Interleukin-26 Has Synergistic Catabolic Effects with Palmitate in Human Articular Chondrocytes via the TLR4-ERK1/2-c-Jun Signaling Pathway.

The inflammatory cytokine interleukin-26 (IL-26) is highly expressed in the serum and synovial fluid of patients with inflammatory arthritis. The effect of IL-26 on human articular chondrocytes (HACs) remains unclear. Obesity is associated with disability of patients with rheumatoid arthritis and disease activity in those with ankylosing spondylitis. The saturated free fatty acid palmitate with IL-1ß can synergistically induce catabolic effects in HACs. The aim of this study was to evaluate the effects of IL-26 and palmitate in HACs. In this study, palmitate markedly synergizes the IL-26-induced proinflammatory effects and matrix protease, including COX-2, IL-6, and MMP-1, in HACs via the toll-like receptor 4 (TLR4)-ERK1/2-c-Jun signal transduction pathway. The synergistic catabolic effects of palmitate and IL-26 were attenuated by inhibitors of TLR4 (TAK242), ERK1/2 (U0126), or c-Jun (SP600125) in HACs and cartilage matrix. In addition, metformin, a potential inhibitor of TLR4, also decreased expression of COX-2 and IL-6 induced by co-incubation with IL-26 and palmitate. IL-26 and palmitate synergistically induced expression of inflammatory and catabolic mediators, resulting in articular cartilage matrix breakdown. The present study also revealed a possible mechanism and therapeutic targets against articular cartilage degradation by increased saturated fatty acids in patients with inflammatory arthritis.

Calcineurin A gamma and NFATc3/SRPX2 axis contribute to human embryonic stem cell differentiation.

Our understanding of signaling pathways regulating the cell fate of human embryonic stem cells (hESCs) is limited. Calcineurin-NFAT signaling is associated with a wide range of biological processes and diseases. However, its role in controlling hESC fate remains unclear. Here, we report that calcineurin A gamma and the NFATc3/SRPX2 axis control the expression of lineage and epithelial-mesenchymal transition (EMT) markers in hESCs. Knockdown of PPP3CC, the gene encoding calcineurin A gamma, or NFATC3, downregulates certain markers both at the self-renewal state and during differentiation of hESCs. Furthermore, NFATc3 interacts with c-JUN and regulates the expression of SRPX2, the gene encoding a secreted glycoprotein known as a ligand of uPAR. We show that SRPX2 is a downstream target of NFATc3. Both SRPX2 and uPAR participate in controlling expression of lineage and EMT markers. Importantly, SRPX2 knockdown diminishes the upregulation of multiple lineage and EMT markers induced by co-overexpression of NFATc3 and c-JUN in hESCs. Together, this study uncovers a previously unknown role of calcineurin A gamma and the NFATc3/SRPX2 axis in modulating the fate determination of hESCs.

Coupling Computational and Intracellular Screening and Selection Toward Co-compatible cJun and cFos Antagonists.

Basic leucine-zipper (bZIP) proteins represent difficult, yet compelling, oncogenic targets since numerous cell-signaling cascades converge upon them, where they function to modulate the transcription of specific gene targets. bZIPs are widely recognized as important regulators of cellular processes that include cell proliferation, apoptosis, and differentiation. Once such validated transcriptional regulator, activator protein-1, is typically composed of heterodimers of Fos and Jun family members, with cFos-cJun being the best described. It has been shown to be key in the progression and development of a number of different diseases. As a proof-of-principle for our approach, we describe the first use of a novel combined in silico/in cellulo peptide-library screening platform that facilitates the derivation of a sequence that displays high selectivity for cJun relative to cFos, while also avoiding homodimerization. In particular, >60 million peptides were computationally screened and all potential on/off targets ranked according to predicted stability, leading to a reduced size library that was further refined by intracellular selection. The derived sequence is predicted to have limited cross-talk with a second previously derived peptide antagonist that is selective for cFos in the presence of cJun. The study provides new insight into the use of multistate screening with the ability to combine computational and intracellular approaches in evolving multiple cocompatible peptides that are capable of satisfying conflicting design requirements.

Activator protein-1 contributes to the NaCl-induced expression of VEGF and PlGF in RPE cells.

