Endoplasmic reticulum localization of phosphoinositide specific phospholipase C enzymes in U73122 cultured human osteoblasts

Abstract

<abstract> <p>Different signal transduction pathways contribute to the differentiation and metabolic activities of osteoblasts, with special regard to the calcium-related pathway of phosphoinositide specific phospholipase C (PLC) enzyme family. PLC enzymes were demonstrated to be involved in the differentiation of osteoblasts and differently localize in the nucleus, cytoplasm or both depending on the isoform. The amino-steroid molecule U-73122 inhibits the enzymes belonging to the PLC family. In addition to the temporary block of the enzymatic activity, U-73122 promotes off-target effects, including modulation of the expression of selected <italic>PLC</italic> genes and different localization of PLC enzymes, depending on the cell line, in different cell lines.</p> <p>In order to evaluate possible off-target effects of the molecule in human osteoblasts, we investigated the expression of <italic>PLC</italic> genes and the localization of PLC enzymes in cultured human osteoblasts (hOBs) in the presence of low dose U-73122.</p> <p>Our results confirm that all <italic>PLC</italic> genes are transcribed in hOBs, that probably splicing variants of selected <italic>PLC</italic> genes are expressed and that all PLC enzymes are present in hOBs, except for PLC δ3 in quiescent hOBs at seeding. Our results confirm literature data excluding toxicity of U-73122 on cell survival. Our results indicate that U-73122 did not significantly affect the transcription of <italic>PLC</italic> genes. It acts upon the localization of PLC enzymes, as PLC enzymes are detected in cell protrusions or pseudopodia-like structures, at the nuclear or the plasma membrane, in membrane ruffles and/or in the endoplasmic reticulum.</p> </abstract>