Salicylate-induced growth arrest is associated with inhibition of p70s6k and down-regulation of c-myc, cyclin D1, cyclin A, and proliferating cell nuclear antigen.

Salicylate and its pro-drug form aspirin are widely used medicinally for their analgesic and anti-inflammatory properties, and more recently for their ability to protect against colon cancer and cardiovascular disease. Despite the wide use of salicylate, the mechanisms underlying its biological activities are largely unknown. Recent reports suggest that salicylate may produce some of its effects by modulating the activities of protein kinases. Since we have previously shown that the farnesyltransferase inhibitor l-744, 832 inhibits cell proliferation and p70(s6k) activity, and salicylate inhibits cell proliferation, we examined whether salicylate affects p70(s6k) activity. We find that salicylate potently inhibits p70(s6k) activation and phosphorylation in a p38 MAPK-independent manner. Interestingly, low salicylate concentrations (/=5 mm) are required to block p70(s6k) activation by epidermal growth factor + insulin-like growth factor-1. These data suggest that salicylate may selectively inhibit p70(s6k) activation in response to specific stimuli. Inhibition of p70(s6k) by salicylate occurs within 5 min, is independent of the phosphatidylinositol 3-kinase pathway, and is associated with dephosphorylation of p70(s6k) on its major rapamycin-sensitive site, Thr(389). A rapamycin-resistant mutant of p70(s6k) is resistant to salicylate-induced Thr(389) dephosphorylation.

BMP type II receptor is required for gastrulation and early development of mouse embryos.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta superfamily, play a variety of roles during mouse development. BMP type II receptor (BMPR-II) is a type II serine/threonine kinase receptor, which transduces signals for BMPs through heteromeric complexes with type I receptors, including activin receptor-like kinase 2 (ALK2), ALK3/BMPR-IA, and ALK6/BMPR-IB. To elucidate the function of BMPR-II in mammalian development, we generated BMPR-II mutant mice by gene targeting. Homozygous mutant embryos were arrested at the egg cylinder stage and could not be recovered at 9.5 days postcoitum. Histological analysis revealed that homozygous mutant embryos failed to form organized structure and lacked mesoderm. The BMPR-II mutant embryos are morphologically very similar to the ALK3/BMPR-IA mutant embryos, suggesting that BMPR-II is important for transducing BMP signals during early mouse development. Moreover, the epiblast of the BMPR-II mutant embryo exhibited an undifferentiated character, although the expression of tissue-specific genes for the visceral endoderm was essentially normal. Our results suggest that the function of BMPR-II is essential for epiblast differentiation and mesoderm induction during early mouse development.

Identification of STRAP, a novel WD domain protein in transforming growth factor-beta signaling.

Transforming growth factor-beta1 (TGF-beta1) is the prototype of a large family of proteins that regulate a variety of biological processes. The pleiotropic responses to TGF-beta are mediated via ligand-induced heteromeric complex formation by type I (TbetaR-I) and type II (TbetaR-II) serine-threonine kinase receptors. Several studies have shown that TbetaR-II acts as a primary receptor, binding TGF-beta and phosphorylating TbetaR-I, whose kinase activity then propagates the signals. Therefore, intracellular proteins that interact with type I receptors are likely to play important roles in TGF-beta signaling. We have identified a novel WD domain-containing protein, designated STRAP (serine-threonine kinase receptor-associated protein), which interacts with TbetaR-I in a yeast two-hybrid system. STRAP associates with both functional TbetaR-I and TbetaR-II in vivo. Overexpression of STRAP leads to inhibition of TGF-beta-mediated transcriptional activation. It also shows synergistic inhibition of TGF-beta signaling in concert with Smad7, but not with Smad6, as measured by TGF-beta-dependent transcriptional reporters. The existence of the STRAP gene from yeast to mammals indicates an evolutionarily conserved function in eukaryotes. The data suggest a potential role for STRAP in TGF-beta signal transduction.

Mammary tumor suppression by transforming growth factor beta 1 transgene expression.