Purpose: Systemic hypertension is a risk factor of neovascular age-related macular degeneration; consumption of dietary salt resulting in extracellular hyperosmolarity is a main cause of hypertension. Extracellular hyperosmolarity was shown to induce expression of angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and placental growth factor (PlGF), in RPE cells. The aim of the present study was to determine whether the hyperosmotic expression of growth factor genes in RPE cells is mediated by activator protein-1 (AP-1), and whether c-Fos and c-Jun genes are regulated by extracellular osmolarity. Methods: Hyperosmotic media were made up with the addition of NaCl or sucrose. Gene expression was quantified with real-time reverse transcription (RT)-PCR, and protein secretion was investigated with enzyme-linked immunosorbent assay (ELISA). Nuclear factor of activated T cell 5 (NFAT5) was depleted with siRNA. DNA binding of AP-1 protein was evaluated with electrophoretic mobility shift assay (EMSA). Results: High NaCl and the addition of sucrose triggered expression of the c-Fos gene, but not of the c-Jun gene. High NaCl also increased the levels of c-Fos and phosphorylated c-Jun proteins and the level of DNA binding of AP-1. Hypoosmolarity decreased the expression of the c-Fos and c-Jun genes. NaCl-induced expression of the c-Fos gene was in part mediated by NFAT5. Autocrine/paracrine activation of fibroblast growth factor and adenosine A1 receptors is involved in mediating NaCl-induced expression of the c-Fos gene. Pharmacological inhibition of the AP-1 activity decreased the NaCl-induced expression of the HIF-1α, NFAT5, VEGF, PlGF, and TGF-ß2 genes, and prevented the NaCl-induced secretion of PlGF but not of VEGF. Conclusions: The data indicate that AP-1 is activated in RPE cells in response to extracellular hyperosmolarity and mediates in part via the NaCl-induced expression of VEGF and PlGF, and secretion of PlGF. It is suggested that high consumption of dietary salt may exacerbate the angiogenic response of RPE cells in part via activation of AP-1.

Together JUN and DDIT3 (CHOP) control retinal ganglion cell death after axonal injury.

BACKGROUND: Optic nerve injury is an important pathological component in neurodegenerative diseases such as traumatic optic neuropathies and glaucoma. The molecular signaling pathway(s) critical for retinal ganglion cell (RGC) death after axonal insult, however, is/are not fully defined. RGC death after axonal injury is known to occur by BAX-dependent apoptosis. Two transcription factors JUN (the canonical target of JNK) and DDIT3 (CHOP; a key mediator of the endoplasmic reticulum stress response) are known to be important apoptotic signaling molecules after axonal injury, including in RGCs. However, neither Jun nor Ddit3 deficiency provide complete protection to RGCs after injury. Since Jun and Ddit3 are important apoptotic signaling molecules, we sought to determine if their combined deficiency might provide additive protection to RGCs after axonal injury. METHODS: To determine if DDIT3 regulated the expression of JUN after an axonal insult, mice deficient for Ddit3 were examined after optic nerve crush (ONC). In order to critically test the importance of these genes in RGC death after axonal injury, RGC survival was assessed at multiple time-points after ONC (14, 35, 60, and 120 days after injury) in Jun, Ddit3, and combined Jun/Ddit3 deficient mice. Finally, to directly assess the role of JUN and DDIT3 in axonal degeneration, compound actions potentials were recorded from Jun, Ddit3, and Jun/Ddit3 deficient mice after ONC. RESULTS: Single and combined deficiency of Jun and Ddit3 did not appear to alter gross retinal morphology. Ddit3 deficiency did not alter expression of JUN after axonal injury. Deletion of both Jun and Ddit3 provided significantly greater long-term protection to RGCs as compared to Jun or Ddit3 deficiency alone. Finally, despite the profound protection to RGC somas provided by the deficiency of Jun plus Ddit3, their combined loss did not lessen axonal degeneration. CONCLUSIONS: These results suggest JUN and DDIT3 are independently regulated pro-death signaling molecules in RGCs and together account for the vast majority of apoptotic signaling in RGCs after axonal injury. Thus, JUN and DDIT3 may represent key molecular hubs that integrate upstream signaling events triggered by axonal injury with downstream transcriptional events that ultimately culminate in RGC apoptosis.

Transcriptomic analysis of the PI3K/Akt signaling pathway reveals the dual role of the c-Jun oncogene in cytotoxicity and the development of resistance in HL-60 leukemia cells in response to arsenic trioxide.

