Dominant-negative P38 Research Articles

Role of PI3-Kinase in Angiotensin II-Induced Cardiac Hypertrophy: Class I Versus Class III.

Cardiac hypertrophy is an adaptive response to cardiac overload initially but turns into a decompensated condition chronically, leading to heart failure and sudden cardiac death. The molecular mechanisms involved in cardiac hypertrophy and the signaling pathways that contribute to the switch from compensation to decompensation are not fully clear. The aim of the current study was to examine the role of PI3-kinases Class I (PI3KC1) and Class III (PI3KC3) in angiotensin (Ang) II-induced cardiac hypertrophy. The results demonstrate that treatment of cardiomyocytes with Ang II caused dose-dependent increases in autophagy, with an increasing phase followed by a decreasing phase. Ang II-induced autophagic increases were potentiated by inhibition of PI3KC1 with LY294002, but were impaired by inhibition of PI3KC3 with 3-methyladenine (3-MA). In addition, blockade of PI3KC1 significantly attenuated Ang II-induced ROS production and cardiomyocyte hypertrophy. In contrast, blockade of PI3KC3 potentiated Ang II-induced ROS production and cardiac hypertrophy. Moreover, blockade of PI3KC1 by overexpression of dominant negative p85 subunit of PI3KC1 significantly attenuated Ang II-induced cardiac hypertrophy in normotensive rats. Taken together, these results demonstrate that both PI3KC1 and PI3KC3 are involved in Ang II-induced cardiac hypertrophy by different mechanisms. Activation of PI3KC1 impairs autophagy activity, leading to accumulation of mitochondrial ROS, and, hence, cardiac hypertrophy. In contrast, activation of PI3KC3 improves autophagy activity, thereby reducing mitochondrial ROS and leads to a protective effect on Ang II-induced cardiac hypertrophy.

Bone secreted factors induce cellular quiescence in prostate cancer cells

Disseminated tumor cells (DTCs) undergo a dormant state in the distant metastatic site(s) before becoming overt metastatic diseases. In prostate cancer (PCa), bone metastasis can occur years after prostatectomy, suggesting that bone may provide dormancy-inducing factors. To search for these factors, we prepared conditioned media (CM) from calvariae. Using live-cell imaging, we found that Calvarial-CM treatment increased cellular quiescence in C4-2B4 PCa cells. Mass spectrometry analysis of Calvarial-CM identified 132 secreted factors. Western blot and ELISA analyses confirmed the presence of several factors, including DKK3, BMP1, neogenin and vasorin in the Calvarial-CM. qRT-PCR analysis of total calvariae versus isolated osteoblasts showed that DKK3, BMP1, vasorin and neogenin are mainly expressed by osteoblasts, while MIA, LECT1, NGAL and PEDF are expressed by other calvarial cells. Recombinant human DKK3, BMP1, vasorin, neogenin, MIA and NGAL treatment increased cellular quiescence in both C4-2b and C4-2B4 PCa cells. Mechanistically, DKK3, vasorin and neogenin, but not BMP1, increased dormancy through activating the p38MAPK signaling pathway. Consistently, DKK3, vasorin and neogenin failed to induce dormancy in cells expressing dominant-negative p38αMAPK while BMP1 remained active, suggesting that BMP1 uses an alternative dormancy signaling pathway. Thus, bone secretes multiple dormancy-inducing factors that employ distinct signaling pathways to induce DTC dormancy in bone.

Oleate inhibits hepatic autophagy through p38 mitogen-activated protein kinase (MAPK)

Hepatic autophagy plays an important role in lipid metabolism, especially in nonalcoholic fatty liver disease. The relationship between Oleate acid and autophagy is not yet clear. In this work, using mouse epithelial cell hepa1c1c7, we investigated the role of Oleate acid on autophagy and explored its potential mechanisms. The exposure of hepatic cells to Oleate acid resulted in a significant reduction of LC3 accumulation together with enhancement of p62 protein expression and the mRNA levels of ATG7 and BECN1 were reduced as well. Mechanistically, the inhibitory effects of Oleate acid on rapamycin-induced autophagy were completely blocked by treatment with dominant negative p38α and p38 inhibitor SB203580. Furthermore, ATF-2, downstream of p38, was activated by Oleate treatment. Oleate treatment also inhibited the ULK1 promoter and decreased the ULK1 mRNA level. Our data therefore suggest that Oleate activated the ATF-2 via p38 kinase which inhibited the ULK1 via binding to ULK1 promoter, and eventually the rapamycin-induced autophagy was suppressed.

