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Cultured Preadipocytes Undergoing Stable Transfection with Cyclooxygenase-1 in the Antisense Direction Accelerate Adipogenesis During the Maturation Phase of Adipocytes

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Abstract

The arachidonate cyclooxygenase (COX) pathway is involved in the generation of several types of endogenous prostaglandins (PGs) with opposite effects on adipogenesis at different life stages of adipocytes. However, the specific role of COX isoforms, the rate-limiting enzymes for the pathway, remains elusive in the regulation of the endogenous synthesis of PGs. This study was aimed at the selective suppression of the constitutive COX-1 in cultured preadipocytes by the isolation of cloned preadipocytes transfected stably with a mammalian expression vector harboring cDNA encoding mouse COX-1 in the antisense direction. The gene expression analysis revealed that the transcript and protein levels of the constitutive COX-1 were substantially suppressed in the isolated cloned transfectants with antisense COX-1. By contrast, the expression of the inducible COX-2 was not affected in the stable transfectants with antisense COX-1. All of the cloned stable transfectants with antisense COX-1 exhibited a significant reduction in the immediate synthesis of PGE2 serving as an anti-adipogenic factor. The sustained expression of COX-1 in the antisense direction induced the appreciable stimulation of fat storage in adipocytes during the maturation phase, which was associated with the higher expression levels of adipocyte-specific genes, indicating the positive regulation of adipogenesis program. Moreover, the up-regulation of adipogenesis is accompanied by a higher production of J2 series PGs including 15-deoxy-Δ12,14-PGJ2 and Δ12-PGJ2, known as pro-adipogenic factors by the transfectants with antisense COX-1. The results suggest that the inducible COX-2 can contribute to the endogenous synthesis of PGJ2 derivatives acting as autocrine mediators to simulate adipogenesis during the maturation phase by way of compensation for the suppressed expression of the constitutive COX-1.

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Abbreviations

COX:

Cyclooxygenase

PG:

Prostaglandin

15d-PGJ2 :

15-Deoxy-Δ12,14-PGJ2

PPARγ:

Peroxisome proliferator-activated receptor γ

AA:

Arachidonic acid

DME-HEPES:

Dulbecco's modified Eagle's medium with 25 mM HEPES

PMA:

Phorbol 12-myristate 13-acetate

FBS:

Fetal bovine serum

IBMX:

3-Isobutyl-1-methylxanthine

G418:

Geneticin disulfate

RT:

Reverse transcriptase

PCR:

Polymerase chain reaction

ELISA:

Enzyme-linked immunosorbent assay

GM:

Growth medium

DM:

Differentiation medium

MM:

Maturation medium

C/EBPα:

CCAAT/enhancer-binding protein α

GLUT-4:

Glucose transporter-4

LPL:

Lipoprotein lipase

aP2:

Adipocyte protein 2

SDS-PAGE:

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene difluoride

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Authors and Affiliations

  1. Department of Life Science and Biotechnology, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane, 690-8504, Japan

    Mohammad Sharifur Rahman, Pinky Karim Syeda, Ferdous Khan, Mitsuo Jisaka, Tsutomu Nagaya & Kazushige Yokota

  2. Department of Molecular and Functional Genomics, Center for Integrated Research in Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane, 690-8504, Japan

    Kohji Nishimura

  3. Department of Clinical Pharmacy, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima-shi, Tokushima, 770-8514, Japan

    Fumiaki Shono

Authors
  1. Mohammad Sharifur Rahman
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  2. Pinky Karim Syeda
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Correspondence to Kazushige Yokota.

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Rahman, M.S., Syeda, P.K., Khan, F. et al. Cultured Preadipocytes Undergoing Stable Transfection with Cyclooxygenase-1 in the Antisense Direction Accelerate Adipogenesis During the Maturation Phase of Adipocytes. Appl Biochem Biotechnol 171, 128–144 (2013). https://doi.org/10.1007/s12010-013-0347-3

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