Breast cancer frequently metastasizes to the skeleton, and the associated bone destruction is mediated by the osteoclast. Growth factors, including transforming growth factor-β (TGF-β), released from bone matrix by the action of osteoclasts, may foster metastatic growth. Because TGF-β inhibits growth of epithelial cells, and carcinoma cells are often defective in TGF-β responses, any role of TGF-β in metastasis is likely to be mediated by effects on the surrounding normal tissue. However, we present evidence that TGF-β promotes breast cancer metastasis by acting directly on the tumor cells. Expression of a dominant–negative mutant (TβRIIΔcyt) of the TGF-β type II receptor rendered the human breast cancer cell line MDA-MB-231 unresponsive to TGF-β. In a murine model of bone metastases, expression of TβRIIΔcyt by MDA-MB-231 resulted in less bone destruction, less tumor with fewer associated osteoclasts, and prolonged survival compared with controls. Reversal of the dominant–negative signaling blockade by expression of a constitutively active TGF-β type I receptor in the breast cancer cells increased tumor production of parathyroid hormone–related protein (PTHrP), enhanced osteolytic bone metastasis, and decreased survival. Transfection of MDA-MB-231 cells that expressed the dominant–negative TβRIIΔcyt with the cDNA for PTHrP resulted in constitutive tumor PTHrP production and accelerated bone metastases. These data demonstrate an important role for TGF-β in the development of breast cancer metastasis to bone, via the TGF-β receptor–mediated signaling pathway in tumor cells, and suggest that the bone destruction is mediated by PTHrP.

The bone morphogenetic proteins (BMPs), TGFβ superfamily members, play diverse roles in embryogenesis, but how the BMPs exert their action is unclear and how different BMP receptors (BMPRs) contribute to this process is not known. Here we demonstrate that the two type I BMPRs, BMPR-IA and BMPR-IB, regulate distinct processes during chick limb development. BmpR-IB expression in the embryonic limb prefigures the future cartilage primordium, and its activity is necessary for the initial steps of chondrogenesis. During later chondrogenesis, BmpR-IA is specifically expressed in prehypertrophic chondrocytes. BMPR-IA regulates chondrocyte differentiation, serving as a downstream mediator of Indian Hedgehog (IHH) function in both a local signaling loop and a longer-range relay system to PTHrP. BMPR-IB also regulates apoptosis: Expression of activated BMPR-IB results in increased cell death, and we showed previously that dominant-negative BMPR-IB inhibits apoptosis. Our studies indicate that in TGFβ signaling systems, different type I receptor isoforms are dedicated to specific functions during embryogenesis.

Transforming growth factor beta (TGF-beta) and activin bind to receptor complexes that contain two distantly related transmembrane serine/threonine kinases known as receptor types I and II. The type II receptors determine ligand binding specificity, and each interacts with a distinct repertoire of type I receptors. Here we identify a new type I receptor for activin, ActR-IB, whose kinase domain is nearly identical to that of the recently cloned TGF-beta type I receptor, T beta R-I. ActR-IB has the structural and binding properties of a type I receptor: it binds activin only in the presence of an activin type II receptor and forms a heteromeric noncovalent complex with activin type II receptors. In Mv1Lu lung epithelial cells, ActR-IB and T beta R-I signal a common set of growth-inhibitory and transcriptional responses in association with their corresponding ligands and type II receptors. The transcriptional responses include elevated expression of fibronectin and plasminogen activator inhibitor 1. Although T beta R-I and ActR-IB are nearly identical in their kinase domains (90% amino acid sequence identity), their corresponding type II receptor kinase domains are very different from each other (42% amino acid sequence identity). Therefore, signaling of a specific set of responses by TGF-beta and activin correlates with the presence of similar type I kinases in their complex. Indeed, other TGF-beta and activin type I receptors (TSR-I and ActR-I) whose kinase domains significantly diverge from those of T beta R-I and ActR-IB do not substitute as mediators of these growth-inhibitory and extracellular matrix transcriptional responses. Hence, we conclude that the type I receptor subunits are primary specifiers of signals sent by TGF-beta and activin receptor complexes.

The Raf kinases play a key role in relaying signals elicited by mitogens or oncogenes. Here, we report that c‐raf‐1^−/− embryos are growth retarded and die at midgestation with anomalies in the placenta and in the fetal liver. Although hepatoblast proliferation does not appear to be impaired, c‐raf‐1^−/− fetal livers are hypocellular and contain numerous apoptotic cells. Similarly, the poor proliferation of Raf‐1^−/− fibroblasts and hematopoietic cells cultivated in vitro is due to an increase in the apoptotic index of these cultures rather than to a cell cycle defect. Furthermore, Raf‐1‐ deficient fibroblasts are more sensitive than wild‐ type cells to specific apoptotic stimuli, such as actinomycin D or Fas activation, but not to tumor necrosis factor‐α. MEK/ERK activation is normal in Raf‐1‐deficient cells and embryos, and is probably mediated by B‐Raf. These results indicate that the essential function of Raf‐1 is to counteract apoptosis rather than to promote proliferation, and that effectors distinct from the MEK/ERK cascade must mediate the anti‐apoptotic function of Raf‐1.

The transforming growth factor beta (TGF-beta) type II receptor (T beta R-II) is a transmembrane serine/threonine kinase that contains two inserts in the kinase region and a serine/threonine-rich C-terminal extension. T beta R-II is required for TGF-beta binding to the type I receptor, with which it forms a heteromeric receptor complex, and its kinase activity is required for signaling by this complex. We investigated the role of various cytoplasmic regions in T beta R-II by altering or deleting these regions and determining the signaling activity of the resulting products in cell lines made resistant to TGF-beta by inactivation of the endogenous T beta R-II. TGF-beta binding to receptor I and responsiveness to TGF-beta in these cells can be restored by transfection of wild-type T beta R-II. Using this system, we show that the kinase insert 1 and the C-terminal tail of T beta R-II, in contrast to the corresponding regions in most tyrosine kinase receptors, are not essential to specify ligand-induced responses. Insert 2 is necessary to support the catalytic activity of the receptor kinase, and its deletion yields a receptor that is unable to mediate any of the responses tested. However, substitution of this insert with insert 2 from the activin receptor, ActR-IIB, does not diminish the ability of T beta R-II to elicit these responses. A truncated T beta R-II lacking the cytoplasmic domain still binds TGF-beta, supports ligand binding to receptor I, and forms a complex with this receptor. However, TGF-beta binding to receptor I facilitated by this truncated T beta R-II fails to inhibit cell proliferation, activate extracellular matrix protein production, or activate transcription from a promoter containing TGF-beta-responsive elements. We conclude that the transcriptional and antiproliferative responses to TGF-beta require both components of a heteromeric receptor complex that differs from tyrosine kinase receptors in its mode of signaling.