Cytokine signaling through Stat3 activates integrins, promotes adhesion, and induces growth arrest in the myeloid cell line 32D

Hematopoietic cell development and function is dependent on cytokines and on intercellular interactions with the microenvironment. Although the intracellular signaling pathways stimulated by cytokine receptors are well described, little is known about the mechanisms through which these pathways modulate hematopoietic cell adhesion events in the microenvironment. Here we show that cytokine-activated Stat3 stimulates the expression and function of cell surface adhesion molecules in the myeloid progenitor cell line 32D. We generated an erythropoietin receptor (EpoR) isoform (ER343/401-S3) that activates Stat3 rather than Stat5 by substituting the Stat3 binding/activation sequence motif from gp130 for the sequences surrounding tyrosines 343 and 401 in the receptor cytoplasmic region. Activation of Stat3 leads to homotypic cell aggregation, increased expression of intercellular adhesion molecule 1 (ICAM-1), CD18, and CD11b, and activation of signaling through CD18-containing integrins. Unlike the wild type EpoR, ER343/401-S3 is unable to support long term Epo-dependent proliferation in 32D cells. Instead, Epo-treated ER343/401-S3 cells undergo G(1) arrest and express elevated levels of the cyclin-dependent kinase inhibitor p27(Kip1). Sustained activation of Stat3 in these cells is required for their altered morphology and growth properties since constitutive SOCS3 expression abrogates homotypic cell aggregation, signaling through CD18-containing integrins, G(1) arrest, and accumulation of p27(Kip1). Collectively, our results demonstrate that cytokine-activated Stat3 stimulates the expression and function of cell surface adhesion molecules, indicating that a role for Stat3 is to regulate intercellular contacts in myeloid cells.     

Unique structural determinants for Stat3 recruitment and activation by the granulocyte colony-stimulating factor receptor at phosphotyrosine ligands 704 and 744

G-CSFR cytoplasmic tyrosine (Y) residues (Y704, Y729, Y744, and Y764) become phosphorylated upon ligand binding and recruit specific Src homology 2 domain-containing proteins that link to distinct yet overlapping programs for myeloid cell survival, differentiation, proliferation, and activation. The structural basis for recruitment specificity is poorly understood but could be exploited to selectively target deleterious G-CSFR-mediated signaling events such as aberrant Stat3 activation demonstrated in a subset of acute myeloid leukemia patients with poor prognosis. Recombinant Stat3 bound to G-CSFR phosphotyrosine peptide ligands pY704VLQ and pY744LRC with similar kinetics. Testing of three models for Stat3 Src homology 2-pY ligand binding in vitro and in vivo revealed unique determinants for Stat3 recruitment and activation by the G-CSFR, the side chain of Stat3 R609, which interacts with the pY ligand phosphate group, and the peptide amide hydrogen of E638, which bonds with oxygen/sulfur within the + 3 Q/C side chain of the pY ligand when it assumes a beta turn. Thus, our findings identify for the first time the structural basis for recruitment and activation of Stat3 by the G-CSFR and reveal unique features of this interaction that can be exploited to target Stat3 activation for the treatment of a subset of acute myeloid leukemia patients.     

Stat3 phosphorylation mediates resistance of primary human T cells to regulatory T cell suppression

Human autoimmune diseases are characterized by systemic T cell dysfunction, resulting in chronically activated Th1 and Th17 cells that are inadequately suppressed by regulatory T cells (Tregs). IL-6, which is overexpressed in tissue and serum of patients with autoimmune diseases, inhibits human Treg function. We sought to determine the mechanism for the antitolerogenic properties of IL-6 by examining the signaling pathways downstream of IL-6R in primary human T cells. Inhibition of Stat3 signaling in MLCs containing IL-6 restores Treg-mediated suppression, demonstrating that IL-6-mediated loss of Treg suppression requires phosphorylation of Stat3. Cultures in which either effector T cells (Teffs) or Tregs were pretreated with Stat3 inhibitors indicate that phosphorylated (p)Stat3 is required in both T cell populations for IL-6-mediated reversal of Treg function. IL-21, which signals preferentially through pStat3, also reverses Treg suppression, in contrast to IL-27 and IFN-γ, which signal preferentially through Stat1 and do not inhibit Treg function. Interestingly, both Teffs and Tregs respond to IL-6 stimulation through strong Stat3 phosphorylation with minimal MAPK/Erk activation and moderate Stat1 phosphorylation. Finally, Teffs stimulated strongly through the TCR are also resistant to suppression by Tregs and show concurrent Stat3 phosphorylation. In these cultures, inhibition of pStat3 restores functional suppression by Tregs. Taken together, our findings suggest that an early dominance of Stat3 signaling, prior to subsequent T cell activation, is required for the loss of functional Treg suppression and that kinase-specific inhibitors may hold therapeutic promise in the treatment of autoimmune and chronic inflammatory diseases.     