Tumor p38MAPK signaling enhances breast carcinoma vascularization and growth by promoting expression and deposition of pro-tumorigenic factors.

The breast carcinoma microenvironment strikingly influences cancer progression and response to therapy. Various cell types in the carcinoma microenvironment show significant activity of p38 mitogen-activated protein kinase (MAPK), although the role of p38MAPK in breast cancer progression is still poorly understood. The present study examined the contribution of tumor p38MAPK to breast carcinoma microenvironment and metastatic capacity. Inactivation of p38MAPK signaling in metastatic breast carcinoma cells was achieved by forced expression of the kinase-inactive mutant of p38/MAPK14 (a dominant-negative p38, dn-p38). Disruption of tumor p38MAPK signaling reduced growth and metastases of breast carcinoma xenografts. Importantly, dn-p38 markedly decreased tumor blood-vessel density and lumen sizes. Mechanistic studies revealed that p38 controls expression of pro-angiogenic extracellular factors such as matrix protein Fibronectin and cytokines VEGFA, IL8, and HBEGF. Tumor-associated fibroblasts enhanced tumor growth and vasculature as well as increased expression of the pro-angiogenic factors. These effects were blunted by dn-p38. Metadata analysis showed elevated expression of p38 target genes in breast cancers and this was an unfavorable marker of disease recurrence and poor-outcome. Thus, our study demonstrates that tumor p38MAPK signaling promotes breast carcinoma growth, invasive and metastatic capacities. Importantly, p38 enhances carcinoma vascularization by facilitating expression and deposition of pro-angiogenic factors. These results argue that p38MAPK is a valuable target for anticancer therapy affecting tumor vasculature. Anti-p38 drugs may provide new therapeutic strategies against breast cancer, including metastatic disease.

Activation of PI3K in response to high glucose leads to regulation of SOCS-3 and STAT1/3 signals and induction of glomerular mesangial extracellular matrix formation.

Excessive deposition of extracellular matrix (ECM) in the glomerulus contributed by mesangial cells is the hallmark of diabetic nephropathy, eventually leading to glomerulosclerosis. In this study, we examined the regulatory signals involved in the high glucose (HG)-induced overproduction of ECM in rat mesangial cells (RMCs). We disclosed excessive fibronectin and collagen IV production, tyrosine phosphorylation of signal transducer and activator of transcription 1 and 3 (STAT1/3), and up-regulation of suppressor of cytokine signaling-3 (SOCS-3) expression in HG-treated RMCs. STAT1/STAT3 binding element was essential for SOCS-3 promoter activity stimulated by HG. HG was capable of promoting the specific DNA binding activities to an oligonucleotide probe containing the SOCS-3 sequence. The selective phosphoinositide 3-kinase (PI3K) inhibitor LY294002 and dominant negative p85 vector (DNΔp85) transfection effectively abolished these HG-induced responses. Moreover, HG markedly increased the cyclin kinase inhibitor p27Kip1 protein expression, which could be inhibited by LY294002 or transfection of DNΔp85. Taken together, these results suggest that HG-induced SOCS-3 upregulation depends upon the presence of STAT-binding element in the SOCS-3 promoter, which is specifically activated by STAT1/3. The PI3K/STAT1/3 signaling pathway mediated HG-triggered ECM accumulation and SOCS-3 upregulation in RMCs.

Inflammatory cytokines induce caveolin-1/β-catenin signalling in rat nucleus pulposus cell apoptosis through the p38 MAPK pathway.

