Certain solid tumors metastasize to bone and cause osteolysis and abnormal new bone formation.The respective phenotypes of dysregulated bone destruction and bone formation represent two ends of a spectrum, and most patients will have evidence of both. The mechanisms responsible for tumor growth in bone are complex and involve tumor stimulation of the osteoclast and the osteoblast as well as the response of the bone microenvironment. Furthermore, factors that increase bone resorption, independent of tumor, such as sex steroid deficiency, may contribute to this vicious cycle of tumor growth in bone. This article discusses mechanisms and therapeutic implications of osteolytic and osteoblastic bone metastases.Certain solid tumors, such as breast and prostate cancer, have a propensity to metastasize to bone and cause osteolysis and abnormal new bone formation (1, 2). The respective phenotypes of dysregulated bone destruction and bone formation represent two ends of a spectrum, and most patients will have evidence of both. In fact, bone metastases are heterogeneous: data gleaned from a rapid autopsy program indicate that the same prostate cancer patient often has evidence of osteolytic and osteoblastic disease as shown by histologic examination (3). The mechanisms responsible for tumor growth in bone are complex and involve tumor stimulation of the osteoclast and the osteoblast as well as the response of the bone microenvironment. Furthermore, factors that increase bone resorption, independent of tumor, such as sex steroid deficiency, may contribute to this vicious cycle of tumor growth in bone, illustrated in Fig. 1. This article discusses mechanisms and therapeutic implications of osteolytic and osteoblastic bone metastases. Breast Cancer: The Prototypic OsteolyticTumorBreast cancer commonly metastasizes to and destroys bone, causing pain and fracture. Tumors produce many factors that stimulate osteolysis: parathyroid hormone-related protein (PTHrP), interleukin (IL)-11, IL-8, IL-6, and receptor activator of nuclear factor-nB ligand (RANKL;. Substantial data support a role for bone-derived transforming growth factor-h (TGF-h) and tumor-derived osteolytic factors, such as PTHrP, in a vicious cycle of local bone destruction in osteolytic metastases. Bone matrix stores several immobilized growth factors, particularly TGF-h, which is released in active form during osteoclastic resorption (10) and stimulates PTHrP production by tumor cells. PTHrP in turn mediates bone destruction by stimulating osteoclasts. A dominant-negative mutant of the type II TGF-h receptor inhibited TGF-h-induced PTHrP secretion in vitro and development of bone metastases in an MDA-MB-231 experimental metastasis model (5, 6). In addition, TGF-h regulates several genes that are responsible for enhanced bone metastases in MDA-MB-231: IL-11 and connective tissue growth factor (CTGF; refs. 8, 9). Collectively, these studies provided proof of principle to support a role for TGF-h blockade in the treatment of breast cancer bone metastases.SD-20...

During development, growth factors and hormones cooperate to establish the unique sizes, shapes and material properties of individual bones. Among these, TGF-β has been shown to developmentally regulate bone mass and bone matrix properties. However, the mechanisms that control postnatal skeletal integrity in a dynamic biological and mechanical environment are distinct from those that regulate bone development. In addition, despite advances in understanding the roles of TGF-β signaling in osteoblasts and osteoclasts, the net effects of altered postnatal TGF-β signaling on bone remain unclear. To examine the role of TGF-β in the maintenance of the postnatal skeleton, we evaluated the effects of pharmacological inhibition of the TGF-β type I receptor (TβRI) kinase on bone mass, architecture and material properties. Inhibition of TβRI function increased bone mass and multiple aspects of bone quality, including trabecular bone architecture and macro-mechanical behavior of vertebral bone. TβRI inhibitors achieved these effects by increasing osteoblast differentiation and bone formation, while reducing osteoclast differentiation and bone resorption. Furthermore, they induced the expression of Runx2 and EphB4, which promote osteoblast differentiation, and ephrinB2, which antagonizes osteoclast differentiation. Through these anabolic and anti-catabolic effects, TβRI inhibitors coordinate changes in multiple bone parameters, including bone mass, architecture, matrix mineral concentration and material properties, that collectively increase bone fracture resistance. Therefore, TβRI inhibitors may be effective in treating conditions of skeletal fragility.

