Jae-Hyuck Shim

T-bet represses T H 17 differentiation by preventing Runx1-mediated activation of the gene encoding RORγt

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.Overactive responses by interleukin 17 (IL-17)-producing helper T cells (TH17 cells) are tightly linked to the development of autoimmunity, yet the factors that negatively regulate the differentiation of this lineage remain unknown. Here we report that the transcription factor T-bet suppressed development of the TH17 cell lineage by inhibiting transcription of Rorc (which encodes the transcription factor RORγt). T-bet interacted with the transcription factor Runx1, and this interaction blocked Runx1-mediated transactivation of Rorc. T-bet Tyr304 was required for formation of the T-bet–Runx1 complex, for blockade of Runx1 activity and for inhibition of the TH17 differentiation program. Our data reinforce the idea 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 p38 MAPK pathway is essential for skeletogenesis and bone homeostasis in mice

Nearly every extracellular ligand that has been found to play a role in regulating bone biology acts, at least in part, through MAPK pathways. Nevertheless, much remains to be learned about the contribution of MAPKs to osteoblast biology in vivo. Here we report that the p38 MAPK pathway is required for normal skeletogenesis in mice, as mice with deletion of any of the MAPK pathway member-encoding genes MAPK kinase 3 (Mkk3), Mkk6, p38a, or p38b displayed profoundly reduced bone mass secondary to defective osteoblast differentiation. Among the MAPK kinase kinase (MAP3K) family, we identified TGF-beta-activated kinase 1 (TAK1; also known as MAP3K7) as the critical activator upstream of p38 in osteoblasts. Osteoblast-specific deletion of Tak1 resulted in clavicular hypoplasia and delayed fontanelle fusion, a phenotype similar to the cleidocranial dysplasia observed in humans haploinsufficient for the transcription factor runt-related transcription factor 2 (Runx2). Mechanistic analysis revealed that the TAK1-MKK3/6-p38 MAPK axis phosphorylated Runx2, promoting its association with the coactivator CREB-binding protein (CBP), which was required to regulate osteoblast genetic programs. These findings reveal an in vivo function for p38beta and establish that MAPK signaling is essential for bone formation in vivo. These results also suggest that selective p38beta agonists may represent attractive therapeutic agents to prevent bone loss associated with osteoporosis and aging.

BET1, BOS1, and SEC22 are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the Golgi complex

A subset of the genes required for transport from the endoplasmic reticulum (ER) to the Golgi complex in Saccharomyces cerevisiae was found to interact genetically. While screening a yeast genomic library for genes complementing the ER-accumulating mutant bet1 (A. Newman and S. Ferro-Novick, J. Cell Biol. 105: 1587-1594, 1987), we isolated BET1 and BOS1 (bet one suppressor). BOS1 suppresses bet1-1 in a gene dosage-dependent manner, providing greater suppression when it is introduced on a multicopy vector than when one additional copy is present. The BET1 and BOS1 genes are not functionally equivalent; overproduction of BOS1 does not alleviate the lethality associated with disruption of BET1. We also identified a pattern of genetic interactions among these genes and another gene implicated in transport from the ER to the Golgi complex: SEC22. Overproduction of either BET1 or BOS1 suppresses the growth and secretory defects of the sec22-3 mutant over a wide range of temperatures. Further evidence for genetic interaction was provided by the finding that a bet1 sec22 double mutant is inviable. Another mutant which is blocked in transport from the ER to the Golgi complex, sec21-1, demonstrates a more limited ability to be suppressed by the BET1 gene. The interactions we observed are specific for genes required for transport from the ER to the Golgi complex. The products of the genes involved are likely to have a direct role in transport, as bet1-1 and sec22-3 begin to display their mutant phenotypes within 5 min of a shift to the restrictive temperature.