Apoptosis of nucleus pulposus (NP) cells is a major cause of intervertebral disc degeneration. To elucidate relationships between caveolin-1 and cytokine-induced apoptosis, we investigated the role of caveolin-1 in cytokine-induced apoptosis in rat NP cells and the related signalling pathway. Rat NP cells were treated with interleukin (IL)-1β or tumour necrosis factor alpha (TNF-α), and knockdown of caveolin-1 and β-catenin was achieved using specific siRNAs. Then, apoptotic level of rat NP cells and expression and activation of caveolin-1/β-catenin signalling were assessed by flow cytometric analysis, qRT-PCR, western blotting and luciferase assays. The relationship between the mitogen-activated protein kinase (MAPK) pathway and caveolin-1 promoter activity was also determined by luciferase assays. IL-1β and TNF-α induced apoptosis, upregulated caveolin-1 expression and activated Wnt/β-catenin signalling in rat NP cells, while the induction effect of cytokines was reversed by caveolin-1 siRNA and β-catenin siRNA. Promotion of rat NP cell apoptosis and nuclear translocation of β-catenin induced by caveolin-1 overexpression were abolished by β-catenin siRNA. Furthermore, pretreatment with a p38 MAPK inhibitor or dominant negative-p38, blocked cytokine-dependent induction of caveolin-1/β-catenin expression and activity. The results revealed the role of p38/caveolin-1/β-catenin in inflammatory cytokine-induced apoptosis in rat NP cells. Thus, controlling p38/caveolin-1/β-catenin activity seemed to regulate IL-1β- and TNF-α-induced apoptosis in the NP during intervertebral disc degeneration.

P38 MAPK-induced MDM2 degradation confers paclitaxel resistance through p53-mediated regulation of EGFR in human lung cancer cells

Paclitaxel (PTX) is a chemotherapeutic agent that is used to treat a variety of cancers, including non-small cell lung cancer (NSCLC). However, the emergence of drug resistance limits the utility of PTX. This study determined the signaling pathway that contributes to PTX resistance. We first established PTX resistant cell lines (H460/R and 226B/R) using a dose-escalating maintenance of PTX. We found that p38 MAPK and epidermal growth factor receptor (EGFR) were constitutively activated in these cell lines. The inhibition of p38 MAPK activity by SB203580 treatment or the transfection of dominant-negative p38 MAPK sensitized both cell lines to PTX treatment. Erlotinib, an EGFR inhibitor, also increased PTX-induced apoptosis in PTX resistant cells, which suggests a role for p38 MAPK and EGFR in the development of PTX resistance. We demonstrated that p38 MAPK enhanced EGFR expression via the induction of the rapid degradation of mouse double-minute 2 homolog (MDM2) and the consequent stabilization of p53, a transcription factor of EGFR. These results suggest for the first time that the p38 MAPK/p53/EGFR axis is crucial for the facilitation of PTX resistance in NSCLCs. We also propose a mechanism for the role of the tumor-suppressor p53 in drug resistance. These results provide a foundation for the future development of potential therapeutic strategies to regulate the p38 MAPK/p53/EGFR pathway for the treatment of lung cancer patients with PTX resistance.

G Protein-coupled Receptor 40 (GPR40) and Peroxisome Proliferator-activated Receptor γ (PPARγ): AN INTEGRATED TWO-RECEPTOR SIGNALING PATHWAY

Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been widely used to treat type 2 diabetes mellitus. However, knowledge of PPARγ signaling remains incomplete. In addition to PPARγ, these drugs also activate G protein-coupled receptor 40 (GPR40), a Gαq-coupled free fatty acid receptor linked to MAPK networks and glucose homeostasis. Notably, p38 MAPK activation has been implicated in PPARγ signaling. Here, rosiglitazone (RGZ) activation of GPR40 and p38 MAPK was found to boost PPARγ-induced gene transcription in human endothelium. Inhibition or knockdown of p38 MAPK or expression of a dominant negative (DN) p38 MAPK mutant blunted RGZ-induced PPARγ DNA binding and reporter activity in EA.hy926 human endothelial cells. GPR40 inhibition or knockdown, or expression of a DN-Gαq mutant likewise blocked activation of both p38 MAPK and PPARγ reporters. Importantly, RGZ induction of PPARγ target genes in primary human pulmonary artery endothelial cells (PAECs) was suppressed by knockdown of either p38 MAPK or GPR40. GPR40/PPARγ signal transduction was dependent on p38 MAPK activation and induction of PPARγ co-activator-1 (PGC1α). Silencing of p38 MAPK or GPR40 abolished the ability of RGZ to induce phosphorylation and expression of PGC1α in PAECs. Knockdown of PGC1α, its essential activator SIRT1, or its binding partner/co-activator EP300 inhibited RGZ induction of PPARγ-regulated genes in PAECs. RGZ/GPR40/p38 MAPK signaling also led to EP300 phosphorylation, an event that enhances PPARγ target gene transcription. Thus, GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development.

