Tgfbeta signaling directly induces Arf promoter remodeling by a mechanism involving Smads 2/3 and p38 MAPK.

We have investigated how the Arf gene product, p19(Arf), is activated by Tgfß during mouse embryo development to better understand how this important tumor suppressor is controlled. Taking advantage of new mouse models, we provide genetic evidence that Arf lies downstream of Tgfß signaling in cells arising from the Wnt1-expressing neural crest and that the anti-proliferative effects of Tgfß depend on Arf in vivo. Tgfß1, -2, and -3 (but not BMP-2, another member of the Tgfß superfamily) induce p19(Arf) expression in wild type mouse embryo fibroblasts (MEFs), and they enhance Arf promoter activity in Arf(lacZ/lacZ) MEFs. Application of chemical inhibitors of Smad-dependent and -independent pathways show that SB431542, a Tgfß type I receptor (TßrI) inhibitor, and SB203580, a p38 MAPK inhibitor, impede Tgfß2 induction of Arf. Genetic studies confirm the findings; transient knockdown of Smad2, Smad3, or p38 MAPK blunt Tgfß2 effects, as does Cre recombinase treatment of Tgfbr2(fl/fl) MEFs to delete Tgfß receptor II. Chromatin immunoprecipitation reveals that Tgfß rapidly induces Smads 2/3 binding and histone H3 acetylation at genomic DNA proximal to Arf exon 1ß. This is followed by increased RNA polymerase II binding and progressively increased Arf primary and mature transcripts from 24 through 72 h, indicating that increased transcription contributes to p19(Arf) increase. Last, Arf induction by oncogenic Ras depends on p38 MAPK but is independent of TßrI activation of Smad 2. These findings add to our understanding of how developmental and tumorigenic signals control Arf expression in vivo and in cultured MEFs.

Expression of the Arf tumor suppressor gene is controlled by Tgfbeta2 during development.

The Arf tumor suppressor (also known as Cdkn2a) acts as an oncogene sensor induced by ;abnormal' mitogenic signals in incipient cancer cells. It also plays a crucial role in embryonic development: newborn mice lacking Arf are blind due to a pathological process resembling severe persistent hyperplastic primary vitreous (PHPV), a human eye disease. The cell-intrinsic mechanism implied in the oncogene sensor model seems unlikely to explain Arf regulation during embryo development. Instead, transforming growth factor beta2 (Tgfbeta2) might control Arf expression, as we show that mice lacking Tgfbeta2 have primary vitreous hyperplasia similar to Arf(-/-) mice. Consistent with a potential linear pathway, Tgfbeta2 induces Arf transcription and p19(Arf) expression in cultured mouse embryo fibroblasts (MEFs); and Tgfbeta2-dependent cell cycle arrest in MEFs is maintained in an Arf-dependent manner. Using a new model in which Arf expression can be tracked by beta-galactosidase activity in Arf(lacZ/+) mice, we show that Tgfbeta2 is required for Arf transcription in the developing vitreous as well as in the cornea and the umbilical arteries, two previously unrecognized sites of Arf expression. Chemical and genetic strategies show that Arf promoter induction depends on Tgfbeta receptor activation of Smad proteins; the induction correlates with Smad2 phosphorylation in MEFs and Arf-expressing cells in vivo. Chromatin immunoprecipitation shows that Smads bind to genomic DNA proximal to Arf exon 1beta. In summary, Tgfbeta2 and p19(Arf) act in a linear pathway during embryonic development. We present the first evidence that p19(Arf) expression can be coupled to extracellular cues in normal cells and suggest a new mechanism for Arf control in tumor cells.

The identification and characterisation of novel KIT transcripts in aggressive mast cell malignancies and normal CD34+ cells.

KIT mutations have been identified in several malignancies, including acute myeloid leukemia (AML) and systemic mastocytosis (SM). Mast cell leukemia (MCL) is the most aggressive mast cell neoplasm, but has not been well studied due to its rarity. We identified novel KIT transcripts in two patients with MCL and two patients with SM with an associated hematological disorder, but not from two patients with SM. Similar novel KIT transcripts were also observed in normal CD34+ cells from bone marrow and umbilical cord blood, suggesting that altered KIT isoforms may be specific to the blast stage of hematopoietic precursors. The novel KIT proteins lack several domains including the ATP binding site, and one was inactive in a functional test for autophosphorylation. Our discovery of novel KIT transcripts underscores the importance of analysing entire protein encoding regions when studying genes of interest.