The Developmentally Regulated Osteoblast Phosphodiesterase GDE3 Is Glycerophosphoinositol-specific and Modulates Cell Growth

Abstract

The glycerophosphodiester phosphodiesterase enzyme family involved in the hydrolysis of glycerophosphodiesters has been characterized in bacteria and recently identified in mammals. Here, we have characterized the activity and function of GDE3, one of the seven mammalian enzymes. GDE3 is up-regulated during osteoblast differentiation and can affect cell morphology. We show that GDE3 is a glycerophosphoinositol (GroPIns) phosphodiesterase that hydrolyzes GroPIns, producing inositol 1-phosphate and glycerol, and thus suggesting specific roles for this enzyme in GroPIns metabolism. Substrate specificity analyses show that wild-type GDE3 selectively hydrolyzes GroPIns over glycerophosphocholine, glycerophosphoethanolamine, and glycerophosphoserine. A single point mutation in the catalytic domain of GDE3 (GDE3R231A) leads to loss of GroPIns enzymatic hydrolysis, identifying an arginine residue crucial for GDE3 activity. After heterologous GDE3 expression in HEK293T cells, phosphodiesterase activity is detected in the extracellular medium, with no effect on the intracellular GroPIns pool. Together with the millimolar concentrations of calcium required for GDE3 activity, this predicts an enzyme topology with an extracellular catalytic domain. Interestingly, GDE3 ectocellular activity is detected in a stable clone from a murine osteoblast cell line, further confirming the activity of GDE3 in a more physiological context. Finally, overexpression of wild-type GDE3 in osteoblasts promotes disassembly of actin stress fibers, decrease in growth rate, and increase in alkaline phosphatase activity and calcium content, indicating a role for GDE3 in induction of differentiation. Thus, we have identified the GDE3 substrate GroPIns as a candidate mediator for osteoblast proliferation, in line with the GroPIns activity observed previously in epithelial cells.

A Novel Glycerophosphodiester Phosphodiesterase, GDE5, Controls Skeletal Muscle Development via a Non-enzymatic Mechanism

“…As mentioned previously, recent reports have shown the remarkable characteristics of GroPIns as a water-soluble and bioactive intracellular derivative of phosphatidylinositol that is involved in a variety of cellular signaling pathways (9,19). GDE1 and GDE3 can hydrolyze GroPIns (16,18); however, the catalytic domain of these proteins is localized extracellularly and is enzymatically active in the extracellular space (13,16). These observations raise the possibility that GDE5 is a candidate for the direct regulation of the intracellular GroPIns levels.…”

“…Furthermore, it is reported that GDE3 is regulated during osteoblast development and induces osteo-* This work was supported in part by a grant-in-aid for scientific research from blast differentiation and morphological changes in cultured mammalian cells (13). In addition, our recent study demonstrates that GDE3 is a GroPIns phosphodiesterase that hydrolyzes GroPIns to glycerol and inositol 1-phosphate, unlike the bacterial GP-PDEs and GDE1 (18). In mammalian cells, GroPIns is being increasingly recognized as an important intracellular messenger that is involved in various cellular signaling pathways such as cell proliferation, transformation, and differentiation (9,18,19).…”

“…In addition, our recent study demonstrates that GDE3 is a GroPIns phosphodiesterase that hydrolyzes GroPIns to glycerol and inositol 1-phosphate, unlike the bacterial GP-PDEs and GDE1 (18). In mammalian cells, GroPIns is being increasingly recognized as an important intracellular messenger that is involved in various cellular signaling pathways such as cell proliferation, transformation, and differentiation (9,18,19). Although these observations strongly suggest a central role of mammalian GP-PDEs in regulating intracellular GP concentrations, previous reports on the existence of proteins that interact with mammalian GP-PDEs, such as RGS16, PRAF2 (also known as JM4), and Prdx1 (12,20,21), have prompted us to consider that GP-PDEs might also act via a non-enzymatic mechanism.…”

New Members of the Mammalian Glycerophosphodiester Phosphodiesterase Family

“…Moreover, our recent study demonstrated that GDE5 is a unique cytosolic protein that can regulate intracellular GroPCho concentration and myogenic differentiation (10). Previous work showed that both GDE1 and GDE3 can hydrolyze GroPIns (11,12) and that the biological function of GDE3 in osteoblast proliferation and differentiation appears to be mediated by GroPIns. Indeed, GroPIns has been shown to regulate cell growth in thyroid cells (4), and induction of its hydrolysis through GDE3 expression results in reduced osteoblast proliferation and the appearance of markers of osteoblast differentiation (12).…”

“…Previous work showed that both GDE1 and GDE3 can hydrolyze GroPIns (11,12) and that the biological function of GDE3 in osteoblast proliferation and differentiation appears to be mediated by GroPIns. Indeed, GroPIns has been shown to regulate cell growth in thyroid cells (4), and induction of its hydrolysis through GDE3 expression results in reduced osteoblast proliferation and the appearance of markers of osteoblast differentiation (12). Thus, mammalian GP-PDEs have an intriguing feature; they show restricted substrate specificities, which prompted us to consider the possibility that mammalian GP-PDEs modulate GroPCho and/or GroPIns concentrations, because these intracellular GPs are increasingly recognized as bioactive molecules that are involved in a variety of cellular events (6).…”