Targeted disruption and subcellular distribution of phospholipase C 3

Abstract: Phosphatidylinositol specific phospholipase C (PLC) isoforms are key enzymes in the signal transduction pathway that control many cellular processes including cell proliferation, hormone secretion and egg fertilization. The PLCβ3 gene, assigned to the multiple endocrine neoplasia type 1 (MEN1) locus, has been implicated in endocrine tumorigenesis due to the low expression in several endocrine tumors. In order to study the biological function of PLCβ3, we have isolated and characterized the mouse cDNA as well as the genomic DNA. We found that the gene encompasses 31 exons, encodes a protein of 1235 amino acids. We have also mapped the gene to mouse chromosome 19B and disrupted the gene. Homozygous inactivation of the gene was lethal at embryonic day 2.5. PLCβ3 deficient embryos, cultured in vitro, showed poor embryonicorganization as well as reduction of cell numbers and failed to form blastocoele cavities. PLCβ3 protein was expressed in unfertilized mouse eggs, 3-cell and egg cylinder stages of wild type embryos as detected by immunohistochemistry. Although expression of the protein in the heterozygous mice was reduced by approximately 50% compared to wild type littermates, they showed no specific phenotype and remained healthy for at least two generations.The subcellular distribution of four different PLC isozymes in rodent pancreas and gastric mucosa was examined by electron microscope immunocytochemistry. PLCβ1 was exclusively expressed in zymogen granules of the pancreatic acinar cells. PLCβ2 and PLCβ3 were located in the endoplasmic reticulum (ER) of acinar and gastric mucosa cells. Furthermore, both PLCβ2 and PLCβ3 were highly expressed in the heterochromatin areas of the gastric mucosa cell nuclei. In neurons of the mouse brain, PLCβ3 exhibited an expression pattern similar to that of the gastric mucosa. PLCβ3 displayed a weak labeling in nuclei and BR of pancreatic endocrine cells. In contrast to PLCβ3, PLCβ2 was present in secretory granules of pancreatic acinar cells and endocrine cells. PLCγ1 was present in the secretory granules of both pancreatic acinar cells and endocrine cells.