In cell culture, type alpha transforming growth factor (TGF-alpha) stimulates epithelial cell growth, whereas TGF-beta 1 overrides this stimulatory effect and is growth inhibitory. Transgenic mice that overexpress TGF-alpha under control of the mouse mammary tumor virus (MMTV) promoter/enhancer exhibit mammary ductal hyperplasia and stochastic development of mammary carcinomas, a process that can be accelerated by administration of the chemical carcinogen 7,12-dimethylbenz[a]anthracene. MMTV-TGF-beta 1 transgenic mice display mammary ductal hypoplasia and do not develop mammary tumors. We report that in crossbreeding experiments involving the production of mice carrying both the MMTV-TGF-beta 1 and MMTV-TGF-alpha transgenes, there is marked suppression of mammary tumor formation and that MMTV-TGF-beta 1 transgenic mice are resistant to 7,12-dimethylbenz[a]anthracene-induced mammary tumor formation. These data demonstrate that overexpression of TGF-beta 1 in vivo can markedly suppress mammary tumor development.

Cloning of a novel type II serine/threonine kinase receptor through interaction with the type I transforming growth factor-beta receptor.

The transforming growth factor-beta (TGF-beta) superfamily comprises a number of molecules that are involved in a wide variety of biological processes. Specific receptors for several members of this family have been molecularly identified, forming a new category of transmembrane serine/threonine kinase receptors. The type I and type II receptor interact both physically and functionally, thereby cooperating to generate intracellular signals. The yeast two-hybrid system was used to identify proteins that can interact with the cytoplasmic region of the type I TGF-beta receptor. One of the proteins identified encodes a novel putative serine/threonine kinase receptor. Sequence analysis suggests that this molecule belongs to the type II receptor class. This receptor, however, is distinct from other type II receptors in having an extraordinarily long C-terminal tail region. The pattern of expression in adult tissues is different from that of other known type II receptors; it is highly expressed in heart and liver. In the yeast system, the cytoplasmic regions of different combinations of type I and type II receptors heterodimerize, providing a new cloning strategy for the large number of serine/threonine kinase receptors likely to exist for the many ligands of the TGF-beta superfamily.

Serum alpha 1-antitrypsin deficiency associated with the common S-type (Glu264----Val) mutation results from intracellular degradation of alpha 1-antitrypsin prior to secretion.

The S-type alpha 1-antitrypsin (alpha 1AT) deficiency allele differs from the normal M1(Val213) allele by a single amino acid substitution (Glu264----Val). To evaluate the molecular pathophysiology responsible for the reduced serum levels of alpha 1AT associated with the S-type allele, alpha 1AT gene expression was examined in blood monocytes, cells which normally produce alpha 1AT, as well as murine fibroblasts modified by retroviral gene transfer to express the S-type and normal M-type human alpha 1AT genes. Northern analysis and S1 protection analysis demonstrated that monocytes of M and S homozygotes both express 1.8-kilobase alpha 1AT mRNA transcripts in comparable levels and similar in structure. Pulse-chase labeling studies demonstrated that both M and S monocytes synthesized and secreted a 52-kDa protein, but the S monocytes secreted significantly less. The cellular lysates of both M and S monocytes contained a newly synthesized 50-kDa precursor form of alpha 1AT, but the S monocytes contained reduced amounts. Pulse-chase labeling in the presence of tunicamycin, an inhibitor of core oligosaccharide addition, demonstrated that S monocytes exhibited a selective inhibition of secretion of 45-kDa nonglycosylated alpha 1AT not observed in M monocytes. Consistent with these observations, murine fibroblasts modified by retroviral gene transfer to contain an integrated human S-type alpha 1AT cDNA demonstrated reduced secretion of alpha 1AT compared with fibroblasts containing an integrated human M-type alpha 1AT cDNA and also reproduced the abnormality of alpha 1AT biosynthesis observed with S-type monocytes. Furthermore, in the presence of leupeptin, an inhibitor of cellular proteinases, the S-type modified fibroblasts demonstrated a selective augmentation of human alpha 1AT secretion not observed for the M-type. Together, these observations are consistent with the concept that the single A----T mutation of the S-type alpha 1AT gene results in reduced cellular secretion of alpha 1AT because the newly synthesized S-type alpha 1AT protein is degraded intracellularly prior to secretion.

