Arachidonic Acid Release from Mammalian Cells Transfected with Human Groups IIA and X Secreted Phospholipase A2 Occurs Predominantly during the Secretory Process and with the Involvement of Cytosolic Phospholipase A2-α

Carine M Mounier,Farideh Ghomashchi,Margaret R Lindsay,Scott James,Alan G Singer,Robert G Parton,Michael H Gelb

doi:10.1074/jbc.m313019200

Carine M Mounier, Farideh Ghomashchi + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m313019200

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Abstract

Stable expression of human groups IIA and X secreted phospholipases A(2) (hGIIA and hGX) in CHO-K1 and HEK293 cells leads to serum- and interleukin-1beta-promoted arachidonate release. Using mutant CHO-K1 cell lines, it is shown that this arachidonate release does not require heparan sulfate proteoglycan- or glycosylphosphatidylinositol-anchored proteins. It is shown that the potent secreted phospholipase A(2) inhibitor Me-Indoxam is cell-impermeable. By use of Me-Indoxam and the cell-impermeable, secreted phospholipase A(2) trapping agent heparin, it is shown that hGIIA liberates free arachidonate prior to secretion from the cell. With hGX-transfected CHO-K1 cells, arachidonate release occurs before and after enzyme secretion, whereas all of the arachidonate release from HEK293 cells occurs prior to enzyme secretion. Immunocytochemical studies by confocal laser and electron microscopies show localization of hGIIA to the cell surface and Golgi compartment. Additional results show that the interleukin-1beta-dependent release of arachidonate is promoted by secreted phospholipase A(2) expression and is completely dependent on cytosolic (group IVA) phospholipase A(2). These results along with additional data resolve the paradox that efficient arachidonic acid release occurs with hGIIA-transfected cells, and yet exogenously added hGIIA is poorly able to liberate arachidonic acid from mammalian cells.

Highlights

Stable expression of human groups IIA and X secreted phospholipases A2 in CHO-K1 and HEK293 cells leads to serum- and interleukin-1␤-promoted arachidonate release
The behavior of human group IIA and human group X sPLA2s when transfected in HEK293 and CHO cells has been extensively studied. hGIIA is secreted from HEK293 cells, and most of the extracellular enzyme is associated with the cell surface
Arachidonate Release in CHO-K1 Cells Transfected with hGIIA and human group X (hGX)—CHO-K1 cells were transfected with plasmids driving constitutive expression of hGIIA and hGX, and 12 stable transfectants were established

Summary

EXPERIMENTAL PROCEDURES

Materials—The cPLA2-␣ inhibitors pyrrophenone [26], pyrrolidine-1 [27], and AZ-1 (compound 22 of [28]) and the sPLA2 inhibitor MeIndoxam [29] were prepared as described. The cells were washed three times with complete medium (medium carefully removed with a pipette rather than by aspiration) and covered with 1 ml of medium containing the desired additives (described in the figure legends). After removing the blocking agent, the cells were incubated for 2 h at room temperature with 1% BSA containing anti-hGIIA antiserum (1/500 dilution, antiserum prepared as described [2]). The cells were washed four times with PBS at room temperature and prepared for confocal microscopy as described above. Cell Permeability of Me-Indoxam—Uptake of Me-Indoxam into HEK293 cells was measured by incubating cells with [3H]Me-Indoxam (680 Ci/mol, prepared as described in the Supplemental Material) followed by centrifugation of the cells through a layer of serum to rapidly separate them from the extracellular medium [33]. Immunoblot Analysis of cPLA2-␣—This is described in the Supplemental Material

RESULTS

DISCUSSION

Arachidonic Acid Release from Mammalian Cells Transfected with Human Groups