BACKGROUND: Arsenic trioxide (ATO) is a well-recognized antileukemic drug used for the treatment of newly diagnosed and relapsed acute promyelocytic leukemia (APL). A major drawback of therapy with ATO is the development of APL cell resistance, the mechanisms of which are still not clear. OBJECTIVES: The aim of this study was to investigate the role of the PI3K/Akt signaling pathway in ATOtreated human acute myeloid leukemia (HL-60) cells and in ATO-resistant clones. MATERIAL AND METHODS: The cytotoxicity of ATO was assessed using Trypan blue staining or a WST-1 reduction assay. The Akt phosphorylation level was measured by immunofluorescent staining and flow cytometry. Gene expression analysis was performed using real-time polymerase chain reaction (PCR). RESULTS: The clones derived by culturing for 8-12 weeks in the presence of 1.75, 2.5, and 5 µM ATO were characterized by high viability but a slower growth rate compared to the parental HL-60 cells. The flow cytometry analysis showed that in the parental cells the levels of p-Akt were undetectable or very low, and that ATO had no effect on the level of p-Akt in either the ATO-treated parental cells or the clones. The gene expression analysis revealed that some of the genes involved in the Akt pathway may play a key role in the induction of resistance to ATO, e.g., genes encoding cyclin D1 (CCND1), fork head box O1 (FOXO1), Jun oncogene (JUN), protein kinase C isoform B1 (PRKCB1), because their expression profiles were predominantly changed in the clones and/or the ATO-treated parental HL-60 cells. CONCLUSIONS: The overall results indicate that CCND1, FOXO1, and JUN may contribute to the induction of resistance to ATO, and that the C-Jun N-terminal kinase (JNK) signaling pathway may have greater significance than the phosphoinositide 3-kinase (PI3K)/Akt pathway in mediating the cytotoxic effects of ATO and the development of resistance to ATO in the HL-60 cell line.

Transcriptome of the inner circular smooth muscle of the developing mouse intestine: Evidence for regulation of visceral smooth muscle genes by the hedgehog target gene, cJun.

BACKGROUND: Digestion is facilitated by coordinated contractions of the intestinal muscularis externa, a bilayered smooth muscle structure that is composed of inner circular muscles (ICM) and outer longitudinal muscles (OLM). We performed transcriptome analysis of intestinal mesenchyme tissue at E14.5, when the ICM, but not the OLM, is present, to investigate the transcriptional program of the ICM. RESULTS: We identified 3967 genes enriched in E14.5 intestinal mesenchyme. The gene expression profiles were clustered and annotated to known muscle genes, identifying a muscle-enriched subcluster. Using publically available in situ data, 127 genes were verified as expressed in ICM. Examination of the promoter and regulatory regions for these co-expressed genes revealed enrichment for cJUN transcription factor binding sites, and cJUN protein was enriched in ICM. cJUN ChIP-seq, performed at E14.5, revealed that cJUN regulatory regions contain characteristics of muscle enhancers. Finally, we show that cJun is a target of Hedgehog (Hh), a signaling pathway known to be important in smooth muscle development, and identify a cJun genomic enhancer that is responsive to Hh. CONCLUSIONS: This work provides the first transcriptional catalog for the developing ICM and suggests that cJun regulates gene expression in the ICM downstream of Hh signaling. Developmental Dynamics 245:614-626, 2016. © 2016 Wiley Periodicals, Inc.

The intriguing role of fibroblasts and c-Jun in the chemopreventive and therapeutic effect of finasteride on xenograft models of prostate cancer.

In a large clinical trial, finasteride reduced the rate of low-grade prostate cancer (PCa) while increasing the incidence of high-grade cancer. Whether finasteride promotes the development of high-grade tumors remains controversial. We demonstrated the role of fibroblasts and c-Jun in chemopreventive and therapeutic effect of finasteride on xenograft models of PCa. LNCaP (PC3) cells or recombinants of cancer cells and fibroblasts were implanted in male athymic nude mice treated with finasteride. Tumor growth, cell proliferation, apoptosis, p-Akt, and p-ERK1/2 were evaluated. In LNCaP (PC3) mono-grafted models, finasteride did not change the tumor growth. In recombinant-grafted models, fibroblasts and c-Jun promoted tumor growth; finasteride induced proliferation of LNCaP cells and repressed PC3 cell apoptosis. When c-Jun was knocked out, fibroblasts and/or finasteride did not promote the tumor growth. Finasteride inhibited p-Akt and p-ERK1/2 in mono-culture cancer cells while stimulating the same signaling molecules in the presence of fibroblasts. Reduced p-Akt and p-ERK1/2 were noted in the presence of c-Jun-/- fibroblasts. Fibroblasts and c-Jun promote PCa growth; finasteride further stimulates tumor growth with promoted proliferation, repressed apoptosis, and up-regulated pro-proliferative molecular pathway in the presence of fibroblasts and c-Jun. Stromal-epithelial interactions play critical roles in finasteride's therapeutic effects on PCa. Our findings have preliminary implications in using finasteride as a chemopreventive or therapeutic agent for PCa patients.