BMPs signal alternately through a SMAD or FRAP-STAT pathway to regulate fate choice in CNS stem cells

The ability of stem cells to generate distinct fates is critical for the generation of cellular diversity during development. Central nervous system (CNS) stem cells respond to bone morphogenetic protein (BMP) 4 by differentiating into a wide variety of dorsal CNS and neural crest cell types. We show that distinct mechanisms are responsible for the generation of two of these cell types, smooth muscle and glia. Smooth muscle differentiation requires BMP-mediated Smad1/5/8 activation and predominates where local cell density is low. In contrast, glial differentiation predominates at high local densities in response to BMP4 and is specifically blocked by a dominant-negative mutant Stat3. Upon BMP4 treatment, the serine-threonine kinase FKBP12/rapamycin-associated protein (FRAP), mammalian target of rapamycin (mTOR), associates with Stat3 and facilitates STAT activation. Inhibition of FRAP prevents STAT activation and glial differentiation. Thus, glial differentiation by BMP4 occurs by a novel pathway mediated by FRAP and STAT proteins. These results suggest that a single ligand can regulate cell fate by activating distinct cytoplasmic signals.     

Mammary gland remodeling depends on gp130 signaling through Stat3 and MAPK

The interleukin-6 (IL6) family of cytokines signals through the common receptor subunit gp130, and subsequently activates Stat3, MAPK, and PI3K. Stat3 controls cell death and tissue remodeling in the mouse mammary gland during involution, which is partially induced by IL6 and LIF. However, it is not clear whether Stat3 activation is mediated solely through the gp130 pathway or also through other receptors. This question was explored in mice carrying two distinct mutations in the gp130 gene; one that resulted in the complete ablation of gp130 and one that led to the loss of Stat3 binding sites (gp130Delta/Delta). Deletion of gp130 specifically from mammary epithelium resulted in a complete loss of Stat3 activity and resistance to tissue remodeling comparable to that seen in the absence of Stat3. A less profound delay of mammary tissue remodeling was observed in gp130Delta/Delta mice. Stat3 tyrosine and serine phosphorylation was still detected in these mice suggesting that Stat3 activation could be the result of gp130 interfacing with other receptors. Experiments in primary mammary epithelial cells and transfected COS-7 cells revealed a p44/42 MAPK and EGFR-dependent Stat3 activation. Moreover, the gp130-dependent EGFR activation was independent of EGF ligands, suggesting a cytoplasmic interaction and cross-talk between these two receptors. These experiments establish that two distinct Stat3 signaling pathways emanating from gp130 are utilized in mammary tissue.     

Stat3 links activated keratinocytes and immunocytes required for development of psoriasis in a novel transgenic mouse model

Here we report that epidermal keratinocytes in psoriatic lesions are characterized by activated Stat3. Transgenic mice with keratinocytes expressing a constitutively active Stat3 (K5.Stat3C mice) develop a skin phenotype either spontaneously, or in response to wounding, that closely resembles psoriasis. Keratinocytes from K5.Stat3C mice show upregulation of several molecules linked to the pathogenesis of psoriasis. In addition, the development of psoriatic lesions in K5.Stat3C mice requires cooperation between Stat3 activation in keratinocytes and activated T cells. Finally, abrogation of Stat3 function by a decoy oligonucleotide inhibits the onset and reverses established psoriatic lesions in K5.Stat3C mice. Thus, targeting Stat3 may be potentially therapeutic in the treatment of psoriasis.     

The rapid inactivation of nuclear tyrosine phosphorylated Stat1 depends upon a protein tyrosine phosphatase.

After interferon-gamma (IFN-gamma) treatment of cells the appearance of tyrosine phosphorylated Stat1 in the nucleus was maximal within 20-30 min, remained for 2-2.5 h and activated molecules disappeared by 4 h. In the absence of continued signaling from the receptor (imposed by staurosporine treatment) previously activated Stat1 disappeared completely within 60 min, implying continuous generation and removal of active molecules during extended IFN-gamma treatment. Proteasome inhibitors prolonged the time of activation of Stat1 by prolonging signaling from the receptor but not by blocking removal of already activated Stat1 molecules. By analyzing with 35S labeling the distribution of total Stat1 and activated Stat1, we concluded that the Stat1 molecules promptly cycle into the nucleus as tyrosine phosphorylated molecules and later return quantitatively to the cytoplasm as non-phosphorylated molecules. Therefore, the removal of the activated Stat1 molecules from the nucleus appears not to be proteolytic but must depend on a protein tyrosine phosphatase(s).