Signal transduction pathways in the clinical approach: where are we going?

invited editoria

Phosphoinositide signal transduction pathway and osteosarcoma metastases

Metastasis spreading confers worse prognosis to the clinical outcome in patients affected with osteosarcoma, the most common primary bone tumour in childhood and adolescence. The identification of the molecules involved in spreading might help to understand the mechanisms of tumour dissemination, opening the way to novel therapeutic strategies. The activation of ezrin-radixin-moesin (ERM) family proteins was suggested to occur after interaction with molecules belonging to Phosphoinositide signal transduction pathway. The Phosphatydil inositol (4,5) bisphosphate (PIP2), a crucial molecule in PI pathway, was indicated to be involved in the stabilization of ERM proteins or a more efficient receptor binding. The levels of PIP2 in the pathway represent a critical element for regulation of a number of cell events. PIP2 levels are regulated by means of enzymes, including the PI-specific Phospholipase C family. The reduction of PIP2 levels induces ERM protein dissociation from the membrane. PI-PLC enzymes contribute to regulate this event. The role of PI signal transduction molecules in osteosarcoma metastases will be discussed

Phosphoinositide-specific Phospholipases C in psychiatric diseases and suicide

Mood disorders represent a major medical need requiring chronic treatment. About one million people die by suicide worldwide each year, both as a consequence of major depression or not. Multiple deficits, including cell atrophy and loss, were described in the brains of mood disorders affected patients and in experimental animal models. Numerous changes in gene expression and activity were described in limbic and cortical brain regions. Available therapies probably regulate many of these changes. Different signal transduction pathways play a role in the pathogenesis of schizoaffective disorders, namely the cyclic‐AMP, phosphoinositides (PI), mitogen‐activated protein kinase, and glycogen synthase kinase cascades. Neurobiology studies focused upon abnormalities of signalling mechanisms with special regard to the serotonin system and related PI signalling system. Involvement of PI-specific Phospholipase C (PLC) enzymes was also described. In suicide brains the overall PLC expression was altered due to a complex reorganization of the isoforms, and PLC 1 isoform was suggested to be involved in schizophrenia and bipolar disorder. The knowledge of the complex network of neurobiological molecules and interconnected signal transduction pathways in the brain might help to understand the natural history and the pathogenesis of mood disorders, as well as of the suicidal behaviour. Moreover, it might widen the panel of available therapeutic tools, also gaining prognostic suggestions in order to prevent suicide

Ezrin-related phosphoinositide pathway modifies RhoA and Rac1 in human osteosarcoma cell lines

Selected Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes occupy the convergence point of the broad range of pathways that promote Rho and Ras GTPase mediated signalling, which also regulate the activation of ezrin, a member of the ezrin-radixin-moesin (ERM) proteins family involved in the metastatic osteosarcoma spread. Previous studies described that in distinct human osteosarcoma cell lines ezrin networks the PI-PLC with complex interplay controlling the expression of the PLC genes, which codify for PI-PLC enzymes. In the present study, we analyzed the expression and the sub-cellular distribution of RhoA and Rac1 respectively after ezrin silencing and after PI-PLC ε silencing, in order to investigate whether ezrin-RhoGTPAses signalling might involve one or more specific PI-PLC isoforms in cultured 143B and Hs888 human osteosarcoma cell lines. In the present experiments, both ezrin and PLCE gene silencing had different effects upon RhoA and Rac1 expression and sub-cellular localization. Displacements of Ezrin and of RhoA localization were observed, probably playing functional roles

Expression and localization of Phosphoinositide-specific Phospholipase C enzymes in polarized macrophages

The phenotypic and functional diversity of macrophages depends on differentiating programs being developed during the cells’ lives. Great interest was addressed to identify the signal transduction pathways acting in macrophage polarization, including the phosphoinositide (PI) system and related phospholipase C (PLC) family of enzymes. Enzymes belonging to the PLC family are strictly tissue specific and the expression panel, as well as the subcellular localization differs in quiescent cells compared to the pathological counterpart. We analyzed the expression of PLC enzymes in unpolarized (M0), M1 and M2 macrophages to list the isoforms expressed in the polarized macrophages and their subcellular localization. Our results confirmed that macrophages express a wide number of PLC isoforms. All PLC enzymes were detected within both M1 and M2 cells, but not in M0 cells. M0, as well as M1 and M2 cells own a specific panel of expression, different for both genes’ mRNA expression and intracellular localization of PLC enzymes. PLC enzymes might play a complex role in macrophages during inflammation and probably also during polarization