The ATP-Binding Cassette Transporter-2 (ABCA2) Overexpression Modulates Sphingosine Levels and Transcription of the Amyloid Precursor Protein (APP) Gene.

The ATP-binding cassette transporter-2 (ABCA2) is a member of a family of multipass transmembrane proteins that use the energy of ATP hydrolysis to transport substrates across membrane bilayers. ABCA2 has also been genetically linked with Alzheimer's disease but the molecular mechanisms are unknown. In this report, we hypothesized that ABCA2 modulation of sphingolipid metabolism activates a signaling pathway that regulates amyloid precursor protein transcription. We found that ABCA2 overexpression in N2a cells was associated with increased mass of the sphingolipid sphingosine, derived from the catabolism of ceramide. ABCA2 overexpression increased in vitro alkaline and acid ceramidase activity. Sphingosine is a physiological inhibitor of protein kinase C (PKC) activity. Pharmacological inhibition of ceramidase activity or activation PKC activity with 12-myristate 13-acetate (PMA) or diacylglycerol (DAG) decreased endogenous APP mRNA levels in ABCA2 overexpressing cells. Treatment with PMA also decreased the expression of a transfected human APP promoter reporter construct, while treatment with a general PKC inhibitor, GF109203x, increased APP promoter activity. In N2a cells, chromatin immunoprecipitation experiments revealed that a repressive complex forms at the AP-1 site in the human APP promoter, consisting of c-jun, c-jun dimerization protein 2 (JDP2) and HDAC3 and this complex was reduced in ABCA2 overexpressing cells. Activation of the human APP promoter in A2 cells was directed by the upstream stimulatory factors USF-1 and USF-2 that bound to an E-box element in vivo. These findings indicate that ABCA2 overexpression modulates sphingosine levels and regulates transcription of the endogenous APP gene.

Sulforaphane inhibition of TPA-mediated PDCD4 downregulation contributes to suppression of c-Jun and induction of p21-dependent Nrf2 expression.

Programmed cell death 4 (PDCD4) is a bona fide tumor suppressor protein and plays a critical role in controlling the rate of protein synthesis. Here, we show that TPA selectively activated the S6K1 and ERK1/2 kinases, contributing to PDCD4 proteolysis and Pdcd4 mRNA degradation in HepG2 cells, respectively. In addition, we observed that sulforaphane suppression of TPA-induced S6K1 and ERK1/2 activation played a critical role in attenuating PDCD4 poly-ubiquitination and Pdcd4 mRNA downregulation. Moreover, we observed that silencing Pdcd4 led to not only an increased expression of c-Jun, but also a decreased expression of p21, the latter of which contributed to suppression of Keap1-dependent Nrf2 poly-ubiquitination. Finally, we demonstrate that the expression of PDCD4, p21 and Nrf2 is higher, but that of c-Jun is lower in normal human liver tissues, compared with hepatoma tissues. Collectively, our study illustrates that attenuating the rate of PDCD4 proteolysis and Pdcd4 mRNA degradation serves as a novel anti-inflammatory and cytoprotective mechanism of sulforaphane.

Roles of binding elements, FOXL2 domains, and interactions with cJUN and SMADs in regulation of FSHß.

