Mesenchymal stem cells (MSCs) are a pluripotent cell type that can differentiate into several distinct lineages. Two key transcription factors, Runx2 and peroxisome proliferator-activated receptor gamma (PPARgamma), drive MSCs to differentiate into either osteoblasts or adipocytes, respectively. How these two transcription factors are regulated in order to specify these alternate cell fates remains a pivotal question. Here we report that a 14-3-3-binding protein, TAZ (transcriptional coactivator with PDZ-binding motif), coactivates Runx2-dependent gene transcription while repressing PPARgamma-dependent gene transcription. By modulating TAZ expression in model cell lines, mouse embryonic fibroblasts, and primary MSCs in culture and in zebrafish in vivo, we observed alterations in osteogenic versus adipogenic potential. These results indicate that TAZ functions as a molecular rheostat that modulates MSC differentiation.

Cell lineage specification depends on both gene activation and gene silencing, and in the differentiation of T helper progenitors to Th1 or Th2 effector cells, this requires the action of two opposing transcription factors, T-bet and GATA-3. T-bet is essential for the development of Th1 cells, and GATA-3 performs an equivalent role in Th2 development. We report that T-bet represses Th2 lineage commitment through tyrosine kinase-mediated interaction between the two transcription factors that interferes with the binding of GATA-3 to its target DNA. These results provide a novel function for tyrosine phosphorylation of a transcription factor in specifying alternate fates of a common progenitor cell.

Overactive TH17 responses are tightly linked to the development of autoimmunity, yet the factors that negatively regulate differentiation of this lineage remain unknown. Here, we report that T-bet suppresses the development of the TH17 cell lineage by inhibiting the transcription of Rorc. T-bet interacts with the transcription factor Runx1 and this interaction blocks Runx1-mediated transactivation of Rorc. T-bet residue Tyr304 is required for T-bet-Runx1 complex formation, for blocking Runx1 activity and for inhibiting the TH17 differentiation program. These data reinforce the concept of master regulators that shape immune responses by simultaneously activating one genetic program while silencing the activity of competing regulators in a common progenitor cell.

The osteopontin (Opn) glycoprotein has been implicated in diverse physiological processes, including vascularization, bone formation, and inflammatory responses. Studies of its role in immune responses has suggested that Opn can set the early stage of type-1 immune (cell-mediated) responses through differential regulation of IL-12 and IL-10 cytokine gene expression in macrophages. Although Opn has been suggested to play a role in the development of type-1 immunity, little is known about control of Opn gene expression. Here, we report that Opn gene expression in activated T cells, but not macrophages, is regulated by T-bet, a transcription factor that controls CD4 ؉ T helper (Th1) cell lineage commitment. We also find that T-bet-dependent expression of Opn in T cells is essential for efficient skewing of CD4 ؉ T and CD8 ؉ T cells toward the Th1 and type 1 CD8 ؉ T cells (Tc1) pathway, respectively. Taken together, these findings begin to delineate the genetic basis of Opn expression in T cells and further clarify the role of Opn in Th and Tc1 development.genetic programming ͉ T helper 1 development ͉ type-1 immune response T he osteopontin (Opn) glycoprotein has been independently identified and studied by investigators from numerous scientific fields in view of its role in immune responses, vascularization, and bone formation through interactions with mononuclear, endothelial, and bone cells, respectively. Analysis of its contribution to immune responses has suggested that Opn expression can set the stage for protective type-1 immune responses after viral and bacterial infection through differential regulation of IL-12 and IL-10 cytokine production (1-3).Studies of Opn-deficient (Opn Ϫ/Ϫ ) mice have indicated that Opn contributes to host resistance against diverse microbial pathogens including herpes simplex virus 1, Listeria monocytogenes (4), and rotavirus (5). Opn expression is also essential for effective Th1-dependent granuloma formation and a positive clinical outcome in patients suffering from mycobacterial infection (6), whereas ectopic Opn expression has been implicated in the granulomatous lesions of Crohn's disease (7). Dysregulated Opn expression has also been implicated in several autoimmune disorders, including murine experimental autoimmune encephalomyelitis (EAE) (8, 9), multiple sclerosis (10), rheumatoid arthritis (11), and atherosclerosis (12, 13). Dysregulated Opn expression has been correlated with excessive Th1 polarization of CD4 ϩ T cells in these disorders (7,14) opening the possibility that Opn gene expression may directly contribute to Th1͞type 1 CD8 ϩ T cells (Tc1) genetic programming. However, the genetic basis of Opn expression in Th-cell subsets is not well understood.T-bet, a member of the T box family of transcription factors, is the master coordinator of gene expression in T cells that initiate type-1 immunity and is essential for Th1 cell polarization (15). Thus, T-bet deficiency reduces IFN-␥ production by activated CD4 ϩ T cells and T cell antigen receptor (TCR)-transgenic CD8...

Shear stress activates cellular signaling involved in cellular proliferation, differentiation, and migration. However, the mechanisms of mesenchymal stem cell (MSC) differentiation under interstitial flow are not fully understood. Here, we show the increased osteogenic differentiation of MSCs under exposure to constant, extremely low shear stress created by osmotic pressure-induced flow in a microfluidic chip. The interstitial level of shear stress in the proposed microfluidic system stimulated nuclear localization of TAZ (transcriptional coactivator with PDZ-binding motif), a transcriptional modulator of MSCs, activated TAZ target genes such as CTGF and Cyr61, and induced osteogenic differentiation. TAZ-depleted cells showed defects in shear stress-induced osteogenic differentiation. In shear stress induced cellular signaling, Rho signaling pathway was important forthe nuclear localization of TAZ. Taken together, these results suggest that TAZ is an important mediator of interstitial flow-driven shear stress signaling in osteoblast differentiation of MSCs.

