Mitochondrial injury induced by a Salmonella genotoxin triggers the proinflammatory senescence-associated secretory phenotype.

Bacterial genotoxins damage host cells by targeting their chromosomal DNA. In the present study, we demonstrate that a genotoxin of Salmonella Typhi, typhoid toxin, triggers the senescence-associated secretory phenotype (SASP) by damaging mitochondrial DNA. The actions of typhoid toxin disrupt mitochondrial DNA integrity, leading to mitochondrial dysfunction and disturbance of redox homeostasis. Consequently, it facilitates the release of damaged mitochondrial DNA into the cytosol, activating type I interferon via the cGAS-STING pathway. We also reveal that the GCN2-mediated integrated stress response plays a role in the upregulation of inflammatory components depending on the STING signaling axis. These SASP factors can propagate the senescence effect on T cells, leading to senescence in these cells. These findings provide insights into how a bacterial genotoxin targets mitochondria to trigger a proinflammatory SASP, highlighting a potential therapeutic target for an anti-toxin intervention.

TRAF3-EWSR1 signaling axis acts as a checkpoint on germinal center responses.

The formation of germinal centers (GCs) is crucial for humoral immunity and vaccine efficacy. Constant stimulation through microbiota drives the formation of constitutive GCs in Peyer's patches (PPs), which generate B cells that produce antibodies against gut antigens derived from commensal bacteria and infectious pathogens. However, the molecular mechanism that regulates this persistent process is poorly understood. We report that Ewing Sarcoma Breakpoint Region 1 (EWSR1) is a brake to constitutive GC generation and immunoglobulin G (IgG) production in PPs, vaccination-induced GC formation, and IgG responses. Mechanistically, EWSR1 suppresses Bcl6 upregulation after antigen encounter, thereby negatively regulating induced GC B cell generation and IgG production. We further showed that tumor necrosis factor receptor-associated factor (TRAF) 3 serves as a negative regulator of EWSR1. These results established that the TRAF3-EWSR1 signaling axis acts as a checkpoint for Bcl6 expression and GC responses, indicating that this axis is a therapeutic target to tune GC responses and humoral immunity in infectious diseases.

Dapl1 controls NFATc2 activation to regulate CD8+ T cell exhaustion and responses in chronic infection and cancer.

CD8+ T cells are central mediators of immune responses against infections and cancer. Here we identified Dapl1 as a crucial regulator of CD8+ T cell responses to cancer and infections. Dapl1 deficiency promotes the expansion of tumour-infiltrating effector memory-like CD8+ T cells and prevents their functional exhaustion, coupled with increased antitumour immunity and improved efficacy of adoptive T cell therapy. Dapl1 controls activation of NFATc2, a transcription factor required for the effector function of CD8+ T cells. Although NFATc2 mediates induction of the immune checkpoint receptor Tim3, competent NFATc2 activation prevents functional exhaustion of CD8+ T cells. Interestingly, exhausted CD8+ T cells display attenuated NFATc2 activation due to Tim3-mediated feedback inhibition; Dapl1 deletion rescues NFATc2 activation and thereby prevents dysfunction of exhausted CD8+ T cells in chronic infection and cancer. These findings establish Dapl1 as a crucial regulator of CD8+ T cell immunity and a potential target for cancer immunotherapy.

Matriptase-2/NR4A3 axis switches TGF-ß action toward suppression of prostate cancer cell invasion, tumor growth, and metastasis.

Dysregulation of pericellular proteolysis is strongly implicated in cancer metastasis through alteration of cell invasion and the microenvironment. Matriptase-2 (MT-2) is a membrane-anchored serine protease which can suppress prostate cancer (PCa) cell invasion. In this study, we showed that MT-2 was down-regulated in PCa and could suppress PCa cell motility, tumor growth, and metastasis. Using microarray and biochemical analysis, we found that MT-2 shifted TGF-ß action towards its tumor suppressor function by repressing epithelial-to-mesenchymal transition (EMT) and promoting Smad2 phosphorylation and nuclear accumulation to upregulate two TGF-ß1 downstream effectors (p21 and PAI-1), culminating in hindrance of PCa cell motility and malignant growth. Mechanistically, MT-2 could dramatically up-regulate the expression of nuclear receptor NR4A3 via iron metabolism in PCa cells. MT-2-induced NR4A3 further coactivated Smad2 to activate p21 and PAI-1 expression. In addition, NR4A3 functioned as a suppressor of PCa and mediated MT-2 signaling to inhibit PCa tumorigenesis and metastasis. These results together indicate that NR4A3 sustains MT-2 signaling to suppress PCa cell invasion, tumor growth, and metastasis, and serves as a contextual factor for the TGF-ß/Smad2 signaling pathway in favor of tumor suppression via promoting p21 and PAI-1 expression.

