J. Jillian Zhang

Orai1 and STIM1 Are Critical for Breast Tumor Cell Migration and Metastasis

Tumor metastasis is the primary cause of death of cancer patients. Understanding the molecular mechanisms underlying tumor metastasis will provide potential drug targets. We report here that Orai1 and STIM1, both of which are involved in store-operated calcium entry, are essential for breast tumor cell migration in vitro and tumor metastasis in mice. Reduction of Orai1 or STIM1 by RNA interference in highly metastatic human breast cancer cells or treatment with a pharmacological inhibitor of store-operated calcium channels decreased tumor metastasis in animal models. Our data demonstrate a role for Orai1 and STIM1 in tumor metastasis and suggest store-operated calcium entry channels as potential cancer therapeutic targets.

Crystal structure of oligomeric β1-adrenergic G protein–coupled receptors in ligand-free basal state

G protein–coupled receptors (GPCRs) mediate transmembrane signaling. Before ligand binding, GPCRs exist in a basal state. Crystal structures of several GPCRs bound with antagonists or agonists have been solved. However, the crystal structure of the ligand-free basal state of a GPCR, the starting point of GPCR activation and function, had not yet been determined. Here we report the X-ray crystal structure of the ligand-free basal state of a GPCR in a lipid membrane–like environment. Oligomeric turkey β1-adrenergic receptors display two dimer interfaces. One interface involves the transmembrane domain (TM) 1, TM2, the C-terminal H8 and extracellular loop 1. The other interface engages residues from TM4, TM5, intracellular loop 2 and extracellular loop 2. Structural comparisons show that this ligand-free state is in an inactive conformation. This provides the structural basis of GPCR dimerization and oligomerization.

Migrastatin analogues target fascin to block tumour metastasis

Tumour metastasis is the primary cause of death of cancer patients. Development of new therapeutics preventing tumour metastasis is urgently needed. Migrastatin is a natural product secreted by Streptomyces, and synthesized migrastatin analogues such as macroketone are potent inhibitors of metastatic tumour cell migration, invasion and metastasis. Here we show that these migrastatin analogues target the actin-bundling protein fascin to inhibit its activity. X-ray crystal structural studies reveal that migrastatin analogues bind to one of the actin-binding sites on fascin. Our data demonstrate that actin cytoskeletal proteins such as fascin can be explored as new molecular targets for cancer treatment, in a similar manner to the microtubule protein tubulin.

Dosage-dependent switch from G protein-coupled to G protein-independent signaling by a GPCR

G‐protein‐coupled receptors (GPCRs) mostly signal through heterotrimeric G proteins. Increasing evidence suggests that GPCRs could function in a G‐protein‐independent manner. Here, we show that at low concentrations of an agonist, β2‐adrenergic receptors (β2‐ARs) signal through Gαs to activate the mitogen‐activated protein kinase pathway in mouse embryonic fibroblast cells. At high agonist concentrations, signals are also transduced through β2‐ARs via an additional pathway that is G‐protein‐independent but tyrosine kinase Src‐dependent. This new dosage‐dependent switch of signaling modes of GPCRs has significant implications for GPCR intrinsic properties and desensitization.

Requirement of Ca2+ and CaMKII for Stat1 Ser-727 phosphorylation in response to IFN-γ

In response to IFN-γ, the latent cytoplasmic protein signal transducers and activators of transcription 1 (Stat1) becomes phosphorylated on Y701, dimerizes, and accumulates in the nucleus to activate transcription of IFN-γ-responsive genes. For maximal gene activation, S727 in the transcription activation domain of Stat1 also is inducibly phosphorylated by IFN-γ. We previously purified a group of nuclear proteins that interact specifically with the Stat1 transcription activation domain. In this report, we identified one of them as the multifunctional Ca2+/calmodulin-dependent kinase (CaMK) II. We demonstrate that IFN-γ mobilizes a Ca2+ flux in cells and activates CaMKII. CaMKII can interact directly with Stat1 and phosphorylate Stat1 on S727 in vitro. Inhibition of Ca2+ flux or CaMKII results in a lack of S727 phosphorylation and Stat1-dependent gene activation, suggesting in vivo phosphorylation of Stat1 S727 by CaMKII. Thus two different cellular signaling events, IFN-γ receptor occupation and Ca2+ flux, are required for Stat1 to achieve maximal transcriptional activation through regulation of phosphorylation.

Identification of Novel Direct Stat3 Target Genes for Control of Growth and Differentiation

Signal transducer and activator of transcription 3 (Stat3) is a key regulator of gene expression in response to signaling of the glycoprotein 130 (gp130) family cytokines, including interleukin 6, oncostatin M, and leukemia inhibitory factor. Many efforts have been made to identify Stat3 target genes and to understand the mechanism of how Stat3 regulates gene expression. Using the microarray technique, hundreds of genes have been documented to be potential Stat3 target genes in different cell types. However, only a small fraction of these genes have been proven to be true direct Stat3 target genes. Here we report the identification of novel direct Stat3 target genes using a genome-wide screening procedure based on the chromatin immunoprecipitation method. These novel Stat3 target genes are involved in a diverse array of biological processes such as oncogenesis, cell growth, and differentiation. We show that Stat3 can act as both a repressor and activator on its direct target genes. We further show that most of the novel Stat3 direct target genes are dependent on Stat3 for their transcriptional regulation. In addition, using a physiological cell system, we demonstrate that Stat3 is required for the transcriptional regulation of two of the newly identified direct Stat3 target genes important for muscle differentiation.

