Eun Jung Jang

TM-25659 enhances osteogenic differentiation and suppresses adipogenic differentiation by modulating the transcriptional co-activator TAZ.

BACKGROUND AND PURPOSE The transcriptional co‐activator with PDZ‐binding motif (TAZ) is characterized as a transcriptional modulator of mesenchymal stem cell differentiation into osteoblasts and adipocytes. Moreover, increased TAZ activity in the nucleus enhances osteoblast differentiation and suppresses adipocyte development by interacting with runt‐related transcription factor 2 (RUNX2) and PPARγ, respectively. Therefore, it would be of interest to identify low MW compounds that modulate nuclear TAZ activity.

In vivo tumor suppression activity by T cell-specific T-bet restoration†

T‐box‐containing protein expressed in T cells (T‐bet) is a master transcription factor for the development of interferon (IFN) γ‐producing T helper 1 (Th1) cells and also functions in other immune cells including natural killer (NK), cytotoxic T lymphocytes and dendritic cells. T‐bet‐deficient mice increased susceptibility to viral infection and tumor development due to the defective functions of immune cells. T‐bet is known to play a key role in NK‐mediated antimetastatic response; however, it remains to be characterized whether T‐bet is essential for in vivo tumor suppression mediated by T cells. Here, we have investigated in vivo tumor suppression effect of T‐bet‐restored T cells using T cell‐specific and inducible T‐bet transgenic mice generated in a T‐bet‐deficient background. T‐bet‐null mice increased susceptibility to tumor development, whereas induction of T cell‐specific T‐bet expression upon melanoma cell injection substantially suppressed tumor development by inducing IFNγ production in T cells and tumor cell apoptosis. Late induction of T‐bet expression in tumor‐bearing mice produced comparable amounts of IFNγ with control and significantly decreased tumor volume. In addition, increased melanoma lung metastasis in T‐bet‐deficient mice was strikingly inhibited by T‐bet restoration in T cells. Intravenous injection of activated Th1 cells, not T‐bet‐null Th1 cells, attenuated metastatic melanoma progression, in addition, restoration of T‐bet in T‐bet‐null Th1 cells certainly retrieved antimetastatic activity. These results suggest that T‐bet expression in T cells is crucial for the control of tumor development and antimetastatic activity.

Augmentation of PPARγ-TAZ interaction contributes to the anti-adipogenic activity of KR62980

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that plays a pivotal role in the modulation of gene expression involved in adipocyte differentiation and insulin sensitivity. It has been previously established that thiazolidinedione (TZD) PPARgamma ligands such as rosiglitazone have potent anti-diabetic and adipogenic activities. A novel non-TZD ligand for PPARgamma, KR62980 has recently been characterized to increase insulin sensitivity and to be weakly adipogenic in 3T3-L1 cells or anti-adipogenic in rosiglitazone-induced adipocyte differentiation. In this study, we have confirmed that KR62980 substantially suppresses rosiglitazone-induced adipocyte differentiation and attenuates adipogenic gene expression via an induced reduction in PPARgamma activity. KR62980 increased the nuclear localization of TAZ, a PPARgamma suppressor, and also enhanced the interaction between PPARgamma and TAZ, thus resulting in the TAZ-mediated suppression of PPARgamma activity. Furthermore, KR62980 failed to suppress PPARgamma-mediated adipogenic gene expression and adipocyte differentiation in TAZ knockdown 3T3-L1 cells, thus indicating a TAZ-dependent suppressive activity of KR62980 on PPARgamma-mediated function. These findings strongly suggest that the novel PPARgamma ligand, KR62980, may prove to be beneficial to anti-adipogenic function through the suppression of PPARgamma-mediated adipocyte differentiation by activating TAZ.