Mitogen-Activated Protein Kinase Pathways in Osteoblasts

Mitogen-activated protein kinases (MAPKs) are ancient signal transducers well characterized as mediators of inflammation and neoplastic transformation. Recent work has expanded our understanding of their developmental functions, particularly in the regulation of bone mass via control of osteoblast differentiation. Here, we review the functions of MAPK pathways in osteoblasts, including a consideration of MAPK substrates. In particular, MAPKs function to regulate the key transcriptional mediators of osteoblast differentiation, with ERK and p38 MAPKs phosphorylating RUNX2, the master regulator of osteoblast differentiation. ERK also activates RSK2, which in turn phosphorylates ATF4, a transcriptional regulator of late-stage osteoblast synthetic functions. The MAP3Ks and MAP2Ks upstream of MAPKs have also been investigated, and significant differences have been found in the wiring of MAPK pathways in osteoblasts relative to other tissues. Thus, the investigation of MAPKs in osteoblasts has both revealed critical mechanisms for the maintenance of bone mass and added to our understanding of how the individual components of MAPK pathways function in concert in a complex in vivo system.

TAK1 is an Essential Regulator of BMP Signalling in Cartilage

TGFβ activated kinase 1 (TAK1), a member of the MAPKKK family, controls diverse functions ranging from innate and adaptive immune system activation to vascular development and apoptosis. To analyse the in vivo function of TAK1 in cartilage, we generated mice with a conditional deletion of Tak1 driven by the collagen 2 promoter. Tak1col2 mice displayed severe chondrodysplasia with runting, impaired formation of secondary centres of ossification, and joint abnormalities including elbow dislocation and tarsal fusion. This phenotype resembled that of bone morphogenetic protein receptor (BMPR)1 and Gdf5‐deficient mice. BMPR signalling was markedly impaired in TAK1‐deficient chondrocytes as evidenced by reduced expression of known BMP target genes as well as reduced phosphorylation of Smad1/5/8 and p38/Jnk/Erk MAP kinases. TAK1 mediates Smad1 phosphorylation at C‐terminal serine residues. These findings provide the first in vivo evidence in a mammalian system that TAK1 is required for BMP signalling and functions as an upstream activating kinase for Smad1/5/8 in addition to its known role in regulating MAP kinase pathways. Our experiments reveal an essential role for TAK1 in the morphogenesis, growth, and maintenance of cartilage.

CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis

Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5 −/− cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5 −/− cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.

H2O2-dependent Hyperoxidation of Peroxiredoxin 6 (Prdx6) Plays a Role in Cellular Toxicity via Up-regulation of iPLA2 Activity

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase activity and Ca2+-independent phospholipase A2 (iPLA2) activity. Here, we report that H2O2-induced cellular toxicity acts through Prdx6 hyperoxidation. Under high concentrations of H2O2 (>100 μm), Prdx6, and 2-Cys Prdxs were hyperoxidized. Contrary to hyperoxidation of 2-Cys Prdxs, hyperoxidation of Prdx6 was irreversible in vivo. Surprisingly, H2O2-induced cell cycle arrest at the G2/M transition correlated with hyperoxidation and increased iPLA2 activity of Prdx6. This arrest was also associated with up-regulation of p53 and p21 and with down-regulation of cyclin B1. Furthermore, the H2O2-mediated increase in iPLA2 activity was dramatically abolished in a hyperoxidation mutant (C47A), an iPLA2 mutant (S32A), and a double mutant (C47A/S32A) of Prdx6, demonstrating the essential requirement of Prdx6 C47 hyperoxidation for its iPLA2 activity. Together, our results demonstrate that H2O2-mediated hyperoxidation of Prdx6 induces cell cycle arrest at the G2/M transition through up-regulation of iPLA2 activity.

The E3 ubiquitin ligase Wwp2 regulates craniofacial development through mono-ubiquitylation of Goosecoid

Craniofacial anomalies (CFAs) are the most frequently occurring human congenital disease, and a major cause of infant mortality and childhood morbidity. Although CFAs seems to arise from a combination of genetic factors and environmental influences, the underlying gene defects and pathophysiological mechanisms for most CFAs are currently unknown. Here we reveal a role for the E3 ubiquitin ligase Wwp2 in regulating craniofacial patterning. Mice deficient in Wwp2 develop malformations of the craniofacial region. Wwp2 is present in cartilage where its expression is controlled by Sox9. Our studies demonstrate that Wwp2 influences craniofacial patterning through its interactions with Goosecoid (Gsc), a paired-like homeobox transcription factor that has an important role in craniofacial development. We show that Wwp2-associated Gsc is a transcriptional activator of the key cartilage regulatory protein Sox6. Wwp2 interacts with Gsc to facilitate its mono-ubiquitylation, a post-translational modification required for optimal transcriptional activation of Gsc. Our results identify for the first time a physiological pathway regulated by Wwp2 in vivo, and also a unique non-proteolytic mechanism through which Wwp2 controls craniofacial development.

