Phospho-STAT5 and phospho-Akt expression in chronic myeloproliferative neoplasms.

The majority of Myeloproliferative Neoplasms (MPNs) are characterised by mutations in genes encoding molecules or receptors involved in cell signalling, the most common being the JAK2 V617F mutation. This mutation leads to ligand-independent activation of downstream signalling pathways by constitutive phosphorylation. The signalling pathways affected include the Janus kinase-signal transducers and activators of transcription (JAK-STAT) and phosphotidylinositide-3 kinase (PI3K) pathways, which regulate cell survival and apoptosis respectively. Monoclonal antibodies to phospho-STAT5 and phospho-Akt were generated and assessed by immunocytochemistry on bone marrow biopsies of MPN patients with JAK2 V617F, JAK2 exon 12, MPL exon 10 and KIT D816V mutations. JAK2 V617F mutation was associated with significantly increased levels of phosphorylated STAT5 and Akt in haemopoietic cells, most marked in megakaryocytes. In contrast, JAK2 exon 12 and MPL exon 10 mutations did not affect the level of phosphorylation. In systemic mastocytosis with KIT D618V mutation there was significantly increased expression of phosphorylated STAT5 and Akt in neoplastic mast cells although there was no change in the expression in other haemopoietic cells. JAK2 V617F is associated with upregulated phosphorylation of STAT5 and Akt in megakaryocytes, and to a lesser extent in other haemopoietic cells. Immunocytochemistry of bone marrow trephines for these phospho-proteins can be used as a supplementary diagnostic test with a high negative predictive value.

TGF-beta activates Erk MAP kinase signalling through direct phosphorylation of ShcA.

Erk1/Erk2 MAP kinases are key regulators of cell behaviour and their activation is generally associated with tyrosine kinase signalling. However, TGF-beta stimulation also activates Erk MAP kinases through an undefined mechanism, albeit to a much lower level than receptor tyrosine kinase stimulation. We report that upon TGF-beta stimulation, the activated TGF-beta type I receptor (TbetaRI) recruits and directly phosphorylates ShcA proteins on tyrosine and serine. This dual phosphorylation results from an intrinsic TbetaRI tyrosine kinase activity that complements its well-defined serine-threonine kinase function. TGF-beta-induced ShcA phosphorylation induces ShcA association with Grb2 and Sos, thereby initiating the well-characterised pathway linking receptor tyrosine kinases with Erk MAP kinases. We also found that TbetaRI is tyrosine phosphorylated in response to TGF-beta. Thus, TbetaRI, like the TGF-beta type II receptor, is a dual-specificity kinase. Recruitment of tyrosine kinase signalling pathways may account for aspects of TGF-beta biology that are independent of Smad signalling.

The comparative role of activator protein 1 and Smad factors in the regulation of Timp-1 and MMP-1 gene expression by transforming growth factor-beta 1.

The balance between matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), is pivotal in the remodeling of extracellular matrix. TGF-beta has profound effects on extracellular matrix homeostasis, in part via its ability to alter this balance at the level of gene expression. The intracellular signaling pathways by which TGF-beta mediates its actions include the Smad pathway, specific to the TGF-beta superfamily, but also, for example, mitogen-activated protein kinase pathways; furthermore, cross-talk between the Smads and other signaling pathways modifies the TGF-beta response. The reciprocal effect of TGF-beta on the expression of Timp-1 and MMP-1 supports its role in matrix anabolism, yet the mechanisms by which TGF-beta induces Timp-1 and represses induced MMP-1 have remained opaque. Here, we (i) investigate the mechanism(s) by which TGF-beta1 induces expression of the Timp-1 gene and (ii) compare this with TGF-beta1 repression of phorbol ester-induced MMP-1 expression. We report that the promoter-proximal activator protein 1 (AP1) site is essential for the response of both Timp-1 and MMP-1 to TGF-beta (induction and repression, respectively). c-Fos, JunD, and c-Jun are essential for the induction of Timp-1 gene expression by TGF-beta1, but these AP1 factors transactivate equally well from both Timp-1 and MMP-1 AP1 sites. Smad-containing complexes do not interact with the Timp-1 AP1 site, and overexpression of Smads does not substitute or potentiate the induction of the gene by TGF-beta1; furthermore, Timp-1 is still induced by TGF-beta1 in Smad knockout cell lines, although to varying extents. In contrast, Smads do interact with the MMP-1 AP1 site and mediate repression of induced MMP-1 gene expression by TGF-beta1.

Cooperative efforts improve compliance with acute stroke guidelines.

OBJECTIVE: Guidelines for emergency treatment of stroke are not always known or followed. Florida Medical Quality Assurance, Inc. collaborated with hospitals to determine how closely the current American Heart Association (AHA) and the Florida Agency for Health Care Administration (AHCA) guidelines were being followed and to improve compliance with these guidelines. METHODS: Medical records of patients admitted for acute stroke to 32 hospitals were retrospectively reviewed for compliance with six quality indicators (QIs) on the basis of AHA and AHCA guidelines. Hospitals were provided feedback on their levels of guideline compliance, and they subsequently implemented measures to improve compliance. After 6 months, the records of patients admitted after the provision of feedback were reviewed for compliance with the same six QIs. RESULTS: Compliance improved with regard to all QIs and was statistically significant for three of them. CONCLUSION: Feedback on performance, coupled with proactive collaboration with emergency department staff, resulted in improved compliance with the stroke guidelines.