Signaling via Class IA Phosphoinositide 3-Kinases (PI3K) in Human,Breast-Derived Cell Lines

We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K) in human breast-derived MCF10a (and iso-genetic derivatives) and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ) and p50–101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P₃) that can activate effectors, eg protein kinase B (PKB), and responses, eg migration. The PtdIns(3,4,5)P₃-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K), basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN^−/− cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN^−/− MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely modulating PI3K-activity-independent mechanisms. Finally, we demonstrate that there is not a universal mechanism that up-regulates p110β function in the absence of PTEN.     

Isoform-specific regulation of adipocyte differentiation by Akt/protein kinase Balpha

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway tightly regulates adipose cell differentiation. Here we show that loss of Akt1/PKBα in primary mouse embryo fibroblast (MEF) cells results in a defect of adipocyte differentiation. Adipocyte differentiation in vitro and ex vivo was restored in cells lacking both Akt1/PKBα and Akt2/PKBβ by ectopic expression of Akt1/PKBα but not Akt2/PKBβ. Akt1/PKBα was found to be the major regulator of phosphorylation and nuclear export of FoxO1, whose presence in the nucleus strongly attenuates adipocyte differentiation. Differentiation-induced cell division was significantly abrogated in Akt1/PKBα-deficient cells, but was restored after forced expression of Akt1/PKBα. Moreover, expression of p27^Kip1, an inhibitor of the cell cycle, was down regulated in an Akt1/PKBα-specific manner during adipocyte differentiation. Based on these data, we suggest that the Akt1/PKBα isoform plays a major role in adipocyte differentiation by regulating FoxO1 and p27^Kip1.     

Biochemical characterization and lysosomal localization of the mannose-6-phosphate protein p76 (hypothetical protein LOC196463)

Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110alpha isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signalling. These include a p110alpha-specific inhibitor (PIK-75), a p110alpha-selective inhibitor (PI-103), a p110beta-specific inhibitor (TGX-221) and a p110delta-specific inhibitor (IC87114). Although we find that p110alpha is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110beta or p110delta alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110alpha inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110alpha, p110beta or p110delta. These results provide evidence that p110beta and p110delta can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.     

IRS-4 mediates protein kinase B signaling during insulin stimulation without promoting antiapoptosis

Insulin receptor substrate (IRS) proteins are tyrosine phosphorylated and mediate multiple signals during activation of the receptors for insulin, insulin-like growth factor 1 (IGF-1), and various cytokines. In order to distinguish common and unique functions of IRS-1, IRS-2, and IRS-4, we expressed them individually in 32D myeloid progenitor cells containing the human insulin receptor (32D(IR)). Insulin promoted the association of Grb-2 with IRS-1 and IRS-4, whereas IRS-2 weakly bound Grb-2; consequently, IRS-1 and IRS-4 enhanced insulin-stimulated mitogen-activated protein kinase activity. During insulin stimulation, IRS-1 and IRS-2 strongly bound p85alpha/beta, which activated phosphatidylinositol (PI) 3-kinase, protein kinase B (PKB)/Akt, and p70(s6k), and promoted the phosphorylation of BAD. IRS-4 also promoted the activation of PKB/Akt and BAD phosphorylation during insulin stimulation; however, it weakly bound or activated p85-associated PI 3-kinase and failed to mediate the activation of p70(s6k). Insulin strongly inhibited apoptosis of interleukin-3 (IL-3)-deprived 32D(IR) cells expressing IRS-1 or IRS-2 but failed to inhibit apoptosis of cells expressing IRS-4. Consequently, 32D(IR) cells expressing IRS-4 proliferated slowly during insulin stimulation. Thus, the activation of PKB/Akt and BAD phosphorylation might not be sufficient to inhibit the apoptosis of IL-3-deprived 32D(IR) cells unless p85-associated PI 3-kinase or p70(s6k) are strongly activated.     