Mitogen-activated protein kinase p38 induces HDAC4 degradation in hypertrophic chondrocytes

Histone deacetylase 4 (HDAC4) is a critical negative regulator for chondrocyte hypertrophy by binding to and inhibiting Runx2, a critical transcription factor for chondrocyte hypertrophy. It is unclear how HDAC4 expression and stability are regulated during growth plate development. We report here that inhibition of mitogen-activated protein kinase (MAPK) p38 by dominant negative p38 or p38 inhibitor prevents HDAC4 degradation. Mutation of a potential caspase-2 and 3 cleavage site Asp289 stabilizes HDAC4 in chondrocytes. In contrast, constitutively active MAPK kinase 6 (constitutive activator of p38) transgenic mice exhibit decreased HDAC4 content in vivo. We also observed that p38 stimulates caspase-3 activity in chondrocytes. Inhibition of p38 or caspases reduced HDAC4 degradation. HDAC4 inhibited Runx2 promoter activity in a dose-dependent manner and caspase inhibitors further enhanced this inhibition by preventing HDAC4 degradation. Overall, these results demonstrate that p38 promotes HDAC4 degradation by increasing caspase-mediated cleavage, which releases Runx2 from a repressive influence of HDAC4 and promotes the chondrocyte hypertrophy and bone formation.

Piperine inhibits IL-1β-induced IL-6 expression by suppressing p38 MAPK and STAT3 activation in gastric cancer cells.

Piperine, a kind of natural alkaloid found in peppers, has been reported to exhibit anti-oxidative and anti-tumor activities, both in vitro and in vivo. Interleukin-6 (IL-6) is an important cytokine that activates the signal transduction, promotes tumor cell metastasis, and induces malignancy, including in gastric cancer. However, the effects of piperine on IL-6 expression in gastric cancer cells have not yet been well defined. In this study, we investigated the effects of piperine on the IL-6 expression, and examined the underlying signaling pathways via RT-PCR, promoter studies and Western blotting in human gastric cancer TMK-1 cells. Our results showed that piperine inhibited interleukin-1β (IL-1β)-induced IL-6 expression in a dose-dependent manner. In addition, piperine also inhibited IL-6 promoter activity. Experiments with mitogen-activated protein kinase (MAPK) inhibitors and dominant negative mutant p38 MAPK indicated that p38 MAPK was essential for IL-6 expression in the TMK-1 cells. Additionally, signal transducer and activator of transcription 3 (STAT3) was also involved in the IL-1β-induced IL-6 expression in gastric cancer cells. Piperine inhibited IL-1β-induced p38 MAPK and STAT3 activation and, in turn, blocked the IL-1β-induced IL-6 expression. Furthermore, gastric cancer cells pretreated with IL-1β showed markedly enhanced invasiveness, which was partially abrogated by treatment with IL-6 siRNA, piperine, and inhibitors of p38 MAPK and STAT3. These results suggest that piperine may exert at least part of its anti-cancer effect by controlling IL-6 expression through the suppression of p38 MAPK and STAT3.

P38δ Regulates p53 to Control p21Cip1 Expression in Human Epidermal Keratinocytes

PKCδ suppresses keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). However, the signaling mechanism that mediates this regulation is not well understood. Our present studies suggest that PKCδ activates p38δ leading to increased p21(Cip1) promoter activity and p21(Cip1) mRNA/protein expression. We further show that exogenously expressed p38δ increases p21(Cip1) mRNA and protein and that p38δ knockdown or expression of dominant-negative p38 attenuates this increase. Moreover, p53 is an intermediary in this regulation, as p38δ expression increases p53 mRNA, protein, and promoter activity, and p53 knockdown attenuates the activation. We demonstrate a direct interaction of p38δ with PKCδ and MEK3 and show that exogenous agents that suppress keratinocyte proliferation activate this pathway. We confirm the importance of this regulation using a stratified epidermal equivalent model, which mimics in vivo-like keratinocyte differentiation. In this model, PKCδ or p38δ knockdown results in reduced p53 and p21(Cip1) levels and enhanced cell proliferation. We propose that PKCδ activates a MEKK1/MEK3/p38δ MAPK cascade to increase p53 levels and p53 drives p21(Cip1) gene expression.

