mcl-1, a bcl-2 family member, was originally identified as an early gene induced during differentiation of ML-1 myeloid leukemia cells. In the present study, we demonstrate that Mcl-1 is tightly regulated by the granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathway. Upon deprivation of survival factor from TF-1 myeloid progenitor cells, Mcl-1 levels quickly dropped prior to visible detection of apoptosis of these cells. Upon restimulation of these deprived cells with GM-CSF, the mcl-1 mRNA was immediately induced and its protein product was accordingly resynthesized. Analysis with Ba/F3 cells expressing various truncation mutants of the GM-CSF receptor revealed that the membrane distal region between amino acids 573 and 755 of the receptor ␤ chain was required for mcl-1 induction. Transient-transfection assays with luciferase reporter genes driven by various regions of the mcl-1 promoter demonstrated that the upstream sequence between ؊197 and ؊69 is responsible for cytokine activation of the mcl-1 gene. Overexpression of mcl-1 delayed but did not completely prevent apoptosis of cells triggered by cytokine withdrawal. Its down regulation by antisense constructs overcame, at least partially, the survival activity of GM-CSF and induced the apoptosis of TF-1 cells. Taken together, these results suggest that mcl-1 is an immediate-early gene activated by the cytokine receptor signaling pathway and is one component of the GM-CSF viability response.

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Osthole, an active component of Chinese herbal medicines, reportedly possesses various pharmacological properties and has potential therapeutic applications. This study explored the anti-allergic effects of osthole in asthmatic mice and investigated the immunomodulatory actions of osthole on dendritic cells (DCs) and T cells. Herein, we show that oral administration of osthole to BALB/c mice after ovalbumin (OVA) sensitization ameliorated all of the cardinal features of T helper 2 (Th2)-mediated allergic asthma; namely, the production of OVA-specific immunoglobulin E, airway hyperresponsiveness, airway inflammation and the production of Th2-type cytokines including interleukin (IL)-4, IL-5 and IL-13. Surprisingly, IL-10 production was not inhibited and was even enhanced by osthole treatment. We observed a significant increase in the percentages of IL-10-producing DCs and forkhead box P3-positive regulatory T (Treg) cells in osthole-treated asthmatic mice. Additionally, in vitro analyses revealed that osthole-treated bone-marrow-derived DCs had a partial maturation phenotype, secreting large amounts of IL-10 and low levels of proinflammatory cytokines, such as IL-12, IL-6 and tumor necrosis factor-α, and displaying reduced levels of MHC class II surface molecules. These DCs displayed immunosuppressive capacity by directly inhibiting effector T-cell responses or inducing Treg cells. In addition, osthole directly inhibited the activated CD4 T-cell proliferation and Th1/Th2-type cytokine production in this system. Collectively, these results suggest that DCs and T cells are potential target cells responsible for the action of osthole against allergic asthma.Cellular &Molecular Immunology advance online publication, 7 August 2017; doi:10.1038/cmi.2017.71.

This study investigated the immunomodulatory effects of ferulic acid (FA) on antigen-presenting dendritic cells (DCs) in vitro and its antiallergic effects against ovalbumin- (OVA-) induced Th2-mediated allergic asthma in mice. The activation of FA-treated bone marrow-derived DCs by lipopolysaccharide (LPS) stimulation induced a high level of interleukin- (IL-) 12 but reduced the expression levels of the proinflammatory cytokines IL-1β, IL-6, and tumor necrosis factor- (TNF-) α. Compared to control-treated DCs, FA significantly enhanced the expressions of Notch ligand Delta-like 4 (Dll4), MHC class II, and CD40 molecules by these DCs. Furthermore, these FA-treated DCs enhanced T-cell proliferation and Th1 cell polarization. In animal experiments, oral administration of FA reduced the levels of OVA-specific immunoglobulin E (IgE) and IgG1 and enhanced IgG2a antibody production in serum. It also ameliorated airway hyperresponsiveness and attenuated eosinophilic pulmonary infiltration in dose-dependent manners. In addition, FA treatment inhibited the production of eotaxin, Th2 cytokines (IL-4, IL-5, and IL-13), and proinflammatory cytokines but promoted the Th1 cytokine interferon- (IFN-) γ production in bronchoalveolar lavage fluid (BALF) and the culture supernatant of spleen cells. These findings suggest that FA exhibits an antiallergic effect via restoring Th1/Th2 imbalance by modulating DCs function in an asthmatic mouse model.

