Flipping the cyclooxygenase (Ptgs) genes reveals isoform-specific compensatory functions ,

Abstract

Two prostaglandin (PG) H synthases encoded by genes, colloquially known as cyclooxygenase (COX)-1 and COX-2, catalyze the formation of PG endoperoxide H, the precursor of the major prostanoids. To address the functional interchangeability of these two isoforms and their distinct roles, we have generated COX-2>COX-1 mice whereby is knocked in to the locus. We then "flipped" genes to successfully create the Reversa mouse strain, where knock-in COX-2 is expressed constitutively and knock-in COX-1 is lipopolysaccharide (LPS) inducible. In macrophages, flipping the two genes has no obvious impact on COX protein subcellular localization. COX-1 was shown to compensate for PG synthesis at high concentrations of substrate, whereas elevated LPS-induced PG production was only observed for cells expressing endogenous COX-2. Differential tissue-specific patterns of expression of the knock-in proteins were evident. Thus, platelets from COX-2>COX-1 and Reversa mice failed to express knock-in COX-2 and, therefore, thromboxane (Tx) production in vitro and urinary Tx metabolite formation in COX-2>COX-1 and Reversa mice in vivo were substantially decreased relative to WT and COX-1>COX-2 mice. Manipulation of COXs revealed isoform-specific compensatory functions and variable degrees of interchangeability for PG biosynthesis in cells/tissues.

Differential compensation of two cyclooxygenases in renal homeostasis is independent of prostaglandin‐synthetic capacity under basal conditions

“…A COX-2 . COX-1 (COX-2.1) mouse strain on the C57BL/6 background was established by partial replacement of the coding region of Ptgs1 (exons 3-10) with Ptgs2 exons 2-10, while leaving the Ptgs1 promoter region intact (23). Crossbreeding of COX-1 .…”

“…COX-2 and COX-2 . COX-1 mice produces Reversa mice (22,23). Genotyping was performed routinely by PCR on DNA, isolated from tail biopsies.…”

“…Tissue lysates of the whole kidney or dissected kidney (cortex and medulla) were prepared with T-PER protein extract reagent (Thermo Fisher Scientific, Waltham, MA, USA), plus a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). Western blot analysis was carried out as previously outlined (22,23).…”

“…anti-COX-2 was against mouse COX-2 aa 584-598 SHSRLDDINPTVLIK in the native C-terminus of COX-2. We previously described that we were unable to devise a strategy to differentiate between the knockin and endogenous (native) proteins (23). Therefore, the proteins detected with Western blot analysis or immunohistochemistry were comprised of both endogenous and knockin COX.…”

“…COX-2 (20), COX-2 . COX-1, and Reversa mouse strains (22,23) to study the distinct roles of the 2 COX isoforms in the kidney. We hypothesized that the 2 COX isoforms could preferentially compensate for some renal functions and in turn, differentially maintain homeostasis under basal conditions.…”