Piperlongumine induces autophagy by targeting p38 signaling

Piperlongumine (PL), a natural product isolated from the plant species Piper longum L., can selectively induce apoptotic cell death in cancer cells by targeting the stress response to reactive oxygen species (ROS). Here we show that PL induces cell death in the presence of benzyloxycarbonylvalyl-alanyl-aspartic acid (O-methyl)-fluoro-methylketone (zVAD-fmk), a pan-apoptotic inhibitor, and in the presence of necrostatin-1, a necrotic inhibitor. Instead PL-induced cell death can be suppressed by 3-methyladenine, an autophagy inhibitor, and substantially attenuated in cells lacking the autophagy-related 5 (Atg5) gene. We further show that PL enhances autophagy activity without blocking autophagy flux. Application of N-acetyl-cysteine, an antioxidant, markedly reduces PL-induced autophagy and cell death, suggesting an essential role for intracellular ROS in PL-induced autophagy. Furthermore, PL stimulates the activation of p38 protein kinase through ROS-induced stress response and p38 signaling is necessary for the action of PL as SB203580, a p38 inhibitor, or dominant-negative p38 can effectively reduce PL-mediated autophagy. Thus, we have characterized a new mechanism for PL-induced cell death through the ROS-p38 pathway. Our findings support the therapeutic potential of PL by triggering autophagic cell death.

Curcumin inhibits oxLDL-induced CD36 expression and foam cell formation through the inhibition of p38 MAPK phosphorylation

The uptake of oxidized low density lipoprotein (oxLDL) via scavenger receptors transforms macrophages into foam cells, which are a hallmark of atherosclerosis. OxLDL markedly increases the expression of the CD36 scavenger receptor. Here, we investigated whether curcumin modulate CD36 expression in oxLDL-treated RAW 264.7 murine macrophages. Our results showed that curcumin dramatically inhibits CD36 expression and foam cell formation. Furthermore, oxLDL-induced expression and activity of peroxisome proliferator-activated receptor-gamma (PPAR-γ), which is involved in CD36 expression, is also blocked in curcumin-treated cells. OxLDL activates the mitogen-activated protein kinase (MAPK) signaling transduction pathway, and p38 MAPK is associated with oxLDL-induced CD36 and PPAR-γ expression. Overexpression of dominant negative p38 MAPK blocks oxLDL-induced CD36 and PPAR-γ expression. Furthermore, curcumin markedly inhibits p38 MAPK phosphorylation. Taken together, our results suggest that curcumin modulates oxLDL-induced CD36 expression and foam cell formation via the inhibition of p38 MAPK phosphorylation in RAW 264.7 murine macrophages.

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells.

Ubiquitin C-terminal hydrolase-L5 is required for high glucose-induced transforming growth factor-β receptor I expression and hypertrophy in mesangial cells

Transforming growth factor-β (TGF-β) is pivotal in the pathogenesis of diabetic nephropathy. Type 1 TGF-β receptor (TGF-βR1) is degraded by Smad7-dependent ubiquitination–proteasomal pathway, which is deubiquitinated by ubiquitin C-terminal hydrolase-L5 (UCHL5). Therefore, we studied the role of UCHL5 in high glucose (27.8mM)-induced TGF-βR1 protein expression in mouse mesangial (MES13) cells. UCHL5 short hairpin RNA (shRNA) was used to knock down UCHL5 while LY294002 and the dominant-negative p85 were used to inhibit phosphatidylinositol-3-kinase (PI3K). We found that high glucose increased phospho-Akt, TGF-βR1 mRNA and protein expression. High glucose also increased UCHL5 protein expression, which was attenuated by LY294002, the dominant-negative p85 and the dominant-negative CREB. High glucose-induced TGF-βR1 protein expression and TGF-βR1 protein deubiquitination were attenuated by UCHL5 shRNA. Additionally, high glucose-induced p21WAF1, fibronectin protein expression and cell hypertrophy were attenuated by UCHL5 shRNA. However, high glucose-induced TGF-βR1 mRNA, p27kip1 protein expression and growth inhibition were not affected by UCHL5 shRNA. Finally, glomerular UCHL5 and TGF-βR1 protein expression were increased in streptozotocin-diabetic rats at 8weeks. We conclude that PI3K-dependent UCHL5 is required for high glucose-induced TGF-βR1 protein expression in mesangial cells. UCHL5 is also required for high glucose-induced TGF-βR1 protein deubiquitination, p21WAF1 and fibronectin protein expression and cell hypertrophy.