Myeloid cell TBK1 restricts inflammatory responses.

Proinflammatory cytokine production by innate immune cells plays a crucial role in inflammatory diseases, but the molecular mechanisms controlling the inflammatory responses are poorly understood. Here, we show that TANK-binding kinase 1 (TBK1) serves as a vital regulator of proinflammatory macrophage function and protects against tissue inflammation. Myeloid cell-conditional Tbk1 knockout (MKO) mice spontaneously developed adipose hypertrophy and metabolic disorders at old ages, associated with increased adipose tissue M1 macrophage infiltration and proinflammatory cytokine expression. When fed with a high-fat diet, the Tbk1-MKO mice also displayed exacerbated hepatic inflammation and insulin resistance, developing symptoms of nonalcoholic steatohepatitis. Furthermore, myeloid cell-specific TBK1 ablation exacerbates inflammation in experimental colitis. Mechanistically, TBK1 functions in macrophages to suppress the NF-κB and MAP kinase signaling pathways and thus attenuate induction of proinflammatory cytokines, particularly IL-1ß. Ablation of IL-1 receptor 1 (IL-1R1) eliminates the inflammatory symptoms of Tbk1-MKO mice. These results establish TBK1 as a pivotal anti-inflammatory mediator that restricts inflammation in different disease models.

Peli1 facilitates NLRP3 inflammasome activation by mediating ASC ubiquitination.

Inflammasomes are crucial for innate immunity against infections and, when deregulated, also contribute to inflammatory diseases. Here, we identify a critical function of the E3 ubiquitin ligase Peli1 in regulating the activation of NLRP3 inflammasome. Peli1 deficiency impairs induction of interleukin-1ß (IL-1ß) secretion by different NLRP3 inducers, but not by inducers of the Aim2, NLRP1, and NLRC4 inflammasomes. Peli1-deficient mice have alleviated peritonitis induction by alum and display increased resistance to lipopolysaccharide (LPS) endotoxin shock, coupled with decreased serum concentration of IL-1ß. Peli1 is required for NLRP3-induced caspase-1 activation and IL-1ß maturation. Mechanistically, Peli1 conjugates K63 ubiquitin chain to lysine 55 of the inflammasome adaptor apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), which in turn facilitates ASC/NLRP3 interaction and ASC oligomerization, thereby contributing to inflammasome activation. Peli1 deficiency impairs the ubiquitination of ASC and inhibits inflammasome activation. Our findings establish Peli1 as an important inflammasome regulator and suggest a mechanism by which Peli1 mediates inflammatory responses.

Microglia promote autoimmune inflammation via the noncanonical NF-κB pathway.

Microglia have been implicated in neuroinflammatory diseases, including multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). We demonstrate that microglia mediate EAE disease progression via a mechanism relying on the noncanonical nuclear factor kB (NF-κB) pathway. Microglia-specific deletion of the noncanonical NF-κB-inducing kinase (NIK) impairs EAE disease progression. Although microglial NIK is dispensable for the initial phase of T cell infiltration into the central nervous system (CNS) and EAE disease onset, it is critical for the subsequent CNS recruitment of inflammatory T cells and monocytes. Our data suggest that following their initial CNS infiltration, T cells activate the microglial noncanonical NF-κB pathway, which synergizes with the T cell-derived cytokine granulocyte-macrophage colony-stimulating factor to induce expression of chemokines involved in the second-wave of T cell recruitment and disease progression. These findings highlight a mechanism of microglial function that is dependent on NIK signaling and required for EAE disease progression.

