Identify structural domains of PLCgamma2 required for OC differentiation and understand the mechanism leading to PLCgamma2 activation, but not PLCgamma1, in the bone resorbing cells:
OCs, the principal bone resorbing cells, develop from bone marrow macrophages (BMMs) primarily under the influence of two major regulators: the macrophage colony stimulating factor (M-CSF) and receptor activator of NFkB ligand (RANKL), and less understood costimulatory factors that act via immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors on OC precursor cells. Our data indicate that targeted deletion of PLCgamma2 in mice leads to an osteopetrotic phenotype. PLCgamma2-deficient OC precursors fail to differentiate due to defective activation of AP1 and NF?B and a failure in upregulation of NFATc1, a master transcription factor for osteoclastogenesis. While PLCgamma2 phosphorylation and its catalytic activity are required for ITAM -dependent upregulation of NFATc1, activation of JNK and NF?B pathways may be dependent on the capacity of PLCgamma2 to bind and activate GAB2, an adapter protein shown to be important in RANKL-mediated osteoclastogenesis, and modulate GAB2 recruitment to the RANK signaling complex (See Figure 1).
The non-redundant role of PLCgamma2 in OC differentiation suggests that identifying the structural domains which make PLCgamma2 unique in its capacity to promote OC differentiation/function may unveil novel OC regulatory mechanisms and provide the basis for new antiresorptive therapies.
Thus, in this project we seek to generate PLCgamma2 mutants to clarify the role of the catalytic activity and the adapter function of PLCgamma2 during regulation of OC differentiation. We also aim to determine the role of PLCgamma2 in OC differentiation and inflammatory osteolysis, in vivo, and to test the in vivo osteolytic response in mice harboring mutated forms of PLCgamma2.
To elucidate the role of PLCgamma2 in OC cytoskeletal reorganization during activation and bone resorption
Activation of OC requires mechanisms enabling substrate-specific adhesion, spreading, and migration. These activities are mediated through M-CSF and the transmembrane integrin avß3 which cooperate with RANKL signal-transduced molecules to achieve normal OC activity. We have found that the PLCgamma inhibitor U73122 destroys the actin ring structures in mature resorbing OC, suggesting an important role for PLCgamma2 in regulation of OC cytoskeleton. Keeping with this posture, overexpression of the master osteoclast transcription factor NFAT rescues OC differentiation but not bone resorption, due to aberrant localization of the cytoskeletal proteins c-Src and Pyk2. Considering that PLCgamma2 is also involved in M-CSF signaling, we hypothesized that PLCgamma2 controls actin reorganization in bone resorbing OC by affecting avß3- and, or M-CSF-stimulated signaling pathways that control actin cytoskeletal dynamics. Thus, in this study we will investigate how PLCgamma2 controls membrane Src localization, and determine how PLCgamma2 affects actin dynamics in M-CSF-generated protruding lamellipodia.
Role of PLCgamma2 in the arthritic disease:
Based on the recent evidence that B cells contribute to the antigen induced inflammatory response (see model in figure 2) through production of autoantibodies and as Antigen Presenting Cells (APCs), critical for the activation of autoreactive T cells, we hypothesize that PLCgamma2-/- mice, due to their intrinsic defect in the B cell lineage, display decreased immunological response following antigen-induced arthritis (AIA). Moreover, the essential role of PLCgamma2 in RANKL- and TNF-mediated OC differentiation, leads us to hypothesize that PLCgamma2 null mice are also protected from focal osteolysis. Thus we are seeking to determine the relative PLCgamma2-dependent contribution of B and T cells in the development of inflammation and bone erosion in AIA model.
Role of PLCgamma2 in the progression of breast cancer induced bone metastases:
Bone metastases are common in a number of cancers, and they contribute heavily to morbidity and mortality, most prominently in prostate cancer, breast cancer, and multiple myeloma. During bone metastases, a general accepted concept is that interplay between tumor cells and osteoclasts exist, a condition known as “vicious cycle”. The tumor cells produce factors that directly or indirectly induce the formation of osteoclasts and in turn, bone resorption by osteoclasts releases growth factors from the bone matrix that stimulate tumor growth. This reciprocal interaction between tumor cells and the bone microenvironment results in bone destruction and increased tumor burden. We are currently studying the role of the immune cells in the modulation of the vicious cycle during bone metastasis (See Figure 2).