Interleukin (IL)-2 is the predominant cytokine that is produced by naive Th cells in a primary response. It is required for proliferation and differentiation of Th precursor cells into effector cells. Initial high-level IL-2 production is followed by its decline, and the concomitant induction of cytokines that are typical of the differentiated state. Although the factors that are responsible for the early induction of IL-2 are well defined, the mechanisms that are responsible for its down-regulation in later stages of Th development have not been studied as much. Previous work from our laboratory revealed a repressor function for the T-box transcription factor, T-bet, in IL-2 gene transcription. Here, we report that T-betS508 is required for the optimal repression of IL-2 production in developing Th1 cells. Phosphorylation of T-betS508 by casein kinase I and glycogen synthase kinase-3 kinases accompanies T-bet's interaction with the RelA nuclear factor–κB transcription factor. Heterodimerization of T-bet and RelA interferes with the binding of RelA to the IL-2 promoter, and hence, transcriptional activation of the IL-2 gene by RelA.

TBX21 encodes for the transcription factor T-bet (T-box expressed in T cells), which influences naïve T lymphocyte development and has been implicated in asthma pathogenesis. Specifically, the T-bet knockout mouse spontaneously develops airway hyperresponsiveness and other changes consistent with asthma. Because airway responsiveness is moderated by the use of inhaled corticosteroids in asthma, it is conceivable that genetic variation in TBX21 may alter asthma phenotypes in a treatment-specific fashion. Here we demonstrate that the nonsynonymous variation in TBX21 coding for replacement of histidine 33 with glutamine is associated with significant improvement in the PC 20 (a measure of airway responsiveness) of asthmatic children in a large clinical trial spanning 4 years. We note that this increase occurs only in the children randomized to inhaled corticosteroids and that it dramatically enhances the overall improvement in PC 20 associated with inhaled corticosteroid usage. The average PC20 at trial end for subjects on inhaled corticosteroids possessing a variant allele was in the normal range for nonasthmatics. In cellular models, we show that the TBX21 variant increases T helper 1 and decreases T helper 2 cytokine expression comparably with wild type. TBX21 may thus be an important determinant pharmacogenetic response to the therapy of asthma with inhaled corticosteroids. PC20 ͉ pharmacogenetics ͉ T-bet ͉ interaction C orticosteroids mediate a variety of immunological actions and are commonly used in the treatment of a diverse number of diseases. Inhaled corticosteroids are the most effective and commonly used therapy in the management of asthma (1, 2) but may be associated with serious adverse reactions (3). In evaluating asthma therapy response, measures of lung function, such as forced expiratory volume at 1 second (FEV 1 ), and of airway responsiveness, as measured by the provocative concentration of methacholine causing a 20% decrement in FEV 1 (PC 20 ) are commonly used. However, there is large interindividual variation in the FEV 1 and PC 20 responses to inhaled corticosteroids (4, 5). Thus, identifying those patients most likely to benefit from this treatment would be valuable. Because the intraindividual response to inhaled corticosteroid treatment in patients with asthma is highly repeatable (6) and because both FEV 1 and PC 20 are heritable traits (7, 8), a genetic basis for the heterogeneity of this therapeutic response is plausible.The gene TBX21 (GenBank accession no. NM 013351) encodes for transcription factor T-bet (T-box expressed in T cells), which is responsible for the induction of T helper (Th)1 cells and the repression of Th2 cells from naïve T lymphocytes (9). T-bet has been implicated in the pathogenesis of asthma (10, 11). Because the T-bet knockout mouse develops spontaneous airway hyperresponsiveness (10), a phenotype that is modulated by corticosteroids, we assessed the relationship of TBX21 with PC 20 outcomes in asthma. Only one common (estimated heterozygosity Ն 5%) nonsynonymo...

Emerging evidence indicates that NADPH oxidase (NOX) and its reactive oxygen species (ROS) products modulate a variety of cellular events, including proliferation, differentiation, and apoptosis. In this study, we investigated the functions of NOX2 and ROS in immune modulation using NOX2 knockout (KO) mice. Interestingly, NOX2 KO mice spontaneously developed arthritis with onset at 6-7 wk of age and high incidence (60%) at 15-18 wk of age. Arthritis severity in NOX2 KO mice was proportionally increased with age and higher in females than in males. Bone destruction was confirmed by microcomputed tomography scanning and histological analyses of joints. Inflammatory factors, including TNF-α, IL-1β, and RANKL, and serum level of anti-type II collagen IgG were significantly increased in NOX2 KO mice. In addition, NOX2 deficiency perturbed the immune system upon aging. NOX2 KO mice demonstrated preferred development of CD11b+Gr-1+ myeloid cells with profound production of proinflammatory cytokines and augmented expression of IL-17 through the activation of STAT3 and RORγt in vivo. NOX2 deficiency increased differentiation of effector Th cells in vitro and decreased CD25+FoxP3+ Treg cells both in vitro and in vivo. Furthermore, adoptive transfer of NOX2-deficient CD4 + T cells into RAG KO mice increased arthritic inflammation compared with WT cells. These results demonstrated that NOX2 deficiency affected the development of CD11b+ myeloid cells and Th17/Treg cells, and thus promoted inflammatory cytokine production and inflammatory arthritis development, strongly supporting a crucial role for ROS generation in the modulation of Th17/Treg cell development and its related inflammatory immune response upon aging.rheumatoid arthritis | myeloid-derived suppressor cell