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, Epotreated 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.
SummaryScrape loading Clostridium botulinum C3 exoenzyme into primary peripheral blood human T lymphocytes (PB T cells) efficiently adenosine diphosphate (ADP)-ribosylates and thus inactivates the guanosine triphosphatase (GTPase) Rho. Basal adhesion of PB T cells to the  1 integrin substrate fibronectin (Fn) was not inhibited by inactivation of Rho, nor was upregulation of adhesion using phorbol myristate acetate (PMA; 10 ng/ml) or Mn ϩϩ (1 mM) affected. Whereas untreated PB T cells adherent to Fn remain spherical, C3-treated PB T cells extend F-actin-containing pseudopodia. Inactivation of Rho delayed the kinetics of PMA-dependent PB T cell homotypic aggregation, a process involving integrin ␣ L  2. Although C3 treatment of PB T cells did not prevent adhesion to the  1 integrin substrate Fn, it did inhibit  1 integrin/CD3-mediated costimulation of proliferation. Analysis of intracellular cytokine production at the single cell level demonstrated that ADP-ribosylation of Rho inhibited  1 integrin/ CD3 and CD28/CD3 costimulation of IL-2 production within 6 h of activation. Strikingly, IL-2 production induced by PMA and ionomycin was unaffected by C3 treatment. Thus, the GTPase Rho is a novel regulator of T lymphocyte cytoarchitecture, and functional Rho is required for very early events regulating costimulation of IL-2 production in PB T cells.
In normal lymphocytes an inside-out signal up-regulating integrin adhesion is followed by a ligand-mediated outside-in cell spreading signal. Protein kinase C (PKC) inhibition blocks lymphocyte adherence to and spreading on fibronectin. In contrast, putative PLC inhibitors yield distinct differences with respect to adhesion and morphology. The phosphatidylinositol-specific phospholipase C (PLC) inhibitor neomycin blocked spreading of CD3/CD28-activated T cells on fibronectin by disrupting adhesion. Furthermore, when an additional inside-out signal for fibronectin adhesion is unnecessary such as with HPB-ALL T leukemic or phorbol-myristate-acetate-treated normal T cells, neomycin treatment does not alter adhesion or morphology. However, the phosphatidylcholinespecific PLC inhibitor D609 abrogates cell spreading without affecting adhesion to fibronectin in these cells as well as the CD3/CD28-activated T cells. These results strongly suggest that inside-out signaling for the integrin ␣ 4  1 in lymphocytes proceeds through phosphatidylinositol-specific PLC and PKC, whereas the outside-in signal utilizes phosphatidylcholine-specific PLC and PKC. J. Leukoc. Biol. 65: 127-136; 1999.
Background: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton.
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