Control of late apoptotic events by the p38 stress kinase in l‐glutamine‐deprived mouse hybridoma cells

L-Glutamine (Gln) starvation rapidly triggers apoptosis in Sp2/0-Ag14 (Sp2/0) murine hybridoma cells. Here, we report on the role played by the stress-activated kinase p38 mitogen-activated protein kinase (MAPK) in this process. p38 activation was detected 2 h after Gln withdrawal and, although treatment with the p38 inhibitor SB203580 did not prevent caspase activation in Gln-starved cells, it reduced the occurrence of both nuclear condensation/fragmentation and apoptotic body formation. Similarly, transfection of Sp2/0 cells with a dominant negative p38 MAPK reduced the incidence of nuclear pyknosis and apoptotic body formation following 2 h of Gln starvation. Gln withdrawal-induced apoptosis was blocked by the overexpression of the anti-apoptotic protein Bcl-xL or by the caspase inhibitor Z-VAD-fmk. Interestingly, Bcl-xL expression inhibited p38 activation, but Z-VAD-fmk treatment did not, indicating that activation of this MAPK occurs downstream of mitochondrial dysfunction and is independent of caspases. Moreover, the anti-oxidant N-acetyl-l-cysteine prevented p38 phosphorylation, showing that p38 activation is triggered by an oxidative stress. Altogether, our findings indicate that p38 MAPK does not contribute to the induction of apoptosis in Gln-starved Sp2/0 cells. Rather, Gln withdrawal leads to mitochondrial dysfunction, causing an oxidative stress and p38 activation, the latter contributing to the formation of late morphological features of apoptotic Sp2/0 cells.

Stress-mediated p38 activation promotes somatic cell reprogramming

Environmental stress-mediated adaptation plays essential roles in the evolution of life. Cellular adaptation mechanisms usually involve the regulation of chromatin structure, transcription, mRNA stability and translation, which eventually lead to efficient changes in gene expression. Global epigenetic change is also involved in the reprogramming of somatic cells into induced pluripotent stem (iPS) cells by defined factors. Here we report that environmental stress such as hyperosmosis not only facilitates four factor-mediated reprogramming, but also enhances two or one factor-induced iPS cell generation. Hyperosmosis-induced p38 activation plays a critical role in this process. Constitutive active p38 mimics the positive effect of hyperosmosis, while dominant negative p38 and p38 inhibitor block the effect of hyperosmosis. Further study indicates stress-mediated p38 activation may promote reprogramming by reducing the global DNA methylation level and enhancing the expression of pluripotency genes. Our results demonstrate how simple environmental stress like hyperosmosis helps to alter the fate of cells via intracellular signaling and epigenetic modulation.

Interleukin-28A triggers wound healing migration of bladder cancer cells via NF-κB-mediated MMP-9 expression inducing the MAPK pathway