DYRK1a mediates BAFF-induced noncanonical NF-κB activation to promote autoimmunity and B-cell leukemogenesis.

B-cell-activating factor (BAFF) mediates B-cell survival and, when deregulated, contributes to autoimmune diseases and B-cell malignancies. The mechanism connecting BAFF receptor (BAFFR) signal to downstream pathways and pathophysiological functions is not well understood. Here we identified DYRK1a as a kinase that responds to BAFF stimulation and mediates BAFF-induced B-cell survival. B-cell-specific DYRK1a deficiency causes peripheral B-cell reduction and ameliorates autoimmunity in a mouse model of lupus. An unbiased screen identified DYRK1a as a protein that interacts with TRAF3, a ubiquitin ligase component mediating degradation of the noncanonical nuclear factor (NF)-κB-inducing kinase (NIK). DYRK1a phosphorylates TRAF3 at serine-29 to interfere with its function in mediating NIK degradation, thereby facilitating BAFF-induced NIK accumulation and noncanonical NF-κB activation. Interestingly, B-cell acute lymphoblastic leukemia (B-ALL) cells express high levels of BAFFR and respond to BAFF for noncanonical NF-κB activation and survival in a DYRK1a-dependent manner. Furthermore, DYRK1a promotes a mouse model of B-ALL through activation of the noncanonical NF-κB pathway. These results establish DYRK1a as a critical BAFFR signaling mediator and provide novel insight into B-ALL pathogenesis.

Afatinib Exerts Immunomodulatory Effects by Targeting the Pyrimidine Biosynthesis Enzyme CAD.

Current clinical trials of combined EGFR-tyrosine kinase inhibitors (TKI) and immune checkpoint blockade (ICB) therapies show no additional effect. This raises questions regarding whether EGFR-TKIs attenuate ICB-enhanced CD8+ T lymphocyte function. Here we show that the EGFR-TKI afatinib suppresses CD8+ T lymphocyte proliferation, and we identify CAD, a key enzyme of de novo pyrimidine biosynthesis, to be a novel afatinib target. Afatinib reduced tumor-infiltrating lymphocyte numbers in Lewis lung carcinoma (LLC)-bearing mice. Early afatinib treatment inhibited CD8+ T lymphocyte proliferation in patients with non-small cell lung cancer, but their proliferation unexpectedly rebounded following long-term treatment. This suggests a transient immunomodulatory effect of afatinib on CD8+ T lymphocytes. Sequential treatment of afatinib with anti-PD1 immunotherapy substantially enhanced therapeutic efficacy in MC38 and LLC-bearing mice, while simultaneous combination therapy showed only marginal improvement over each single treatment. These results suggest that afatinib can suppress CD8+ T lymphocyte proliferation by targeting CAD, proposing a timing window for combined therapy that may prevent the dampening of ICB efficacy by EGFR-TKIs. SIGNIFICANCE: This study elucidates a mechanism of afatinib-mediated immunosuppression and provides new insights into treatment timing for combined targeted therapy and immunotherapy. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/12/3270/F1.large.jpg.

TRAF2 regulates T cell immunity by maintaining a Tpl2-ERK survival signaling axis in effector and memory CD8 T cells.

Generation and maintenance of antigen-specific effector and memory T cells are central events in immune responses against infections. We show that TNF receptor-associated factor 2 (TRAF2) maintains a survival signaling axis in effector and memory CD8 T cells required for immune responses against infections. This signaling axis involves activation of Tpl2 and its downstream kinase ERK by NF-κB-inducing kinase (NIK) and degradation of the proapoptotic factor Bim. NIK mediates Tpl2 activation by stimulating the phosphorylation and degradation of the Tpl2 inhibitor p105. Interestingly, while NIK is required for Tpl2-ERK signaling under normal conditions, uncontrolled NIK activation due to loss of its negative regulator, TRAF2, causes constitutive degradation of p105 and Tpl2, leading to severe defects in ERK activation and effector/memory CD8 T cell survival. Thus, TRAF2 controls a previously unappreciated signaling axis mediating effector/memory CD8 T cell survival and protective immunity.