Signal Transducers and Activators of Transcription 3 (STAT3) Directly Regulates Cytokine-induced Fascin Expression and Is Required for Breast Cancer Cell Migration

Background: The goal is to understand the molecular mechanism of metastasis and the roles of IL-6/OSM, STAT3, and fascin. Results: STAT3 binds to the fascin promoter and is essential for its expression and cell migration in response to IL-6/OSM. Conclusion: STAT3 plays a central role in cell migration through direct control of fascin expression. Significance: Drug targets are identified to block tumor metastasis. The cytokines oncostatin M (OSM) and IL-6 promote breast cancer cell migration and metastasis. Both cytokines activate STAT3, a member of the STAT (signal transducers and activators of transcription) family of transcription factors. Through transcriptional regulation of its target genes, STAT3 controls a wide range of cellular processes, including cellular proliferation, oncogenesis, and cancer metastasis. Fascin is an actin-bundling protein involved in cell migration. Elevated levels of fascin expression are found in many metastatic cancers, and inhibition of fascin function by small chemical compounds leads to a block of tumor metastasis. In this work, we demonstrate that fascin is a direct STAT3 target gene in response to OSM and IL-6 in both mouse and human breast cancer cells. We show that NFκB also binds to the fascin promoter in response to cytokine treatment and this binding is STAT3-dependent. Both STAT3 and NFκB are required for the cytokine-induced expression of fascin in cancer cells. Furthermore, we demonstrate that STAT3, in directly controlling fascin expression, is both necessary and sufficient for breast cancer cell migration.

Stimulation of Guanylyl Cyclase-D by Bicarbonate

Guanylyl cyclases (GCs) catalyze the conversion of GTP to the second messenger cGMP. While some transmembrane GCs are receptors for extracellular ligands, other transmembrane GCs such as retinal-specific GC-E and GC-F are stimulated by cellular proteins. GC-D is expressed in a special group of olfactory sensory neurons. However, the direct regulatory mechanism of GC-D activity is not completely understood. Here we have demonstrated that bicarbonate directly increases the activity of purified GC-D. Bicarbonate also increases the cGMP levels in cells expressing GC-D. These results identify bicarbonate as a small molecule that regulates GC-D.

Distinct Transcriptional Activation Functions of STAT1α and STAT1β on DNA and Chromatin Templates

Interferon-induced transcription depends upon tyrosine phosphorylation, subsequent dimerization, and binding to DNA of STAT1. Other factors, including but not necessarily limited to CBP/p300, then bind within the C-terminal 38 amino acid transactivation domain (TAD) to activate transcription. We show that both tyrosine-phosphorylated STAT1α (full-length wild-type protein) and STAT1β (lacking the TAD) stimulate in vitro transcription on a naked DNA template. Furthermore, in a system with purified proteins and naked DNA, STAT1α- and STAT1β-dependent transcription is stimulated by the TRAP/Mediator co-activator complex. Thus STAT1, through some site other than the C-terminal TAD, can interact with TRAP/Mediator or some intermediate protein. Although both STAT1α and STAT1β bind to known STAT sites within in vitro assembled chromatin templates, only STAT1α, and not STAT1β, in cooperation with p300 and acetyl-CoA, stimulated in vitro transcription from chromatin. After interferon-γ treatment, cells recruit STAT1α or -β to the chromosomal interferon-1 gene, but only STAT1α-containing cells recruit p300 and stimulate transcription. We conclude that chromatin remodeling by p300 in vivo makes TRAP/Mediator effective in stimulating transcription.

Stat3 Directly Controls the Expression of Tbx5, Nkx2.5, and GATA4 and Is Essential for Cardiomyocyte Differentiation of P19CL6 Cells

The transcription factor Stat3 (signal transducer and activator of transcription 3) mediates many physiological processes, including embryogenesis, stem cell self-renewal, and postnatal survival. In response to gp130 receptor activation, Stat3 becomes phosphorylated by the receptor-associated Janus kinase, forms dimers, and enters the nucleus where it binds to Stat3 target genes and regulates their expression. In this report, we demonstrate that Stat3 binds directly to the promoters and regulates the expression of three genes that are essential for cardiac differentiation: Tbx5, Nkx2.5, and GATA4. We further demonstrate that Tbx5, Nkx2.5, and GATA4 expression is dependent on Stat3 in response to ligand treatment and during ligand-independent differentiation of P19CL6 cells into cardiomyocytes. Finally, we show that Stat3 is necessary for the differentiation of P19CL6 cells into beating cardiomyocytes. All together, these results demonstrate that Stat3 is required for the differentiation of cardiomyocytes through direct transcriptional regulation of Tbx5, Nkx2.5, and GATA4.