Lysine 313 of T-box Is Crucial for Modulation of Protein Stability, DNA Binding, and Threonine Phosphorylation of T-bet

A T-box–containing protein expressed in T cells (T-bet) is a key transcription factor involved in the regulation of Th cell differentiation. Although T-bet–deficient CD4+ T cells fail to produce IFN-γ and typically differentiate into Th2 cells in vitro, ectopic overexpression of T-bet elevates IFN-γ and suppresses production of IL-2 and Th2 cytokines through different mechanisms. Despite the importance of the T-bet protein level, the regulatory mechanisms that control T-bet protein stability are largely unknown. In this study, we found that T-bet underwent proteasomal degradation via ubiquitination at Lys-313. Despite its robust accumulation following lysine mutation, T-betK313R failed to increase IFN-γ production because of diminished DNA binding activity, as demonstrated in the crystal structure of T-bet–DNA complex. Strikingly, T-betK313R entirely lost the ability to suppress IL-2 production and Th2 cell development; this was due to loss of its interaction with NFAT1. We further identified that the T-betK313R reduced the phosphorylation of T-bet at Thr-302, and that threonine phosphorylation was essential for T-bet interaction with NFAT1 and suppression of NFAT1 activity. Retroviral transduction of T-betT302A into T-bet–deficient cells restored IFN-γ levels compared with those induced by wild-type T-bet, but this mutant failed to inhibit IL-2 and Th2 cytokine production. Collectively, these data show that Lys-313 in the T-box domain is essential for controlling T-bet protein stability via ubiquitin-dependent degradation, T-bet binding to the IFN-γ promoter, and for the interaction with and suppression of NFAT1. Thus, multiple posttranslational modifications of T-bet are involved in fine-tuning cytokine production during Th cell development.

TAZ Suppresses NFAT5 Activity through Tyrosine Phosphorylation

ABSTRACT Transcriptional coactivator with PDZ-binding motif (TAZ) physically interacts with a variety of transcription factors and modulates their activities involved in cell proliferation and mesenchymal stem cell differentiation. TAZ is highly expressed in the kidney, and a deficiency of this protein results in multiple renal cysts and urinary concentration defects; however, the molecular functions of TAZ in renal cells remain largely unknown. In this study, we examined the effects of osmotic stress on TAZ expression and activity in renal cells. We found that hyperosmotic stress selectively increased protein phosphorylation at tyrosine 316 of TAZ and that this was enhanced by c-Abl activation in response to hyperosmotic stress. Interestingly, phosphorylated TAZ physically interacted with nuclear factor of activated T cells 5 (NFAT5), a major osmoregulatory transcription factor, and subsequently suppressed DNA binding and transcriptional activity of NFAT5. Furthermore, TAZ deficiency elicited an increase in NFAT5 activity in vitro and in vivo, which then reverted to basal levels following restoration of wild-type TAZ but not mutant TAZ (Y316F). Collectively, the data suggest that TAZ modulates cellular responses to hyperosmotic stress through fine-tuning of NFAT5 activity via tyrosine phosphorylation.

Ablation of peroxiredoxin II attenuates experimental colitis by increasing FoxO1-induced Foxp3+ regulatory T cells.

Peroxiredoxin (Prx) II is an intracellular antioxidant molecule that eliminates hydrogen peroxide, employing a high substrate-binding affinity. PrxII deficiency increases the levels of intracellular reactive oxygen species in many types of cells, which may increase reactive oxygen species–mediated inflammation. In this study, we investigated the susceptibility of PrxII knockout (KO) mice to experimentally induced colitis and the effects of PrxII on the immune system. Wild-type mice displayed pronounced weight loss, high mortality, and colon shortening after dextran sulfate sodium administration, whereas colonic inflammation was significantly attenuated in PrxII KO mice. Although macrophages were hyperactivated in PrxII KO mice, the amount of IFN-γ and IL-17 produced by CD4+ T cells was substantially reduced. Foxp3+ regulatory T (Treg) cells were elevated, and Foxp3 protein expression was increased in the absence of PrxII in vitro and in vivo. Restoration of PrxII into KO cells suppressed the increased Foxp3 expression. Interestingly, endogenous PrxII was inactivated through hyperoxidation during Treg cell development. Furthermore, PrxII deficiency stabilized FoxO1 expression by reducing mouse double minute 2 homolog expression and subsequently activated FoxO1-mediated Foxp3 gene transcription. PrxII overexpression, in contrast, reduced FoxO1 and Foxp3 expression. More interestingly, adoptive transfer of naive CD4+ T cells from PrxII KO mice into immune-deficient mice attenuated T cell–induced colitis, with a reduction in mouse double minute 2 homolog expression and an increase in FoxO1 and Foxp3 expression. These results suggest that inactivation of PrxII is important for the stability of FoxO1 protein, which subsequently mediates Foxp3+ Treg cell development, thereby attenuating colonic inflammation.

A FoxO1-dependent, but NRF2-independent induction of heme oxygenase-1 during muscle atrophy.