We previously identified FOXL2 as a critical component in FSHß gene transcription. Here, we show that mice deficient in FOXL2 have lower levels of gonadotropin gene expression and fewer LH- and FSH-containing cells, but the same level of other pituitary hormones compared to wild-type littermates, highlighting a role of FOXL2 in the pituitary gonadotrope. Further, we investigate the function of FOXL2 in the gonadotrope cell and determine which domains of the FOXL2 protein are necessary for induction of FSHß transcription. There is a stronger induction of FSHß reporter transcription by truncated FOXL2 proteins, but no induction with the mutant lacking the forkhead domain. Specifically, FOXL2 plays a role in activin induction of FSHß, functioning in concert with activin-induced SMAD proteins. Activin acts through multiple promoter elements to induce FSHß expression, some of which bind FOXL2. Each of these FOXL2-binding sites is either juxtaposed or overlapping with a SMAD-binding element. We determined that FOXL2 and SMAD4 proteins form a higher order complex on the most proximal FOXL2 site. Surprisingly, two other sites important for activin induction bind neither SMADs nor FOXL2, suggesting additional factors at work. Furthermore, we show that FOXL2 plays a role in synergistic induction of FSHß by GnRH and activin through interactions with the cJUN component of the AP1 complex that is necessary for GnRH responsiveness. Collectively, our results demonstrate the necessity of FOXL2 for proper FSH production in mice and implicate FOXL2 in integration of transcription factors at the level of the FSHß promoter.

Nonuniform molecular features of myelinating Schwann cells in models for CMT1: distinct disease patterns are associated with NCAM and c-Jun upregulation.

We investigated three models for Charcot-Marie-Tooth type 1 (CMT1) neuropathy, comprising mice lacking connexin 32 (Cx32def), mice with reduced myelin protein zero (P0) expression (P0het) and transgenic mouse mutants overexpressing peripheral myelin protein 22 (PMP22tg), with regard of the expression of the developmentally regulated molecules NCAM, L1, the low-affinity NGF-receptor p75 (p75(NTR) ) and the transcription factor component c-Jun. We found that all molecules were uniformly expressed by myelin deficient and supernumerary Schwann cells. The mutant myelinating Schwann cells of PMP22tg mice showed a robust NCAM-immunoreactivity in Schmidt-Lanterman incisures (SLI) that accompanies other early onset abnormalities, such as the presence of supernumerary Schwann cells and impaired myelin formation in some fibers. In line with this, Cx32def and P0het mice, which represent demyelinating models, only rarely express NCAM in SLI. Surprisingly, c-Jun immunoreactivity displayed a mosaic-like pattern with mostly negative and some weakly or moderately positive nuclei both in myelinating Schwann cells and Remak cells of wildtype (wt), P0het and PMP22tg mice. However, c-Jun expression was substantially upregulated in myelinating Schwann cells of Cx32def mice and spatially associated with axon perturbation, a typical predemyelinating feature of Cx32 deficiency. Additionally, c-Jun upregulation was correlated with an elevated level of GDNF, possibly causally linked to the typical compensatory sprouting of axons in Cx32def mice and CMT1X patients. Our findings suggest that in myelinating Schwann cells of distinct models of CMT1, c-Jun upregulation is a marker for predemyelinating axonal perturbation while myelin-related NCAM expression is indicative for early Schwann cell abnormalities.

Traumatic scratch injury in astrocytes triggers calcium influx to activate the JNK/c-Jun/AP-1 pathway and switch on GFAP expression.

Astrocyte activation is a hallmark of central nervous system injuries resulting in glial scar formation (astrogliosis). The activation of astrocytes involves metabolic and morphological changes with complex underlying mechanisms, which should be defined to provide targets for astrogliosis intervention. Astrogliosis is usually accompanied by an upregulation of glial fibrillary acidic protein (GFAP). Using an in vitro scratch injury model, we scratched primary cultures of cerebral cortical astrocytes and observed an influx of calcium in the form of waves spreading away from the wound through gap junctions. Using the calcium blocker BAPTA-AM and the JNK inhibitor SP600125, we demonstrated that the calcium wave triggered the activation of JNK, which then phosphorylated the transcription factor c-Jun to facilitate the binding of AP-1 to the GFAP gene promoter to switch on GFAP upregulation. Blocking calcium mobilization with BAPTA-AM in an in vivo stab wound model reduced GFAP expression and glial scar formation, showing that the calcium signal, and the subsequent regulation of downstream signaling molecules, plays an essential role in brain injury response. Our findings demonstrated that traumatic scratch injury to astrocytes triggered a calcium influx from the extracellular compartment and activated the JNK/c-Jun/AP-1 pathway to switch on GFAP expression, identifying a previously unreported signaling cascade that is important in astrogliosis and the physiological response following brain injury.

Intrathecal application of short interfering RNA knocks down c-jun expression and augments spinal motoneuron death after root avulsion in adult rats.