Treatment with IFN‐γ prevents insulin‐dependent PKB,p70S6k phosphorylation and protein synthesis in mouse C2C12 myogenic cells

The purpose of the present study was to examine the potential effect of IFN‐γ (interferon‐γ) on the cellular content and phosphorylation of PKB (protein kinase B), p70^S6k (p70 S6 kinase) and MAPK (mitogen‐activated protein kinase), and on the ability of insulin to stimulate the glucose uptake and protein synthesis in mouse C2C12 myotubes. Insulin (100 nmol/l) stimulated glucose uptake in C2C12 myotubes by 203.4%. Glucose uptake in cells differentiated in the presence of IFN‐γ (10 ng/ml) was increased by 165.8% and was not further significantly modified by the addition of insulin (183.4% of control value). Insulin increased the rate of protein synthesis by 198.8%. The basal rate of protein synthesis was not affected by IFN‐γ; however, this cytokine abolished the insulin effect. Cellular levels of PKB, p70^S6k, p42^MAPK and p44^MAPK were not modified by IFN‐γ. Insulin caused the phosphorylation of PKB and the activation of p70^S6k, but not p42^MAPK and p44^MAPK. In cells differentiated in the presence of IFN‐γ, the insulin‐mediated PKB phosphorylation was significantly diminished, whereas the phosphorylation of p70^S6k was completely prevented. Pretreatment of C2C12 myogenic cells with IFN‐γ led to the marked increase in p42^MAPK phosphorylation. Exposure of C2C12 myoblasts to IFN‐γ impaired MyoD and myogenin expression and decreased the fusion index on the fifth day of differentiation. In conclusion, (i) IFN‐γ present in the extracellular environment during C2C12 myoblast differentiation prevents the stimulatory action of insulin on protein synthesis; (ii) IFN‐γ‐induced insulin resistance of protein synthesis in myogenic cells can be associated with the decreased phosphorylation of PKB and p70^S6k, as well as with the augmented basal phosphorylation of p42^MAPK; (iii) this cytokine effect can be partly explained by alterations in the differentiation process.     

The Pleckstrin Homology and Phosphotyrosine Binding Domains of Insulin Receptor Substrate 1 Mediate Inhibition of Apoptosis by Insulin

Insulin and insulin-like growth factor 1 (IGF-1) evoke diverse biological effects through receptor-mediated tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. We investigated the elements of IRS-1 signaling that inhibit apoptosis of interleukin 3 (IL-3)-deprived 32D myeloid progenitor cells. 32D cells have few insulin receptors and no IRS proteins; therefore, insulin failed to inhibit apoptosis during IL-3 withdrawal. Insulin stimulated mitogen-activated protein kinase in 32D cells expressing insulin receptors (32D^IR) but failed to activate the phosphatidylinositol 3 (PI 3)-kinase cascade or to inhibit apoptosis. By contrast, insulin stimulated the PI 3-kinase cascade, inhibited apoptosis, and promoted replication of 32D^IR cells expressing IRS-1. As expected, insulin did not stimulate PI 3-kinase in 32D^IR cells, which expressed a truncated IRS-1 protein lacking the tail of tyrosine phosphorylation sites. However, this truncated IRS-1 protein, which retained the NH₂-terminal pleckstrin homology (PH) and phosphotyrosine binding (PTB) domains, mediated phosphorylation of PKB/akt, inhibition of apoptosis, and replication of 32D^IR cells during insulin stimulation. These results suggest that a phosphotyrosine-independent mechanism mediated by the PH and PTB domains promoted antiapoptotic and growth actions of insulin. Although PI 3-kinase was not activated, its phospholipid products were required, since LY294002 inhibited these responses. Without IRS-1, a chimeric insulin receptor containing a tail of tyrosine phosphorylation sites derived from IRS-1 activated the PI 3-kinase cascade but failed to inhibit apoptosis. Thus, phosphotyrosine-independent IRS-1-linked pathways may be critical for survival and growth of IL-3-deprived 32D cells during insulin stimulation.     