The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses.

Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor (TCR) signal and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased tumor-infiltrating CD4 and CD8 T cells. The Peli1-deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild-type T cells. Peli1 controls the activation of a metabolic kinase, mTORC1, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through regulation of the mTORC1-inhibitory proteins, TSC1 and TSC2. Peli1 mediates non-degradative ubiquitination of TSC1, thereby promoting TSC1-TSC2 dimerization and TSC2 stabilization. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1-mediated actions on T cell metabolism and antitumor immunity.

Intestinal Epithelial TBK1 Prevents Differentiation of T-helper 17 Cells and Tumorigenesis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) regulate intestinal immune cells, particularly development of T-helper 17 (Th17) cells. Deregulation of this process leads to intestinal inflammation and tumorigenesis, via unknown mechanisms. TANK-binding kinase 1 (TBK1) is expressed by IECs and cells in the innate immune system. We studied the functions of TBK1 in the intestinal immune response and tumorigenesis in mice. METHODS: We performed studies of wild-type mice, mice with conditional disruption of Tbk1 (Tbk1IEC-KO), Tbk1IEC-KO mice crossed with ApcMin/+ mice, and Mt-/- mice crossed with ApcMin/+ mice. Some mice were given intraperitoneal injections of a neutralizing antibody against interleukin 17 (IL17) or IL1ß. Intestine tissues were collected from mice and analyzed by histology, for numbers of adenomas and Th17 cells, and expression of inflammatory cytokines by real-time PCR. IECs were isolated from wild-type and Tbk1IEC-KO mice, stimulated with lipopolysaccharide, co-cultured for with bone marrow-derived macrophages, and analyzed by RNA sequencing and biochemical analyses. RESULTS: Compared to ApcMin/+Tbk1WT mice, ApcMin/+Tbk1IEC-KO mice had significant increases in number and size of intestinal polyps, and significantly more Th17 cells in lamina propria. Administration of an antibody against IL17 reduced the number of intestinal polyps in ApcMin/+Tbk1IEC-KO mice to that observed in ApcMin/+Tbk1WT mice. In culture, TBK1-deficient IECs promoted expression of IL1ß by macrophages, which induced differentiation of naïve CD4+ T cells into Th17 cells. RNA sequencing analysis revealed that the TBK1-deficient IECs had increased expression of metallothionein 1 (MT1), an immune regulator that promotes intestinal inflammation. Intestine tissues from ApcMin/+Mt-/- mice had significant fewer Th17 cells than ApcMin/+Mt+/+ mice, and a significantly lower number of polyps. Analyses of colorectal tumors in the Cancer Genome Atlas found colorectal tumors with high levels of MT1 and IL17 mRNAs to be associated with reduced survival times of patients. CONCLUSIONS: Expression of TBK1 by IECs suppresses expression of MT1 and prevents expression of IL1ß by macrophages and differentiation of Th17 cells, to prevent inflammation and tumorigenesis. Strategies to block this pathway might be developed for colorectal tumorigenesis.

Inhibition of TMPRSS2 by HAI-2 reduces prostate cancer cell invasion and metastasis.

TMPRSS2 is an important membrane-anchored serine protease involved in human prostate cancer progression and metastasis. A serine protease physiologically often comes together with a cognate inhibitor for execution of proteolytically biologic function; however, TMPRSS2's cognate inhibitor is still elusive. To identify the cognate inhibitor of TMPRSS2, in this study, we applied co-immunoprecipitation and LC/MS/MS analysis and isolated hepatocyte growth factor activator inhibitors (HAIs) to be potential inhibitor candidates for TMPRSS2. Moreover, the recombinant HAI-2 proteins exhibited a better inhibitory effect on TMPRSS2 proteolytic activity than HAI-1, and recombinant HAI-2 proteins had a high affinity to form a complex with TMPRSS2. The immunofluorescence images further showed that TMPRSS2 was co-localized to HAI-2. Both KD1 and KD2 domain of HAI-2 showed comparable inhibitory effects on TMPRSS2 proteolytic activity. In addition, HAI-2 overexpression could suppress the induction effect of TMPRSS2 on pro-HGF activation, extracellular matrix degradation and prostate cancer cell invasion. We further determined that the expression levels of TMPRSS2 were inversely correlated with HAI-2 levels during prostate cancer progression. In orthotopic xenograft animal model, TMPRSS2 overexpression promoted prostate cancer metastasis, and HAI-2 overexpression efficiently blocked TMPRSS2-induced metastasis. In summary, the results together indicate that HAI-2 can function as a cognate inhibitor for TMPRSS2 in human prostate cancer cells and may serve as a potential factor to suppress TMPRSS2-mediated malignancy.