The immediate-early gene, c-jun, is expressed in spinal motoneurons after spinal root avulsion. The expression of c-jun was suggested to be necessary for motoneuron survival and regeneration after avulsion. In the present study, a small interfering RNA (siRNA) was delivered intrathecally to the injured spinal segments immediately after root avulsion in rats to knock down expression of the c-jun gene in injured spinal motoneurons so as to explore the role of c-jun in the motoneurons in vivo. Our results showed that the siRNA not only inhibited the expression of both c-jun mRNA and protein but also augmented the death of injured motoneurons at day 14 post-injury. These findings indicated that induction of c-jun gene expression plays a pivotal role in the survival of injured motoneurons. Meanwhile, these results suggest that siRNAs applied intrathecally can effectively mediate the expression of the c-jun gene in injured motoneurons.

Prostaglandin F2α regulation of mRNA for activating protein 1 transcriptional factors in porcine corpora lutea (CL): lack of induction of JUN and JUND in CL without luteolytic capacity.

Porcine corpora lutea (CL) develop sensitivity to regression by prostaglandin F2α (PGF2α), termed luteolytic capacity, about 13 d after estrus. We postulated that PGF2α regulation of activating protein 1 (AP-1) transcriptional factor expression underlies acquisition of luteolytic capacity. CL were collected from gilts on day 9 (estrous cycle) or day 17 (pseudopregnancy) before or after PGF2α treatment with mRNA measured for FOS, FOSB, FOSL1, FOSL2, JUN, JUNB, and JUND and the AP-1 target genes CCL2 and SERPINE1. At 0.5 h after PGF2α, both day-9 and day-17 CL had increased (P < 0.01) mRNA for FOS (2,225% and 1,817%), JUNB (237% and 358%), and FOSB (1,060% and 925%). Intriguingly, at 0.5 h after PGF2α there was increased (P < 0.01) mRNA encoding JUN (1,099%) and JUND (300%) in day-17 but not day-9 CL. At 10 h after PGF2α there was elevated FOSB mRNA in day-17 (771%) but not day-9 CL and no PGF2α-induced change in FOS, JUN, JUND, and JUNB mRNA in day-9 or day-17 CL. Treatment with PGF2α increased mRNA for AP-1-responsive genes, CCL2 at 0.5 h (202%) and CCL2 and SERPINE1 at 10 h (719% and 1,515%), only in day-17 CL. Thus, many of the fos family of transcription factors are dramatically induced by PGF2α in CL with or without luteolytic capacity. However, PGF only induced JUN and JUND expression in CL with luteolytic capacity, a finding that may be key for understanding the acquisition of luteolytic capacity, given that JUN is the only AP-1 family member with strong N-terminal trans-activation activity.

Pro-life role for c-Jun N-terminal kinase and p38 mitogen-activated protein kinase at rostral ventrolateral medulla in experimental brain stem death.

BACKGROUND: Based on an experimental brain stem death model, we demonstrated previously that activation of the mitogen-activated protein kinase kinase 1/2 (MEK1/2)/extracellular signal-regulated kinase 1/2 (ERK1/2)/ mitogen-activated protein kinase signal-interacting kinase 1/2 (MNK1/2) cascade plays a pro-life role in the rostral ventrolateral medulla (RVLM), the origin of a life-and-death signal detected from systemic arterial pressure, which sequentially increases (pro-life) and decreases (pro-death) to reflect progressive dysfunction of central cardiovascular regulation during the advancement towards brain stem death in critically ill patients. The present study assessed the hypothesis that, in addition to ERK1/2, c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38MAPK), the other two mammalian members of MAPKs that are originally identified as stress-activated protein kinases, are activated specifically by MAPK kinase 4 (MAP2K4) or MAP2K6 and play a pro-life role in RVLM during experimental brain stem death. We further delineated the participation of phosphorylating activating transcriptional factor-2 (ATF-2) and c-Jun, the classical transcription factor activated by JNK or p38MAPK, in this process. RESULTS: An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos (Mev; 10 nmol) bilaterally into RVLM of Sprague-Dawley rats was used, alongside cardiovascular, pharmacological and biochemical evaluations. Results from ELISA showed that whereas the total JNK, p38MAPK, MAP2K4 and MAP2K6 were not affected, augmented phosphorylation of JNK at Thr183 and Tyr185 and p38MAPK at Thr180 and Tyr182, accompanied by phosphorylation of their upstream activators MAP2K4 at Ser257 and Thr261 and MAP2K6 at Ser207 and Thr211 in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Moreover, the activity of transcription factors ATF-2 at Thr71 and c-Jun at Ser73, rather than Elk-1 at Ser383 in RVLM were also augmented during the pro-life phase. Furthermore, pretreatment by microinjection into the bilateral RVLM of specific JNK inhibitors, JNK inhibitor I (100 pmol) or SP600125 (5 pmol), or specific p38MAPK inhibitors, p38MAPK inhibitor III (500 pmol) or SB203580 (2 nmol), exacerbated the depressor effect and blunted the augmented life-and-death signal exhibited during the pro-life phase. On the other hand, pretreatment with the negative control for JNK or p38MAPK inhibitor, JNK inhibitor I negative control (100 pmol) or SB202474 (2 nmol), was ineffective in the vehicle-controls and Mev-treatment groups. CONCLUSIONS: Our results demonstrated that activation of JNK or p38MAPK in RVLM by their upstream activators MAP2K4 or MAP2K6 plays a preferential pro-life role by sustaining the central cardiovascular regulatory machinery during experimental brain stem death via phosphorylation and activation of nuclear transcription factor ATF-2 or c-Jun.