A Limited Role for PI(3,4,5)P3 Regulation in Controlling Skeletal Muscle Mass in Response to Resistance Exercise

Background

Since activation of the PI3K/(protein kinase B; PKB/akt) pathway has been shown to alter muscle mass and growth, the aim of this study was to determine whether resistance exercise increased insulin like growth factor (IGF) I/phosphoinositide 3-kinase (PI3K) signalling and whether altering PI(3,4,5)P₃ metabolism genetically would increase load induced muscle growth.

Methodology/Principal Findings

Acute and chronic resistance exercise in wild type and muscle specific PTEN knockout mice were used to address the role of PI(3,4,5)P₃ regulation in the development of skeletal muscle hypertrophy. Acute resistance exercise did not increase either IGF-1 receptor phosphorylation or IRS1/2 associated p85. Since insulin/IGF signalling to PI3K was unchanged, we next sought to determine whether inactivation of PTEN played a role in load-induced muscle growth. Muscle specific knockout of PTEN resulted in small but significant increases in heart (PTEN^+/+  = 5.00±0.02 mg/g, PTEN^−/−  = 5.50±0.09 mg/g), and TA (PTEN^+/+  = 1.74±0.04 mg/g, PTEN^−/−  = 1.89 ±0.03) muscle mass, while the GTN, SOL, EDL and PLN remain unchanged. Following ablation, hypertrophy of the PLN, SOL or EDL muscles was similar between PTEN^−/− and PTEN^+/+ animals. Even though there were some changes in overload-induced PKB and S6K1 phosphorylation, 1 hr following acute resistance exercise there was no difference in the phosphorylation state of S6K1 Thr389 between genotypes.

Conclusions/Significance

These data suggest that physiological loading does not lead to the enhanced activation of the PI3K/PKB/mTORC1 axis and that neither PI3K activation nor PTEN, and by extension PI(3,4,5)P₃ levels, play a significant role in adult skeletal muscle growth.     

Aggressive neuroblastomas have high p110alpha but low p110delta and p55alpha/p50alpha protein levels compared to low stage neuroblastomas

Background

The phosphoinositide 3-kinase (PI3K)/Akt pathway is involved in neuroblastoma development where Akt/PKB activation is associated with poor prognosis. PI3K activity subsequently activates Akt/PKB, and as mutations of PI3K are rare in neuroblastoma and high levels of PI3K subunit p110delta is associated with favorable disease with low p-Akt/PKB, the levels of other PI3K subunits could be important for Akt activation.

Methods

Protein levels of Type IA PI3K catalytic and regulatory subunits were investigated together with levels of phosphorylated Akt/PKB and the PI3K negative regulator PTEN in primary neuroblastoma tumors. Relation between clinical markers and protein levels were evaluated through t-tests.

Results

We found high levels of p-Akt/PKB correlating to aggressive disease and p-Akt/PKB (T308) showed inverse correlation to PTEN levels. The regulatory isomers p55alpha/p50alpha showed higher levels in favorable neuroblastoma as compared with aggressive neuroblastoma. The PI3K-subunit p110alpha was found mainly in advanced tumors while p110delta showed higher levels in favorable neuroblastoma.

Conclusions

Activation of the PI3K/Akt pathway is seen in neuroblastoma tumors, however the contribution of the different PI3K isoforms is unknown. Here we show that p110alpha is preferentially expressed in aggressive neuroblastomas, with high p-Akt/PKB and p110delta is mainly detected in favorable neuroblastomas, with low p-Akt/PKB. This is an important finding as PI3K-specific inhibitors are suggested for enrollment in treatment of neuroblastoma patients.

