Background and Purpose-Brain-derived neurotrophic factor (BDNF) is involved in neuronal survival, synaptic plasticity, learning and memory, and neuroplasticity. Further, exogenous treatment with BDNF or exposing animals to enrichment and exercise regimens, which also increase BDNF, enhances behavioral recovery after brain injury. Thus, the beneficial effects of rehabilitation in promoting recovery after stroke may also depend on BDNF. We tested this hypothesis by evaluating the contribution of BDNF to motor skill relearning after endothelin-1-induced middle cerebral artery occlusion in rats. Methods-Antisense BDNF oligonucleotide, which blocks the expression of BDNF (or saline vehicle) was infused into the contralateral lateral ventricle for 28 days after ischemia. Animals received either a graduated rehabilitation program, including running exercise and skilled reaching training, which simulates clinical practice, or no rehabilitation. Functional recovery was assessed with a battery of tests that measured skilled reaching, forelimb use asymmetry, and foraging ability. Results-Rehabilitation significantly improved skilled reaching ability in the staircase task. Antisense BDNF oligonucleotide effectively blocked BDNF mRNA, and negated the beneficial effects of rehabilitation on recovery of skilled reaching. Importantly, antisense BDNF oligonucleotide did not affect reaching with the unaffected limb, body weight, infarct size, or foraging ability, indicating the treatment was specific to relearning of motor skill after ischemia. Conclusions-This study is the first to identify a critical role for BDNF in rehabilitation-induced recovery after stroke, and our results suggest that new treatments to enhance BDNF would constitute a promising therapy for promoting recovery of function after stroke.

Transforming growth factor-B (TGF-B) stimulates cellular proliferation and transformation to a myofibroblast phenotype in vivo and in a subset of fibroblast cell lines. As the Smad pathway is activated by TGF-B in essentially all cell types, it is unlikely to be the sole mediator of cell type-specific outcomes to TGF-B stimulation. In the current study, we determined that TGF-B receptor signaling activates phosphatidylinositol 3-kinase (PI3K) in several fibroblast but not epithelial cultures independently of Smad2 and Smad3. PI3K activation occurs in the presence of dominant-negative dynamin and is required for p21-activated kinase-2 kinase activity and the increased proliferation and morphologic change induced by TGF-B in vitro. (Cancer Res 2005; 65(22): 10431-40)

Members of the transforming growth factor ␤ (TGF-␤) family of proteins signal through cell surface transmembrane serine/threonine protein kinases known as type I and type II receptors. The TGF-␤ signal is extended through phosphorylation of receptor-associated Smad proteins by the type I receptor. Although numerous investigations have established the sequence of events in TGF-␤ receptor (TGF-␤R) activation, none have examined the role of the endocytic pathway in initiation and/or maintenance of the signaling response. In this study we investigated whether TGF-␤R internalization modulates type I receptor activation, the formation of a functional receptor/Smad/SARA complex, Smad2/3 phosphorylation or nuclear translocation, and TGF-␤-dependent reporter gene activity. Our data provide evidence that, whereas type I receptor phosphorylation and association of SARA and Smad2 with the TGF-␤R complex take place independently of clathrin lattice formation, Smad2 or Smad3 activation and downstream signaling only occur after endocytic vesicle formation. Thus, TGF-␤R endocytosis is not simply a way to dampen the signaling response but instead is required to propagate signaling via the Smad pathway.

The Transforming Growth Factor-Beta (TGF-β) family is involved in regulating a variety of cellular processes such as apoptosis, differentiation, and proliferation. TGF-β binding to a Serine/Threonine kinase receptor complex causes the recruitment and subsequent activation of transcription factors known as smad2 and smad3. These proteins subsequently translocate into the nucleus to negatively or positively regulate gene expression. In this study, we define a second signaling pathway leading to TGF-β receptor activation of Extracellular Signal Regulated Kinase (Erk) in a cell-type dependent manner. TGF-β induced Erk activation was found in phenotypically normal mesenchymal cells, but not normal epithelial cells. By activating phosphotidylinositol 3-kinase (PI3K), TGF-β stimulates p21-activated kinase2 (Pak2) to phosphorylate c-Raf, ultimately resulting in Erk activation. Activation of Erk was necessary for TGF-β induced fibroblast replication. In addition, Erk phosphorylated the linker region of nuclear localized smads, resulting in increased half-life of C-terminal phospho-smad 2 and 3 and increased duration of smad target gene transcription. Together, these data show that in mesenchymal cell types the TGF-β/PI3K/Pak2/Raf/MEK/Erk pathway regulates smad signaling, is critical for TGF-β-induced growth and is part of an integrated signaling web containing multiple interacting pathways rather than discrete smad/non-smad pathways.

Transforming growth factor beta (TGF beta) family ligands initiate a cascade of events capable of modulating cellular growth and differentiation. The receptors responsible for transducing these cellular signals are referred to as the type I and type II TGF beta receptors. Ligand binding to the type II receptor results in the transphosphorylation and activation of the type I receptor. This heteromeric complex then propagates the signal(s) to downstream effectors. There is presently little data concerning the fate of TGF beta receptors after ligand binding, with conflicting reports indicating no change or decreasing cell surface receptor numbers. To address the fate of ligand-activated receptors, we have used our previously characterized chimeric receptors consisting of the ligand binding domain from the granulocyte/macrophage colony-stimulating factor alpha or beta receptor fused to the transmembrane and cytoplasmic domain of the type I or type II TGF beta receptor. This system not only provides the necessary sensitivity and specificity to address these types of questions but also permits the differentiation of endocytic responses to either homomeric or heteromeric intracellular TGF beta receptor oligomerization. Data are presented that show, within minutes of ligand binding, chimeric TGF beta receptors are internalized. However, although all the chimeric receptor combinations show similar internalization rates, receptor down-regulation occurs only after activation of heteromeric TGF beta receptors. These results indicate that effective receptor down-regulation requires cross-talk between the type I and type II TGF beta receptors and that TGF beta receptor heteromers and homomers show distinct trafficking behavior.