Induction of dsRNA-activated protein kinase links mitochondrial unfolded protein response to the pathogenesis of intestinal inflammation.

OBJECTIVE: Inflammatory bowel diseases (IBDs) feature multiple cellular stress responses, including endoplasmic reticulum (ER) unfolded protein responses (UPRs). UPRs represent autoregulatory pathways that adjust organelle capacity to cellular demand. A similar mechanism, mitochondrial UPR (mtUPR), has been described for mitochondria. ER UPR in intestinal epithelial cells (IECs) contributes to the development of intestinal inflammation, and since mitochondrial alterations and dysfunction are implicated in the pathogenesis of IBDs, the authors characterised mtUPR in the context of intestinal inflammation. METHODS: Truncated ornithine transcarbamylase was used to selectively induce mtUPR in a murine IEC line. Dextran sodium sulphate (DSS) was administered to PKR (double-stranded-RNA-activated protein kinase) knockout mice to induce IEC stress in vivo and to test for their susceptibility to DSS-induced colitis. Expression levels of the mitochondrial chaperone chaperonin 60 (CPN60) and PKR were quantified in IECs from patients with IBDs and from murine models of colitis using immunohistochemistry and Western blot analysis. RESULTS: Selective mtUPR induction by truncated ornithine transcarbamylase transfection triggered the phosphorylation of eukaryotic translation initiation factor (eIF) 2α and cJun through the recruitment of PKR. Using pharmacological inhibitors and small inhibitory RNA, the authors identified mtUPR-induced eIF2α phosphorylation and transcription factor activation (cJun/AP1) as being dependent on the activities of the mitochondrial protease ClpP and the cytoplasmic kinase PKR. Pkr(-/-) mice failed to induce CPN60 in IECs upon DSS treatment at early time points and subsequently showed an almost complete resistance to DSS-induced colitis. Under inflammatory conditions, primary IECs from patients with IBDs and two murine models of colitis exhibited a strong induction of the mtUPR marker protein CPN60 associated with enhanced expression of PKR. CONCLUSION: PKR integrates mtUPR into the disease-relevant ER UPR via eIF2α phosphorylation and AP1 activation. Induction of mtUPR and PKR was observed in IECs from murine models and patients with IBDs. The authors' results indicate that PKR might link mitochondrial stress to intestinal inflammation.

LPA stimulates intestinal DRA gene transcription via LPA2 receptor, PI3K/AKT, and c-Fos-dependent pathway.