Skeletal muscle plays key roles in metabolic homeostasis. Loss of muscle mass, called muscle atrophy exacerbates disease‐associated metabolic perturbations. In this study, we characterized the molecular functions and mechanisms underlying regulation of skeletal muscle atrophy induced by denervation. Denervation significantly increased the expression of heme oxygenase‐1 (HO‐1) and atrogenes in skeletal muscle. Forkhead box protein O1 (FoxO1) drastically increased in atrophied muscle and selectively stimulated HO‐1 gene transcription through direct DNA binding. Lack of HO‐1 substantially attenuated muscle atrophy, whereas HO‐1 overexpression caused muscle damage in vitro and in vivo. Collectively, HO‐1 induced by FoxO1 may cause skeletal muscle atrophy.

(-)-Epicatechin gallate (ECG) stimulates osteoblast differentiation via runt-related transcription factor 2 (Runx2) and transcriptional coactivator with PDZ-binding motif (TAZ)-mediated transcriptional activation

Background: Catechins in green tea have a beneficial effect in bone formation, but the detailed mechanism is not fully understood. Results: ECG, a major compound of green tea, stimulates TAZ- and RUNX2-mediated osteogenic gene transcription through PP1A. Conclusion: ECG stimulates osteoblast differentiation through a transcriptional activation. Significance: A novel mechanism for green tea-stimulated osteoblast differentiation is revealed. Osteoporosis is a degenerative bone disease characterized by low bone mass and is caused by an imbalance between osteoblastic bone formation and osteoclastic bone resorption. It is known that the bioactive compounds present in green tea increase osteogenic activity and decrease the risk of fracture by improving bone mineral density. However, the detailed mechanism underlying these beneficial effects has yet to be elucidated. In this study, we investigated the osteogenic effect of (−)-epicatechin gallate (ECG), a major bioactive compound found in green tea. We found that ECG effectively stimulates osteoblast differentiation, indicated by the increased expression of osteoblastic marker genes. Up-regulation of osteoblast marker genes is mediated by increased expression and interaction of the transcriptional coactivator with PDZ-binding motif (TAZ) and Runt-related transcription factor 2 (RUNX2). ECG facilitates nuclear localization of TAZ through PP1A. PP1A is essential for osteoblast differentiation because inhibition of PP1A activity was shown to suppress ECG-mediated osteogenic differentiation. Taken together, the results showed that ECG stimulates osteoblast differentiation through the activation of TAZ and RUNX2, revealing a novel mechanism for green tea-stimulated osteoblast differentiation.

Suppression of Inflammatory cytokine production by ar-Turmerone isolated from Curcuma phaeocaulis.

Rhizomes of Curcuma phaeocaulis Valeton (Zingiberaceae) have traditionally been used for controlling inflammatory conditions. Numerous studies have aimed to isolate and characterize the bioactive constituents of C. phaeocaulis. It has been reported that its anti‐inflammatory properties are a result of cyclooxygenase‐2 inhibition; however, its effect on the T‐cell function remains to be elucidated. In this study, four known sesquiterpenoids, viz., ar‐turmerone (TM), germacrone (GM), (+)‐(4S,5S)‐germacrone‐4,5‐epoxide (GE), and curzerenone (CZ), were isolated from C. phaeocaulis rhizomes and evaluated for their effects on the CD4+ T‐cell function. While GM, GE, and CZ had no effect on the activation of splenic T cells or CD4+ T cells, TM suppressed the interferon (IFN)‐γ production, without affecting the interleukin (IL)‐4 expression. TM also decreased the expression of IL‐2 in CD4+ T cells, but did not change their cell‐division rates upon stimulation. These results suggest that TM, a major constituent of C. phaeocaulis rhizomes selectively exerts potent anti‐inflammatory effects via suppression of the inflammatory cytokines IFN‐γ and IL‐2.

Novel TAZ modulators enhance myogenic differentiation and muscle regeneration.

The transcriptional co‐activator with PDZ‐binding motif (TAZ) is a key controller of mesenchymal stem cell differentiation through its nuclear localization and subsequent interaction with master transcription factors. In particular, TAZ directly associates with myoblast determining protein D (MyoD) and activates MyoD‐induced myogenic gene expression, thereby enhancing myogenic differentiation. Here, we have synthesized and characterized low MW compounds modulating myogenic differentiation via induction of TAZ nuclear localization.