Stem cell factor (SCF) has been suggested as essential for optimal production of various hematopoietic lineages mainly because of its apoptosis prevention function when it costimulates with other cytokines. However, the underlying mechanism of this synergism of apoptosis prevention is largely unknown. The present study examined the expression of some Bcl-2 family members, including Bcl-2, Bcl-XL, Mcl-1, and Bax, in response to cytokine stimulation in TF-1 and JYTF-1 cells in which SCF costimulation is differentially required for optimal proliferation. The results revealed that only the expression of Mcl-1 highly correlated with the antiapoptotic activity of interleukin-5 (IL-5) and the synergistic effect of SCF. In TF-1 cells, the defect of IL-5 in apoptosis suppression and Mcl-1 induction was associated with the incapability to highly phosphorylate Janus kinases (JAK1, JAK2), signal transducer and activator of transcription-5 (STAT5), mitogen-activated protein kinase (MAPK), and Akt/PKB, whereas SCF costimulation restored the potent phosphorylation of MAPK and Akt/PKB, but not STAT5. The importance of MAPK and Akt/PKB signaling pathways in regulating the expression of Mcl-1 and cell survival was further supported by the observation that inhibition of MEK by PD98059 or phosphatidylinositol-3 kinase (PI-3K) by LY294002 independently resulted in the reduction of Mcl-1 expression and loss of cell viability. Therefore, the data suggest that Mcl-1 is a common antiapoptotic target of both early-stage cytokine SCF and late-stage cytokine IL-5. Both MEK/MAPK and PI-3K/Akt signaling pathways are essential in the regulation of Mcl-1 expression and apoptosis prevention.

Activin A is a member of the transforming growth factor (TGF)-beta superfamily that regulates cell proliferation and differentiation. Using the p38 inhibitor SB203580, our previous studies demonstrated that p38 was involved in activin A-mediated hemoglobin (Hb) synthesis in K562 cells. SB203580 is an inhibitor of p38alpha and p38beta isoforms. In this study, we show that p38alpha and p38beta mRNA were expressed in K562 cells and that activin A activated the kinase activities of these isoforms. To investigate the roles of p38alpha and p38beta isoforms in activin A-mediated erythroid differentiation, we generated stable clones that over-expressed the dominant negative p38 isoforms p38alpha(AF) and p38beta(AF) in K562 cells. The expressions of either p38alpha(AF) or p38beta(AF) reduced activin A-induced p38 activation, Hb synthesis, and zeta-globin promoter activity. Similarly, down-regulation of either p38alpha or p38beta by isoform-specific siRNAs also reduced activin A-induced zeta-globin promoter activity. Co-expressions of p38alpha(AF) and p38beta(AF), together, greatly inhibited the transcription activity of the zeta-globin promoter. Conversely, expression of mitogen-activated protein kinase kinase (MKK) 6b(E), a constitutive activator of p38, significantly activated zeta-globin promoter. Co-expressions of either p38alpha or p38beta with MKK6b had a similar activation of zeta-globin promoter. Activin A induction of erythroid differentiation was inhibited by follistatin. Activin A-induced phosphorylation of MKK6 and p38 was also inhibited by follistatin. Moreover, over-expression of MKK6b(E) reverted follistatin inhibition of activin A-induced zeta-globin promoter activity. These results demonstrate that activin A induces erythroid differentiation of K562 cells through activation of MKK6-p38alpha/p38beta pathway and follistatin inhibits those effects.

Type II collagen is known to modulate chondrogenesis of mesenchymal stem cells (MSCs). In this study, MSCs from human bone marrow aspirates were used to study the modulating effects of type II collagen on MSC differentiation during the early stages of osteogenesis and adipogenesis. With osteogenic induction, MSCs cultured on the type II collagen-coated surface showed an enhanced calcium deposition level with increasing mRNA expressions of RUNX2, osteocalcin, and alkaline phosphatase. A synthetic integrin binding peptide, which specifically interacts with the I-domain of α(1)β(1)/α(2)β(1) integrins significantly blocks the mineralization-enhancing effect of type II collagen. MSCs attached on the type II collagen-coated plates exhibited expanded cell morphology with increasing spreading area, and the pretreatment of cells with integrin α(1)β(1) or α(2)β(1)-blocking antibody reduced the effect. The phosphorylation levels of FAK, ERK, and JNK significantly increased in the MSCs that attached on the type II collagen-coated plates. On the contrary, the mineralization-enhancing effect of type II collagen was diminished by JNK and MEK inhibitors. Furthermore, type II collagen blocked the adipogenic differentiation of MSCs, and this effect is rescued by JNK and MEK inhibitors. In conclusion, type II collagen facilitates osteogenesis and suppresses adipogenesis during early stage MSC differentiation. Such effects are integrin binding-mediated and conducted through FAK-JNK and/or FAK-ERK signaling cascades. These results inspire a novel strategy encompassing type II collagen in bone tissue engineering.

The results suggest that FIP-fve can inhibit IL-5-mediated survival of eosinophils through the modulation of cytokine receptor expression and apoptotic signal protein production. The modulatory effect of FIP-fve on eosinophil apoptosis in vitro indicates that it may have some therapeutic effect on eosinophil-related allergic inflammation in vivo.