Gpat2: A gene associated with cell proliferation that is expressed in undifferentiated cells

PELLON-MAISON, M.; CATTANEO, E.R.; COLEMAN, R.A.; GONZALEZ BARO, M.R.

Waterville Valley, NH, USA.

Conferencia; Gordon Research Conference “Molecular and Cellular Biology of Lipids”; 2011

Gordon Research Conferences

The first step in the de novo glycerolipid biosynthesis is catalyzed by glycerol-3-phosphate acyltransferase (GPAT).  Four genes encoding GPATs have been cloned; and their products present different tissue and cellular locations.  Gpat2 encodes a mitochondrial protein mainly expressed in testis.  We have shown that Gpat2 is expressed in the spermatic cells and GPAT2 overexpression in CHOK1 cells increases triacylglycerol synthesis and storage, as well as cellular proliferation. To identify which metabolites product of GPAT2 activity stimulate cellular proliferation, our aim was to establish GPAT2’s substrate specificity and also determine which spermatic cells express Gpat2 and analyze the gene expression in other differentiating and tumor cells. GPAT2 was transiently transfected to CHOK1 cells. GPAT2-overexpressing cells specifically esterified arachidonoyl-CoA using both glycerol-3-phosphate and 1-oleoyl-glycerol-3-phosphate as substrates.  GPAT and acyl-glycerol-3-phosphate acyltransferase (AGPAT) activities increased 2-fold compared to control cells.  When palmitoyl-CoA, oleoyl-CoA, linoleoyl-CoA and docosahexanoyl-CoA were assayed, GPAT activity was similar in GPAT2 overexpressing cells than in control cells.  Consistent with the preference of GPAT2 for arachidonoyl-CoA and its role in TAG synthesis, TAG from GPAT2 overexpressing cells incubated for 24 h in media plus 10% FBS contained ~6% arachidonic acid whereas arachidonate could not be detected in TAG from control cells.  In GPAT2 overexpressing cells and after a 3-h incubation with 1-[14C] arachidonate, label incorporation in TAG increased by 40% and in PE decreased by the same percentage.  After 6-h and 24-h incubations significant label incorporation in the lysophosphatidylcholine fraction was observed. To identify the specific spermatogenic cell types that endogenously express Gpat2, we performed in situ mRNA hybridization using an anti-sense probe on adult mouse testis sections.  Gpat2 mRNA was unequivocally detected only in primary spermatocytes, and the signal was absent in cells undergoing further meiotic and differentiation phases.  Consistently, the expression of GPAT2 protein was highest in spermatocytes from adult rats, and the signal decreased in the spermatides, suggesting that Gpat2 is expressed in the less differentiated cell types.  In silico transcriptome analysis corroborated our hypothesis.  Because Gpat2 was expressed in several undifferentiated cells and its expression was decreased in different models of cellular differentiation, we analyzed Gpat2 mRNA expression in 3T3L1 cells differentiating to adipocytes.  Contrary to the other three GPAT isoform expression, Gpat2 mRNA did not increase during differentiation, indicating that the cellular role of GPAT2 strongly differs from the other isoforms.  We showed by RT-PCR that Gpat2 is highly expressed in the human-breast-cancer-derived cell line MDA-MB231, but no expression was detected in more differentiated breast-cancer cell lines. We conclude that Gpat2 expresses in spermatocytes in normal conditions and it is overexpressed in undifferentiated tumor cells.  Some arachidonic-acid-rich metabolites product of its activity may act as signals for cellular proliferation or survival. Support: NIH (TW8698 and DK56598), CONICET, Argentina (PIP0527)