Author Correction: The Kunitz Domain I of Hepatocyte Growth Factor Activator Inhibitor-2 Inhibits Matriptase Activity and Invasive Ability of Human Prostate Cancer Cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

TBKBP1 and TBK1 form a growth factor signalling axis mediating immunosuppression and tumourigenesis.

TANK-binding kinase 1 (TBK1) responds to microbial stimuli and mediates the induction of type I interferon (IFN). Here, we show that TBK1 is also a central mediator of growth factor signalling; this function of TBK1 relies on a specific adaptor-TBK-binding protein 1 (TBKBP1). TBKBP1 recruits TBK1 to protein kinase C-theta (PKCθ) through a scaffold protein, CARD10. This enables PKCθ to phosphorylate TBK1 at Ser 716, a crucial step for TBK1 activation by growth factors but not by innate immune stimuli. Although the TBK1-TBKBP1 signalling axis is not required for the induction of type I IFN, it mediates mTORC1 activation and oncogenesis. Conditional deletion of either TBK1 or TBKBP1 in lung epithelial cells inhibits tumourigenesis in a mouse model of lung cancer. In addition to promoting tumour growth, the TBK1-TBKBP1 axis facilitates tumour-mediated immunosuppression through a mechanism that involves induction of the checkpoint molecule PD-L1 and stimulation of glycolysis. These findings suggest a PKCθ-TBKBP1-TBK1 growth factor signalling axis that mediates both tumour growth and immunosuppression.

Activation of sphingosine kinase by lipopolysaccharide promotes prostate cancer cell invasion and metastasis via SphK1/S1PR4/matriptase.

Gram-negative bacteria have been found to be a major population in prostatitis and prostate cancer (PCa) tissues. Lipopolysaccharide (LPS), a major compound of Gram-negative bacteria, with stimulatory activities in some cancer types, but has not been fully studied in PCa. In this study, we examined the effect of LPS on the invasion of PCa cells. Interestingly, LPS can enhance the invasiveness of PCa, but had no significant effect on PCa cell viability. Using protease inhibitor screening and biochemical analyses, matriptase, a member of the membrane-anchored serine protease family, is found to play a key role in LPS-induced PCa cell invasion. Mechanistically, Toll-like receptor 4 (TLR4, LPS receptor)-sphingosine kinase 1 (SphK1) signaling underlies LPS-induced matriptase activation and PCa cell invasion. Specifically, LPS induced the S225 phosphorylation of SphK1 and the translocation of SphK1 to plasma membrane, leading to the production of sphingosine 1-phosphate (S1P), ERK1/2 and matriptase activation via S1P receptor 4 (S1PR4). This phenomenon is further validated using the patient-derived explant (PDE) model. Indeed, there is a significant correlation among the elevated SphK1 levels, the Gleason grades of PCa specimens, and the poor survival of PCa patients. Taken together, this study demonstrates a potential impact of LPS on PCa progression. Our results provide not only a new finding of the role of bacterial infection in PCa progression but also potential therapeutic target(s) associated with PCa metastasis.

HAI-2 as a novel inhibitor of plasmin represses lung cancer cell invasion and metastasis.

