Elizabeth Renee Cattaneo

Glycerol-3-phosphate acyltranferase-2 behaves as a cancer testis gene and promotes growth and tumorigenicity of the breast cancer MDA-MB-231 cell line.

The de novo synthesis of glycerolipids in mammalian cells begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferase (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions. Because it is aberrantly expressed in multiple myeloma, it has been proposed as a novel cancer testis gene. Using a bioinformatics approach, we found that GPAT2 is highly expressed in melanoma, lung, prostate and breast cancer, and we validated GPAT2 expression at the protein level in breast cancer by immunohistochemistry. In this case GPAT2 expression correlated with a higher histological grade. 5-Aza-2′ deoxycytidine treatment of human cells lines induced GPAT2 expression suggesting epigenetic regulation of gene expression. In order to evaluate the contribution of GPAT2 to the tumor phenotype, we silenced its expression in MDA-MB-231 cells. GPAT2 knockdown diminished cell proliferation, anchorage independent growth, migration and tumorigenicity, and increased staurosporine-induced apoptosis. In contrast, GPAT2 over-expression increased cell proliferation rate and resistance to staurosporine-induced apoptosis. To understand the functional role of GPAT2, we performed a co-expression analysis in mouse and human testis and found a significant association with semantic terms involved in cell cycle, DNA integrity maintenance, piRNA biogenesis and epigenetic regulation. Overall, these results indicate the GPAT2 would be directly associated with the control of cell proliferation. In conclusion, we confirm GPAT2 as a cancer testis gene and that its expression contributes to the tumor phenotype of MDA-MB-231 cells.

Glycerol-3-phosphate acyltransferase-2 is expressed in spermatic germ cells and incorporates arachidonic acid into triacylglycerols.

Background De novo glycerolipid synthesis begins with the acylation of glycerol-3 phosphate catalyzed by glycerol-3-phosphate acyltransferase (GPAT). In mammals, at least four GPAT isoforms have been described, differing in their cell and tissue locations and sensitivity to sulfhydryl reagents. In this work we show that mitochondrial GPAT2 overexpression in CHO-K1 cells increased TAG content and both GPAT and AGPAT activities 2-fold with arachidonoyl-CoA as a substrate, indicating specificity for this fatty acid. Methods and Results Incubation of GPAT2-transfected CHO-K1 cells with [1-14C]arachidonate for 3 h increased incorporation of [14C]arachidonate into TAG by 40%. Consistently, arachidonic acid was present in the TAG fraction of cells that overexpressed GPAT2, but not in control cells, corroborating GPAT2s role in synthesizing TAG that is rich in arachidonic acid. In rat and mouse testis, Gpat2 mRNA was expressed only in primary spermatocytes; the protein was also detected in late stages of spermatogenesis. During rat sexual maturation, both the testicular TAG content and the arachidonic acid content in the TAG fraction peaked at 30 d, matching the highest expression of Gpat2 mRNA and protein. Conclusions These results strongly suggest that GPAT2 expression is linked to arachidonoyl-CoA incorporation into TAG in spermatogenic germ cells.

Methylation of the Gpat2 promoter regulates transient expression during mouse spermatogenesis

We studied the expression pattern and the mechanisms governing the transcription of GPAT2, an enzyme that is highly expressed in testis. GPAT2 is regulated epigenetically and its expression correlates with the initiation of meiosis.

Glycerol-3-phosphate acyltransferase 2 expression modulates cell roughness and membrane permeability: An atomic force microscopy study

In mammalian cells, de novo glycerolipid synthesis begins with the acylation of glycerol-3-phosphate, catalyzed by glycerol-3-phosphate acyltransferases (GPAT). GPAT2 is a mitochondrial isoform primarily expressed in testis under physiological conditions, and overexpressed in several types of cancers and cancer-derived human cell lines where its expression contributes to the tumor phenotype. Using gene silencing and atomic force microscopy, we studied the correlation between GPAT2 expression and cell surface topography, roughness and membrane permeability in MDA-MB-231 cells. In addition, we analyzed the glycerolipid composition by gas-liquid chromatography. GPAT2 expression altered the arachidonic acid content in glycerolipids, and the lack of GPAT2 seems to be partially compensated by the overexpression of another arachidonic-acid-metabolizing enzyme, AGPAT11. GPAT2 expressing cells exhibited a rougher topography and less membrane damage than GPAT2 silenced cells. Pore-like structures were present only in GPAT2 subexpressing cells, correlating with higher membrane damage evidenced by lactate dehydrogenase release. These GPAT2-induced changes are consistent with its proposed function as a tumor-promoting gene, and might be used as a phenotypic differentiation marker. AFM provides the basis for the identification and quantification of those changes, and demonstrates the utility of this technique in the study of cancer cell biology.

Glycerol-3-phosphate acyltransferase 2 is essential for normal spermatogenesis

Glycerol-3-phosphate acyltransferases (GPATs) catalyze the first and rate-limiting step in the de novo glycerolipid synthesis. The GPAT2 isoform differs from the other isoforms because its expression is restricted to male germ cells and cancer cells. It has been recently reported that GPAT2 expression in mouse testis fluctuates during sexual maturation and that it is regulated by epigenetic mechanisms in combination with vitamin A derivatives. Despite progress made in this field, information about GPAT2 role in the developing male germ cells remains unclear. The aim of the present study was to confirm the hypothesis that GPAT2 is required for the normal physiology of testes and male germ cell maturation. The gene was silenced in vivo by inoculating lentiviral particles carrying the sequence of a short-hairpin RNA targeting Gpat2 mRNA into mouse testis. Histological and gene expression analysis showed impaired spermatogenesis and arrest at the pachytene stage. Defects in reproductive fitness were also observed, and the analysis of apoptosis-related gene expression demonstrated the activation of apoptosis in Gpat2-silenced germ cells. These findings indicate that GPAT2 protein is necessary for the normal development of male gonocytes, and that its absence triggers apoptotic mechanisms, thereby decreasing the number of dividing germ cells.