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Related Experiment Videos

Human cyclooxygenase-2 cDNA.

T Hla1, K Neilson

  • 1Department of Molecular Biology, Holland Laboratory, American Red Cross, Rockville, MD 20855.

Proceedings of the National Academy of Sciences of the United States of America
|August 15, 1992
PubMed
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Researchers cloned the human cyclooxygenase-2 (Cox-2) gene, revealing it encodes a 70-kDa protein. This new Cox-2 enzyme is differentially regulated and suggests a role in inflammatory conditions.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Immunology

Background:

  • Cyclooxygenase (Cox) enzymes, also known as prostaglandin (PG) H synthase, are crucial for synthesizing inflammatory prostaglandins.
  • Cox activity is a major regulatory point in PG biosynthesis, influenced by growth factors, cytokines, and tumor promoters.

Purpose of the Study:

  • To clone and characterize the second form of the cyclooxygenase gene, designated Cox-2, from human umbilical vein endothelial cells (HUVEC).
  • To investigate the expression and regulation of the newly identified Cox-2 gene and its corresponding mRNA.

Main Methods:

  • Cloning of the human Cox-2 gene from HUVEC.
  • In vitro translation of human Cox-2 (hCox-2) transcript and immunoprecipitation.
  • Expression of hCox-2 in Cos-7 cells to assess cyclooxygenase activity.

Related Experiment Videos

  • Hybridization analysis of hCox-2 and hCox-1 cDNA with mRNA species.

    • Main Results:

    • The hCox-2 cDNA encodes a 604-amino acid polypeptide, 61% identical to human Cox-1 (hCox-1).
    • In vitro translation produced a 70-kDa hCox-2 protein recognized by anti-ovine Cox antiserum.
    • Expression of hCox-2 in Cos-7 cells conferred cyclooxygenase activity.
    • hCox-2 mRNA is a 4.5-kilobase species, distinct from hCox-1 mRNA (3- and 5.3-kilobase species).
    • Both Cox-1 and Cox-2 mRNAs are expressed in various cell types, including HUVEC, vascular smooth muscle cells, monocytes, and fibroblasts.
    • Cox-2 mRNA showed preferential induction by phorbol 12-myristate 13-acetate and lipopolysaccharide in endothelial cells and monocytes.

    Conclusions:

    • The cyclooxygenase enzyme is encoded by at least two genes (Cox-1 and Cox-2) that are expressed and differentially regulated.
    • The high-level induction of hCox-2 transcript in inflammatory cells suggests a significant role in inflammatory processes.