ABSTRACT
Phosphoinositide-specific phospholipase Cγ1 (PLCγ1) is activated downstream of many receptor tyrosine kinases to promote cell motility. Inhibition of this protein is being explored as a therapeutic strategy for blocking cancer cell invasion and metastasis. The clinical development of such cytostatic therapies requires the implementation of pharmacodynamic biomarkers of target modulation. In this study, we use magnetic resonance spectroscopy to explore metabolic biomarkers of PLCγ1 down-regulation in PC3LN3 prostate cancer cells. We show that inhibition of PLCγ1 via an inducible short hairpin RNA system causes a reduction in phosphocholine levels by up to 50% relative to the control as detected by (1)H and (31)P magnetic resonance spectroscopy analyses. This correlated with a rounded-up morphology and reduced cell migration. Interestingly, the fall in phosphocholine levels was not recorded in cells with constitutive PLCγ1 knockdown where the rounded-up phenotype was no longer apparent. This study reveals alterations in metabolism that accompany the cellular effects of PLCγ1 knockdown and highlights phosphocholine as a potential pharmacodynamic biomarker for monitoring the action of inhibitors targeting PLCγ1 signaling.
Subject(s)
Biomarkers, Tumor , Gene Expression Regulation, Neoplastic , Phospholipase C gamma/metabolism , Phosphorylcholine/metabolism , Prostatic Neoplasms/metabolism , Animals , Cell Line , Cell Movement/genetics , Down-Regulation , Gene Knockdown Techniques , Humans , Male , Mice , Phospholipase C gamma/genetics , Prostatic Neoplasms/pathology , RNA InterferenceABSTRACT
Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.
Subject(s)
Isoenzymes/metabolism , Phosphoinositide Phospholipase C/metabolism , Alternative Splicing , Animals , Catalytic Domain , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Mice , Mice, Knockout , Phosphoinositide Phospholipase C/chemistry , Phosphoinositide Phospholipase C/geneticsABSTRACT
Cell motility is a critical event in many processes and is underlined by complex signalling interactions. Although many components have been implicated in different forms of cell migration, identification of early key mediators of these events has proved difficult. One potential signalling intermediate, PLCgamma1, has previously been implicated in growth-factor-mediated chemotaxis but its position and roles in more-complex motility events remain poorly understood. This study links PLCgamma1 to early, integrin-regulated changes leading to cell motility. The key role of PLCgamma1 was supported by findings that specific depletion of PLCgamma1 by small interfering (si)RNA, or by pharmacological inhibition, or the absence of this isoform in PLCgamma1(-/-) cells resulted in the failure to form cell protrusions and undergo cell spreading and elongation in response to integrin engagement. This integrin-PLCgamma1 pathway was shown to underlie motility processes involved in morphogenesis of endothelial cells on basement membranes and invasion of cancer cells into such three-dimensional matrices. By combining cellular and biochemical approaches, we have further characterized this signalling pathway. Upstream of PLCgamma1 activity, beta1 integrin and Src kinase are demonstrated to be essential for phosphorylation of PLCgamma1, formation of protein complexes and accumulation of intracellular calcium. Cancer cell invasion and the early morphological changes associated with cell motility were abolished by inhibition of beta1 integrin or Src. Our findings establish PLCgamma1 as a key player in integrin-mediated cell motility processes and identify other critical components of the signalling pathway involved in establishing a motile phenotype. This suggests a more general role for PLCgamma1 in cell motility, functioning as a mediator of both growth factor and integrin-initiated signals.