Expression and localization of Phosphoinositide-specific Phospholipases C in cultured, differentiating and stimulated human osteoblasts

The osteoblasts contribute to bone homeostasis maintaining the bone mass, and intervene in bone injuries repair. The limited number of available therapeutic agents promoting osteogenesis aroused the greatest interest in the control of osteoblasts’ activity. Insights in the events leading to the proliferation and differentiation of osteoblasts might allow uncover potential molecular targets to control the complex mechanisms underlying bone remodeling. Oscillations of calcium act crucially during this remodeling, affecting both the differentiation and proliferation of osteoblasts. Signal transduction pathways contribute to the differentiation and metabolic activities of osteoblasts, with special regard to calcium-related signaling, including the Phosphoinositide (PI) pathway and related Phospholipases C (PLCs). In order to evaluate the role of PLC enzymes’ family in human osteoblasts (HOBs), we analyzed the expression of PLC genes and the localization of PLC enzymes in cultured HOBs and in in vitro differentiating HOBs after 3, 10, 17 and 23 days, and in HOBs stimulated with Lipopolysaccharide, which affects the differentiation of osteoblasts, after 3, 6, 24 and 48 hours. Our results confirm the transcription of most PLC genes and the presence of a number of PLC enzymes in HOBs, differently localized in the nucleus, in the cytoplasm or both, as well as in cell protrusions. The localization of PLC enzymes within the cell suggests the activation of both the PI nuclear and of the cytoplasmic cycle in HOBs. Depending on the experimental conditions, transcripts of splicing variants of selected PLC genes were detected and the localization of most PLC enzymes varied, with special regard to enzymes belonging to the PLC , and sub-families. Further studies addressed to elucidate the complex network involving the signal transduction of PLCs might provide further insights into the complex signal transduction network in bone remodeling, also offering the opportunity to identify promising molecular targets

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

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 PLC genes and different localization of PLC enzymes, depending on the cell line, in different cell lines. In order to evaluate possible off-target effects of the molecule in human osteoblasts, we investigated the expression of PLC genes and the localization of PLC enzymes in cultured human osteoblasts (hOBs) in the presence of low dose U-73122. Our results confirm that all PLC genes are transcribed in hOBs, that probably splicing variants of selected PLC 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 PLC 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

Unexpected Absence of Skeletal Responses to Dietary Magnesium Depletion: Basis for Future Perspectives?

It's known that a magnesium (Mg)-deficient diet is associated with an increased risk of osteoporosis. The aim of this work is to investigate, by a histological approach, the effects of a Mg-deprived diet on the bone of 8-weeks-old C57BL/6J male mice. Treated and control mice were supplied with a Mg-deprived or normal diet for 8 weeks, respectively. Body weight, serum Mg concentration, expression of kidney magnesiotropic genes, and histomorphometry on L5 vertebrae, femurs, and tibiae were evaluated. Body weight gain and serum Mg concentration were significantly reduced, while a trend toward increase was found in gene expression in mice receiving the Mg-deficient diet, suggesting the onset of an adaptive response to Mg depletion. Histomorphometric parameters on the amount of trabecular and cortical bone, number of osteoclasts, and thickness of the growth plate in femoral distal and tibial proximal metaphyses did not differ between groups; these findings partially differ from most data present in the literature showing that animals fed a Mg-deprived diet develop bone loss and may be only in part explained by differences among the experimental protocols. However, the unexpected findings we recorded on bones could be attributed to genetic differences that may have developed after multiple generations of inbreeding

The role of the Phosphoinositide signal transduction pathway in the endometrium

The regulation of calcium concentration triggers physiological events in all cell types (Berridge 1993). Unregulated elevation in calcium concentrations is often cytotoxic (Annunziato 2003). In fact, uncontrolled calcium levels alter proteins’ function, apoptosis regulation, as well as proliferation, secretion and contraction. Calcium levels are tightly regulated by (Berridge 1993; Berridge 1994). A great interest was paid to signal transduction pathways for their role in mammalian reproduction. The role of PI signal transduction pathway and related Phosphoinositide-specific Phospholipase C (PI-PLC) enzymes in the regulation of calcium levels was actively studied and characterized. However, the role of PI signaling and PI-PLC enzymes in the endometrium is far to be completely highlighted. In the present review the role of PI, the expression of selected PI-PLC enzymes and the crosstalk with further signaling systems in the endometrium will be discussed