Interleukin (IL)-28A, also called IFN-λ2, is a member of the classIIcytokine family and has demonstrated anti-proliferative and anti-viral signals. The present study demonstrated migration inducement of IL-28A-treated bladder cancer cells — a novel function. RNA microarray analysis showed an enhanced expression of IL-28A and its receptor IL-28AR1 in muscle invasive urothelial carcinoma in a human bladder. Strong expression of IL-28A and IL-28AR1 was detected in bladder cancer tissues and cell lines (5637, T-24, and HT1376 cells), as determined by real-time PCR and immunoblot analysis. IL-28A treatment induced migration of bladder cancer cells, independent of the cell growth. IL-28AR1-specific small interfering RNA (si-IL-28AR1) inhibited the induction of migration in IL-28A-treated cells. IL-28A treatment stimulated the expression of matrix metalloproteinases-9 (MMP-9) via activation of transcription factor NF-κB. Gene knockdown for MMP-9 and the p65 subunit of NF-κB, using siRNA transfection, suppressed wound healing migration in IL-28A-treated bladder cancer cells. Also, treatment with IL-28A induced activation of mitogen-activated protein kinase (MAPK) in bladder cancer cells. MAPK function blockage by a MAPK-specific inhibitor showed that MAPK phosphorylation is required for IL-28A-induced MMP-9 expression through activation of NF-κB. The transient transfection of bladder cancer cells with ERK1/2 knock down (si-ERK1/2) and dominant negative p38 (DNp38) suppressed IL-28A-induced migration. IL-28A-induced induction of MMP-9 expression, MAPK activation, and DNA binding activity of NF-κB was abolished in the presence of IL-28A neutralizing antibody or by transfection of si-IL-28AR1. These results show that IL-28A/IL-28AR1 dyad-induced wound healing migration requires NF-κB-mediated MMP-9 expression by MAPK activation.

Regulation of phosphatase and tensin homolog on chromosome 10 in response to hypoxia

Phosphatase and tensin homolog on chromosome 10 (PTEN) is downregulated during hypertrophic and cancerous cell growth, leading to activation of the prosurvival Akt pathway. However, PTEN regulation in cardiac myocytes upon exposure to hypoxia remains unclear. We explored the role of PTEN in response to hypoxia/ischemia in the myocardium. We validated that PTEN is a transcriptional target of activating transcription factor 2 (ATF-2) and is positively regulated via a p38/ATF-2 signaling pathway. Accordingly, hypoxia-induced upregulation of phosphorylation of ATF-2 and PTEN were reversed by a dominant negative mutant p38. Inhibition of PTEN in cardiomyocytes attenuated hypoxia-induced cell death and apoptosis. Cardiac-specific knockout of PTEN resulted in increased phosphorylation of Akt and forkhead box O 1 (forkhead transcription factors), limited infarct size in animals exposed to ischemia-reperfusion injury, and ameliorated deterioration of left ventricular function and remodeling following permanent coronary artery occlusion. In addition, the activation of Bim, FASL, and caspase was coupled with PTEN activation, all of which were attenuated by PTEN inhibition. In conclusion, cardiomyocyte-specific conditional PTEN deletion limited myocardial infarct size in an in vivo model of ischemia-reperfusion injury and attenuated adverse remodeling in a model of chronic permanent coronary artery ligation.

Insulin inhibits hepatocyte iNOS expression induced by cytokines by an Akt-dependent mechanism

Hepatocyte inducible nitric oxide synthese (iNOS) expression is a tightly controlled pathway that mediates hepatic inflammation and hepatocyte injury in a variety of disease states. We have shown that cyclic adenosine monophosphate (cAMP) regulates cytokine-induced hepatocyte iNOS expression through mechanisms that involve protein kinase B/Akt. We hypothesized that insulin, which activates Akt signaling in hepatocytes, as well as signaling through p38 and MAPK p42/p44, would regulate iNOS expression during inflammation. In primary rat hepatocytes, insulin inhibited cytokine-stimulated nitrite accumulation and iNOS expression in a dose-dependent manner. Inhibition of MAPK p42/p44 with PD98059 had no effect on iNOS activation, whereas SB203580 to block p38 reversed insulin's inhibitory effect. However, insulin did not increase p38 activation and inhibition of p38 signaling with a dominant negative p38 plasmid had no effect on cytokine- or insulin-mediated effects on iNOS. We found that SB203580 blocked insulin-induced Akt activation. Inhibition of Akt signaling with LY294002 or a dominant negative Akt plasmid increased cytokine-stimulated nitrite production and iNOS protein expression and blocked the inhibitory effects of insulin. NF-κB induces iNOS expression and can be regulated by Akt, but insulin had no effect on cytokine-mediated IκBα levels or NF-κB p65 translocation. Our data demonstrate that insulin inhibits cytokine-stimulated hepatocyte iNOS expression and does so through effects on Akt-mediated signaling.