BACKGROUND: Dysregulation of pericellular proteolysis usually accounts for cancer cell invasion and metastasis. Isolation of a cell-surface protease system for lung cancer metastasis is an important issue for mechanistic studies and therapeutic target identification. METHODS: Immunohistochemistry of a tissue array (n = 64) and TCGA database (n = 255) were employed to assess the correlation between serine protease inhibitors (SPIs) and lung adenocarcinoma progression. The role of SPI in cell motility was examined using transwell assays. Pulldown and LC/MS/MS were performed to identify the SPI-modulated novel protease(s). A xenografted mouse model was harnessed to demonstrate the role of the SPI in lung cancer metastasis. RESULTS: Hepatocyte growth factor activator inhibitor-2 (HAI-2) was identified to be downregulated following lung cancer progression, which was related to poor survival and tumour invasion. We further isolated a serum-derived serine protease, plasmin, to be a novel target of HAI-2. Downregulation of HAI-2 promotes cell surface plasmin activity, EMT, and cell motility. HAI-2 can suppress plasmin-mediated activations of HGF and TGF-ß1, EMT and cell invasion. In addition, downregulated HAI-2 increased metastasis of lung adenocarcinoma via upregulating plasmin activity. CONCLUSION: HAI-2 functions as a novel inhibitor of plasmin to suppress lung cancer cell motility, EMT and metastasis.

The Kunitz Domain I of Hepatocyte Growth Factor Activator Inhibitor-2 Inhibits Matriptase Activity and Invasive Ability of Human Prostate Cancer Cells.

Dysregulation of pericellular proteolysis is often required for tumor invasion and cancer progression. It has been shown that down-regulation of hepatocyte growth factor activator inhibitor-2 (HAI-2) results in activation of matriptase (a membrane-anchored serine protease), human prostate cancer cell motility and tumor growth. In this study, we further characterized if HAI-2 was a cognate inhibitor for matriptase and identified which Kunitz domain of HAI-2 was required for inhibiting matriptase and human prostate cancer cell motility. Our results show that HAI-2 overexpression suppressed matriptase-induced prostate cancer cell motility. We demonstrate that HAI-2 interacts with matriptase on cell surface and inhibits matriptase proteolytic activity. Moreover, cellular HAI-2 harnesses its Kunitz domain 1 (KD1) to inhibit matriptase activation and prostate cancer cell motility although recombinant KD1 and KD2 of HAI-2 both show an inhibitory activity and interaction with matriptase protease domain. The results together indicate that HAI-2 is a cognate inhibitor of matriptase, and KD1 of HAI-2 plays a major role in the inhibition of cellular matritptase activation as well as human prostate cancer invasion.

Ketamine Increases Permeability and Alters Epithelial Phenotype of Renal Distal Tubular Cells via a GSK-3ß-Dependent Mechanism.

Ketamine, a dissociative anesthetic, is misused and abused worldwide as an illegal recreational drug. In addition to its neuropathic toxicity, ketamine abuse has numerous effects, including renal failure; however, the underlying mechanism is poorly understood. The process called epithelial phenotypic changes (EPCs) causes the loss of cell-cell adhesion and cell polarity in renal diseases, as well as the acquisition of migratory and invasive properties. Madin-Darby canine kidney cells, an in vitro cell model, were subjected to experimental manipulation to investigate whether ketamine could promote EPCs. Our data showed that ketamine dramatically decreased transepithelial electrical resistance and increased paracellular permeability and junction disruption, which were coupled to decreased levels of apical junctional proteins (ZO-1, occludin, and E-cadherin). Consistent with the downregulation of epithelial markers, the mesenchymal markers N-cadherin, fibronectin, and vimentin were markedly upregulated following ketamine stimulation. Of the E-cadherin repressor complexes tested, the mRNA levels of Snail, Slug, Twist, and ZEB1 were elevated. Moreover, ketamine significantly enhanced migration and invasion. Ketamine-mediated changes were at least partly caused by the inhibition of GSK-3ß activity through Ser-9 phosphorylation by the PI3K/Akt pathway. Inhibiting PI3K/Akt with LY294002 reactivated GSK-3ß and suppressed ketamine-enhanced permeability, EPCs, and motility. These findings were recapitulated by the inactivation of GSK-3ß using the inhibitor 3F8. Taken together, these results provide evidence that ketamine induces renal distal tubular EPCs through the downregulation of several junction proteins, the upregulation of mesenchymal markers, the activation of Akt, and the inactivation of GSK-3ß.