Estrogen inhibits GH signaling by suppressing GH-induced JAK2 phosphorylation, an effect mediated by SOCS-2

Oral estrogen administration attenuates the metabolic action of growth hormone (GH) in humans. To investigate the mechanism involved, we studied the effects of estrogen on GH signaling through Janus kinase (JAK)2 and the signal transducers and activators of transcription (STATs) in HEK293 cells stably expressing the GH receptor (293GHR), HuH7 (hepatoma) and T-47D (breast cancer) cells. 293GHR cells were transiently transfected with an estrogen receptor-α expression plasmid and luciferase reporters with binding elements for STAT3 and STAT5 or the β-casein promoter. GH stimulated the reporter activities by four- to sixfold. Cotreatment with 17β-estradiol (E2) resulted in a dose-dependent reduction in the response of all three reporters to GH to a maximum of 49-66% of control at 100 nM (P < 0.05). No reduction was seen when E2 was added 1-2 h after GH treatment. Similar inhibitory effects were observed in HuH7 and T-47D cells. E2 suppressed GH-induced JAK2 phosphorylation, an effect attenuated by actinomycin D, suggesting a requirement for gene expression. Next, we investigated the role of the suppressors of cytokine signaling (SOCS) in E2 inhibition. E2 increased the mRNA abundance of SOCS-2 but not SOCS-1 and SOCS-3 in HEK293 cells. The inhibitory effect of E2 was absent in cells lacking SOCS-2 but not in those lacking SOCS-1 and SOCS-3. In conclusion, estrogen inhibits GH signaling, an action mediated by SOCS-2. This paper provides evidence for regulatory interaction between a sex steroid and the GH/JAK/STAT pathway, in which SOCS-2 plays a central mechanistic role

Structural insights into the function of the catalytically active human Taspase1

19 pags., 7 figs., 2 tabs.Taspase1 is an Ntn-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse models of glioblastoma. Taspase1 is synthesized as an inactive proenzyme, becoming active upon intramolecular cleavage. The activation process changes the conformation of a long fragment at the C-terminus of the α subunit, for which no full-length structural information exists and whose function is poorly understood. We present a cloning strategy to generate a circularly permuted form of Taspase1 to determine the crystallographic structure of active Taspase1. We discovered that this region forms a long helix and is indispensable for the catalytic activity of Taspase1. Our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases, suggesting that it could be a potential target for the design of inhibitors with potential to be developed into anticancer therapeutics.This project has been funded in whole with Federal funds from the National Cancer Institute (NCI), National Institutes of Health (NIH), under Chemical Biology Consortium contract no. HHSN261200800001E

Lung Epithelial Injury by B. Anthracis Lethal Toxin Is Caused by MKK-Dependent Loss of Cytoskeletal Integrity

Bacillus anthracis lethal toxin (LT) is a key virulence factor of anthrax and contributes significantly to the in vivo pathology. The enzymatically active component is a Zn2+-dependent metalloprotease that cleaves most isoforms of mitogen-activated protein kinase kinases (MKKs). Using ex vivo differentiated human lung epithelium we report that LT destroys lung epithelial barrier function and wound healing responses by immobilizing the actin and microtubule network. Long-term exposure to the toxin generated a unique cellular phenotype characterized by increased actin filament assembly, microtubule stabilization, and changes in junction complexes and focal adhesions. LT-exposed cells displayed randomly oriented, highly dynamic protrusions, polarization defects and impaired cell migration. Reconstitution of MAPK pathways revealed that this LT-induced phenotype was primarily dependent on the coordinated loss of MKK1 and MKK2 signaling. Thus, MKKs control fundamental aspects of cytoskeletal dynamics and cell motility. Even though LT disabled repair mechanisms, agents such as keratinocyte growth factor or dexamethasone improved epithelial barrier integrity by reducing cell death. These results suggest that co-administration of anti-cytotoxic drugs may be of benefit when treating inhalational anthrax

Pak1 regulates focal adhesion strength, myosin IIA distribution, and actin dynamics to optimize cell migration

p21-activated kinases are essential for spatial and temporal coordination of cytoskeletal dynamics with cellular adhesion during cell migration

p21-activated kinase signaling in breast cancer

The p21-activated kinases signal through a number of cellular pathways fundamental to growth, differentiation and apoptosis. A wealth of information has accumulated at an impressive pace in the recent past, both with regard to previously identified targets for p21-activated kinases that regulate the actin cytoskeleton and cellular stress pathways and with regard to newly identified targets and their role in cancer. Emerging data also provide new clues towards a previously unappreciated link between these various cellular processes. The present review attempts to provide a quick tutorial to the reader about the evolving significance of p21-activated kinases and small GTPases in breast cancer, using information from mouse models, tissue culture studies, and human materials

Regulation of growth hormone signaling by SIRP(alpha) and the protein tyrosine phosphatase SHP-2.

Reversible tyrosyl phosphorylation of proteins is a key regulatory mechanism that cells utilize to transduce signals in response to a variety of polypeptide hormones and growth factors, which regulate cellular growth, movement, or metabolism. Growth hormone (GH) is a key regulator of body growth and metabolism, and GH exerts its physiological actions by binding to GH receptor (GHR) and rapidly and transiently inducing tyrosyl phosphorylation and activation of the Janus tyrosine kinase JAK2. Activation of JAK2 is required for tyrosyl phosphorylation and/or activation of signaling molecules such as GHR, JAK2, the signal transducer and activator of transcription STAT5B and extracellular signal regulated kinases ERKs 1 and 2. Although the events which initiate GH signaling (such as JAK2 activation and tyrosyl phosphorylation) are beginning to be understood, the events which terminate GH signaling (such as dephosphorylation of signaling proteins) are poorly understood. In this dissertation, the protein tyrosine phosphatase SHP-2 and its substrate SIRP a (signal regulatory protein a ) are identified as negative regulators of GH signaling. GH induces tyrosyl phosphorylation of SHP-2 and SIRP a and association of SHP-2 with both SIRP a and GHR. Although JAK2 can associate with and tyrosyl phosphorylate SIRP a in a COS cell overexpression system, neither LiF nor IFN g , ligands that activate JAK2, induces tyrosyl phosphorylation of SIRP a or association of SHP-2 with SIRP a . Overexpression of wild-type SIRP a , but not SIRP a which lacks all potential SHP-2 binding sites, reduces tyrosyl phosphorylation of STAT5B, ERK, and a subset of JAK2 associated with SIRP a . To explore further the role of SHP-2 in GH signaling, the role of the association of SHP-2 with GHR was examined. SHP-2 associates with tyrosine 595 of GHR via the SH2 domains of SHP2, and mutation of tyrosine 595 prolongs GH-promoted tyrosyl phosphorylation of STAT5B and GHR, but not JAK2. These data indicate that SHP-2 and SIRP a may negatively regulate GH signal transduction.Ph.D.Biological SciencesMicrobiologyMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/131798/2/9929962.pd