TRAFs in RANK signaling.

Members of the tumor necrosis factor (TNF) family govern many diverse physiological and cellular responses including cellular proliferation, differentiation, and apoptosis. Ligands of this family interact through a distinct set of specific receptors that lack enzymatic activity and therefore are dependent on the association of adaptor molecules. One receptor/ligand pair known as receptor activator of nuclear factor-kappa B (RANK) and RANK ligand (RANKL) regulates bone remodeling, mammary gland development, and lymph node organogenesis. RANK interacts with five members of the TNF receptor-associated factor (TRAF) family, of which TRAF6 is indispensable for its signaling capability. An accumulation of evidence from various research laboratories indicates TRAFs, but more importantly TRAF6, is the key to understanding how RANKL links cytoplasmic signaling to the nuclear transcriptional program.

Inhibition of RANKL-mediated osteoclast differentiation by selective TRAF6 decoy peptides.

RANK and RANKL are essential mediators of osteoclastogenesis. RANK interacts with members of the tumor necrosis factor receptor-associated factor (TRAF) family, of which TRAF6 is the critical signaling molecule. We identified a unique TRAF6-binding motif in RANK, which was subsequently co-crystallized with TRAF6 revealing distinct molecular interactions. A cell-permeable TRAF6 decoy peptide (T6DP) was shown to specifically target TRAF6 and inhibit RANKL-mediated signaling. In this study, we identified a core motif for binding to TRAF6 by generating a series of deletion mutants linked via palmitate as a means to internalize the peptide, thus making a smaller scaffold for intracellular delivery. The core motif of RKIPTEDEY inhibited RANKL-mediated osteoclastogenesis and bone resorption. In contrast, TRAF2/5 decoy peptides appeared to have no affect. Thus, disruption of the RANK-TRAF6 interaction may prove useful as a novel target for the development of a small molecule therapeutic agent for the treatment of bone-related diseases.

Inhibition of prostate tumor growth and bone remodeling by the vascular targeting agent VEGF121/rGel.

The pathophysiology of tumor growth following skeletal metastases and the poor response of this type of lesion to therapeutic intervention remains incompletely understood. Vascular endothelial growth factor (VEGF)-A and its receptors play a role in both osteoclastogenesis and tumor growth. Systemic (i.v.) treatment of nude mice bearing intrafemoral prostate (PC-3) tumors with the vascular ablative agent VEGF(121)/recombinant gelonin (rGel) strongly inhibited tumor growth. Fifty percent of treated animals had complete regression of bone tumors with no development of lytic bone lesions. Immunohistochemical analysis showed that VEGF(121)/rGel treatment suppressed tumor-mediated osteoclastogenesis in vivo. In vitro treatment of murine osteoclast precursors, both cell line (RAW264.7) and bone marrow-derived monocytes (BMM), revealed that VEGF(121)/rGel was selectively cytotoxic to osteoclast precursor cells rather than mature osteoclasts. VEGF(121)/rGel cytotoxicity was mediated by Flt-1, which was down-regulated during osteoclast differentiation. Analysis by flow cytometry and reverse transcription-PCR showed that both BMM and RAW264.7 cells display high levels of Flt-1 but low levels of Flk-1. Internalization of VEGF(121)/rGel into osteoclast precursor cells was suppressed by pretreatment with an Flt-1 neutralizing antibody or by placenta growth factor but not with an Flk-1 neutralizing antibody. Thus, VEGF(121)/rGel inhibits osteoclast maturation in vivo and it seems that this process is important in the resulting suppression of skeletal osteolytic lesions. This is a novel and unique mechanism of action for this class of agents and suggests a potentially new approach for treatment or prevention of tumor growth in bone.

Bcl-xL overexpression blocks bax-mediated mitochondrial contact site formation and apoptosis in rod photoreceptors of lead-exposed mice.

Photoreceptor apoptosis and resultant visual deficits occur in humans and animals with inherited and disease-, injury-, and chemical-induced retinal degeneration. A clinically relevant mouse model of progressive rod photoreceptor-selective apoptosis was produced by low-level developmental lead exposure and studied in combination with transgenic mice overexpressing Bcl-x(L) only in the photoreceptors. A multiparametric analysis of rod apoptosis and mitochondrial structure-function was performed. Mitochondrial cristae topography and connectivity, matrix volume, and contact sites were examined by using 3D electron tomography. Lead-induced rod-selective apoptosis was accompanied by rod Ca(2+) overload, rhodopsin loss, translocation of Bax from the cytosol to the mitochondria, decreased rod mitochondrial respiration and membrane potential, mitochondrial cytochrome c release, caspase-3 activation, and an increase in the number of mitochondrial contact sites. These effects occurred without mitochondrial matrix swelling, outer membrane rupture, caspase-8 activation, or Bid cleavage. Bcl-x(L) overexpression completely blocked all apoptotic events, except Ca(2+) overload, and maintained normal rod mitochondrial function throughout adulthood. This study presents images of mitochondrial contact sites in an in vivo apoptosis model and shows that Bcl-x(L) overexpression blocks increased contact sites and apoptosis. These findings extend our in vitro retinal studies with Pb(2+) and Ca(2+) and suggest that developmental lead exposure produced rod-selective apoptosis without mitochondrial swelling by translocating cytosolic Bax to the mitochondria, which likely sensitized the Pb(2+) and Ca(2+) overloaded rod mitochondria to release cytochrome c. These results have relevance for therapies in a wide variety of progressive retinal and neuronal degenerations where Ca(2+) overload, lead exposure, andor mitochondrial dysfunction occur.