     

Phosphorylation of p27Kip1 regulates assembly and activation of cyclin D1-Cdk4

p27 mediates Cdk2 inhibition and is also found in cyclin D1-Cdk4 complexes. The present data support a role for p27 in the assembly of D-type cyclin-Cdk complexes and indicate that both cyclin D1-Cdk4-p27 assembly and kinase activation are regulated by p27 phosphorylation. Prior work showed that p27 can be phosphorylated by protein kinase B/Akt (PKB/Akt) at T157 and T198. Here we show that PKB activation and the appearance of p27pT157 and p27pT198 precede p27-cyclin D1-Cdk4 assembly in early G₁. PI3K/PKB inhibition rapidly reduced p27pT157 and p27pT198 and dissociated cellular p27-cyclin D1-Cdk4. Mutant p27 allele products lacking phosphorylation at T157 and T198 bound poorly to cellular cyclin D1 and Cdk4. Cellular p27pT157 and p27pT198 coprecipitated with Cdk4 but were not detected in Cdk2 complexes. The addition of p27 to recombinant cyclin D1 and Cdk4 led to cyclin D1-Cdk4-p27 complex formation in vitro. p27 phosphorylation by PKB increased p27-cyclin D1-Cdk4 assembly in vitro but yielded inactive Cdk4. In contrast, Src pretreatment of p27 did not affect p27-cyclin D1-Cdk4 complex formation. However, Src treatment led to tyrosine phosphorylation of p27 and catalytic activation of assembled cyclin D1-Cdk4-p27 complexes. Thus, while PKB-dependent p27 phosphorylation appears to increase cyclin D1-Cdk4-p27 assembly or stabilize these complexes in vitro, cyclin D1-Cdk4-p27 activation requires the tyrosine phosphorylation of p27. Constitutive activation of PKB and Abl or Src family kinases in cancers would drive p27 phosphorylation, increase cyclin D1-Cdk4 assembly and activation, and reduce the cyclin E-Cdk2 inhibitory function of p27. Combined therapy with both Src and PI3K/PKB inhibitors may reverse this process.     

AMPK-independent induction of autophagy by cytosolic Ca2+ increase

Autophagy is a eukaryotic lysosomal bulk degradation system initiated by cytosolic cargo sequestration in autophagosomes. The Ser/Thr kinase mTOR has been shown to constitute a central role in controlling the initiation of autophagy by integrating multiple nutrient-dependent signaling pathways that crucially involves the activity of PI3K class III to generate the phosphoinositide PI(3)P. Recent reports demonstrate that the increase in cytosolic Ca²⁺ can induce autophagy by inhibition of mTOR via the CaMKK-α/β-mediated activation of AMPK. Here we demonstrate that Ca²⁺ signaling can additionally induce autophagy independently of the Ca²⁺-mediated activation of AMPK. First, by LC3-II protein monitoring in the absence or presence of lysosomal inhibitors we confirm that the elevation of cytosolic Ca²⁺ induces autophagosome generation and does not merely block autophagosome degradation. Further, we demonstrate that Ca²⁺-chelation strongly inhibits autophagy in human, mouse and chicken cells. Strikingly, we found that the PI(3)P-binding protein WIPI-1 (Atg18) responds to the increase of cytosolic Ca²⁺ by localizing to autophagosomal membranes (WIPI-1 puncta) and that Ca²⁺-chelation inhibits WIPI-1 puncta formation, although PI(3)P-generation is not generally affected by these Ca²⁺ flux modifications. Importantly, using AMPK-α1^?/?α2^?/? MEFs we show that thapsigargin application triggers autophagy in the absence of AMPK and does not involve complete mTOR inhibition, as detected by p70S6K phosphorylation. In addition, STO-609-mediated CaMKK-α/β inhibition decreased the level of thapsigargin-induced autophagy only in AMPK-positive cells. We suggest that apart from reported AMPK-dependent regulation of autophagic degradation, an AMPK-independent pathway triggers Ca²⁺-mediated autophagy, involving the PI(3)P-effector protein WIPI-1 and LC3.     