DRA (downregulated in adenoma) or SLC26A3 is the major apical anion exchanger mediating Cl(-) absorption in intestinal epithelial cells. Disturbances in DRA function and expression have been implicated in diarrheal conditions such as congenital chloride diarrhea and inflammatory bowel diseases. Previous studies have shown that DRA is subject to regulation by short-term and transcriptional mechanisms. In this regard, we have recently shown that short-term treatment by lysophosphatidic acid (LPA), an important bioactive phospholipid, stimulates Cl(-)/HCO(3)(-)(OH(-)) exchange activity via an increase in DRA surface levels in human intestinal epithelial cells. However, the long-term effects of LPA on DRA at the level of gene transcription have not been examined. The present studies were aimed at investigating the effects of LPA on DRA function and expression as well as elucidating the mechanisms underlying its transcriptional regulation. Long-term LPA treatment increased the Cl(-)/HCO(3)(-) exchange activity in Caco-2 cells. LPA treatment (50-100 µM) of Caco-2 cells significantly stimulated DRA mRNA levels and DRA promoter activity (-1183/+114). This increase in DRA promoter activity involved the LPA2 receptor and phosphatidylinositol 3-kinase (PI3K)/AKT pathways. Progressive deletions from -1183/+114 to -790/+114 abrogated the stimulatory effects of LPA, indicating that the -1183/-790 promoter region harbors LPA response elements. Utilizing EMSA and mutational studies, our results showed that LPA induced the DRA promoter activity in a c-Fos-dependent manner. LPA also increased the protein expression of c-Fos and c-Jun in Caco-2 cells. Furthermore, overexpression of c-Fos but not c-Jun enhanced the DRA promoter activity. This increase in DRA transcription in response to LPA indicates that LPA may act as an antidiarrheal agent and could be exploited for the treatment of diarrhea associated with inflammatory or infectious diseases of the gut.

Genes involved in the immediate early response and epithelial-mesenchymal transition are regulated by adipocytokines in the female reproductive tract.

Obesity increases the risk of female reproductive tract cancers, but the underlying mechanistic link between the two is ill-defined. Thus, the objective of the current study was to identify obesity-dependent changes in the expression of immediate early (IE) genes that contribute to cell proliferation and differentiation, and epithelial-mesenchymal transition (EMT) genes that promote cell migration. When HeLa cells were treated for 0-48 hr with IGF-1, leptin, TNFα, or IL-6, each individual adipocytokine altered the abundance of IE (cJUN, cFOS, and cMYC) and EMT (SNAI1, SNAI2, and TWIST1) mRNA abundance. For example, IGF-1 increased cJUN and cFOS and decreased cMYC; leptin increased cFOS; IL-6 increased cFOS and cMYC; and TNFα increased cJUN and cFOS mRNA abundance. Likewise, EMT gene expression was altered by IGF-1, TNFα, and IL-6. SNAI1 was increased by IGF-1 and IL-6; SNAI2 was increased by IGF-1 and TNFα; and TWIST1 was increased by TNFα and IL-6. Chronic exposure to adipocytokines also altered EMT gene expression in the whole uterus of obese compared to normal-weight mice. Specifically, there was no difference in cJun, cFos, or cMyc mRNA abundance between normal-weight and obese animals. Snai1, Snai2, and Twist1 mRNA abundance, however, was increased in the uterus of obese females and correlated with increased circulating IGF-1 levels. These data indicate that obesity-dependent alterations in adipocytokine levels regulate the expression of genes associated with cell proliferation and migration, and therefore may provide a plausible mechanism for obesity-dependent increases in cancers of the female reproductive tract.

CD44 mediates oligosaccharides of hyaluronan-induced proliferation, tube formation and signal transduction in endothelial cells.

Oligosaccharides of hyaluronan (o-HA) can induce angiogenesis and the growth and tube formation of vascular endothelial cells (ECs) in particular. As the major o-HA receptor, CD44 has been implicated in EC function, but its role in mediating o-HA-induced EC proliferation and tube formation remains unclear. In this study, we investigated the role of CD44 in o-HA-induced proliferation and tube formation of human umbilical vein endothelial cells (HUVECs) and explored the molecular mechanisms underlying the angiogenesis process. A CD44 siRNA was delivered into HUVECs by electroporation and o-HA-induced proliferation and tube formation capacity of CD44-silenced or control HUVECs were assessed by methylthiazolyldiphenyl-tetrazolium bromide (MTT) and Matrigel assays. Furthermore, the changes in Src, focal adhesion kinase (FAK) and extracellular signal-regulated kinase1 and 2 (ERK1/2) phosphorylation, as well as the expression of c-jun and c-fos were examined by Western blot and realtime-polymerase chain reaction assays. Our results demonstrated that 10 µg/mL o-HA obviously induced the proliferation and tube formation in HUVECs, and stimulated the phosphorylation of Src, FAK and ERK1/2 and upregulation of c-jun and c-fos, which could be inhibited by CD44 silencing. Altogether our data suggest that CD44 functions to initiate tyrosine phosphorylation of Src, FAK and ERK1/2, and upregulates the expression of c-jun and c-fos, thus mediating o-HA-induced proliferation and tube formation in HUVECs.