Clonal gene therapy: transplanted mouse fibroblast clones express human alpha 1-antitrypsin gene in vivo.

A retroviral vector was used to insert human alpha 1-antitrypsin (alpha 1AT) complementary DNA into the genome of mouse fibroblasts to create a clonal population of mouse fibroblasts secreting human alpha 1AT. After demonstrating that this clone of fibroblasts produced alpha 1AT after more than 100 population doublings in the absence of selection pressure, the clone was transplanted into the peritoneal cavities of nude mice. When the animals were evaluated 4 weeks later, human alpha 1AT was detected in both sera and the epithelial surface of the lungs. The transplanted clone of fibroblasts could be recovered from the peritoneal cavities of those mice and demonstrated to still be producing human alpha 1AT. Thus, even after removal of selective pressure, a single clone of retroviral vector-infected cells that expressed an exogenous gene in vitro, continued to do so in vivo, and when recovered, continued to produce the product of the exogenous gene.

Production of glycosylated physiologically "normal" human alpha 1-antitrypsin by mouse fibroblasts modified by insertion of a human alpha 1-antitrypsin cDNA using a retroviral vector.

Alpha 1-Antitrypsin (alpha 1AT) deficiency is a hereditary disorder characterized by reduced serum levels of alpha 1AT, resulting in destruction of the lower respiratory tract by neutrophil elastase. As an approach to augment alpha 1AT levels in this disorder with physiologically normal human alpha 1AT, we have integrated a full-length normal human alpha 1AT cDNA into the genome of mouse fibroblasts. To accomplish this, the retroviral vector N2 was modified by inserting the simian virus 40 early promoter followed by the alpha 1AT cDNA. Southern analysis demonstrated that the intact cDNA was present in the genome of selected clones of the transfected murine fibroblasts psi 2 and infected NIH 3T3. The clones produced three mRNA transcripts (5.8, 4.8, and 2.4 kilobases) containing human alpha 1AT sequences, secreted an alpha 1AT molecule recognized by an anti-human alpha 1AT antibody, with the same molecular mass (52 kDa) as normal human alpha 1AT and that complexed with and inhibited human neutrophil elastase. The psi 2 produced alpha 1AT was glycosylated, and when infused intravenously into mice, it had a serum half-life similar to normal alpha 1AT purified from human plasma and markedly longer than that of nonglycosylated human alpha 1AT cDNA-directed yeast-produced alpha 1AT. These studies demonstrate the feasibility of using a retroviral vector to insert the normal human alpha 1AT cDNA into non-alpha 1AT-producing cells, resulting in the synthesis and secretion of physiologically "normal" human alpha 1AT.

In vitro characterization of a thermolabile herpes simplex virus DNA-binding protein.

The major herpes simplex virus DNA-binding protein, ICP8, was purified from cells infected with the herpes simplex virus type 1 temperature-sensitive strain tsHA1. tsHA1 ICP8 bound single-stranded DNA in filter binding assays carried out at room temperature and exhibited nonrandom binding to single-stranded bacteriophage fd DNA circles as determined by electron microscopy. The filter binding assay results and the apparent nucleotide spacing of the DNA complexed with protein were identical, within experimental error, to those observed with wild-type ICP8. Thermal inactivation assays, however, showed that the DNA-binding activity of tsHA1 ICP8 was 50% inactivated at approximately 39 degrees C as compared with 45 degrees C for the wild-type protein. Both wild-type and tsHA1 ICP8 were capable of stimulating viral DNA polymerase activity at permissive temperatures. The stimulatory effect of both proteins was lost at 39 degrees C.