A drug targeting only p110α can block phosphoinositide 3-kinase signalling and tumour growth in certain cell types

Genetic alterations in PI3K (phosphoinositide 3-kinase) signalling are common in cancer and include deletions in PTEN (phosphatase and tensin homologue deleted on chromosome 10), amplifications of PIK3CA and mutations in two distinct regions of the PIK3CA gene. This suggests drugs targeting PI3K, and p110α in particular, might be useful in treating cancers. Broad-spectrum inhibition of PI3K is effective in preventing growth factor signalling and tumour growth, but suitable inhibitors of p110α have not been available to study the effects of inhibiting this isoform alone. In the present study we characterize a novel small molecule, A66, showing the S-enantiomer to be a highly specific and selective p110α inhibitor. Using molecular modelling and biochemical studies, we explain the basis of this selectivity. Using a panel of isoform-selective inhibitors, we show that insulin signalling to Akt/PKB (protein kinase B) is attenuated by the additive effects of inhibiting p110α/p110β/p110δ in all cell lines tested. However, inhibition of p110α alone was sufficient to block insulin signalling to Akt/PKB in certain cell lines. The responsive cell lines all harboured H1047R mutations in PIK3CA and have high levels of p110α and class-Ia PI3K activity. This may explain the increased sensitivity of these cells to p110α inhibitors. We assessed the activation of Akt/PKB and tumour growth in xenograft models and found that tumours derived from two of the responsive cell lines were also responsive to A66 in vivo. These results show that inhibition of p110α alone has the potential to block growth factor signalling and reduce growth in a subset of tumours.     

Deletion of the FHL2 gene attenuates intima‐media thickening in a partially ligated carotid artery ligated mouse model

The four and a half LIM domain protein 2 (FHL2) is a member of the four and a half LIM domain (FHL) gene family, and it is associated with cholesterol‐enriched diet‐promoted atherosclerosis. However, the effect of FHL2 protein on vascular remodelling in response to hemodynamic alterations remains unclear. Here, we investigated the role of FHL2 in a model of restricted blood flow‐induced atherosclerosis. To promote neointimal hyperplasia in vivo, we subjected FHL2^+/+ and FHL2^?/? mice to partial ligation of the left carotid artery (LCA). The expression of p‐ERK and p‐AKT was decreased in FHL2^?/? mice. FHL2 bound to AKT regulated AKT phosphorylation and led to Rac1‐GTP inactivation. FHL2 silencing in human aortic smooth muscle cells down‐regulated the PDGF‐induced phosphorylation of ERK and AKT. Furthermore, FHL2 silencing reduced cytoskeleton conformational changes and caused cell cycle arrest. We concluded that FHL2 is essential for the regulation of arterial smooth muscle cell function. FHL2 modulates proliferation and migration via mitogen‐activated protein kinase (MAPK) and PI3K‐AKT signalling, leading to arterial wall thickening and thus neointimal hyperplasia.     

Effects of rapamycin on cell proliferation and phosphorylation of mTOR and p70 in HepG2 and HepG2 cells overexpressing constitutively active Akt/PKB

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that plays an important role in the regulation of cell proliferation and protein synthesis through the activation of its downstream target ribosomal p70 S6 kinase (p70^S6K). The levels of p-mTOR are regulated by the protein kinase B (Akt/PKB). Therefore, the effects of insulin and rapamycin (an inhibitor of mTOR) on the phosphorylation of mTOR (Ser 2448) and p70^S6K (Thr 389) as well as on cell proliferation in parental HepG2 cells and HepG2 cells overexpressing constitutively active Akt/PKB (HepG2-CA-Akt/PKB) were studied. Insulin increased the levels of phosphorylated mTOR and p70^S6K in both the cell lines. Rapamycin treatment partially decreased the phosphorylation of mTOR but completely abolished the phosphorylation of p70^S6K in the absence as well as presence of insulin in both cell lines. The effect of insulin and rapamycin on the cell proliferation in both cell lines was further studied. In the presence of serum, parental HepG2 cells and HepG2-CA-Akt/PKB showed an increase in cell proliferation until 120 and 168 h respectively. Rapamycin inhibited cell proliferation under all experimental conditions more evident under serum deprived conditions. Parental HepG2 cells showed decline in the cell proliferation after 48 h and the presence of insulin prolonged cell survival until 120 h and this effect were also inhibited by rapamycin under serum deprived conditions. On the contrary, HepG2-CA-Akt/PKB cells continued proliferation until 192 h. The effects of insulin on cell proliferation were more pronounced in parental HepG2 cells as compared to HepG2-CA-Akt/PKB cells. Long term effects of rapamcyin significantly decreased the levels of p-mTOR (Ser 2448) both in the presence and absence of insulin in these cells. A positive correlation between the levels of p-mTOR (Ser2448) and cell proliferation was observed (99% confidence interval, r² = 0.525, p < 0.0001). These results suggest that rapamycin causes a decline in the cell growth through the inhibition of mTOR.     