Identification of selective anchoring proteins responsible for specialized localization of specific signaling proteins has led to the identification of new inhibitors of signal transduction, inhibitors of anchoring protein-ligand interactions. RACK1, the first receptor for activated C kinase identified in our lab, is a selective anchoring protein for ␤II protein kinase C (␤IIPKC). We previously found that at least part of the RACK1-binding site resides in the C2 domain of ␤IIPKC (Ron, D., Luo, J., and Mochly-Rosen, D. (1995) J. Biol. Chem. 270, 24180 -24187). Here we show that the V5 domain also contains part of the RACK1-binding site in ␤IIPKC. In neonatal rat cardiac myocytes, the ␤IIV5-3 peptide (amino acids 645-650 in ␤IIPKC) selectively inhibited phorbol 12-myristate 13-acetate (PMA)-induced translocation of ␤IIPKC and not ␤IPKC. In addition, the ␤IIV5-3 peptide inhibited cardiac myocyte hypertrophy in PMAtreated cells. Interestingly, ␤IV5-3 (646 -651 in ␤IPKC), a selective translocation inhibitor of ␤IPKC, also inhibited PMA-induced cardiac myocyte hypertrophy, demonstrating that both ␤I-and ␤IIPKC are essential for this cardiac function. Therefore, the ␤IIV5 domain contains part of the RACK1-binding site in ␤IIPKC; a peptide corresponding to this site is a selective inhibitor of ␤IIPKC and, hence, enables the identification of ␤IIPKC-selective functions.The localization of signaling enzymes within cells is highly specific and often regulated by selective anchoring proteins (1, 2). A number of these proteins have recently been identified; some anchor and coordinate multiple enzymes in the same signaling cascade (3, 4) and can bind to their selective proteins or enzymes depending on their activation state (2). Selective localization of signaling enzymes in cells results in tethering them in the proper subcellular location for their function. Disruption of the selective protein-protein interactions between the signaling enzymes and their anchoring proteins alters the specialized localization of the signaling enzymes and thus disrupts their function (5).We have studied the mechanism leading to selective localization of protein kinase C (PKC).1 PKC isozymes are a family of serine/threonine, phospholipid-dependent protein kinases (6) that translocate after stimulation to select subcellular sites where they bind their corresponding selective anchoring proteins, RACKs (receptor for activated C kinase) (2). RACKs bind only the active form of their respective PKCs. Our lab has identified some of the RACK-binding sites on ␤, ⑀, and ␦PKC and demonstrated that RACK binding is essential for both proper localization and function of these PKC isozymes (5). So far we have cloned and characterized two RACKs and demonstrated that RACK1 is selective for ␤IIPKC (7, 8), whereas RACK2, also known as ␤ЈCOP (a coatomer protein involved in vesicle transport) is selective for ⑀PKC (9).␤I-and ␤IIPKC, members of the classical family of PKCs, are differentially spliced products of the same gene and therefore differ only in their C-ter...

A yeast two-hybrid approach was used to discern possible new effectors for the ␤␥ subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to G␤, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, coimmunoprecipitate with G␤␥ using an anti-G␤ antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with G␤ 1 ␥ 1 but also unexpectedly with the transducin heterotrimer G␣ t ␤ 1 ␥ 1 . G␣ t alone does not interact, but it must contribute to the interaction because the apparent EC 50 value of RACK1 for G␣ t ␤ 1 ␥ 1 is 3-fold greater than that for G␤ 1 ␥ 1 (0.1 versus 0.3 M). RACK1 is a scaffold that interacts with several proteins, among which are activated ␤IIPKC and dynamin-1 (1). ␤IIPKC and dynamin-1 compete with G␤ 1 ␥ 1 and G␣ t ␤ 1 ␥ 1 for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that G␤␥ interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.Heterotrimeric G proteins are a family of proteins that transduce an extracellular signal to an intracellular response via a seven helical transmembrane G protein-coupled receptor (GPCR).1 Upon activation, the receptor facilitates the exchange of GDP for GTP in the G␣ subunit. G␣ is then thought to dissociate from the G␤␥ heterodimer allowing both complexes to individually activate a number of effectors (2, 3). Free G␤␥ interacts with a large assortment of effector proteins, including phospholipases (4), adenylyl cyclases (5), ion channels (6), and G protein-coupled receptor kinases (7). There are, however, G protein-coupled receptor responses, such as MAP kinase activation (8 -10), receptor internalization (11, 12), and organelle transport (13-15) that are mediated through the G␤␥ subunit but that have not been definitively linked to known G␤␥ effectors.G␤ is the prototypical member of a family of proteins known as WD40 repeat proteins, which seem to function as adaptors and enzyme regulators (16,17). G␤ is the only WD40 repeat protein whose three-dimensional structure is known, and it exhibits a toroidal bladed ␤-propeller structure, with each blade consisting of 4 anti-parallel ␤-strands (18). Because the WD repeat motif is a structural element of the ␤-propeller, all of these proteins are thought to be ␤-propeller proteins with a variable number of blades. Furthermore, G␤ subunits are known to interact with G␥ subunits, proteins containing a G␥-like domain (19), a pleckstrin homology domain (20), a QXXER domain (found in adenylyl cyclases) (21), and a domain contained within phosducin and its relatives (22). In this work we propose that G␤␥ also interacts with certain other WD40 repeat prote...

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