Roles for TAB1 in regulating the IL-1-dependent phosphorylation of the TAB3 regulatory subunit and activity of the TAK1 complex

The protein kinase TAK1 (transforming growth factor-beta-activated kinase 1), which has been implicated in the activation of MAPK (mitogen-activated protein kinase) cascades and the production of inflammatory mediators by LPS (lipopolysaccharide), IL-1 (interleukin 1) and TNF (tumour necrosis factor), comprises the catalytic subunit complexed to the regulatory subunits, termed TAB (TAK1-binding subunit) 1 and either TAB2 or TAB3. We have previously identified a feedback-control mechanism by which p38alpha MAPK down-regulates TAK1 and showed that p38alpha MAPK phosphorylates TAB1 at Ser(423) and Thr(431). In the present study, we identified two IL-1-stimulated phosphorylation sites on TAB2 (Ser(372) and Ser(524)) and three on TAB3 (Ser(60), Thr(404) and Ser(506)) in human IL-1R cells [HEK-293 (human embryonic kidney) cells that stably express the IL-1 receptor] and MEFs (mouse embryonic fibroblasts). Ser(372) and Ser(524) of TAB2 are not phosphorylated by pathways dependent on p38alpha/beta MAPKs, ERK1/2 (extracellular-signal-regulated kinase 1/2) and JNK1/2 (c-Jun N-terminal kinase 1/2). In contrast, Ser(60) and Thr(404) of TAB3 appear to be phosphorylated directly by p38alpha MAPK, whereas Ser(506) is phosphorylated by MAPKAP-K2/MAPKAP-K3 (MAPK-activated protein kinase 2 and 3), which are protein kinases activated by p38alpha MAPK. Studies using TAB1(-/-) MEFs indicate important roles for TAB1 in recruiting p38alpha MAPK to the TAK1 complex for the phosphorylation of TAB3 at Ser(60) and Thr(404) and in inhibiting the dephosphorylation of TAB3 at Ser(506). TAB1 is also required to induce TAK1 catalytic activity, since neither IL-1 nor TNFalpha was able to stimulate detectable TAK1 activity in TAB1(-/-) MEFs. Surprisingly, the IL-1 and TNFalpha-stimulated activation of MAPK cascades and IkappaB (inhibitor of nuclear factor kappaB) kinases were similar in TAB1(-/-), MEKK3(-/-) [MAPK/ERK (extracellular-signal-regulated kinase) kinase kinase 3] and wild-type MEFs, suggesting that another MAP3K (MAPK kinase kinase) may mediate the IL-1/TNFalpha-induced activation of these signalling pathways in TAB1(-/-) and MEKK3(-/-) MEFs.

Immunosuppressive effects of tautomycetin in vivo and in vitro via T cell-specific apoptosis induction

Tautomycetin (TMC) was identified as an immunosuppressor of activated T cells. Inhibition of T cell proliferation with TMC was observed at concentrations 100-fold lower than those needed to achieve maximal inhibition with cyclosporin A (CsA). TMC specifically blocked tyrosine phosphorylation of intracellular signal mediators downstream of Src tyrosine kinases in a T cell-specific manner, leading to apoptosis due to cleavage of Bcl-2, caspase-9, caspase-3, and poly(ADP-ribose) polymerase, but not caspase-1. In TMC-treated rats that received a heterotopic cardiac allograft, the graft survived more than 160 days, comparable to graft survival in allografted rats treated with CsA. Thus, TMC, whose mechanism of action is different from that of CsA or FK506, can be used as a potent T cell-specific immunosuppressor.