Mechanism of Activation of PKB/Akt by the Protein Phosphatase Inhibitor Calyculin A

The protein phosphatase inhibitor calyculin A activates PKB/Akt to ~50% of the activity induced by insulin-like growth factor 1 (IGF1) in HeLa cells promoting an evident increased phosphorylation of Ser473 despite the apparent lack of Thr308 phosphorylation of PKB. Nevertheless, calyculin A-induced activation of PKB seems to be dependent on basal levels of Thr308 phosphorylation, since a PDK1-dependent mechanism is required for calyculin A-dependent PKB activation by using embryonic stem cells derived from PDK1 wild-type and knockout mice. Data shown suggest that calyculin A-induced phosphorylation of Ser473 was largely blocked by LY294002 and SB-203580 inhibitors, indicating that both PI3-kinase/TORC2-dependent and SAPK2/p38-dependent protein kinases contributed to phosphorylation of Ser473 in calyculin A-treated cells. Additionally, our results suggest that calyculin A blocks the IGF1-dependent Thr308 phosphorylation and activation of PKB, likely due to an enhanced Ser612 phosphorylation of insulin receptor substrate 1 (IRS1), which can be inhibitory to its activation of PI3-kinase, a requirement for PDK1-induced Thr308 phosphorylation and IGF1-dependent activation of PKB. Our data suggest that PKB activity is most dependent on the level of Ser473 phosphorylation rather than Thr308, but basal levels of Thr308 phosphorylation are a requirement. Additionally, we suggest here that calyculin A regulates the IGF1-dependent PKB activation by controlling the PI3-kinase-associated IRS1 Ser/Thr phosphorylation levels.     

Divergent results induced by different types of septic shock in transglutaminase 2 knockout mice

Acute sepsis can be induced by cytokines such as TNF-α and biological products such as LPS. All of these agents cause systemic inflammation, which is characterized by hemodynamic shock and liver toxicity. However, the outcomes of different septic shock models were totally opposite in transglutaminase 2 knockout (TGase 2^?/?) mice. The aim of our study was to clarify the role of TGase 2 in liver injury. Therefore, we explored the role of TGase 2 in liver damage using two different stress models: LPS-induced endotoxic shock and TNF-α/actinomycin D (ActD)-induced sepsis. TNF-α-dependent septic shock resulted in increased liver damage in TGase 2^?/? mice compared with wild-type (WT) mice, and was accompanied by increased levels of caspase 3 and cathepsin D (CTSD) in the damaged liver. Conversely, LPS-induced septic shock resulted in ablation of inflammatory endotoxic shock in TGase 2^?/? mice and decreased liver injury. We found that TGase 2 protected liver tissue from TNF-α-dependent septic shock by reducing the expression of caspase 3 and CTSD. However, TGase 2 differently participated in increased the hemodynamic shock in LPS-induced septic shock through macrophage activation rather than protecting direct liver damage. Therefore, these findings demonstrate that septic shock caused by different agents may induce different results in TGase 2^?/? mice depending on the primary target organs affected.