphenolyruvate-carboxy-kinase, C/EBPα, lipoprotein lipase, and fatty acid syntase are positively regulated by PPARγ (38, 39) and PPARγ ligands. However, few adipocyte-specific genes such as resistin (40) and leptin (41, 42, 43) are regulated by PPARγ-induced inhibition of transcription. Although PPARγ is necessary for adipocyte differentiation, ligand-induced activation of PPARγ can also lead to an inhibition of adipocytic genes such as leptin (44). Thus, CORS-26 represents a further adipocytic gene negatively regulated by PPARγ activation. Rare data on the transcriptional regulation of CORS-26 suggest some similarities to the transcriptional regulation of the leptin gene. Because leptin levels are partly associated with a linkage locus containing the CORS-26 gene, a similar regulation might be reasonable. In this context, both leptin (45, 46, 47) and CORS-26 (13) are regulated, in part, by the transcription factor SP-1. These data suggest an additional and transcriptional repression of CORS-26 in response to PPRE ligands with mainly PPARγ specificity.

Both adiponectin and CORS-26 represent adipocyte secretory proteins that are induced during late adipocyte differentiation. Although adiponectin and CORS-26 have an homology score of only 65% concerning the nucleotide sequence, both genes show some similar and striking features: adiponectin is composed of 244 amino acids containing a short noncollagenous N-terminal domain followed by a collagen-like sequence with 22 perfect Gly-X-Pro motifs. This collagen domain is followed by a C-terminal globular domain that forms a so-called “bouquet of flowers” structure of the oligomerization complex that has striking homology to the complement component C1q (1).

The open reading frame of CORS-26 (5) predicts a polypeptide sequence of 246 amino acids corresponding to a calculated molecular mass of 26 kDa (adiponectin: 244 amino acids). The presence of a signal peptide of 22 amino acid residues with a predicted cleavage site at Cys 22 suggests that CORS-26 is a secretory protein, as is the case with adiponectin. Using the BLASTp search program, a COOH-terminal globular domain with homology to the C1q complement domains in adiponectin and C1q A, B, C chains was found to be present (5). A feature of the protein is that the NH2-terminal part contains uninterrupted collagen-like Gly-X-Y repeats (21 repeats) immediately downstream of a short noncollagenous sequence at amino acid positions 51–111. NH2-terminal collagen-triplet repeats have been shown to be present not only in the CORS-26 gene (5), but also in the genes encoding adiponectin (2), complement protein C1q A, B, and C chains (48), and chipmunks hibernation-specific proteins (49).

The homologies concerning both proteins are depicted in Figure 8A. Because of these structural features and in homology to “adipo-nectin,” the name “carto-nectin” is suggested for the protein encoded by the CORS-26 gene sequence (Figure 8B). Additionally, the positioning of CORS-26 among the other adipocytokines is depicted in Figure 8C.

image

Figure 8. Comparison of adiponectin and CORS-26 protein structures as a basis for nomenclature. (A) Adiponectin and CORS-26 protein structure. (B) Aspects for nomenclature. (C) Positioning of CORS-26 among adipocytokines.

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Summary. These data suggest cartonectin, encoded by the CORS-26 gene, to represent a new adipokine belonging to the C1q-complement family of proteins with a N-terminal collagenous domain and a C-terminal globular domain. Because cartonectin is specifically induced during late adipocyte differentiation and because cartonectin induces the adipocytic secretion of adiponectin and resistin, a putative role in the context of insulin resistance, type 2 diabetes, and metabolic syndrome can be suspected.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

The technical assistance of K. Winkler and N. Smolnikow is highly appreciated. This work was supported by the 2003 EULAR-AMGEN Young Investigator Award and by grants of the German Research Association (SCHA 789/2-3).

Footnotes
  • 1

    Nonstandard abbreviations: CORS-26, collagenous repeat containing sequence of 26-kDa protein; PPRE, peroxisome proliferator response element; DMEM, Dulbecco's modified eagle medium; RT-PCR, reverse transcriptase-polymerase chain reaction; PBS, phosphate-buffered saline; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; PPAR, peroxisome proliferator-activated receptor; EMSA, electrophoretic mobility shift assay; RLU, relative light units; 1-D-PGJ2, 15-deoxy-prostaglandin-J2; NE, nuclear extract.

  • The costs of publication of this article were defrayed, in part, by the payment of page charges. This article must, therefore, be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Research Methods and Procedures
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References