Loss of functional cell surface transforming growth factor beta (TGF-beta) type 1 receptor correlates with insensitivity to TGF-beta in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries, and there is significant variability in survival within CLL clinical stages. Earlier studies showed that CLL cells produce and are usually growth inhibited by transforming growth factor beta type 1 (TGF-beta1), suggesting a mechanism for the clinically indolent course of most CLL. Here we studied the mechanism by which CLL cells from about one-third of the patients are insensitive to TGF-beta1. Of the 13 patients studied, CLL cells isolated from the peripheral blood of 8 patients were sensitive to growth inhibition by TGF-beta1, as determined by incorporation of tritiated thymidine, whereas those from 5 patients were completely resistant to TGF-beta1. As judged by binding of radiolabeled TGF-beta1 followed by cross-linking and immunoprecipitation with anti-receptor antisera, CLL cells sensitive to TGF-beta1 exhibited normal cell surface expression of both types 1 and 2 TGF-beta receptors. In contrast, all CLL cells resistant to TGF-beta1 exhibited no detectable surface type I receptors able to bind TGF-beta1, but normal expression of type II receptors. Both TGF-beta1-sensitive and TGF-beta1-resistant CLL cells contained normal amounts of both type 1 and type 2 receptor mRNAs. Specific loss of type 1 receptor expression represents a new mechanism by which cells acquire resistance to TGF-beta1-mediated growth inhibition in the development and progression of human lymphoproliferative malignancies.

Biosynthesis of the type I and type II TGF-beta receptors. Implications for complex formation.

The TGF-beta type I and type II receptors (TbetaRI and TbetaRII) are signaling receptors that form heteromeric cell surface complexes with the TGF-betas as one of the earliest events in the cellular response to these multifunctional growth factors. Using TGF-beta-responsive mink lung epithelial cells (Mv1Lu), we have determined the half-lives of the endoplasmic reticulum (ER) and mature forms of these receptors. In metabolically labeled cells, approximately 90% of newly synthesized type II receptor undergoes modification of N-linked sugars in the Golgi, with a half-life of 30-35 min; the Golgi-processed form of the receptor has a relatively short metabolic half-life of 2.5 h. In contrast, only 50% of pulse-labeled type I receptor is converted to the Golgi-processed and therefore endoglycosidase H-resistant form, and the endoglycosidase H-sensitive ER form has a half-life of 2.8-3 h. Addition of 100 pM TGF-beta1 causes the Golgi-processed type II receptor to become less stable, with a half-life of 1.7 h, and also destabilizes the Golgi-processed type I receptor. TGF-beta1 binding and cross-linking experiments on cells treated with tunicamycin for various times confirm different ER to cell surface processing times for TbetaRI and TbetaRII. Our results, which suggest that stable complexes between type I and II TGF-beta receptors do not form until the proteins reach a post-ER compartment (presumably the cell surface), have important implications for our understanding of complex formation and receptor regulation.

A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T-cell lymphoma.

In many cancers, inactivating mutations in both alleles of the transforming growth factor beta (TGF-beta) type 11 receptor (TbetaRII) gene occur and correlate with loss of sensitivity to TGF-beta. Here we describe a novel mechanism for loss of sensitivity to growth inhibition by TGF-beta in tumor development. Mac-1 cells, isolated from the blood of a patient with an indolent form of cutaneous T-cell lymphoma, express wild-type TbetaRII and are sensitive to TGF-beta. Mac-2A cells, clonally related to Mac-1 and isolated from a skin nodule of the same patient at a later, clinically aggressive stage of lymphoma, are resistant to TGF-beta. They express both the wild-type TbetaRII and a receptor with a single point mutation (Asp-404-Gly [D404G]) in the kinase domain (D404G-->TbetaRII); no TbetaRI or TbetaRII is found on the plasma membrane, suggesting that D404G-TbetaRII dominantly inhibits the function of the wild-type receptor by inhibiting its appearance on the plasma membrane. Indeed, inducible expression, under control of a tetracycline-regulated promoter, of D404G-TbetaRII in TGF-beta- sensitive Mac-1 cells as well as in Hep3B hepatoma cells results in resistance to TGF-beta and disappearance of cell surface TbetaRI and TbetaRII. Overexpression of wild-type TbetaRII in Mac-2A cells restores cell surface TbetaRI and TbetaRH and sensitivity to TGF-beta. The ability of the D404G-TbetaRH to dominantly inhibit function of wild-type TGF-beta receptors represents a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.

Synaptic activity and connective tissue remodeling in denervated frog muscle.

Denervation of skeletal muscle results in dramatic remodeling of the cellular and molecular composition of the muscle connective tissue. This remodeling is concentrated in muscle near neuromuscular junctions and involves the accumulation of interstitial cells and several extracellular matrix molecules. Given the role of extracellular matrix in neurite outgrowth and synaptogenesis, we predict that this remodeling of the junctional connective tissue directly influences the regeneration of the neuromuscular junction. As one step toward understanding the role of this denervation-induced remodeling in synapse formation, we have begun to look for the signals that are involved in initiating the junctional accumulations of interstitial cells and matrix molecules. Here, the role of muscle inactivity as a signal was examined. The distributions of interstitial cells, fibronectin, and tenascin were determined in muscles inactivated by presynaptic blockade of muscle activity with tetrodotoxin. We found that blockade of muscle activity for up to 4 wk produced neither the junctional accumulation of interstitial cells nor the junctional concentrations of tenascin and fibronectin normally present in denervated frog muscle. In contrast, the muscle inactivity induced the extrajunctional appearance of two synapse-specific molecules, the acetylcholine receptor and a muscle fiber antigen, mAb 3B6. These results demonstrate that the remodeling of the junctional connective tissue in response to nerve injury is a unique response of muscle to denervation in that it is initiated by a mechanism that is independent of muscle activity. Thus connective tissue remodeling in denervated skeletal muscle may be induced by signals released from or associated with the nerve other than the evoked release of neurotransmitter.